Bioregulators for Pest Control 9780841209107, 9780841211070, 0-8412-0910-3

Table of contents :
Title Page ......Page 1
Half Title Page ......Page 3
Copyright ......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
1 Physiological Basis of Phloem Transport of Agrichemicals......Page 9
Physiological Basis of Phloem Translocation......Page 10
Agrichemical Transport......Page 16
Acknowledgment......Page 19
Literature Cited......Page 20
2 Interference by Herbicides with Photosynthetic Electron Transfer......Page 21
Herbicide Binding Experiments......Page 22
Photoaffinity Labeling of the Herbicide Binding Proteins......Page 24
Herbicide/Quinone Interactions......Page 28
An Azidoplastoquinone Photoaffinity Label......Page 29
The 34 kDa Herbicide Binding Protein: The Molecular Level......Page 30
Literature Cited......Page 33
3 New Approaches to Chemical Control of Plant Pathogens......Page 36
Second Generation Fungicides......Page 37
Third Generation Fungicides......Page 41
Resistance Mechanisms......Page 42
Conclusions......Page 44
Literature Cited......Page 45
Protection of Plants Against Pests......Page 47
Statement of Thesis......Page 50
Methods of Sensitization......Page 51
Non-specificity of Protection......Page 52
Dynamics of Induced Resistance......Page 54
Chemically Induced Resistance......Page 60
Summary......Page 63
Literature Cited......Page 64
5 Use of Subtoxic Herbicide Pretreatments to Improve Crop Tolerance to Herbicides......Page 69
Mode of Action of R-25788......Page 70
Increasing Crop Tolerance to Herbicides with Herbicide Pretreatments......Page 74
Effect of CDAA pretreatments on subsequent CDAA metabolism......Page 79
Effects of R-25788 and EPTC on the GSH/GSH-S-transferase System......Page 80
Literature Cited......Page 83
6 Regulation of Plant Growth and Development by Endogenous Hormones......Page 85
Auxins......Page 86
Gibberellins......Page 87
Cytokinins......Page 88
Abscisic Acid......Page 90
Anthesins......Page 91
Literature Cited......Page 92
7 Plant Bioregulators: Overview, Use, and Development......Page 94
What substances and what principles of action are available?......Page 95
How have plant bioregulators been used so far in crop production and where are they employed in particular?......Page 101
What factors will have a major influence on determining the development of new plant bioregulators and on opening up further possibilities for use in crop production?......Page 103
Literature Cited......Page 105
8 Effects of Allelopathic Chemicals on Crop Productivity......Page 107
Actions and Interactions of Allelochemicals......Page 110
Allelochemical Interference in Agricultural Fields......Page 113
Exploiting Allelopathy and Allelochemicals......Page 118
Conclusions......Page 122
Literature Cited......Page 123
9 Use of Transition-State Theory in the Development of Bioactive Molecules......Page 129
Transition State Theory......Page 130
Transition State Analogs (TSA)......Page 137
Application of TS Theory to Organophosphate and Carbamate Insecticides......Page 142
TSA as Inhibitors of Juvenile Hormone Esterase(s)......Page 143
Literature Cited......Page 152
10 Role of Mixed-Function Oxidases in Insect Growth and Development......Page 155
Xenobiotic Metabolism......Page 156
Role of Mixed-function Oxidases in Steroidogenic Reactions......Page 157
Steroidogenesis in Insects......Page 159
Juvenile Hormone......Page 165
Summary......Page 167
Literature Cited......Page 169
11 Inhibition of Reproduction in Insect Control......Page 171
Compounds that Interfere with the Development and/or Function of the Gonads......Page 172
Compounds that Prevent the Development of Progeny......Page 173
Literature Cited......Page 174
12 Potent Insect Antifeedants from the African Medicinal Plant Bersama abyssinica......Page 177
Structure of abyssinin......Page 179
Structure of Abyssinol A, B, and C......Page 187
Antifeedant Activity......Page 190
Literature Cited......Page 194
13 Cockroach Control with Juvenoids......Page 195
Assay for Comparative Activity......Page 196
Residue test on latex painted surface......Page 201
Persistence of hydroprene residues on various household surface substrates......Page 202
Vapor test......Page 204
Aquarium and chamber experiments......Page 205
Field Experiments......Page 206
Discussion......Page 208
Literature cited......Page 211
14 Some Chemical Ecological Approaches to the Control of Stored-Product Insects and Mites......Page 213
Control of Feeding Behavior......Page 214
Control of Mating Behavior......Page 216
Control of Oviposition Behavior......Page 217
Literature Cited......Page 218
15 Phytochemical Disruption of Insect Development and Behavior......Page 219
Insect Phytohormones......Page 220
Insect Phytophreromones......Page 224
Summary......Page 226
Literature Cited......Page 228
16 Proallatocidins......Page 231
Chemical Stabilization of Precocene Structures......Page 232
Modification of transport properties......Page 233
Chemical Studies of 3,4-epoxyprecocenes......Page 234
Acknowledgments......Page 236
Literature cited......Page 237
17 Propionate and Methyl Malonate Metabolism in Insects......Page 238
Sources of Propionate and Methylmalonate......Page 239
Metabolism of Propionate......Page 240
Summary......Page 244
Literature Cited......Page 245
18 Suicidal Destruction of Cytochrome P-450 In the Design of Inhibitors of Insect Juvenile Hormone Biosynthesis......Page 247
Materials and Methods......Page 250
Results......Page 251
Discussion......Page 255
Literature Cited......Page 257
19 Detoxification Enzyme Relationships in Arthropods of Differing Feeding Strategies......Page 259
Enzyme Associations with Herbivore Status......Page 260
Relevance of Detoxification Dissimilarities to In Vivo Toxicosis......Page 266
Literature Cited......Page 269
20 δ-Endotoxin of Bacillus thuringiensis var. israelensis Broad-Spectrum Toxicity and Neural Response Elicited in Mice and Insects......Page 271
SDS-PAGE Analysis of BTK and BTI δ-Endotoxin......Page 272
δ-Endotoxin Toxicity in Mice and Insects......Page 273
Neural Toxicity of BTI δ-Endotoxin......Page 277
Conclusions......Page 282
Literature Cited......Page 283
21 Bioassay of Anti Juvenile Hormone Compounds: An Alternative Approach......Page 285
The Bioassay......Page 286
Discussion......Page 294
Literature Cited......Page 297
General ELISA Methodology......Page 299
Examples of Pesticide Immunoassays......Page 301
Paraquat......Page 302
Conclusions......Page 307
Literature Cited ......Page 308
23 Role of Natural Product Chemistry......Page 312
Biorational Approaches......Page 313
Natural Products in Pest Control......Page 314
Economic Aspects......Page 315
Resistant Plants and Ecological Chemistry......Page 316
Natural Products as Leads to New Pesticides......Page 318
Behavioural Compounds......Page 320
Conclusion......Page 322
Literature Cited......Page 323
24 Do Plants "Psychomanipulate" Insects?......Page 325
The Psychomanipulation Scenario......Page 326
Action in the CNS of Plant-derived Compounds......Page 328
Candidate Psychomanipulants of Plant Origin......Page 331
Summary......Page 337
Literature Cited......Page 338
Uses of Attractants......Page 340
Tephritid Attractants......Page 342
Experimental Approach, Results, and Discussion......Page 345
Acknowledgements......Page 350
Literature Cited......Page 351
26 Beetles: Pheromonal Chemists par Excellence......Page 354
Scolytidae......Page 356
Curculionidae......Page 358
Scarabaeidae......Page 359
Chrysomelidae......Page 360
Other Families......Page 363
Literature Cited......Page 365
27 Sexual Messages of Moths: Chemical Themes Are Known and New Research Challenges Arise......Page 368
Literature Cited......Page 377
28 Alkaloidal Ant Venoms: Chemistry and Biological Activities......Page 379
Alkalodial Chemistry of Ant Venoms......Page 380
Pyrrolidines.......Page 381
Piperidines.......Page 382
Piperideines.......Page 385
Pyrazines.......Page 386
Insecticidal Activities.......Page 388
Repellent (Deterrent) Activities.......Page 390
Conclusions......Page 391
Literature Cited......Page 392
Control of reproduction......Page 395
Using hormones as insect growth regulators......Page 396
Ivermectin feeding studies......Page 400
Safety......Page 406
The need for integrated control programs......Page 407
Immunological methods......Page 408
Use of oils and allelochemicals......Page 412
Legend of Symbols......Page 413
Literature cited......Page 416
30 Insect Antifeedant Terpenoids in Wild Sunflower A Possible Source of Resistance to the Sunflower Moth......Page 419
Terpenoids from wild species of Helianthus......Page 420
Toxicity and antifeedant activity of Helianthus sesquiterpene lactones to sunflower insects......Page 421
Melanoplus sanguinipes (Orthoptera: Acrididae ) = the migratory grasshopper.......Page 424
Possible role of sesquiterpene lactones in resistance to the sunflower moth.......Page 427
Literature Cited......Page 429
31 Insect Feeding Deterrents from Semiarid and Arid Land Plants......Page 433
Sesquiterpene Lactones as Insect Feeding Deterrents......Page 434
Literature Cited......Page 438
32 Secondary Metabolites from Plants and Their Allelochemic Effects......Page 440
Literature Cited......Page 452
33 Insect Antifeedants from the Peruvian Plant Alchornea triplinervia......Page 454
Tobacco Budworm Larval Growth Bioassay......Page 455
Extraction of Alchornea triplinervia Leaves......Page 456
Tobacco Budworm Growth Study......Page 459
Conclusion......Page 460
Literature Cited......Page 461
34 Biotechnology in Crop Improvement......Page 462
Acknowledgments......Page 494
Literature Cited......Page 495
Insecticyanin......Page 496
Hemolymph Lipoprotein......Page 497
Adult Lipophorin......Page 503
Literature Cited......Page 506
Author Index......Page 507
A......Page 508
Β......Page 509
C......Page 510
Ε......Page 512
G......Page 513
H......Page 514
I......Page 515
M......Page 516
O......Page 517
Ρ......Page 518
R......Page 519
S......Page 520
V......Page 521
Y ......Page 522

Citation preview

Bioregulators for Pes Contro

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

ACS

SYMPOSIUM

SERIES

Bioregulators for Pest Control Paul A. Hedin, EDITOR U.S. Department of Agriculture ASSOCIATE EDITORS

Horace G. Cutler, Bruce D. Hammock, Julius J. Menn, Donald E. Moreland, and Jack R. Plimmer

Based on a symposium sponsored by the Division of Pesticide Chemistry at the Division of Pesticide Chemistry Special Conference II, Snowbird, Utah, June 24-29, 1984

American Chemical Society, Washington, D.C. 1985

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

276

Library of Congress Cataloging in Publication Data Bioregulators for pest control. ( A C S symposium series, I S S N 0097-6156; 276) "Based on a symposium sponsored by the Division of Pesticide Chemistry at the Division of Pesticide Chemistry Special Conference II, Snowbird, Utah, June 24-29, 1984." I. Pesticides—Congresses. 2. Congresses. I. Hedin, Paul A . (Paul Arthur), 1926 II. American Chemical Society. Division of Pesticide Chemistry. SB950.93.B56 1985 I S B N 0-8412-0910-3

632'.95

85-6087

Copyright © 1985 American Chemical Society A l l Rights Reserved. T h e appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., 21 Congress Street, Salem, MA 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names a n d / o r names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

ACS Symposium Series M . Joan Comstock, Series Editor Advisory Board Robert Baker U.S. Geological Survey Martin L . Gorbaty Exxon Research and Engineering Co.

, Regiona Research Center Geoffrey D. Parfitt Carnegie-Mellon University

Roland F. Hirsch U.S. Department of Energy

James C. Randall Phillips Petroleum Company

Herbert D. Kaesz University of California—Los Angeles

Charles N. Satterfield Massachusetts Institute of Technology

Rudolph J. Marcus Office of Naval Research

W. D. Shults Oak Ridge National Laboratory

Vincent D. McGinniss Battelle Columbus Laboratories

Charles S. Tuesday General Motors Research Laboratory

Donald E . Moreland USDA, Agricultural Research Service

Douglas B. Walters National Institute of Environmental Health

W. H . Norton J. T. Baker Chemical Company

C. Grant Willson IBM Research Department

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

FOREWORD The ACS S Y M P O S I U M S E R I E S was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the Serie IN C H E M I S T R Y

SERIE

papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

PREFACE PESTICIDES ARE BIOREGULATORS because they regulate various aspects of life processes. We are moving into a new era of regulating growth in a broad spectrum of pests that includes plants, insects, and diseases. Bioregulators can be characterized as being "endogenous," that is, originating within the organism, or "exogenous" where the agent obtained from an outside source acts to induce a desirable response in a treated species. These bioregulators may have a natural or synthetic origin. Included in the specia Chemistry were symposia Growth," "Control of Pests with Natural Products," and "Molecular Biology and Genetic Engineering." The first three symposia comprise the three major sections of the book. Additionally, a chapter on "The Impact of Biotechnology on Crop Improvement" is included. The "Control of Plant Growth" symposium, organized by Donald E. Moreland, highlighted the current status of knowledge on the mechanisms of action of herbicides, fungicides, and endogenous plant hormones. Other topics that were discussed included the phloem transport system of plants, development of pathogen resistance to fungicides, strategies for controlling pathogens by manipulation or potentiation of the plant's defense mechanisms, integration of exogenous plant bioregulators into crop production practices, methods for increasing tolerance of crop plants to herbicides, and the effects of allelochemicals on plant growth and development. The "Control of Insect Growth" symposium, organized by Julius Menn, highlighted recent advances in the biochemistry of regulation of development by insect growth regulators, anti juvenile hormones, and behavior modification governed by antifeedants, pheromones, and defensive secretions. The "Control of Pests with Natural Products" symposium was organized by Jack R. Plimmer and moderated by Horace G. Cutler in Plimmer's absence. The role of natural products in the control of pests has increased in recent decades as the chemist has acquired more sophisticated tools with which to elucidate complex structures. This, in turn, has led to explosive growth in the understanding of biochemical processes. Knowledge of metabolism, biosynthetic processes, neurochemistry, regulatory mechanisms, and many other aspects of plant, animal, and insect biochemistry has provided a more complete basis for understanding the modes of action of pesticides. The exploitation of biological information with a chemical basis (i.e., biorational approaches) may lead to the synthesis of a new molecule designed to act at a particular site or to block a key step in a biochemical process.

xi In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Additionally, a poster session was held during the conference under the direction of Bruce D. Hammock that consisted of 15 presentations. From these, seven manuscripts and two abstracts were submitted, of which six and the abstracts are included in the section on "Control of Insect Growth." The other is included in the section on "Control of Pests with Natural Products." Finally, chapters based on the banquet address by John H . Law and on "Biotechnology in Crop Improvement" by John T. Marvel are also included in the section on "Control of Pests with Natural Products." It is the hope of the editors that this book will contribute to the elucidation and subsequent adoption of yet additional "New Concepts" including novel chemical compounds for the control of pests. These compounds should be effective at low concentrations selective in activity against specific pests, o environmentally nonpersistent, and we should have a understanding of their mechanisms of action. It is the further hope of the editors that this book will serve as a document that identifies unifying themes by which research to control pests can be conducted. We thank Nicholas A. Mangan and Henry J. Dishburger for their excellent administrative management of the conference and Gerald G. Still for his organization and chairing of the section on "Molecular Biology and Genetic Engineering." We are also grateful to all of the participants for their contributions that added materially to this book. Finally, I thank the Agricultural Research Service of the U.S. Department of Agriculture for granting me permission to organize the conference and compile the book. PAUL A. HEDIN

U.S. Department of Agriculture Mississippi State, MS 39762 Associate Editors Horace G. Cutler U.S. Department of Agriculture Athens, G A 30613

Bruce D. Hammock University of California Davis, C A 95616

Julius J. Menn Zoecon Corporation Palo Alto, C A 94304

Donald E. Moreland U.S. Department of Agriculture Raleigh, NC 27695-7620 Jack R. Plimmer International Atomic Energy Agency Vienna, Austria

January 17, 1985

xii In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1 Physiological Basis of Phloem Transport of Agrichemicals ROBERT T. GIAQUINTA Experimental Station, Central Research and Development Department, Ε. I. du Pont de Nemours & Co., Inc., Wilmington, DE 19898 Agrichemical transpor terms of the physiological tural bases of assimilate translocation. Specifically, the cellular pathways and mechanisms of phloem loading in source leaves, long distance transport, and phloem unloading in sinks, are used as a framework for examin­ ing the biological basis of the systemic mobility of agrichemicals. Phloem transport is the process responsible for the systemic mobility of agrichemicals in plants. From a practical viewpoint, knowledge of the structural and chemical properties of molecules that are necessary for phloem mobility should have considerable impact on the rational design of systemic agrichemicals with im­ proved efficacy. Unfortunately, l i t t l e practical information exists on structure-activity relationships of agrichemicals with respect to phloem mobility. That is, what is it about a molecule that governs its ability to be translocated in the phloem? In general, several factors ultimately determine whether an agrichemical moves to its site of action in the plant. These include: (1) efficient chemical penetration through the cuticle of the leaves and stems; (2) the ability of the chemical to enter the symplast or metabolic compartment of the cell ( i . e . , crossing the cell membrane); (3) short- and long-distance transport, either cell-to-cell via plasmodesmata or in the xylem, or phloem; (4) metabolism or conju­ gation of an agrichemical to an inactive form; and (5) immobiliza­ tion of the agrichemical at non-active sites (e.g., binding at the cell walls, sequestering in the vacuole, or adsorption to cellular protein). No single factor will dictate whether a chemical is translocated in a l l cases and a complex interrelationship probably exists between a l l of these. The reader is referred to several recent and comprehensive reviews on various aspects of xenobiotic entry and transport within plants (1-5). In this review I address the phloem mobility of agrichemicals from the viewpoint of a phloem physiologist. First, I will present an overview of the physiological basis of translocation by using 0097-6156/85/0276-0007$06.00/0 © 1985 American Chemical Society

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

8

BIOREGULATORS FOR PEST C O N T R O L

what we know about the i n v i v o t r a n s p o r t o f s u c r o s e as a way o f d e s c r i b i n g the c h a r a c t e r i s t i c s o f the t r a n s l o c a t i o n system which are r e l e v a n t t o a g r i c h e m i c a l t r a n s p o r t . T h i s i s i m p o r t a n t because i f we d e s i r e t o r a t i o n a l l y d e s i g n m o l e c u l e s w i t h improved phloem m o b i l i t y , we need t o be aware o f the s t r u c t u r a l , b i o c h e m i c a l , b i o p h y s i c a l , and p h y s i o l o g i c a l a s p e c t s o f the t r a n s p o r t p r o c e s s i t s e l f . The second p a r t o f t h i s r e v i e w w i l l h i g h l i g h t the p r o p e r t i e s o f x e n o b i o t i c movement i n p l a n t s . A l t h o u g h space c o n s t r a i n t s i n e v i t a b l y l i m i t an i n - d e p t h treatment o f t h i s s u b j e c t , I hope the b r o a d - s t r o k e approach p r e s e n t e d here w i l l g e n e r a t e a b e t t e r u n d e r s t a n d i n g o f the b i o l o g i ­ c a l b a s i s f o r t r a n s l o c a t i o n and, more i m p o r t a n t l y , spur a d d i t i o n a l r e s e a r c h i n the r e l a t i v e l y u n c h a r t e d a r e a o f phloem p h y s i o l o g y and agrichemical transport. P h y s i o l o g i c a l B a s i s o f Phloem T r a n s l o c a t i o n The t r a n s l o c a t i o n system i s u s u a l l y d i v i d e d i n t o t h r e e s t r u c t u r a l l y and p h y s i o l o g i c a l l y d i s t i n c photosynthetic leaves producin c o n n e c t i n g s i e v e elements which comprise the c o n d u i t s f o r a s s i m i l a t e f l o w ; and (3) the s i n k , which i s comprised o f a s s i m i l a t e - c o n s u m i n g o r t a r g e t c e l l s ( e . g . , growing, u t i l i z i n g , o r s t o r a g e r e g i o n s ) . In s o u r c e l e a v e s , phloem l o a d i n g i s the p r o c e s s whereby photos y n t h e t i c a l l y - d e r i v e d s u c r o s e produced i n the m e s o p h y l l i s accumu­ l a t e d i n t o the minor v e i n network o f the phloem. This sucrose accu­ m u l a t i o n i n c r e a s e s the s o l u t e p o t e n t i a l o f the s i e v e t u b e s , c a u s i n g water from the s u r r o u n d i n g t i s s u e s t o e n t e r the phloem t o produce h y d r o s t a t i c p r e s s u r e (6^, _7). At the s i n k end, a s s i m i l a t e s e x i t the s i e v e tubes by a v a r i e t y o f mechanisms (see below) t h e r e b y r e d u c i n g the s u c r o s e c o n c e n t r a t i o n i n t h i s p a r t o f the system ( 8 ) . Because o f t h i s p r e s s u r e and c o n c e n t r a t i o n d i f f e r e n c e , water, s u c r o s e , and any o t h e r s u b s t a n c e ( i n c l u d i n g a g r i c h e m i c a l s ) p r e s e n t i n the phloem w i l l move i n b u l k o r mass f l o w from s o u r c e t o s i n k . The d i r e c t i o n o f t h i s o s m o t i c a l l y - d r i v e n flow i n the phloem i s governed s o l e l y by the p o s i t i o n o f s o u r c e s and s i n k s i n the p l a n t . However, the p o s i ­ t i o n o f these s o u r c e s and s i n k s can d i f f e r a t d i f f e r e n t s t a g e s o f l e a f development o r p l a n t ontogeny (_7). Phloem L o a d i n g . How does s u c r o s e which i s produced i n the m e s o p h y l l c e l l s o f s o u r c e l e a v e s e n t e r the t r a n s l o c a t i o n stream and how does t h i s s u c r o s e e x i t from the t r a n s l o c a t i o n stream i n the s i n k r e g i o n s ? More i m p o r t a n t l y , can t h i s t e l l us a n y t h i n g about a g r i c h e m i c a l transport? F i g u r e 1A shows an a u t o r a d i o g r a p h o f a source l e a f f o l l o w i n g the a c c u m u l a t i o n o f - ^ C - s u c r o s e ( l ^ C - l a b e l i s i n w h i t e ) . The l ^ C - l a b e l i s accumulated markedly i n t o the v e i n network com­ p r i s e d o f the minor v e i n phloem. T h i s i s an e x t e n s i v e network about 70 cm veins/cm^ l e a f - and thus i t r e p r e s e n t s an e f f i c i e n t c o l l e c t i n g system f o r b o t h s u c r o s e which i s produced i n the meso­ p h y l l and f o r c h e m i c a l s e n t e r i n g the l e a f . F i g u r e IB i l l u s t r a t e s d i a g r a m m a t i c a l l y a c r o s s s e c t i o n o f a s i n g l e minor v e i n t r a c e d from an e l e c t r o n m i c r o g r a p h . The v a s c u l a r bundle i s composed o f a s i n g l e xylem element and the phloem bundle which c o n t a i n s two c e n t r a l l y l o c a t e d s i e v e tubes surrounded by s p e c i a l i z e d phloem c e l l s c a l l e d e i t h e r companion c e l l s or t r a n s f e r c e l l s depending on the p r e s e n c e

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1.

GIAQUINTA

Phloem Transport of Agrichemicals

F i g u r e 1. Source l e a f minor v e i n phloem. (A) A u t o r a d i o g r a p h o f l e a f t i s s u e s f o l l o w i n g -^C-sucrose a c c u m u l a t i o n showing r a d i o ­ a c t i v i t y (white) i n v e i n s . (B) T r a c i n g o f an e l e c t r o n m i c r o ­ graph o f a c r o s s s e c t i o n o f minor v e i n , x, xylem, vp, v a s c u l a r parenchyma; c c , companion c e l l ; se, s i e v e element; pp, phloem parenchyma, mc, m e s o p h y l l c e l l . Reproduced w i t h p e r m i s s i o n from Ref. 6. C o p y r i g h t 1983. Annual Reviews.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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BIOREGULATORS FOR PEST CONTROL

o f c e l l w a l l ingrowths ( t r a n s f e r c e l l s have w a l l i n g r o w t h s ) . The e n t i r e bundle i s surrounded by m e s o p h y l l c e l l s and phloem parenchyma c e l l s (6). Substances can e n t e r the phloem v i a s e v e r a l r o u t e s , f o r example,from the a p o p l a s t , and c e l l - t o - c e l l (see arrows i n F i g u r e IB). S e v e r a l l i n e s o f e v i d e n c e show t h a t sugars do not s i m p l y d i f f u s e down a c o n c e n t r a t i o n g r a d i e n t from the m e s o p h y l l to phloem. I n s t e a d t h e r e i s a marked c o n c e n t r a t i o n o f sugars i n the s i e v e element-companion c e l l complex i n d i c a t i n g t h a t a c o n c e n t r a t i n g mechanism e x i s t s a t the m e s o p h y l l - p h l o e m i n t e r f a c e . The c u r r e n t body o f e v i d e n c e i n d i c a t e s t h a t p h o t o s y n t h e t i c a l l y d e r i v e d s u c r o s e t r a v e l s s y m p l a s t i c a l l y ( v i a plasmodesmata) to the phloem r e g i o n , then e x i t s the symplasm i n t o the a p o p l a s t where i t i s t h e n a c t i v e l y accumulated a c r o s s the phloem c e l l membranes ( 8 ) . The c u r r e n t w o r k i n g model f o r the mechanism o f s u c r o s e uptake a c r o s s the phloem membranes i s i l l u s t r a t e d i n F i g u r e 2. In t h i s model, s u c r o s e which i s the f r e e space, i n t e r a c t s w i t h a s u c r o s e - s p e c i f i c c a r r i e r on the membrane ( 6 ) . We know v e r y l i t t l e about t h i s p u t a t i v e s u c r o s y l c a r r i e r o t h e r than t h a t is highly selective for The c h a r a c t e r i s t i c s o f the phloem i t s e l f f i g u r e p r o m i n e n t l y i n t h i s proposed mechanism. The phloem i n t e r i o r has a low p r o t o n con­ c e n t r a t i o n (pH 7.5 t o 8.0) r e l a t i v e t o the a p o p l a s t which has a h i g h p r o t o n c o n c e n t r a t i o n (pH 5.5). Thus, a s u b s t a n t i a l p r o t o n g r a d i e n t o f up t o 2-3 pH u n i t s e x i s t s a c r o s s the phloem membranes. More c o r r e c t l y , t h e r e i s an e l e c t r o c h e m i c a l p o t e n t i a l g r a d i e n t a c r o s s the phloem membrane which g i v e s r i s e to an i n t e r i o r n e g a t i v e membrane p o t e n t i a l o f about -150 mv. It i s believed that t h i s electrochem­ i c a l p o t e n t i a l o f p r o t o n s which e x i s t s a c r o s s the phloem membranes i s the d r i v i n g f o r c e f o r s u c r o s e l o a d i n g . The g r a d i e n t i s e s t a b l i s h e d by a "metabolism-dependent" p r o t o n pump, presumably an ATPase enzyme which i s l o c a t e d on the phloem membrane. It i s envisioned t h a t s u c r o s e uptake i s c o u p l e d t o the c o - t r a n s p o r t o f p r o t o n s where­ by the e n e r g e t i c a l l y " d o w n h i l l " movement o f p r o t o n s i n t o the phloem i s c o u p l e d t o the " s e c o n d a r y " a c t i v e t r a n s p o r t o f s u c r o s e i n t o the phloem (6^). As d i s c u s s e d below, t h e s e c h e m i c a l and e l e c t r i c a l prop­ e r t i e s o f the phloem can i n f l u e n c e the a b i l i t y o f a g r i c h e m i c a l s t o e n t e r the phloem. The c h a r a c t e r i s t i c s o f the phloem l o a d i n g system can be sum­ m a r i z e d as f o l l o w s . Sucrose l o a d i n g i s : (1) dependent on metabo­ l i s m ; (2) c a r r i e r - m e d i a t e d ; (3) s e l e c t i v e f o r s u c r o s e ; (4) m a i n t a i n s a h i g h c o n c e n t r a t i o n i n s i d e the phloem which i s the b a s i s f o r the o s m o t i c a l l y - d r i v e n mass flow o f s o l u t i o n s ; and (5) dependent on the f a c t o r s which c o n t r o l a s s i m i l a t e s u p p l y t o the l o a d i n g s i t e s ( e . g . , p h o t o s y n t h e s i s , s u c r o s e s y n t h e s i s , and s u c r o s e movement between l e a f c e l l s , and w i t h i n s u b c e l l u l a r compartments such as the c y t o ­ plasm and v a c u o l e ) (^, 7^). V a s c u l a r Anatomy. One a s p e c t o f the t r a n s l o c a t i o n system t h a t i s o f t e n o v e r l o o k e d i s the i n f l u e n c e o f the s t r u c t u r a l f e a t u r e s o f the p l a n t ' s v a s c u l a r system on s o l u t e t r a n s p o r t . F o r example, a l t h o u g h much o f what i s known about phloem l o a d i n g has been d e r i v e d from a few d i c o t y l e d o n l e a v e s , a l l d i c o t y l e d o n l e a v e s are not s i m i l a r . A n o t a b l e example i s the soybean l e a f . Soybean l e a v e s a r e s p e c i a l i z e d i n t h a t they have a unique c e l l type c a l l e d the p a r a v e i n a l m e s o p h y l l

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1.

GIAQUINTA

Phloem Transport of Agrichemicals

SIEVE ELEMENT COMPANION CELL MEMBRANE

ATPost

pH 5.5 LOWK* LOW SUCROSE

pH 8.5 HIGH K* HIGH SUCROSE

(-)

Figure 2. Model for phloem loading of sucrose. See text for details. Reproduced with permission from Ref. 8. Copyright 1980. Academic Press.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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(PVM) (9^, 10). In c r o s s s e c t i o n the PVM appears as a s i n g l e , d i s ­ c o n t i n u o u s l a y e r o f c e l l s i n the c e n t e r of the l e a f ( F i g u r e 3A). The s i g n i f i c a n c e o f the c e l l type t o t r a n s p o r t , however, i s i n d i c a t e d i n a paradermal s e c t i o n ( F i g u r e 3B) which shows t h a t the PVM forms an i n t e r c o n n e c t i n g network o f c e l l s i n the c e n t e r o f the l e a f t h a t c o n n e c t s t o the phloem. A l l a s s i m i l a t e s produced i n the p a l i s a d e and spongy m e s o p h y l l appear to pass t h r o u g h the PVM b e f o r e they e n t e r the phloem. I t i s a l s o i n t e r e s t i n g t h a t the v a c u o l e s o f these c e l l s accumulate s u b s t a n t i a l amounts o f a g l y c o p r o t e i n d u r i n g c e r ­ t a i n s t a g e s o f l e a f development ( f l o w e r i n g t o e a r l y p o d - f i l l i n soy­ beans) (9^, 10). T h i s p r o t e i n , which p r o v i d e s a n i t r o g e n r e s e r v e f o r seed growth, c o u l d cause s e q u e s t e r i n g o f a g r i c h e m i c a l s a t nonactive sites. A n o t h e r example o f v a s c u l a r d i f f e r e n c e s i n p l a n t s i s r e p r e ­ sented by monocotyledon g r a s s e s such as wheat ( F i g u r e 3C). The v a s c u l a r bundle i n wheat and i n many g r a s s e s i s surrounded by a mestome sheath which i s comprised o f an impermeable, s u b e r i z e d l a y e r o f c e l l s (6). T h i s may r e p r e s e n applied hydrophilic agrichemicals need t o be t a k e n i n t o account when s e e k i n g t o r a t i o n a l l y d e s i g n c r o p s p e c i f i c systemic chemicals. There are even s t r u c t u r a l d i f f e r e n c e s among the d i f f e r e n t g r a s s s p e c i e s . F o r example, i n g r a s s e s t h a t have C-3 p h o t o s y n t h e s i s , such as b a r l e y , o a t s , wheat, and f e s c u e , t h e r e are 11-15 m e s o p h y l l c e l l s between each l o n g i t u d i n a l v a s c u l a r b u n d l e , w i t h a d i s t a n c e between each v a s c u l a r bundle o f about 0.30 mm. In c o n t r a s t , g r a s s e s w i t h C-4 p h o t o s y n t h e s i s , l i k e c o r n , s o r g ­ hum, s u g a r c a n e , f o x t a i l , c r a b g r a s s , b a r n y a r d g r a s s , have o n l y 2 m e s o p h y l l c e l l s between each v a s c u l a r bundle and the v a s c u l a r bun­ d l e s are o n l y 0.1 mm away from each o t h e r (JL1). T h i s s h o r t e r r o u t e o f s u c r o s e t r a n s p o r t from m e s o p h y l l to phloem i n the C-4 s p e c i e s i s thought to be one of the prime reasons why C-4 p l a n t s t r a n s l o c a t e a t a f a s t e r r a t e than C-3 s p e c i e s . I t i s not known i f t h i s i n f l u e n c e s the t r a n s p o r t c h a r a c t e r i s t i c s o f a g r i c h e m i c a l s , but t h e r e are d a t a which suggest t h a t t h e s e s t r u c t u r a l d i f f e r e n c e s may i n f l u e n c e move­ ment. F o r example, M a r t i n and E d g i n g t o n (12) found t h a t o n l y 3% o f the t o t a l amount o f f e n a r i m o l t h a t was t r a n s p o r t e d i n b a r l e y o c c u r ­ r e d s y m p l a s t i c a l l y , whereas i n soybeans t h i s v a l u e was 43%. Simi­ l a r l y , the p e r c e n t o f oxamyl t r a n s p o r t w i t h i n the symplasm was 4 and 31% i n b a r l e y and soybean, r e s p e c t i v e l y . The p e r c e n t o f 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d t h a t was t r a n s p o r t e d s y m p l a s t i c a l l y was 65 and 96% i n b a r l e y and soybean, r e s p e c t i v e l y . The reduced amount of s y m p l a s t i c t r a n s p o r t o f these t h r e e c h e m i c a l s i n b a r l e y compared t o soybean may be r e l a t e d t o the d i f f e r e n c e s i n v a s c u l a r anatomy between t h e s e s p e c i e s . Path and S i n k F e a t u r e s . In the t r a n s l o c a t i o n p a t h ( e . g . , stems and p e t i o l e s ) , a s s i m i l a t e s and s o l u t e s move i n mass flow t h r o u g h the c y l i n d r i c a l s i e v e tubes which have open s i e v e p o r e s . The a b i l i t y o f a c h e m i c a l t o l e a k a c r o s s the membrane from the s i e v e tube d u r i n g t r a n s i t w i l l a f f e c t i t s a b i l i t y t o be t r a n s p o r t e d t h r o u g h the e n t i r e pathway. In s i n k r e g i o n s , t h e r e are e s s e n t i a l l y t h r e e i n v i v o pathways by which s u c r o s e e x i t s the s i e v e tubes ( F i g u r e 4 ) . A l l t h r e e are o p e r a t i n g i n d i f f e r e n t t y p e s o f s i n k s and a l l are m e t a b o l i s m depend-

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Phloem Transport of Agrichemicals

13

F i g u r e 3. L e a f v a s c u l a t u r e anatomy. T r a c i n g s from l i g h t m i c r o ­ graphs o f : (A) soybean l e a f c r o s s s e c t i o n showing PVM ( a r r o w s ) ; (B) paradermal s e c t i o n o f soybean l e a f showing i n t e r c o n n e c t i n g PVM (shaded c e l l s ) ; and (C) c r o s s s e c t i o n o f a wheat l e a f showing mestome sheath c e l l s s u r r o u n d i n g v a s c u l a r t i s s u e . Tracings p r o v i d e d by S h i e l a McKelvey.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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BIOREGULATORS FOR PEST C O N T R O L

ent ( 8 , 1 3 ) . S u c r o s e c a n e x i t t h e s i e v e tube and: (1) e n t e r t h e f r e e space where i t i s h y d r o l y z e d by a c e l l w a l l i n v e r t a s e t o hexose p r i o r t o uptake ( t h i s r o u t e o c c u r s i n c o r n k e r n e l s and sugarcane s t o r a g e s t a l k s ; (2) e n t e r t h e f r e e space and then be accumulated as t h e i n t a c t m o l e c u l e ( e . g . , s u g a r b e e t r o o t , soybean seeds, wheat s e e d s ) ; and (3) e n t e r s i n k c e l l s v i a plasmodesmata c o n n e c t i o n s w i t h o u t l e a v i n g t h e symplasm o r m e t a b o l i c space (growing r o o t s and young l e a v e s ) . D i f f e r e n t pathways e x i s t i n d i f f e r e n t organs and t h i s s h o u l d be r e c o g n i z e d when c o n s i d e r i n g t h e t a r g e t - s p e c i f i c phloem m o b i l i t y o f a g r i c h e m i c a l s . Thus, based on t h e c h a r a c t e r i s t i c s o f the t r a n s p o r t system, s e v e r a l f a c t o r s can be i d e n t i f i e d t h a t i n f l u e n c e t h e e n t r y o f a compound i n t o c e l l s and i t s subsequent t r a n s l o c a t i o n i n t h e phloem. F i r s t , t h e b i n d i n g o f compound t o t h e c e l l w a l l c a n p r e v e n t i n i t i a l e n t r y i n t o the c y t o p l a s m . I n g e n e r a l , because o f the nega­ t i v e charge o f t h e c e l l w a l l s , p o s i t i v e l y charged compounds w i l l b i n d more than uncharged o r n e g a t i v e l y charged ones. Second, p e r ­ m e a b i l i t y b a r r i e r s , suc v a s c u l a r system i n c e r t a i h y d r o p h i l i c c h e m i c a l d i r e c t l y t o phloem. Third, structural features o f t h e v a s c u l a t u r e , such as v e i n d i s t a n c e o r the p a r a v e i n a l meso­ p h y l l i n soybeans, may a l s o i n f l u e n c e movement o f a c h e m i c a l t o t h e vein. Fourth, increased l i p o p h i l i c i t y i s u s u a l l y a p r e r e q u i s i t e f o r phloem m o b i l i t y b u t , as w i l l be d i s c u s s e d below, h i g h l y p e n e t r a t i n g c h e m i c a l s can show v e r y l i t t l e s y s t e m i c movement. F i f t h , t h e chemi­ c a l cannot be a s h o r t - t e r m i n h i b i t o r o f m e t a b o l i s m because phloem l o a d i n g and t r a n s l o c a t i o n a r e h i g h l y dependent on m e t a b o l i c energy. Compounds such as u n c o u p l e r s o f p h o s p h o r y l a t i o n , photosynthetic i n h i b i t o r s , and compounds which i n c r e a s e t h e p e r m e a b i l i t y o f c e l l membranes w i l l a l l i n h i b i t t r a n s l o c a t i o n and thus p r e v e n t compound movement. S i x t h , t h e o v e r a l l d i r e c t i o n o f a s s i m i l a t e movement i s d e t e r m i n e d by t h e r e l a t i v e p o s i t i o n o f s o u r c e s and s i n k s on a p l a n t . Seventh, e n v i r o n m e n t a l f a c t o r s such as l i g h t , t e m p e r a t u r e , and water s t r e s s a f f e c t t r a n s l o c a t i o n . These f a c t o r s s h o u l d be t a k e n i n t o account, p a r t i c u l a r l y during a p p l i c a t i o n of a chemical i n order t o maximize t r a n s l o c a t i o n e f f i c i e n c y . Agrichemical

Transport

The s y s t e m i c m o b i l i t y o f an e f f i c a c i o u s a g r i c h e m i c a l depends on: (1) e f f e c t i v e p e n e t r a t i o n o f the c u t i c l e ; (2) l o n g d i s t a n c e movement w i t h i n t h e p l a n t ; (3) m e t a b o l i c s t a b i l i t y ; and (4) s e l e c t i v e t o x i ­ city. There a r e two components o f t h e p l a n t ' s s t r u c t u r e and volume t h a t a r e important t o l o n g d i s t a n c e x e n o b i o t i c movement: t h e apo­ p l a s t and t h e s y m p l a s t . The a p o p l a s t i s e s s e n t i a l l y t h e nonm e t a b o l i c space r e s i d i n g o u t s i d e t h e c e l l membrane and c o n s i s t s o f the c e l l w a l l s , xylem, and n o n - l i v i n g f i b e r s . I t i s bounded by t h e c u t i c l e on b o t h l e a f s u r f a c e s . S o l u t e movement i n t h e a p o p l a s t i s s t r o n g l y d i r e c t i o n a l and movement i s u s u a l l y by d i f f u s i o n o r by mass flow i n t h e t r a n s p i r a t i o n stream. A l l c h e m i c a l s e n t e r t h e p l a n t v i a the a p o p l a s t . The symplast i s d e f i n e d as t h e m e t a b o l i c o r c y t o ­ p l a s m i c space r e s i d i n g i n s i d e t h e plasmamembrane. I t a l s o i n c l u d e s the phloem. C h e m i c a l s e n t e r t h e symplast by c r o s s i n g t h e c e l l membrane.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1.

GIAQUINTA

Phloem Transport ofAgrichemicals

15

A g r i c h e m i c a l s which t r a v e l m a i n l y i n t h e a p o p l a s t c h a r a c t e r i s ­ t i c a l l y accumulate a t t h e l e a f t i p s and margins o f mature l e a v e s , whereas compounds t h a t t r a v e l i n t h e phloem accumulate a t growing r e g i o n s ( i . e . , new l e a v e s , buds, r o o t t i p s , and s t o r a g e organs). Based on t h e o v e r a l l d i s t r i b u t i o n p a t t e r n i n p l a n t s , c h e m i c a l t r a n s p o r t h i s t o r i c a l l y has been c h a r a c t e r i z e d as b e i n g a p o p l a s t i c o r symplastic. S i n c e t h e mid-1970's i t has been i n c r e a s i n g l y c l e a r t h a t many compounds a r e ambimobile ( 4 ) , i n t h a t t h e s e c h e m i c a l s t r a v e l i n b o t h t h e a p o p l a s t and symplast depending on t h e p h y s i c a l c h a r a c t e r i s t i c s o f the molecule. I n f a c t , most o f t h e c h e m i c a l s t h a t were p r e v i o u s l y c h a r a c t e r i z e d as moving o n l y i n t h e a p o p l a s t o r xylem a r e now r e g a r d e d as ambimobile because they p e n e t r a t e mem­ branes q u i t e r e a d i l y ( 4 ) . The f i r s t c l u e s t h a t " a p o p l a s t i c " o r xylem m o b i l e c h e m i c a l s were n o t l i m i t e d t o t h e xylem came from s e v e r a l a n o m o l i e s . These included the following observations: (1) many a p o p l a s t i c chemicals have s y m p l a s t i c s i t e s o f a c t i o n ( t h e p h o t o s y n t h e s i s inhibitors like d i u r o n and a t r a z i n e hav but a l s o t h e d o u b l e membran t h y l a k o i d membrane); (2) many xylem t r a n s p o r t e d i n s e c t i c i d e s a r e t o x i c t o a p h i d s which f e e d e x c l u s i v e l y on t h e phloem; ( 3 ) b e n z i m i d a z o l e f u n g i c i d e s have c y t o k i n i n - l i k e a c t i v t y s u g g e s t i n g i n t e r a c t i o n w i t h t h e symplasm; (4) many " a p o p l a s t i c " compounds a r e e i t h e r metab­ o l i z e d t o CO2 o r c o n j u g a t e d w i t h amino a c i d s o r s u g a r s ; ( 5 ) "apo­ p l a s t i c " c h e m i c a l s t h a t t r a n s p o r t a c r o s s t h e water impermeable c a s p a r i a n s t r i p s i n t h e r o o t s ; and ( 6 ) t h e b a s i p e t a l t r a n s p o r t o f certain fungicides (4). E d g i n g t o n and P e t e r s o n ( 4 ) have s u b d i v i d e d a p o p l a s t i c xenob i o t i c s i n t o two c l a s s e s . Euapoplastic (only transported i n the a p o p l a s t ) and p s e u d o a p o p l a s t i c ( t r a n s p o r t o c c u r s m a i n l y i n t h e xylem but e n t r y i n t o t h e symplast o c c u r s ) . Most t r a d i t i o n a l " a p o p l a s t i c " c h e m i c a l s a r e now known t o r e a l l y be p s e u d o a p o p l a s t i c chemicals, e.g., a t r a z i n e , d i u r o n , oxamyl, e t c . The u n r e s o l v e d q u e s t i o n i s why don't t h e s e p s e u d o a p o p l a s t i c c h e m i c a l s which c r o s s t h e c e l l mem­ branes and e n t e r t h e symplast remain i n t h e symplasm o f t h e phloem? There have been numerous s t u d i e s f o c u s i n g on t h e m o l e c u l a r r e q u i r e ­ ments f o r phloem m o b i l i t y ( 1 - 5 ) , I n g e n e r a l , t h e r e i s n o t a good c o r r e l a t i o n between phloem m o b i l i t y and water s o l u b i l i t y , m e t a b o l i s m o f t h e x e n o b i o t i c , o r t h e p r e s e n c e o f v a r i o u s s u b s t i t u t i o n groups i n a molecule. "Weak A c i d " and " I n t e r m e d i a t e - D i f f u s i o n " Hypotheses. Two h y p o t h e s e s (which a r e n o t n e c e s s a r i l y m u t u a l l y e x c l u s i v e ) have been p r o p o s e d f o r t h e e n t r y and s y s t e m i c m o b i l i t y o f c h e m i c a l s i n t h e phloem: t h e "weak-acid" h y p o t h e s i s proposed by C r i s p and c o l l e a g u e s (_5), and t h e " i n t e r m e d i a t e - d i f f u s i o n " h y p o t h e s i s proposed by E d g i n g t o n and Peterson (4). These a r e i l l u s t r a t e d i n F i g u r e 5. The weak-acid h y p o t h e s i s proposes t h a t compounds which have a f r e e COOH group on t h e m o l e c u l e w i l l be i n t h e p r o t o n a t e d s t a t e i n the a p o p l a s t because o f t h e low pH o f t h e a p o p l a s t (pH 5 t o 5.5). The uncharged m o l e c u l e w i l l c r o s s t h e phloem membranes because o f increased l i p i d s o l u b i l i t y . Once i n s i d e t h e phloem, where t h e pH i s a l k a l i n e (pH 8) the COOH group w i l l be i o n i z e d . The i o n i z e d s p e c i e s w i l l be r e l a t i v e l y impermeable t o t h e phloem membrane b o t h

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

16

BIOREGULATORS FOR PEST CONTROL

SUCROSE

VACUOLE

PHLOEM

F i g u r e 4. Pathways o f phloem u n l o a d i n g i n s i n k r e g i o n s . See text f o r d e t a i l s . Reproduced w i t h p e r m i s s i o n from R e f . 13. C o p y r i g h t 1983. American S o c i e t y o f P l a n t P h y s i o l o g i s t s .

"Weak-Acid" H y p o t h e s i s R«COOH-

R-COO"

Lipid soluble Uncharged

"Intermediate" Diffusion Hypothesis R

APOPLAST P

H 5

F i g u r e 5. W e a k - a c i d a n d i n t e r m e d i a t e d i f f u s i o n h y p o t h e s e s f o r the e n t r y and s y s t e m i c m o b i l i t y o f c h e m i c a l s i n t h e phloem.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1.

GIAQUINTA

17

Phloem Transport of Agrichemicals

because o f i t s charge and i t s reduced s o l u b i l i t y i n t h e membrane. T h i s i o n i z e d s p e c i e s c a n t h e n move i n mass flow w i t h t h e t r a n s l o c a ­ t i o n stream. Testable features of t h i s hypothesis are that the a g r i c h e m i c a l w i l l tend t o accumulate i n t h e phloem above i t s e x t e r n a l c o n c e n t r a t i o n because o f t h i s " t r a p p i n g " mechanism and t h a t a g r i c h e m i c a l uptake w i l l be ρ_Η dependent ( h i g h e r a t more a c i d i c pH). There i s q u a l i f i e d support f o r t h e weak-acid h y p o t h e s i s , p a r t i ­ c u l a r l y f o r compounds such as 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid. Crisp and Look (_5) compared t h e phloem m o b i l i t y o f s e v e r a l s y n t h e t i c 4c h l o r o p h e n o x y d e r i v a t i v e s . The c a r b o x y l d e r i v a t i v e was loaded and t r a n s p o r t e d i n t h e phloem, whereas d e r i v a t i v e s i n which t h e COOH group was r e p l a c e d by an e t h y l e s t e r , amide, k e t o n e , a l c o h o l , o r amino group were n o t t r a n s l o c a t e d . A l t h o u g h t h e weak a c i d h y p o t h e s i s appears t o e x p l a i n t h e mobi­ l i t y o f compounds such as c h l o r o p h e n o x y d e r i v a t i v e s , t h e r e a r e s e v e r a l e x c e p t i o n s t o t h e weak-acid h y p o t h e s i s (4 JL4 L 5 ) For example, some x e n o b i o t i c and do n o t appear t o b ( e . g . , a m i t r o l e , oxamyl). A l s o , some x e n o b i o t i c s ( e . g . , g l y p h o s a t e ) which have an i o n i z a b l e COOH group a r e loaded i n t o t h e phloem i n d e ­ p e n d e n t l y o f a p o p l a s t pH. These s h o u l d l o s e t h e i r m o b i l i t y under pH c o n d i t i o n s which i o n i z e t h e c h e m i c a l i n t h e f r e e space. Further­ m o r e , a c c u m u l a t i o n o f t h e weak a c i d g l y p h o s a t e a g a i n s t a c o n c e n t r a ­ t i o n g r a d i e n t does n o t o c c u r ( 1 4 ) . The " i n t e r m e d i a t e d i f f u s i o n " h y p o t h e s i s proposes t h a t t h e c r i t i c a l d e t e r m i n a n t o f phloem m o b i l i t y i s t h e optimum p e r m e a b i l i t y c o e f f i c i e n t o f a g i v e n m o l e c u l e , P. Ρ i s c a l c u l a t e d a s :

p

= fr

l

n

1 1

- o k

]

where L i s t h e l e n g t h o f t h e v a s c u l a r system*, 1, t h e l e n g t h o f t h e source o r l o a d i n g r e g i o n ; r , t h e r a d i u s o f t h e s i e v e t u b e s ; and V, t h e average d a i l y t r a n s l o c a t i o n v e l o c i t y . A compound t h a t i s permeable enough t o e n t e r t h e phloem w i l l be t r a n s p o r t e d as l o n g as t h e compound i s n o t so permeable t o t h e phloem membrane t h a t i t l e a k s back out o f t h e phloem i n t o t h e a d j a c e n t and o p p o s i n g xylem stream. The compound has t o have a r e t e n t i o n time l o n g enough t o be c a r r i e d i n t h e phloem. Each compound and each p l a n t have t h e i r own optimum Ρ and Tyree e t a l . (15) propose t h a t i t i s n o t t h e o r e t i c a l l y p o s s i b l e t o d e v i s e a x e n o b i o t i c which i s o p t i m a l l y ambimobile f o r p l a n t s o f a l l s i z e s . In summary, t h e i n t e n t o f t h i s r e v i e w was t o examine t h e s y s t e m i c t r a n s p o r t o f x e n o b i o t i c s from t h e v i e w p o i n t o f a phloem p h y s i o l o g i s t i n order t o h i g h l i g h t c e r t a i n biochemical, p h y s i o l o g i ­ c a l , and s t r u c t u r a l f e a t u r e s o f t h e t r a n s l o c a t i o n system t h a t may be r e l e v a n t t o t h e f u t u r e d e s i g n o f phloem m o b i l e c h e m i c a l s . I hope the r e v i e w has t a k e n a modest s t e p i n t h a t d i r e c t i o n . Acknowledgment The t y p i n g o f t h e m a n u s c r i p t by T. S p a r r e and m i c r o g r a p h t r a c i n g s by S h i e l a McKelvey a r e g r e a t l y a p p r e c i a t e d .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST CONTROL

18

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Christ, R. A. In "Advances in Pesticide Science"; Geissbühler, H., Ed.; Pergammon Press: New York, 1979; Vol. III, p. 420-9. Kirkwood, R. C. In "Herbicides and Fungicides"; McFarlane, N. R., Ed.; Burlington House: London, 1977; p. 67-80. Price, C. E. In "Herbicides and Fungicides"; McFarlane, N. R., Ed.; Burlington House: London, 1977; p. 426. Edgington, L. V.; Peterson, C. A. In "Antifungal Compounds"; Siegal, M. R.; Sisler, H. D.; Eds.; Marcel Dekker, Inc.: New York, 1977; Vol. II, Chap. 2, p. 51-89. Crisp, C. E . ; Look, M. In "Advances in Pesticide Science"; Geissbühler, H., Ed.; Pergammon Press: New York, 1979; Vol. III. p. 430-7. Giaquinta, R. T., Annu. Rev. Plant Physiol. 1983; 34, 347-87. Geiger, D. R.; Giaquinta, R. T. In "Photosynthesis: CO2 Assimilation and Plant Productivity"; Govindjee, Ed.; Academic Press: Ne Giaquinta, R. T. I Ed.; Academic Press: New York, 1980; Vol. III, p. 271-320. Franceschi, V. R.; Giaquinta, R. T. Planta, 1983a, 157, 411-21. Franceschi, V. R.; Giaquinta, R. T. Planta, 1983b, 157, 422-31. Crookston, K. R.; Moss, D. N. Crop Sci. 1974, 14, 123-5. Martin, R. Α.; Edgington, L. V. Pest. Biochem. Physiol. 1981, 16, 87-96. Giaquinta, R. T.; Lin, W.; Sadler, N. L.; Franceschi, V. R. Plant Physiol. 1983, 72, 362-7. Gougler, J . Α.; Geiger, D. R., Plant Physiol. 1981, 68, 668-72. Tyree, M. T.; Peterson, C. Α.; Edgington, L. V. Plant Physiol. 1979, 63, 367-74

RECEIVED

November 15,

1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

2 Interference by Herbicides with Photosynthetic Electron Transfer WALTER OETTMEIER Lehrstuhl Biochemie der Pflanzen, Ruhr-Universität, Postfach 10 21 48, D-4630 Bochum 1, Federal Republic of Germany

Herbicides that inhibi prevent reductio II acceptor complex. The properties of the photosystem II herbicide receptor proteins have been investigated by binding and displacement studies with radiolabeled herbicides. The herbicide receptor proteins have been identified with herbicide-derived photoaffinity l a bels. Herbicides, similar in their mode of action to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) bind to a 34 kDa protein, whereas phenolic herbicides bind to the 43-51 kDa photosystem II reaction center proteins. At these receptor proteins, plastoquinone/herbicide interactions and plastoquinone binding sites have been studied, the latter by means of a plastoquinone-derived photoaffinity label. For the 34 kDa herbicide binding protein, whose amino acid sequence is known, herbicide and plastoquinone binding are discussed at the molecular level. Plastoquinone is one of the most important components of the photosynthetic electron transport chain. It shuttles both electrons and protons across the photosynthetic membrane system of the thylakoid. In photosynthetic electron flow, plastoquinone is reduced at the acceptor side of photosystem II and reoxidized by the cytochrome b /f-complex. Herbicides that interfere with photosynthesis have been shown to specifically and effectively block plastoquinone reduction. However, the mechanisms of action of these herbicides, i . e., how inhibition of plastoquinone reduction is brought about, has not been established. Recent developments have brought a substantial increase to our knowledge in this field and one objective of this article will be to summarize the recent progress. It was originally assumed that the herbicides bind to a protein component of photosystem II (named "B" or "R") (1,2). This protein component was assumed to contain a special bound plastoquinone whose midpoint potential is lowered due to herbicide binding. Consequently, electron flow is interrupted (1,2). The photosystem II 6

0097-6156/85/0276-0019$06.00/0 © 1985 American Chemical Society In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

20

BIOREGULATORS FOR PEST CONTROL

h e r b i c i d e b i n d i n g p r o t e i n component was l a t e r e s t a b l i s h e d t o f u n c ­ t i o n as a " p r o t e i n a c e o u s s h i e l d " f o r p h o t o s y s t e m I I by Renger (3) . The " p r o t e i n a c e o u s s h i e l d " can be removed by t r e a t m e n t w i t h t h e p r o ­ t e o l y t i c enzyme t r y p s i n and, s u b s e q u e n t l y , D C M U - s e n s i t i v i t y o f phot o s y n t h e t i c e l e c t r o n t r a n s p o r t i s l o s t (J3,4) . I n 1979, t h e c o n c e p t o f a p h o t o s y s t e m I I h e r b i c i d e b i n d i n g p r o ­ t e i n with d i f f e r e n t but overlapping binding s i t e s f o r the various p h o t o s y s t e m I I h e r b i c i d e s was s i m u l t a n e o u s l y e s t a b l i s h e d by T r e b s t and Draber {5) and P f i s t e r and A r n t z e n ( 6 ) . T h i s i d e a o f a h e r b i ­ c i d e r e c e p t o r p r o t e i n p r o v e d t o be extremely f r u i t f u l because t h e t e c h n i q u e s o f r e c e p t o r b i o c h e m i s t r y were now a p p l i c a b l e . T i s c h e r and Strotmann Ç7) were t h e f i r s t i n v e s t i g a t o r s t o study b i n d i n g o f radiolabeled herbicides i n isolated thylakoids. Herbicide Binding

Experiments

T y p i c a l r e s u l t s o b t a i n e d i n a b i n d i n g experiment f o r two d i f f e r e n t photosysÇgm I I h e r b i c i d e z i n o n e [ c] m e t r i b u z i n ( r i g h h e r b i c i d e , and t h e p h e n o l i c h e r b i c i d e [ H ] 2 - i o d o - 4 - n i t r o - 6 - i s o b u t y l p h e n o l (8_) ( l e f t formula) . The term "DCMU-type" h e r b i c i d e does n o t denote a c h e m i c a l d e f i n i t i o n , b u t i s a f u n c t i o n a l d e f i n i t i o n because DCMU (diuron) i s t h e most w i d e l y u s e d p h o t o s y n t h e s i s i n h i b i t o r . I n b i n d i n g e x p e r i m e n t s m e t r i b u z i n seems t o s a t u r a t e a t r e l a t i v e l y low c o n c e n t r a t i o n s ( F i g u r e 1 ) . However, t h e b i n d i n g o f m e t r i b u z i n i s i n f a c t b i p h a s i c : i t has a s o - c a l l e d s p e c i f i c (high a f f i n i t y ) b i n d i n g and an u n s p e c i f i c (low a f f i n i t y ) b i n d i n g (Ί)· The l a t t e r shows a l i n e a r dependency on the c o n c e n t r a t i o n . T h i s ( e x t r a p o l a t e d ) unspe­ c i f i c b i n d i n g o f t h e p h e n o l i c h e r b i c i d e i s much h i g h e r , a s compared t o t h a t o f m e t r i b u z i n ( F i g u r e 1, upper dashed l i n e ) . T h i s i s j u s t one o f t h e many d i f f e r e n c e s t h a t can be f o u n d between "DCMU-type" and p h e n o l i c h e r b i c i d e s and which j u s t i f i e s t o view them as two d i f ­ f e r e n t c l a s s e s o f h e r b i c i d e s ( f o r r e v i e w , see (JO ) . The b i n d i n g c u r v e s o f t h e h e r b i c i d e s i n F i g u r e 1, e s p e c i a l l y the one f o r m e t r i b u z i n , l o o k v e r y much l i k e M i c h a e l i s - M e n t e n enzyme k i n e t i c s . Indeed, h e r b i c i d e b i n d i n g can be t r e a t e d i n t h e same way Ç7). F i g u r e 2 p r e s e n t s a Lineweaver-Burk p l o t o f t h e b i n d i n g d a t a f o r 2 - i o d o - 4 - n i t r o - 6 - i s o b u t y l p h e n o l . C l e a r l y , t h e two t y p e s o f b i n d ­ i n g , s p e c i f i c and u n s p e c i f i c b i n d i n g , can be r e c o g n i z e d . T h i s i s even more e v i d e n t i n t h e S c a t c h a r d p l o t o f t h e b i n d i n g d a t a ( i n s e t F i g u r e 2 ) . F u r t h e r m o r e , from these p l o t s , b i n d i n g p a r a m e t e r s , such as t h e b i n d i n g c o n s t a n t Κ, , and number o f b i n d i n g s i t e s , x , can be o b t a i n e d Ç 7 ) . These a r e a l s o l i s t e d i n F i g u r e 2. A c c o r d i n g t o T i s c h e r and Strotmann ( 7 ) , t h e b i n d i n g c o n s t a n t corresponds t o the i n h i b i t i o n c o n s t a n t , i . e. t h e I ^ v a l u e (the c o n c e n t r a t i o n n e c e s s a r y f o r 50% i n h i b i t i o n o f p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t ) , provided the I v a l u e i s e x t r a p o l a t e d t o z e r o c h l o r o p h y l l concen­ t r a t i o n . The v a l u e o f 527 m o l e c u l e s o f c h l o r o p h y l l p e r m o l e c u l e o f bound i n h i b i t o r i n d i c a t e s t h a t r o u g h l y one m o l e c u l e o f h e r b i c i d e b i n d s p e r e l e c t r o n t r a n s p o r t c h a i n , because about 400-600 m o l e c u l e s o f c h l o r o p h y l l a r e c o n s i d e r e d t o be a s s o c i a t e d w i t h each e l e c t r o n transport chain. t

Q

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

OETTMEIER

Photosynthetic Electron Transfer Inhibition

4-nitro-6-isobutylphenol

(•

•)

to i s o l a t e d

spinach thylakoids.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

22

BIOREGULATORS FOR PEST CONTROL

Herbicide-Resistant

Weeds

The i m p o r t a n c e o f b i n d i n g e x p e r i m e n t s w i t h r a d i o l a b e l e d h e r b i c i d e s became i m m e d i a t e l y e v i d e n t i n t h e case o f h e r b i c i d e - r e s i s t a n t weeds. The use o f c e r t a i n s - t r i a z i n e h e r b i c i d e s l i k e a t r a z i n e f o r more t h a n two decades had l e d t o b i o t y p e s t h a t a r e r e s i s t a n t t o n o r m a l l y a p ­ p l i e d doses. B i n d i n g experiments with a t r a z i n e i n t h y l a k o i d s i s o l a t ­ ed from r e s i s t a n t weed p l a n t s d e m o n s t r a t e d t h a t t h e s p e c i f i c b i n d i n g o f a t r a z i n e was c o m p l e t e l y a b s e n t and o n l y some u n s p e c i f i c b i n d i n g was l e f t (10,11). Thus, t h e r e s i s t a n c e i s due t o d e c r e a s e d b i n d i n g o f t h e h e r b i c i d e i n t h e t h y l a k o i d o f t h e r e s i s t a n t p l a n t , which does not i n h i b i t p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t a t concentrations that a r e l e t h a l t o the* s u s c e p t i b l e t y p e . S i m i l a r i l y , t h e s p e c i f i c b i n d i n g o f m e t r i b u z i n a l s o i s c o m p l e t e l y l o s t , whereas t h e b i n d i n g o f u r e a and b i s c a r b a m a t e h e r b i c i d e s i s o n l y s l i g h t l y a f f e c t e d (11) . I n c o n ­ t r a s t , t h e b i n d i n g o f p h e n o l i c h e r b i c i d e s , i n g e n e r a l , i s more p r o ­ nounced i n t h y l a k o i d s o f r e s i s t a n t p l a n t s than i n t h o s e o f t h e s u s ­ c e p t i b l e types (11). H e r b i c i d e Displacement Experiments The p h o t o s y s t e m I I h e r b i c i d e s b i n d r e v e r s i b l y and n o n - c o v a l e n t l y t o t h e i r b i n d i n g s i t e . C o n s e q u e n t l y , a r a d i o l a b e l e d h e r b i c i d e can be d i s p l a c e d from t h e b i n d i n g s i t e by a n o t h e r h e r b i c i d e o r i n h i b i t o r , p r o v i d e d i t h a s an i d e n t i c a l b i n d i n g s i t e . Even a d i s p l a c e m e n t from a d i f f e r e n t b i n d i n g s i t e i s f e a s i b l e , i f both b i n d i n g s i t e s i n t e r a c t w i t h each o t h e r . A Çvpical d i s p l a c e m e n t e x p e r i m e n t i s shown i n F i g ­ u r e 3. E v i d e n t l y , f c ] m e t r i b u z i n i s e a s i l y d i s p l a c e d from t h e t h y ­ l a k o i d membrane by DCMU. S i n c e t h e P I C Q v a l u e s ( n e g a t i v e l o g a r i t h m o f c o n c e n t r a t i o n a c h i e v i n g 50% i n h i b i t i o n ) o f b o t h compounds a r e in t h e same o r d e r o f magnitude , about 50% o f t h e bound m e t r i b u z i n i s removed from t h e membrane a t t h e i s o m o l a r p o i n t , i . e. when t h e c o n c e n t r a t i o n s o f b o t h compounds a r e i d e n t i c a l . F o r t h e p h e n o l i c h e r b i c i d e d i n o s e b ( 2 , 4 - d i n i t r o - 6 - s e c . - b u t y l p h e n o l ) , a much h i g h e r c o n c e n t r a t i o n , 7 χ 10 M, i s n e c e s s a r y t o o b t a i n 50% r e m o v a l . T h i s i s a consequence o f t h e l o w e r p I ^ v a l u e o f d i n o s e b o f 5.5 ( 1 2 ) . Thus, t h e c o n c e n t r a t i o n n e c e s s a r y f o r 50% d i s p l a c e m e n t r o u g h l y c o r ­ responds t o the P l ^ v a l u e . I t i s p o s s i b l e , t h e r e f o r e , t o assay the pl^ v a l u e o f a new compound j u s t by e x a m i n a t i o n o f i t s d i s p l a c e m e n t b e h a v i o u r . I t i s no l o n g e r n e c e s s a r y t o d e t e r m i n e t h e p I c v a l u e by t e s t i n g the i n h i b i t i o n o f a l i g h t - d r i v e n p h o t o r e d u c t i o n . Another very p o t e n t i n h i b i t o r o f p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t , DBMIB (2,5d i b r o m o - 3 - m e t h y l - 6 - i s o p r o p y l - l , 4 - b e n z o q u i n o n e ) (13) , a l m o s t c o m p l e t e ­ l y f a i l s t o d i s p l a c e m e t r i b u z i n from t h e membrane ( F i g u r e 3 ) . T h i s i s due t o t h e f a c t t h a t DBMIB h a s a c o m p l e t e l y d i f f e r e n t s i t e o f a c ­ t i o n a s compared t o t h e p h o t o s y s t e m I I h e r b i c i d e s , i . e. i t i n h i b i t s p l a s t o h y d r o q u i n o n e o x i d a t i o n by a c t i n g a t t h e cytochrome b / f - c o m p l e x Q

0

0

Q

fi

(13) . P h o t o a f f i n i t y Labeling of the Herbicide Binding

Proteins

As a l r e a d y s t r e s s e d , p h o t o s y s t e m I I h e r b i c i d e s b i n d r e v e r s i b l y t o t h e i r binding s i t e . Altough r a d i o l a b e l e d h e r b i c i d e s are a v a i l a b l e , i t i s impossible to i d e n t i f y the h e r b i c i d e receptor p r o t e i n without a chemical m o d i f i c a t i o n o f the h e r b i c i d e t h a t allows f o r covalent

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

2.

Photosynthetic Electron Transfer Inhibition

OETTMEIER

23

% bound 100< 80 60 40 20

10-8

10-

10"

7

6

10-

5

tog cone.

DCMU (7.4)

DINOSEB (5.5)

DBMIB (7.5)

r 1 τ "1 F i g u r e 3. D i s p l a c e m e n t o f L J m e t r i b u z i n from t h e t h y l a k o i d mem­ brane by DCMU ( · ·) , Dinoseb (• •) , and DBMIB (A A) . The numbers i n p a r a n t h e s i s below t h e s t r u c t u r a l f o r m u l a s o f t h e compounds c o r r e s p o n d t o t h e i r ρ Ι values. C

ς η

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

24

BIOREGULATORS FOR PEST CONTROL

attachment o f t h e h e r b i c i d e . T h i s m o d i f i c a t i o n i s a c h i e v e d by i n t r o ­ d u c t i o n o f an a z i d o f u n c t i o n i n t o t h e h e r b i c i d e m o l e c u l e . An o r g a n i c a z i d e upon i l l u m i n a t i o n w i t h v i s i b l e o r UV l i g h t r e a d i l y s p l i t s - o f f m o l e c u l a r n i t r o g e n and forms a n i t r e n e . N i t r e n e s are e x t r e m e l y e l e c t r o p h i l i c compounds and r e a c t i m m e d i a t e l y w i t h any n u c l e o p h i l i c groups i n t h e i r environment. I f the àzidoderivative o f the h e r b i c i d e i s as good an i n h i b i t o r as i t s p a r e n t compound, i t s s p e c i f i c b i n d i n g s h o u l d e x c l u s i v e l y o c c u r a t i t s r e c e p t o r p r o t e i n . C o n s e q u e n t l y , the n i t r e n e s h o u l d form a c o v a l e n t bond t o the r e c e p t o r p r o t e i n . S i n c e the àzidoderivative o f the h e r b i c i d e i s r a d i o l a b e l e d , the r e c e p t o r p r o t e i n can be e a s i l y i d e n t i f i e d because i t becomes r a d i o a c t i v e by the attachment o f the n i t r e n e . The common p r o c e d u r e f o r i d e n t i f i c a ­ t i o n i n c l u d e s d i s r u p t i o n o f the t h y l a k o i d membrane system by d e t e r ­ g e n t t r e a t m e n t , s e p a r a t i o n o f the t h y l a k o i d p r o t e i n s by p o l y a c r y l amide g e l e l e c t r o p h o r e s i s , and a s s a y i n g f o r r a d i o a c t i v i t y e i t h e r by c u t t i n g the g e l i n t o p,ieces, which a r e s o l u b i l i z e d and c o u n t e d i n a l i q u i d s c i n t i l l a t i o n c o u n t e r , o r by e x p o s u r e o f t h e g e l on X - r a y film. So f a r , t h r e e d i f f e r e n h e r b i c i d e s are a v a i l a b l e (Figure 4): azidodinoseb (phenolic) (14), a z i d o a t r a z i n e (15), and a z i d o t r i a z i n o n e (16) (both "DCMU-type" h e r ­ b i c i d e s ) . A z i d o a t r a z i n e i n i s o l a t e d t h y l a k o i d s from s p i n a c h , and the a l g a Ch1amydomonas r e i n h a r d t i i as w e l l l a b e l s a p r o t e i n w i t h an ap­ p a r e n t m o l e c u l a r weight o f 34 kDa (15,17). F u r t h e r m o r e , a z i d o a t r a ­ z i n e i n the weed Amaranthus b i n d s t o the 34 kDa p r o t e i n o n l y i n t h y ­ l a k o i d s from a t r a z i n e - s u s c e p t i b l e and not t o t h y l a k o i d s from a t r a z i n e - r e s i s t a n t p l a n t s (18). I t was c o n c l u d e d , t h e r e f o r e , t h a t t h e 34 kDa p r o t e i n i s the p h o t o s y s t e m I I h e r b i c i d e b i n d i n g p r o t e i n f o r "DCMU-type" h e r b i c i d e s . T h i s 34 kDa h e r b i c i d e b i n d i n g p r o t e i n i s i d e n t i c a l t o the "photogene" o r " r a p i d l y t u r n i n g o v e r " 34 kDa p r o t e i n t h a t s t a n d s o u t amongst a l l o f t h e t h y l a k o i d p r o t e i n s due t o i t s r a p i d d e s t r u c t i o n and de novo b i o s y n t h e s i s (19). The i d e a o f t h e 34 kDa h e r b i c i d e b i n d i n g p r o t e i n has met some c r i t i c i s m by G r e s s e l (20). T h i s c r i t i c i s m i s due t o the f a c t t h a t p h o t o a f f i n i t y l a b e l i n g e x p e r i m e n t s , i n g e n e r a l , a r e n o t unambiguous. I t i s f e a s i b l e , t h a t a p h o t o a f f i n i t y l a b e l does n o t b i n d t o the t a r ­ get p r o t e i n , but to a neighbouring p r o t e i n i n s t e a d . S p e c i f i c a l l y , G r e s s e l * s c r i t i c i s m i s b a s e d on the f a c t t h a t i n t h e a z i d o a t r a z i n e m o l e c u l e , the a z i d o group and the s t r u c t u r a l element g e n e r a l l y r e ­ c o g n i z e d f o r h e r b i c i d a l a c t i v i t y l i e on o p p o s i t e p a r t s o f the mole­ c u l e . T h e r e f o r e , t h e r e i s a p o s s i b i l i t y t h a t the 34 kDa p r o t e i n t h a t i s t a g g e d by a z i d o a t r a z i n e i s n o t the r e a l h e r b i c i d e b i n d i n g p r o t e i n . To c l a r i f y t h i s q u e s t i o n , we r e c e n t l y s y n t h e s i z e d a n o t h e r "DCMU-type" p h o t o a f f i n i t y l a b e l : a z i d o t r i a z i n o n e ( F j g u r e 4) (_16) . F i g u r e 5 shows r e s u l t s of a l a b e l i n g experiment with £ c ] a z i d o t r i a z i n o n e . Only one p r o t e i n i s h e a v i l y l a b e l e d . From the p o s i t i o n o f the marker p r o ­ t e i n s , a m o l e c u l a r w e i g h t o f 34 kDa can be e s t i m a t e d . I f samples o f the t h y l a k o i d l a b e l e d by C] a z i d o a t r a z i n e o r £ c ] a z i d o t r i a z i n o n e a r e run i n a d j a c e n t l a n e s o f a g e l , r a d i o a c t i v i t y i n b o t h c a s e s i s f o u n d i n e x a c t l y the same p o s i t i o n . F u r t h e r m o r e , p r e l a b e l i n g w i t h i n a c t i v e ^ c ] a z i d o t r i a z i n o n e p r e v e n t s l a b e l i n g o f the 34 kDa p r o ­ t e i n by £ c j a z i d o a t r a z i n e (16). A z i d o t r i a z i n o n e i s c o m p l e t e l y d i f ­ f e r e n t i n i t s c h e m i c a l s t r u c t u r e from a z i d o a t r a z i n e . However, b o t h p h o t o a f f i n i t y l a b e l s b i n d t o an i d e n t i c a l 34 kDa p r o t e i n . I t has t o be c o n c l u d e d t h a t G r e s s e l s s u g g e s t i o n t h a t the 34 kDa p r o t e i n i s n o t the h e r b i c i d e b i n d i n g p r o t e i n i s not v a l i d . 1

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

2.

Photosynthetic Electron Transfer Inhibition

OETTMEIER

[ H]Azido-dinoseb 3

w

[ c]Azido-atrazine

[ C]Azido-triazinone K

X

N^N '

F i g u r e 4. S t r u c t u r a l

H

11

NC H H 2

2

5

5

25

I

N(CH )

formulas o f h e r b i c i d a l p h o t o a f f i n i t y

3

2

labels.

F i g u r e 5. Photograph o f a L i - d o d e c y l s u l f a t e p o l y a c r y l a m i d e e l e c ­ t r o p h o r e s i s g e l (10-15%) and r a d i o a c t i v i t y d i s t r i b u t i o n ^ h e r e i n o f s p i n a c h t h y l a k o i d s i s o l a t e d by 20 nmol/mg c h l o r o p h y l l £ c j a z i d o triazinone.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

26

BIOREGULATORS FOR PEST CONTROL

The p h e n o l i c p h o t o a f f i n i t y l a b e l a z i d o d i n o s e b ( F i g u r e 4) b i n d s l e s s s p e c i f i c a l l y t h a n e i t h e r a z i d o a t r a z i n e o r a z i d o t r i a z i n o n e (14). In a d d i t i o n t o o t h e r p r o t e i n s , i t l a b e l s p r e d o m i n a n t l y t h e p h o t o s y s ­ tem I I r e a c t i o n c e n t e r p r o t e i n s ( s p i n a c h : 43 and 47 kDa; Chlamydomonas: 47 and 51 kDa) (17). Because o f the u n s p e c i f i c b i n d i n g o f a z i d o d i n o s e b , t h i s can b e s t be seen i n p h o t o s y s t e m I I p r e p a r a t i o n s (17). Thus, the p h e n o l i c h e r b i c i d e s b i n d p r e d o m i n a n t l y t o the p h o t o ­ system I I r e a c t i o n c e n t e r , which might e x p l a i n many o f the d i f f e r e n ­ ces o b s e r v e d between "DCMU-type" and p h e n o l i c h e r b i c i d e s (9) . The p h o t o s y s t e m I I r e a c t i o n c e n t e r p r o t e i n s and the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n must be l o c a t e d c l o s e l y t o and i n t e r a c t w i t h each o t h e r i n o r d e r t o e x p l a i n t h e mutual displacement of both types of h e r b i c i d e s (8,12,21). F u r t h e r m o r e , i t s h o u l d be n o t e d t h a t f o r phe­ n o l i c h e r b i c i d e s , some e f f e c t s a t the donor s i d e o f p h o t o s y s t e m I I (22) and on c a r o t e n o i d o x i d a t i o n i n the p h o t o s y s t e m I I r e a c t i o n c e n ­ t e r have been found (23). The h e r b i c i d a l p h o t o a f f i n i t y l a b e l s a r e a l s o u s e f u l t o o l s f o r e l u c i d a t i o n of h e r b i c i d t i c p r e p a r a t i o n s . In p h o t o s y s t e s p l i t t i n g enzyme system b o t h , a z i d o a t r a z i n e and a z i d o d i n o s e b b i n d t o t h e i r r e s p e c t i v e p r o t e i n s (9). In c o n t r a s t , i n a p h o t o s y s t e m I I p a r ­ t i c l e w i t h o u t the water s p l i t t i n g enzyme complex, a z i d o a t r a z i n e does n o t b i n d , whereas a z i d o d i n o s e b s t i l l does ( 1 7 ) . T h i s does not i n d i ­ c a t e , however, t h a t the 34 kDa p r o t e i n i s not p r e s e n t i n t h i s p h o t o ­ system I I p a r t i c l e . As a l r e a d y s t r e s s e d , the 34 kDa h e r b i c i d e b i n d ­ i n g p r o t e i n has a h i g h t u r n o v e r r a | g (19). I f Chlamydomonas c e l l s are grown i n a medium c o n t a i n i n g [ c] a c e t a t e and p h o t o s y s t e m I I p a r t i c l e s a r e p r e p a r e d from t h e s e a l g a e , the maximum o f the r a d i o ­ a c t i v i t y i n the g e l i s f o u n d e x a c t l y a t t h a t p o s i t i o n where the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n m i g r a t e s (24). No b i n d i n g o f a z i d o ­ a t r a z i n e i s o b s e r v e d i n n-hexane e x t r a c t e d t h y l a k o i d s , whereas a z i ­ d o d i n o s e b b i n d i n g i s u n a f f e c t e d by t h i s p r o c e d u r e (24). These r e s u l t s i n d i c a t e t h a t the h e r b i c i d e b i n d i n g p r o p e r t i e s o f the 34 kDa p r o t e i n a r e v e r y s e n s i t i v e t o changes i n i t s p r o t e i n or l i p i d environment. Herbicide/Quinone

Interactions

R e c e n t developments have l e d t o a r e v i s i o n o f the o r i g i n a l i d e a t h a t t h e "B" o r "R" p r o t e i n (see above) which i s i d e n t i c a l t o the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n c o n t a i n s a bound p l a s t o q u i n o n e . Velthuys (25) from f l a s h - i n d u c e d absorbance changes o f p l a s t o s e m i q u i n o n e i n the p r e s e n c e o f v a r i o u s i n h i b i t o r s , and Lavergne (26) from f l u o r e s ­ cence e x p e r i m e n t s , i n f e r r e d t h a t t h e r e i s an e l e c t r o n - d e p e n d e n t d i ­ r e c t c o m p e t i t i o n between p l a s t o q u i n o n e and h e r b i c i d e . A p l a s t o q u i n o n e m o l e c u l e from the p l a s t o q u i n o n e p o o l g e t s bound t o the a c c e p t o r com­ p l e x o f p h o t o s y s t e m I I t o become Q . Q^, i n t u r n , g e t s r e d u c e d by v i a the semiquinone a n i o n r a d i c a l t h e p r i m a r y a c c e p t o r o f pho­ tosystem I I , again another s p e c i a l plastoquinone molecule. Q - s t a ­ b i l i z e s upon b i n d i n g t o Q . In a subsequent second s t e p , Q - g e t s r e d u c e d t o p l a s t o h y d r o q u i n o n e which i s exchanged w i t h a n o t h e r p l a s t o ­ quinone from the p o o l . P h o t o s y s t e m I I h e r b i c i d e s compete w i t h p l a s t o ­ q u i n o n e f o r b i n d i n g t o the h e r b i c i d e / q u i n o n e environment. Urbach et_ a l . (27) r e c e n t l y d e m o n s t r a t e d a f l a s h - i n d u c e d b i n a r y o s c i l l a t i o n o f h e r b i c i d e b i n d i n g . H e r b i c i d e b i n d i n g i s h i g h e r i n t h e dark or a t an

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

2.

OETTMEIER

Photosynthetic Electron Transfer Inhibition

27

even number o f f l a s h e s , i . e when Q i s o x i d i z e d , t h a n a t an odd number, when i s s i n g l y reduced. T h e r e f o r e , h e r b i c i d e b i n d i n g can o n l y take p l a c e when t h e b i n d i n g s i t e i s v a c a n t , i . e. n o t o c c u p i e d by Q " I t i s worthy o f s p e c i a l i n t e r e s t t o study d i r e c t l y t h e d i s p l a c e ­ ment o f a h e r b i c i d e by p l a s t o q u i n o n e o r i t s a n a l o g u e s . I n normal t h y ­ l a k o i d s , almost no d i s p l a c e m e n t o f DCMU even by a m i l l i o n - f o l d e x ­ c e s s o f t h e s h o r t - c h a i n p l a s t o q u i n o n e a n a l o g u e p l a s t o q u i n o n e - 1 c a n be o b s e r v e d (28). T h i s may be due t o t h e h i g h endogenous p l a s t o q u i n o n e c o n t e n t o f t h e t h y l a k o i d membrane. I f t h e t h y l a k o i d s a r e d e p l e t e d o f p l a s t o q u i n o n e by means o f n-hexane e x t r a c t i o n , a c o m p e t i t i v e d i s ­ p l a c e m e n t o f DCMU by p l a s t o q u i n o n e - 1 i s o b s e r v e d ( 2 8 ) . T h i s r e s u l t e s t a b l i s h e s a d i r e c t i n t e r a c t i o n between h e r b i c i d e and p l a s t o q u i n o n e , though n o t n e c e s s a r i l y ' a t an i d e n t i c a l b i n d i n g s i t e . From t h e d i s ­ p l a c e m e n t e x p e r i m e n t s , a b i n d i n g c o n s t a n t f o r p l a s t o q u i n o n e - 1 o f 51± 19 μΜ i n p l a s t o q u i n o n e - d e p l e t e d t h y l a k o i d s c a n be c a l c u l a t e d ( 2 8 ) . As compared t o DCMU ( b i n d i n g c o n s t a n t 34 nM (24)) t h e a f f i n i t y o f p l a s t o q u i n o n e - 1 i s more s i m i l a r d i s p l a c e m e n t experimen t r i a z i n e - r e s i s t a n t t h y l a k o i d s , Vermaas e t a_l. (29) found a p l a s t o ­ quinone-1 b i n d i n g c o n s t a n t o f 20 μΜ which i s i n t h e same o r d e r o f magnitude as o u r v a l u e ( 2 8 ) . f

B

In an attempt t o l e a r n more about t h e n a t u r e o f t h e p l a s t o q u i ­ none b i n d i n g s i t e , we have a n a l y z e d t h e d i s p l a c e m e n t b e h a v i o u r o f 25 d i f f e r e n t 1,4-benzoquinones t o DCMU. A q u a n t i t a t i v e s t r u c t u r e a c t i ­ v i t y r e l a t i o n s h i p r e v e a l e d t h a t t h e d i s p l a c i n g a c t i v i t y o f a quinone toward DCMU i s g o v e r n e d by t h e redox p o t e n t i a l and t h e g e o m e t r i c a l c o n f o r m a t i o n o f t h e quinone ( 3 0 ) . An A z i d o p l a s t o q u i n o n e P h o t o a f f i n i t y

Label

To i d e n t i f y p l a s t o q u i n o n e b i n d i n g p r o t e i n s , we have r e c e n t l y s y n t h e ­ s i z e d an a z i d o p l a s t o q u i n o n e p h o t o a f f i n i t y l a b e l ( 3 1 ) . F i g u r e 6 shows a t y p i c a l l a b e l i n g p a t t e r n o f a spinach photosystem I I p r e p a r a t i o n . Only one major p r o t e i n i n t h e 32-34 kDa m o l e c u l a r weight range i s h e a v i l y t a g g e d . A s i m i l a r p i c t u r e i s o b t a i n e d , i f normal t h y l a k o i d s a r e u s e d ( 3 2 ) . L a b e l i n g o f t h e 32-34 kDa p r o t e i n i s p r e v e n t e d , i f t h e samples a r e p r e i n c u b a t e d e i t h e r w i t h DCMU, t h e p h e n o l i c h e r b i c i d e 2 - i o d o - 4 - n i t r o - 6 - i s o b u t y l p h e n o l , o r the photosystem I I i n h i b i t o r tetraiodo-1,4-benzoquinone (33). The q u e s t i o n a r i s e s a s t o whether t h e 3 2 - 3 4 kDa p r o t e i n , a s l a b e l e d by a z i d o p l a s t o q u i n o n e , and t h e 34 kDa p r o t e i n , as l a b e l e d by a z i d o a t r a z i n e o r a z i d o t r i a z i n o n e , a r e i d e n t i c a l . I f samples l a b e l e d by e i t h e r a z i d o p l a s t o q u i n o n e o r a z i d o ­ atrazine are run i n adjacent lanes of a g e l , the R -values o f the s p o t s w i t h t h e maximum amount o f r a d i o a c t i v i t y d i f f e r by 0.05 (2£) . T h i s experiment h a s been r e p e a t e d s e v e r a l t i m e s . I f 4 M u r e a i s i n ­ c l u d e d i n t h e g e l , t h e maxima o f r a d i o a c t i v i t y c o i n c i d e ( 2 4 ) . T h e r e a r e two p o s s i b l e e x p l a n a t i o n s . One i s t h a t t h e p r o t e i n s l a b e l e d by t h e two d i f f e r e n t p h o t o a f f i n i t y l a b e l s a r e i d e n t i c a l . The d i f f e r e n c e s i n t h e R - v a l u e s i n one g e l system may be due t o t h e attachment o f the two d i f f e r e n t m o i e t i e s o r i g i n a t i n g from t h e l a b e l t o t h e p r o t e i n . The second p o s s i b i l i t y would be t h a t t h e two p r o t e i n s a r e r e a l l y d i f ­ f e r e n t , i . e. h e r b i c i d e and p l a s t o q u i n o n e b i n d i n g s i t e s a r e on d i f f e ­ r e n t p r o t e i n s , b u t t h e two p r o t e i n s have v e r y s i m i l a r m o l e c u l a r w e i g h t s . Most r e c e n t l y , e v i d e n c e i s a c c u m u l a t i n g t h a t i n a d d i t i o n t o f

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BIOREGULATORS FOR PEST CONTROL

t h e 34 kDa h e r b i c i d e b i n d i n g p r o t e i n and a 33 kDa l y s i n e - r i c h p r o ­ t e i n , which i s p a r t o f p h o t o s y s t e m I I , b u t i s a s s o c i a t e d w i t h the water s p l i t t i n g enzyme complex, y e t a n o t h e r u n i d e n t i f i e d 32-34 kDa p r o t e i n may p l a y a r o l e i n h e r b i c i d e and p l a s t o q u i n o n e b i n d i n g (34, 35). The

34 kDa

H e r b i c i d e B i n d i n g P r o t e i n : The

Molecular

Level

The 34 kDa h e r b i c i d e b i n d i n g p r o t e i n i s a p l a s t i d encoded p r o t e i n . I n the p r o c e s s o f s e q u e n c i n g the p l a s t i d genome, the DNA sequence o f the gene c o d i n g f o r t h e 34 kDa h e r b i c i d e b i n d i n g p r o t e i n became known and, hence, a l s o i t s amino a c i d sequence ( 3 6 ) . One g r e a t s u r ­ prise a r i s i n g from the amino a c i d sequence i s the complete l a c k o f l y s i n e r e s i d u e s . E x c e p t f o r c o m p a r a t i v e s t u d i e s , the knowledge o f the amino a c i d sequence o f the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n does n o t seem t o y i e l d v e r y much a d d i t i o n a l i n f o r m a t i o n . However, r e c e n t ­ l y K y t e and D o o l i t t l e (31) and A r g o s e t a l . (38) have d e v i s e d a p r o ­ gram t h a t p r o g r e s s i v e l y c i t y of a p r o t e i n along i t v e r y i m p o r t a n t f o r membrane bound p r o t e i n s l i k e the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n because i t a l l o w s one t o p r e d i c t r e g i o n s o f the p r o ­ t e i n t h a t may be embedded w i t h i n the membrane system. I n the K y t e and D o o l i t t l e p r o c e s s (37), each amino a c i d i s a s ­ s i g n e d a h y d r o p a t h y ("strong f e e l i n g about water") v a l u e which ranges from 4.5 f o r i s o l e u c i n e (as tl*«j most h y d r o p h o b i c amino a c i d ) t o -4.5 f o r a r g i n i n e (as t h e most h y d r o p h i l i c amino a c i d ) . Now a c e r t a i n "window", i . e. a c e r t a i n l e n g t h o f t h e amino a c i d sequence i s s e ­ l e c t e d . Assuming a window o f 11 amino a c i d s , the sum o f the h y d r o ­ pathy v a l u e s o f the amino a c i d sequence f o r amino a c i d s 1 t o 11 i s c a l c u l a t e d . Next t h e window i s moved one amino a c i d ahead w i t h i n the sequence, and the sum o f t h e h y d r o p a t h y v a l u e s f o r amino a c i d s 2 t o 12 i s c a l c u l a t e d . T h i s p r o c e s s i s r e p e a t e d f o r sequence 3-13, 4-14 e t c . u n t i l t h e end o f the sequence i s r e a c h e d . The sum o f the h y d r o ­ p a t h y v a l u e s o v e r the number o f the amino a c i d r e s i d u e i s p l o t t e d . Such a h y d r o p a t h y p l o t f o r the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n i s p r e s e n t e d i n F i g u r e 7. ( I t s h o u l d be n o t e d t h a t f o r the h y d r o p a t h y p l o t , the o r i g i n a l amino a c i d sequence as r e p o r t e d by Z u r a w s k i e t a l . (36) was n o t u s e d . I n s t e a d , a sequence s h o r t e r by 36 amino a c i d s which s t a r t s a t the second Met was used ( 3 9 ) ) . The p o s i t i v e (shaded) a r e a s i n F i g u r e 7 c o r r e s p o n d t o r e g i o n s o f h i g h h y d r o p a t h y , the ne­ g a t i v e a r e a s t o r e g i o n s o f low h y d r o p a t h y . Only r e g i o n s o f h i g h hy­ d r o p a t h y o f the p r o t e i n a r e t h o u g h t t o be b u r i e d w i t h i n the membrane system, p r o v i d e d they a r e a p p r o x i m a t e l y 20 amino a c i d s l o n g t o a c ­ count f o r a h e l i c a l span t h r o u g h the l i p i d b i l a y e r o f the membrane. Based on t h i s assumption, seven h e l i c a l spans a r e p r e d i c t e d f o r the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n ( F i g u r e 7 ) . A v e r y s i m i l a r h y d r o ­ pathy p l o t f o r the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n was p r e s e n t e d by A r g o s s group (40) c a l c u l a t e d a c c o r d i n g t o t h e i r method (38). I t d i f f e r s from t h a t i n F i g u r e 7 by the assignments o f spans V and VI (41) . 1

A s c h e m a t i c p i c t u r e o f the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n as i t i s t h o u g h t t o be l o c a t e d i n the membrane i s g i v e n i n F i g u r e 8. The seven h e l i c a l spans t h r o u g h the membrane a r e i n d i c a t e d . F u r t h e r ­ more, F i g u r e 8 p r o v i d e s t h r e e a d d i t i o n a l r e l e v a n t f a c t s or sugges­ t i o n s . The f i r s t one d e a l s w i t h the p o s s i b l e b i n d i n g s i t e o f a z i d o -

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

2.

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29

F i g u r e 6. Photograph o f a L i - d o d e c y l s u l f a t e p o l y a c r y l a m i d e elec­ t r o p h o r e s i s g e l (10-15%) and r a d i o a c t i v i t y d i s t r i b u t i o n t h e r e i n o f a s p i n a c h p h o t o s y s t e m I I p r e p a r a t i o n l a b e l e d by 2 nmol/mg c h l o r o ­ p h y l l [ ËT| a z i d o p l a s t o q u i n o n e .

H N-

-COOH

2

1

M I I

II

I I I

I

I I I I

I

I I I I

1

I M I

100 200 Nr. Amino Acid Residue (window: 11)

Figure

Iιιι ιh 1 300

7. Hydropathy p l o t o f t h e 34 kDa h e r b i c i d e b i n d i n g

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

protein.

30

BIOREGULATORS FOR PEST CONTROL

Possible Plastoquinone Binding Sites: Met-His 158.159

Phe-His-Met 161 , 163

Azido-atrazine Binding Site

Met-His 178,179

Ser 2

2

8

Resistance Gly, Ala

F i g u r e 8. Schematic drawing o f t h e p o s s i b l e l o c a t i o n o f t h e 34 kDa h e r b i c i d e b i n d i n g p r o t e i n w i t h i n t h e t h y l a k o i d membrane.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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atrazine on the 34 kDa herbicide binding protein. From their recent work on proteolytic digestion after azidoatrazine labeling, Wolber and Steinback (42) have concluded that the azidoatrazine binding site presumably is located in the region Pro 105 to Arg 189 of the amino acid sequence (spans IV to VI, Figure 8). The second suggestion relates to herbicide resistance. By sequencing the DNA of the 34 kDa herbicide binding protein from atrazine-resistant Amaranthus, Hirschberg and Mcintosh (39) have found 4 nucleotide differences as compared to the susceptible type. Only one of the differences leads to a change in the amino acid sequence: Ser in position 228 is replaced by Gly. Similarily, in an atrazine-resistant mutant from Chlamydomonas the same Ser in position 228 is exchanged, but this time against Ala (43). The third suggestion concerns possible plastoquinone binding sites in the 34 kDa herbicide binding protein, if there are any. According to a proposal by Hearst and Sauer (^4), the sequence MetHis is a possible quinone binding site, probably with an additional Phe. There are three consecutive Met-His sequences in the 34 kDa herbicide binding protei ever, that the arginine quinone binding (45). Indeed, the arginine-modifying reagent phenylglyoxal was found to decrease atrazine binding (46). In conclusion, observations made in the last few years, especially the binding studies with radiolabeled herbicides, the photoaffinity labeling technique, and the advances of molecular biology have substantially added to our knowledge of the mechanism of action of photosynthetic herbicides. However, many questions also remain to be answered. Acknowledgements This work was supported by Deutsche Forschungsgemeinschaft. I am indebted to Eva Neumann, Dr. Udo Johanningmeier, Klaus Masson, and Hans-Joachim Soli for their help in the experiments. Literature Cited 1. Bouges-Bocquet, B. Biochim. Biophys. Acta 1973, 314, 250-6. 2. Velthuys, B.R.; Amesz, J . Biochim. Biophys. Acta 1974, 333, 85-94. 3. Renger, G. Biochim. Biophys. Acta 1976, 440, 287-300. 4. Regitz, G; Ohad, I. J . Biol. Chem. 1976, 251, 247-52. 5. Trebst, A; Draber, W. In "Advances in Pesticide Science"; Geissbühler, H., Ed.; Pergamon Press: Oxford, New York, 1979; Part 2, p. 223. 6. Pfister, K; Arntzen, C.J. Z. Naturforsch. 1979, 34c, 996-1009. 7. Tischer, W; Strotmann, H. Biochim. Biophys. Acta 1977, 460, 113-25. 8. Oettmeier, W.; Masson, K.; Johanningmeier, U. Biochim. Biophys. Acta 1982, 679, 376-83. 9. Oettmeier, W.; Trebst, A. In "The Oxygen Evolving System of Photosynthesis"; Inoue, Y., et a l . , Eds.; Academic Press: Tokyo, 1983, p. 411. 10. Pfister, K.; Radosevich, S.R.; Arntzen, C.J. Plant Physiol. 1979, 64, 995-9.

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11. Oettmeier, W.; Masson, K.; Fedtke, C . ; Konze, J.; Schmidt, R.R. Pestic. Biochem. Physiol. 1982, 18, 357-67. 12. Oettmeier, W.; Masson, K. Pestic. Biochem. Physiol. 1980, 14, 86-97. 13. Trebst, A; Harth, E . ; Draber, W. Z. Naturforsch. 1970, 25b, 1157-9. 14. Oettmeier, W.; Masson, K.; Johanningmeier, U. FEBS Lett.1980, 118, 267-70. 15. Gardner, G. Science 1981, 211, 937-40. 16. Oettmeier, W.; Masson, K.; Soll, H . J . ; Draber, W. Biochim. Biophys. Acta 1984, in press. 17. Johanningmeier, U.; Neumann, E.; Oettmeier, W. J . Bioenerg. Biomembr. 1983, 15, 43-66. 18. Pfister, K.; Steinback, K . E . ; Gardner, G.; Arntzen, C . J . Proc. Natl. Acad. Sci.USA 1981, 78, 981-5. 19. Mattoo, A . K . ; Pick, U.; Hoffmann-Falk, H . ; Edelman, M. Proc. Natl. Acad. Sci. USA 1981, 78, 1572-6. 20. Gressel, J . Plant Sci 21. Laasch, H.; Pfister 620-31. 22. Pfister, K.; Schreiber, U. Z. Naturforsch. 1984, 39c, 389-92. 23. Mathis, P.; Rutherford, A.W. Biochim. Biophys. Acta 1984, in press. 24. Oettmeier, W.; Soll, H . J . ; Neumann, Ε. Z. Naturforsch. 1984, 39c, 393-6. 25. Velthuys, B.R. FEBS Lett. 1981, 126, 277-81. 26. Lavergne, J . Biochim. Biophys. Acta, 1982, 682, 345-53. 27. Urbach, W.; Laasch, H . ; Schreiber, U. Z. Naturforsch. 1984, 39c, 397-401. 28. Oettmeier, W.; Soll, H.J. Biochim. Biophys. Acta 1983, 724, 287-90. 29. Vermaas, W.F.J.; Renger, G.; Arntzen, C . J . Z. Naturforsch. 1984, 39c, 368-73. 30. Soll, H . J . ; Oettmeier, W. In "Advances in Photosynthesis Re­ search"; Sybesma, C . , Ed.; Martinus Nijhoff/Dr. W. Junk Pub­ lishers: The Hague, 1984, Vol. 4, p. 5. 31. Oettmeier, W.; Masson, K.; Soll, H . J . ; Hurt, E . ; Hauska, G. FEBS Lett. 1982, 144, 313-7. 32. Oettmeier, W.; Masson, K.; Soll, H . J . ; Olschewski, E. In "Advan­ ces in Photosynthesis Research"; Sybesma, C., Ed.; Martinus Nijhoff/ Dr. W. Junk Publishers: The Hague, 1984, Vol. 1, p.469. 33. Oettmeier, W.; Soll, H.J. in preparation. 34. Satoh, K.; Nakatani, H.Y.; Steinback, K . E . ; Watson, J.; Arntzen, C.J. Biochim. Biophys. Acta 1983, 724, 142-50. 35. Renger, G.; Hagemann, R.; Vermaas, W.F.J. Z. Naturforsch. 1984, 39c, 362-7. 36. Zurawski, G.; Bohnert, H . J . ; Whitfield, P.R.; Bottomley, W. Proc. Natl. Acad. Sci. USA 1982, 79, 7699-703. 37. Kyte, J ; Doolitlle, R.F. J . Mol. Biol. 1982, 157, 105-32. 38. Argos, P.; Rao, J.K.M.; Hargrave, P.A. Eur. J . Biochem. 1982, 128, 565-75. 39. Hirschberg, J ; McIntosh, L. Science 1983, 222, 1346-9. 40. Rao, J.K.M.; Hargrave, P.Α.; Argos, P. FEBS Lett. 1983, 156, 165-9.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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41. Trebst, A. In "Proceedings of the Southern Section, American Society of Plant Physiology, 1984, in press. 42. Wolber, P.K.; Steinback, K.E. Z. Naturforsch. 1984, 39c, 425-9. 43. Erickson, J.M; Rahire, M.; Bennoun, P.; Delepelaire, P.; Diner, B.; Rochaix, J.D. Proc. Natl. Acad. Sci. USA 1984, 81, 3617-21. 44. Hearst, J . E . ; Sauer, Κ. Z. Naturforsch. 1984, 39c, 421-4. 45. Shipman, L . L . J . Theor. Biol. 1981, 90, 123-48. 46. Gardner, G.; Allan, C.D.; Paterson, D.R. FEBS Lett. 1983, 164, 191-4. RECEIVED November 8, 1984

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3 New Approaches to Chemical Control of Plant Pathogens 1

2

NANCY N. RAGSDALE and MALCOLM R. SIEGEL 1

Cooperative State Research Service, U.S. Department of Agriculture, Washington, DC 20251 Plant Pathology Department, University of Kentucky, Lexington, ΚY 40546

2

New approaches t emphasize selectiv understanding of the physiological, biochemical, and molecular levels of the modes of action and mechanisms of resistance. Antifungal compounds currently available for disease control may have either non-specific or specific biochemical modes of action. Nearly all systemic fungicides are of the latter type. Compounds with specific modes of action are prone to a loss of efficacy through selection pressure for resistant pathogens. Evidence suggests that chemicals with certain modes of action may be less likely to encounter resistance problems. The possibility of circumventing resistance by chemically affecting metabolic pathways or through stereoselectivity are discussed. In addition, promising methods for plant disease control with chemicals that activate plant defense mechanisms or interfere with pathogenesis are considered. Most of the pests that cause infectious plant diseases may be classified as fungi, bacteria, nematodes, or viruses. Chemicals frequently are either unavailable or impractical to use in controlling viruses and most bacteria. Efforts to achieve nematode control usually involve compounds associated with insect control. Thus, this article w i l l be devoted to the use of chemicals to control fungi that cause plant diseases, a far greater use than for other disease agents. These fungicides, or antifungal agents as they are more correctly called, are but a part of disease control strategy. Plant breeding programs and cultural practices are also part of plant disease management practices. The traditional approach in the development of fungicides has involved large scale laboratory and greenhouse screening tests followed by closer examination of structurally related chemicals to optimize activity. More recently scientists have become aware that this type of assay system has limitations. There is a need to examine vulnerabilities in both the pest and the host, and to design 0097-6156/85/0276-0035$06.00/0 © 1985 American Chemical Society

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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a n t i f u n g a l m a t e r i a l s t h a t have modes o f a c t i o n based on these principals. T h i s w i l l i d e n t i f y c h e m i c a l s t h a t have b o t h d i r e c t and i n d i r e c t a c t i o n s . New approaches emphasize an u n d e r s t a n d i n g a t the p h y s i o l o g i c a l , m o l e c u l a r , and b i o c h e m i c a l l e v e l s of modes of a c t i o n as w e l l as the mechanisms f o r pathogen r e s i s t a n c e . Some o f the more r e c e n t a n t i f u n g a l compounds r e s u l t e d from t h i s type o f a p p r o a c h . Not o n l y w i l l t h e s e a p p r o a c h e s p r o v i d e knowledge f o r the r a t i o n a l development of new a n t i p a t h o g e n i c compounds, they w i l l a l s o p r o v i d e s t r a t e g i e s f o r the use of e s t a b l i s h e d c h e m i c a l s , e s p e c i a l l y i n r e l a t i o n to r e d u c i n g or p r e v e n t i n g the development o f r e s i s t a n t pathogens· T h i s c h a p t e r w i l l endeavor to condense a r a t h e r wide r a n g i n g s u b j e c t i n t o some b a s i c p r i n c i p l e s r e g a r d i n g the types of c h e m i c a l s used t o c o n t r o l p l a n t pathogens; the s t r a t e g i e s b e h i n d t h e s e ; and some thoughts on new approaches to the development and use of c h e m i c a l s t o c o n t r o l f u n g a l d i s e a s e s of p l a n t s . First

Generation

Fungicide

The f i r s t g e n e r a t i o n f u n g i c i d e s a r e p r i m a r i l y a group r e f e r r e d t o as s u r f a c e p r o t e c t a n t s . These compounds a r e not taken up to any d e g r e e by the p l a n t , have a broad a n t i f u n g a l spectrum, and demonstrate m u l t i p l e s i t e s of a c t i o n ( 1 ) . The f u n c t i o n of t h e s e p e s t i c i d e s i s based on t h e i r b e i n g p r e s e n t when the fungus a r r i v e s and b e f o r e i n f e c t i o n occurs. They e x h i b i t a d i f f e r e n t i a l t o x i c i t y ; thus a s p e c i f i c dose w i l l k i l l the pathogen and n o t i n j u r e the h o s t p l a n t . D i f f e r e n t i a l t o x i c i t y i s based p r i m a r i l y on f u n g i r a p i d l y a c c u m u l a t i n g t o x i c c o n c e n t r a t i o n s of the f u n g i c i d e w h i l e p l a n t t i s s u e s do n o t ( 2 ) . The s u r f a c e p r o t e c t a n t s i n c l u d e i n o r g a n i c c h e m i c a l s such as e l e m e n t a l s u l f u r , Bordeaux m i x t u r e and copper o x i d e as w e l l as o r g a n i c f u n g i c i d e s such as d i a l k y l d i t h i o c a r b a m a t e s ( t h i r a m , ferbam), e t h y l e n e b i s d i t h i o c a r b a m a t e s (maneb, z i n e b ) , p h t h a l i m i d e s ( c a p t a n , f o l p e t ) , and c h l o r o t h a l o n i l . Because t h e s e f i r s t g e n e r a t i o n f u n g i c i d e s a r e m u l t i s i t e i n h i b i t o r s , they a r e n o t l i k e l y t o l o s e e f f e c t i v e n e s s because of f u n g a l r e s i s t a n c e . Pathogen r e s i s t a n c e has n o t been a problem w i t h t h i s group and t h e i r c o n t i n u e d use as an i m p o r t a n t p a r t o f d i s e a s e management programs i s h i g h l y likely. Second G e n e r a t i o n

Fungicides

D u r i n g the mid I960's, the s y s t e m i c t o x i c a n t s , or second g e n e r a t i o n f u n g i c i d e s as they a r e c a l l e d , e n t e r e d the m a r k e t . A l t h o u g h most o f the o l d e r m a t e r i a l s a r e s t i l l i n use, c u r r e n t e f f o r t s have s h i f t e d toward d e v e l o p i n g s y s t e m i c compounds which move i n the symplast, a p o p l a s t , o r a r e ambimoble ( 3 ) . Almost a l l the c o m m e r c i a l l y a v a i l a b l e s y s t e m i c f u n g i c i d e s move o n l y i n the a p o p l a s t and a r e t h e r e f o r e dependent on t r a n s p i r a t i o n f o r t h e i r movement and accumulation i n plant t i s s u e . The s y s t e m i c f u n g i c i d e s have a number o f advantages o v e r s u r f a c e p r o t e c t a n t s . Foremost i s t h e i r a b i l i t y to p e n e t r a t e h o s t t i s s u e and c o n t r o l o r e r a d i c a t e an e s t a b l i s h e d infection. I n o r d e r f o r t h e s e compounds to p r o v i d e i n t e r n a l t h e r a p y , they must have c e r t a i n q u a l i t i e s . They o r t h e i r a c t i v e d e r i v a t i v e s must be r e l a t i v e l y s t a b l e w i t h i n the h o s t t i s s u e w i t h e i t h e r a

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mechanism s p e c i f i c f o r f u n g a l c e l l s or a c o n c e n t r a t i o n so as not to produce p h y t o t o x i c e f f e c t s ( 4 ) . In c o n t r a s t to f i r s t g e n e r a t i o n f u n g i c i d e s , they a r e g e n e r a l l y e f f e c t i v e a g a i n s t a narrower range o f pathogens, and s y s t e m i c s have a s p e c i f i c s i t e or a l i m i t e d number of s i t e s of a c t i o n , making them more s u b j e c t to l o s s o f e f f i c a c y due to fungal resistance. F o l l o w i n g d e t e r m i n a t i o n of t h e i r modes of a c t i o n , tremendous c o n t r i b u t i o n s have been made through the use of t h e s e c h e m i c a l s t o e l u c i d a t e v a r i o u s b i o c h e m i c a l pathways and c e l l u l a r f u n c t i o n s . We w i l l l o o k a t a number of examples of t h e s e second g e n e r a t i o n s y s t e m i c s and b r i e f l y d i s c u s s t h e i r modes of a c t i o n . Cycloheximide. C y c l o h e x i m i d e , an a n t i f u n g a l a n t i b i o t i c produced by Streptomyces g r i s e u s , i n h i b i t s p r o t e i n s y n t h e s i s not o n l y i n f u n g i , but i n e u k a r y o t i c organisms i n g e n e r a l ( 5 ) . Due to p h y t o t o x i c i t y problems, c y c l o h e x i m i d e can o n l y be used s u c c e s s f u l l y f o r d i s e a s e c o n t r o l when low c o n c e n t r a t i o n s a r e e f f e c t i v e or on p l a n t s t h a t a r e r e l a t i v e l y i n s e n s i t i v e (6)· to i n h i b i t a number of c e l l u l a n u c l e i c a c i d s y n t h e s i s , t h e s e a r e r e g a r d e d as s e c o n d a r y e f f e c t s t h a t r e s u l t from the p r i m a r y s i t e of a c t i o n , i . e . , r i b o s o m a l p r o t e i n s y n t h e s i z i n g systems (7)· R e s i s t a n c e to c y c l o h e x i m i d e has been c o r r e l a t e d w i t h changes i n r i b o s o m a l s e n s i t i v i t y or i n c e l l u l a r permeability ( 1). Benomyl. Benomyl i s the b e s t known of s e v e r a l b e n z i m i d a z o l e fungicides. T h i s compound i s e f f e c t i v e a g a i n s t a l a r g e number o f f u n g i and p l a y s an i m p o r t a n t r o l e i n d i s e a s e management. Benomyl i n t e r f e r e s w i t h t u b u l i n p o l y m e r i z a t i o n i n f u n g i by b i n d i n g to the beta t u b u l i n subunit ( 8 ) . As a r e s u l t of the f a i l u r e to form m i c r o t u b u l e s , m i t o s i s i s b l o c k e d (9) as w e l l as o t h e r microtubular-dependent processes (10). I t has been shown t h a t a s i n g l e gene m u t a t i o n a l t e r s the a f f i n i t y of the t u b u l i n b i n d i n g s i t e f o r the i n h i b i t o r r e s u l t i n g i n f u n g a l r e s i s t a n c e ( 8 ) . The n a t u r e of t h i s h i g h l y s p e c i f i c mode of a c t i o n and the c o n t r o l of t o x i c i t y t h r o u g h a s i n g l e gene has r e s u l t e d , as one m i g h t e x p e c t , i n the development of problems i n the f i e l d w i t h f u n g a l r e s i s t a n c e . f

Metalaxyl. S i n c e i t s i n t r o d u c t i o n the l a t e 1 9 7 0 s f o r the c o n t r o l o f pathogens such as p o t a t o l a t e b l i g h t , downy mildews and soil-borne Pythium and P h y t o p h t h o r a spp., m e t a l a x y l , an a c y l a l a n i n e f u n g i c i d e , and o t h e r r e l a t e d phenylamide f u n g i c i d e s have seen i n c r e a s i n g u s a g e . A l t h o u g h the p r e c i s e mechanism of i n h i b i t i o n by m e t a l a x y l has not been d e t e r m i n e d , RNA s y n t h e s i s appears to be the most s e n s i t i v e pathway (JUL). F i e l d r e s i s t a n c e to m e t a l a x y l has d e v e l o p e d r a p i d l y i n s i t u a t i o n s t h a t i n v o l v e d e x c l u s i v e and r e p e a t e d use of the compound (11). T h i s has prompted the development of use s t r a t e g i e s s u c h as t h a t of combining c h e m i c a l s which have d i f f e r e n t modes of a c t i o n . Carboxin. C a r b o x i n i s used p r i m a r i l y to c o n t r o l B a s i d i o m y c e t e s s u c h as r u s t s , b u n t s , and smuts. I t i s used p r i m a r i l y f o r seed t r e a t m e n t , but a l s o has s o i l and f o l i a r a p p l i c a t i o n s . The mode o f a c t i o n o f t h i s and r e l a t e d compounds i s p r o b a b l y the b e s t u n d e r s t o o d of any f u n g i t o x i c mechanism. C a r b o x i n b l o c k s the t r a n s f e r o f e l e c t r o n s from

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BIOREGULATORS FOR PEST CONTROL

s u c c i n i c dehydrogenase t o coenzyme Q i n t h e complex I I r e g i o n o f t h e m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t c h a i n (12, 1 3 ) . L a b o r a t o r y s t u d i e s have i n d i c a t e d t h a t r e s i s t a n c e i s s i n g l e - g e n e based and s e v e r a l l o c i a r e i n v o l v e d (11)« Based on t h e h i g h l y s p e c i f i c mode o f a c t i o n and s t u d i e s on l a b o r a t o r y d e v e l o p e d r e s i s t a n t m u t a n t s , i t i s s u r p r i s i n g t h a t i n s p i t e o f q u i t e a few y e a r s ' u s e , r e s i s t a n c e i n t h e f i e l d has not been a p r o b l e m . One would s p e c u l a t e t h a t n a t u r a l mutants must be l e s s f i t o r do n o t have t h e p a t h o g e n i c c a p a b i l i t i e s o f t h e w i l d t y p e . A more l i k e l y e x p l a n a t i o n i s r e l a t e d t o t h e p a t t e r n o f u s e . Because most c a r b o x i n i s used f o r seed t r e a t m e n t , t h e s e l e c t i o n p r e s s u r e i s r a t h e r low. Polyoxins. The p o l y o x i n a n t i b i o t i c s a r e e f f e c t i v e a g a i n s t f u n g i t h a t contain c h i t i n i n their c e l l walls. The r e s i s t a n c e t o p o l y o x i n s i n some f u n g i o f t h i s type a p p e a r s t o be r e l a t e d t o a p e r m e a b i l i t y f a c t o r r a t h e r than t o a change i n the s i t e o f a c t i o n ( 1 4 ) . Polyoxins c o m p e t i t i v e l y i n h i b i t c h i t i n s y n t h e t a s e and thus p r e v e n t t h e incorporation of uridinediphospho-N-acetylglucosamin polymer ( 1 4 ) . E d i f e n p h o s . E d i f e n p h o s , an organophosphorus compound, has a r a t h e r l i m i t e d use because o f i t s h i g h l y s e l e c t i v e a c t i o n a g a i n s t t h e r i c e b l a s t fungus. Some a m b i g u i t y e x i s t s r e g a r d i n g the mode o f a c t i o n . A l t h o u g h t h e compound has been shown t o i n h i b i t c h i t i n s y n t h e t a s e thus b l o c k i n g c e l l w a l l s y n t h e s i s , i t a l s o i n h i b i t s the s y n t h e s i s o f p h o s p h a t i d y l c h o l i n e , a p h o s p h o l i p i d and i m p o r t a n t membrane component (11). The l a t t e r i n h i b i t i o n , w h i c h has been s u g g e s t e d as the p r i m a r y mechanism o f a c t i o n , r e s u l t s from b l o c k i n g t h e a c t i v i t y o f phospholipid-N-methyltransferase, which i s necessary i n the conversion of phosphatidylethanolamine to phosphatidylcholine (11). The e f f e c t s on c h i t i n s y n t h e s i s p r o b a b l y r e s u l t from a l t e r e d membrane properties. R e s i s t a n c e a g a i n s t e d i f e n p h o s e i n the f i e l d has been slow t o d e v e l o p . P o l y e n e s . A l t h o u g h a m p h o t e r i c i n Β and n y s t a t i n , b o t h p o l y e n e a n t i b i o t i c s , a r e v e r y a c t i v e a g a i n s t a wide v a r i e t y o f f u n g i , they a r e i m p r a c t i c a l as a g r i c u l t u r a l f u n g i c i d e s due t o t h e i r r a p i d photodecomposition (14). These compounds a f f e c t membrane p e r m e a b i l i t y through t h e i r i n t e r a c t i o n s with s t e r o l s (15). These i n t e r a c t i o n s cause membrane changes t h a t r e s u l t i n leakage o f s m a l l molecular materials. R e s i s t a n c e may g e n e r a l l y be a t t r i b u t e d t o a change i n s t e r o l q u a l i t y o r growth c o n d i t i o n s . D i c a r b o x i m i d e s and A r o m a t i c H y d r o c a r b o n s . The d i c a r b o x i m i d e f u n g i c i d e s , procymidone, v i n c l o z o l i n and i p r o d i o n e , and a r o m a t i c h y d r o c a r b o n s s u c h as c h l o r o n e b , d i c h l o r a n and q u i n t o z e n e , show a g r e a t d e a l o f s i m i l a r i t y i n t h e i r modes o f t o x i c i t y ( 1 6 ) . Although the e f f e c t s o f t h e s e compounds on a number o f b i o s y n t h e t i c p r o c e s s e s have been i n v e s t i g a t e d , the e x a c t mode of a c t i o n has n o t been r e s o l v e d (4^, 1 1 ) . I n t e r f e r e n c e w i t h n u c l e a r f u n c t i o n , membrane damage, and i n t e r f e r e n c e w i t h c e l l u l a r m o t i l e f u n c t i o n s , such as f l a g e l l a r movement and c y t o p l a s m i c s t r e a m i n g , have been s u g g e s t e d as possibilities. A b e t t e r u n d e r s t a n d i n g o f the mode o f a c t i o n may e x p l a i n why r e s i s t a n c e has p r e s e n t e d few problems i n t h e p r a c t i c a l use o f t h e s e f u n g i c i d e s .

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E r g o s t e r o l Biosynthesis I n h i b i t o r s (EBI). S i n c e the determination t h a t t r i a r i m o l owed i t s a n t i f u n g a l a c t i v i t y to the i n h i b i t i o n of e r g o s t e r o l b i o s y n t h e s i s ( 1 7 ) , a l a r g e number of s t r u c t u r a l l y d i v e r s e compounds t h a t b l o c k e r g o s t e r o l b i o s y n t h e s i s have become a v a i l a b l e f o r p l a n t d i s e a s e c o n t r o l . T h i s group c o n s i s t s o f p y r i m i d i n e s , i m i d a z o l e s , t r i a z o l e s , and m i s c e l l a n e o u s compounds i n the p y r i d i n e , m o r p h o l i n e and p i p e r a z i n e c l a s s e s . These compounds c o n t r o l a wide range of p l a n t d i s e a s e s i n c l u d i n g smuts, r u s t s , and powdery mildews (4). C u r r e n t l y , t h i s i s the l a r g e s t and most i m p o r t a n t group of s y s t e m i c compounds a v a i l a b l e f o r c o n t r o l l i n g f u n g a l d i s e a s e s i n p l a n t s and a n i m a l s . W h i l e t h e s e compounds a r e c o n v e n t i o n a l l y r e f e r r e d to as EBI f u n g i c i d e s , they can b l o c k s t e r o l b i o s y n t h e s i s i n organisms t h a t s y n t h e s i z e s t e r o l s other than e r g o s t e r o l (18). A l l o f the E B I ' s i n t e r f e r e w i t h s t e p s i n the b i o s y n t h e t i c pathway f o l l o w i n g the c y c l i z a t i o n of squalene. A r e c e n t l y developed c l a s s of a n t i f u n g a l a g e n t s f o r a n i m a l use the a l l y l a m i n e s , d e v e l o p e d by m o d i f i c a t i o n o f n a f t i f i n e , b l o c k the pathwa squalene epoxidase (19) b l o c k i n g the cytochrome P-450 dependent s t e r o l C-14 d e m e t h y l a t i o n r e a c t i o n (18). A l t h o u g h t r i d e m o r p h produces e f f e c t s i n f u n g i s i m i l a r to t h o s e produced by compounds t h a t i n h i b i t s t e r o l C-14 d e m e t h y l a t i o n , i t does n o t i n t e r f e r e w i t h t h i s r e a c t i o n but i n h i b i t s the i s o m e r i z a t i o n o f the s t e r o l C-8(9) d o u b l e bond to C-7(8) (20) or the r e d u c t i o n of the s t e r o l C-14(15) d o u b l e bond ( 2 1 ) . I t i s i n t e r e s t i n g t h a t many s t r u c t u r a l l y d i v e r s e compounds a l l i n h i b i t s t e r o l C-14 d e m e t h y l a t i o n . However, w i t h the e x c e p t i o n o f t r i f o r i n e , t h e s e E B I * s have i n common a n i t r o g e n - c o n t a i n i n g h e t e r o c y c l e s u b s t i t u t e d w i t h one l a r g e l i p o p h i l i c g r o u p . I t has been proposed t h a t a n i t r o g e n atom o f the h e t e r o c y c l e i n t e r a c t s w i t h the protohaem i r o n atom of the cytochrome P-450 enzyme(s) i n v o l v e d i n s t e r o l C-14 d e m e t h y l a t i o n and t h a t the l i p o p h i l i c s u b s t i t u e n t i n c r e a s e s b i n d i n g a f f i n i t y through an i n t e r a c t i o n w i t h a nearby r e g i o n o f the enzyme (22, 2 3 ) . E v i d e n t l y , t h e r e can be a wide v a r i a t i o n i n the l i p o p h i l i c s u b s t i t u e n t ; however, v a r i a t i o n i n the s t r u c t u r e c o n t r o l s s p e c i f i c i t y as i n d i c a t e d by the f a c t t h a t compounds which e f f e c t i v e l y i n h i b i t f u n g a l s t e r o l demethylase systems may be r e l a t i v e l y i n e f f e c t i v e a g a i n s t the demethylase system from mammalian c e l l s ( 2 3 ) . The * * * * *

1

E B I s produce the f o l l o w i n g t y p i c a l s e c o n d a r y e f f e c t s ( 1 8 ) : No i n h i b i t i o n of i n i t i a l c e l l growth Morphological abnormalities No immediate e f f e c t on r e s p i r a t i o n No Immediate e f f e c t on RNA, DNA o r p r o t e i n s y n t h e s e s S t e r o l i n t e r m e d i a t e s accumulate F r e e f a t t y a c i d s accumulate The m o r p h o l o g i c a l a b n o r m a l i t i e s r e s u l t i n g from treatment w i t h t h e s e compounds a r e q u i t e s t r i k i n g ( 1 8 ) . S p o r i d i a of U s t i l a g o become m u l t i c e l l e d and o f t e n b r a n c h e d . G e r m i n a t i n g c o n i d i a o f some f u n g i show s w e l l i n g , l e a k a g e , and membrane r u p t u r e . In a d d i t i o n , e f f e c t s on membranes a p p a r e n t l y l e a d t o a b n o r m a l i t i e s i n s y n t h e s i s o f the c e l l w a l l as w e l l as t h a t of o t h e r c e l l u l a r components. Major l i p i d f r a c t i o n s o t h e r than s t e r o l s a r e not i n i t i a l l y inhibited. However, a d e l a y e d a c c u m u l a t i o n of f r e e f a t t y a c i d s β

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characteristically results after sterol inhibition. These are thought to r e s u l t from the breakdown of membranes and t r i g l y c e r i d e s t o g e t h e r w i t h a d i s p r o p o r t i o n a t e s y n t h e s i s and u t i l i z a t i o n o f f a t t y acids (18). Growth r e t a r d a t i o n i n h o s t p l a n t s has been a s s o c i a t e d w i t h the use of a number of t h e s e E B I ' s . T h i s e f f e c t r e s u l t s from the i n h i b i t i o n o f a r e a c t i o n i n the g i b b e r e l l i n b i o s y n t h e t i c pathway t h a t i n v o l v e s cytochrome P-450 enzymes ( 2 4 ) . A l t h o u g h the i n h i b i t i o n of e r g o s t e r o l b i o s y n t h e s i s i s r e g a r d e d as the p r i m a r y mode of a c t i o n , i t i s p o s s i b l e t h a t the s u c c e s s of t h e s e compounds as a n t i f u n g a l a g e n t s i s r e l a t e d to o t h e r e f f e c t s as well. Not a g r e a t d e a l i s known about f u n g a l hormones. A r e c e n t a r t i c l e c o n c e r n i n g human p a t h o g e n i c f u n g i i n d i c a t e d t h a t f u n g a l s t e r o i d s may be a s s o c i a t e d w i t h the p a t h o g e n i c i t y of t h e s e organisms (25). S i n c e the s t e r o i d s a r e s y n t h e s i z e d from s t e r o l p r e c u r s o r s , an i n h i b i t i o n of s t e r o i d b i o s y n t h e s i s i n a d d i t i o n to the e f f e c t s on p r e c u r s o r s t e r o l s may be r e s p o n s i b l e f o r the e f f e c t i v e n e s s of E B I s i n c o n t r o l l i n g plant pathogeni 1

R e s i s t a n c e has n o t thu EBI's. A l t h o u g h numerous r e s i s t a n t mutants have been d e v e l o p e d i n l a b o r a t o r y s t u d i e s , r e s i s t a n c e problems i n the f i e l d have not occurred. However, r e c e n t i n v e s t i g a t i o n s r e l a t e d t o the use of a t r i a z o l e f u n g i c i d e on b a r l e y i n d i c a t e t h a t r e s i s t a n t s t r a i n s o f p a t h o g e n i c f u n g i may be i n c r e a s i n g ( 2 6 ) . The f a c t t h a t t h e r e have not been major problems may be a s s o c i a t e d w i t h the n a t u r e of the mode of a c t i o n and the f a c t t h a t mutants appear to have r e d u c e d f i t n e s s (11, 1 8 ) . In o t h e r words, the g e n e t i c a l t e r a t i o n s c o n d u c i v e to s u r v i v a l i n the p r e s e n c e of t h e s e compounds may a l s o reduce f i t n e s s . T h i r d Generation

Fungicides

The t h i r d g e n e r a t i o n f u n g i c i d e s c o n s i s t of c h e m i c a l s d e s i g n e d t o take advantage of p r i n c i p l e s u n d e r l y i n g host and/or pathogen vulnerabilities. These compounds t y p i c a l l y a r e h i g h l y s p e c i f i c , o f t e n t a r g e t e d f o r a s p e c i f i c d i s e a s e on a s e l e c t e d h o s t . They c h a r a c t e r i s t i c a l l y show l i t t l e or no t o x i c i t y to f u n g i i n v i t r o . They a c t as a n t i p a t h o g e n i c agents and thus a f f e c t the p r o c e s s of p a t h o g e n e s i s . They may a c t on the h o s t t h r o u g h the i n d u c t i o n o f p l a n t r e s i s t a n c e mechanisms such as s t i m u l a t i o n of l i g n i f i c a t i o n o r enhancement o f p h y t o a l e x i n p r o d u c t i o n . ( P l e a s e r e f e r to the c h a p t e r by S a l t and Kuc i n t h i s volume f o r f u r t h e r d i s c u s s i o n of t h i s t y p e o f compound.) They may a c t on the pathogen to a c c e n t u a t e e l i c i t o r r e l e a s e or to p r e v e n t i n f e c t i o n ( h o s t p e n e t r a t i o n ) , c o l o n i z a t i o n ( i n h i b i t i o n o f p h y t o t o x i n s y n t h e s i s , e x t r a c e l l u l a r enzyme p r o d u c t i o n and a c t i o n , or p h y t o a l e x i n d e g r a d a t i o n ) or r e p r o d u c t i o n . M e l a n i n B i o s y n t h e s i s I n h i b i t o r s . The m e l a n i n b i o s y n t h e s i s i n h i b i t o r s , which a r e used to c o n t r o l r i c e b l a s t d i s e a s e , a r e a good example o f compounds t h a t p r e v e n t i n f e c t i o n . I n c l u d e d i n t h i s group a r e t r i c y c l a z o l e , p y r o q u i l o n , f t h a l i d e , and chlorbenthiazone. T r i c y c l a z o l e , w h i c h was the f i r s t of t h e s e compounds i d e n t i f i e d as b e i n g e f f e c t i v e t h r o u g h the i n h i b i t i o n of m e l a n i n s y n t h e s i s ( 2 7 ) , produces no e f f e c t s on f u n g a l growth a t c o n c e n t r a t i o n s o f the chemical that are e f f e c t i v e f o r disease c o n t r o l . Treated fungi

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

3.

RAGSDALE AND SIEGEL

Chemical Control of Plant Pathogens

41

appear brown r a t h e r than g r a y i s h - b l a c k . T r i c y c l a z o l e blocks the c o n v e r s i o n o f 1 , 3 , 8 - t r i h y d r o x y n a p h t h a l e n e t o vermelone and t h e conversion o f 1,3,6,8-tetrahydroxynaphthalene to scytalone i n the p o k l y k e t i d e pathway o f m e l a n i n b i o s y n t h e s i s ( 2 8 ) . The f a i l u r e t o s y n t h e s i z e m e l a n i n was l a t e r t i e d t o f a i l u r e o f t h e fungus t o penetrate the host epidermis (29). A p p a r e n t l y m e l a n i n o r an o x i d i z e d melanin precursor i s i n v o l v e d i n the f u n g a l c e l l w a l l a r c h i t e c t u r e i n s u c h a way as t o p r o v i d e t h e r i g i d i t y n e c e s s a r y f o r h o s t p e n e t r a t i o n (4). Compounds t h a t a f f e c t H o s t R e a c t i o n s . Examples o f compounds w h i c h enhance o r i n d u c e h o s t r e a c t i o n s t o pathogens i n c l u d e 2 , 2 - d i c h l o r o - 3 , 3 - d i m e t h y l - c y c l o p r o p a n e c a r b o x y l i c a c i d (DDCC), p r o b e n a z o l e , and f o s e t y l - A l ( 4 ) . A l t h o u g h t h e s e c h e m i c a l s do n o t s t o p t h e fungus from p e n e t r a t i n g t h e p l a n t , they a r e q u i t e e f f e c t i v e a t p r e v e n t i n g c o l o n i z a t i o n through the enhancement o f the h o s t ' s r e s i s t a n c e mechanisms. F u r t h e r s t u d i e s a r e needed t o e l u c i d a t e how t h e s e r e s i s t a n c e mechanism Resistance

Mechanisms

W i t h t h e use o f second g e n e r a t i o n a n t i f u n g a l c h e m i c a l s t h e r e has been an i n c r e a s i n g awareness o f t h e need t o u n d e r s t a n d n o t o n l y t h e modes o f a c t i o n b u t a l s o t h e mechanisms o f f u n g a l r e s i s t a n c e . I t has l o n g been r e c o g n i z e d t h a t p l a n t s may be bred f o r two t y p e s o f d i s e a s e r e s i s t a n c e , r a c e s p e c i f i c and g e n e r a l . Highly s p e c i f i c disease r e s i s t a n c e i s very e f f e c t i v e f o r a l i m i t e d time. U s u a l l y the s e l e c t i o n p r e s s u r e on t h e d i s e a s e - p r o d u c i n g organism i s such t h a t a new s t r a i n , which c i r c u m v e n t s the h o s t ' s r e s i s t a n c e , emerges. Plants w i t h moderate r e s i s t a n c e g e n e r a l l y r e t a i n t h i s p r o p e r t y f o r l o n g periods. A p a r a l l e l e x i s t s with fungicides (30). Non-specific t y p e s , such as t h e p r e v i o u s l y m e n t i o n e d f i r s t g e n e r a t i o n f u n g i c i d e s , have been e f f e c t i v e f o r many y e a r s whereas the s i t e s p e c i f i c second and t h i r d g e n e r a t i o n compounds a r e more l i k e l y t o e n c o u n t e r r e s i s t a n c e problems. Among t h e b i o c h e m i c a l mechanisms o f f u n g i c i d e r e s i s t a n c e a r e reduced p e r m e a b i l i t y , metabolism ( i n c r e a s e d d e t o x i f i c a t i o n or d e c r e a s e d c o n v e r s i o n t o t h e t o x i c m a t e r i a l ) , and r e d u c e d a f f i n i t y o f the t a r g e t s i t e f o r t h e t o x i n . E f f o r t s a r e now b e i n g made t o a v o i d r e s i s t a n c e problems t h r o u g h s t r a t e g i e s o f u s e i n c o n j u n c t i o n w i t h knowledge on modes o f a c t i o n and mechanisms o f r e s i s t a n c e . A p p l i c a t i o n s t r a t e g i e s i n c l u d e t i m i n g of u s e , r e d u c t i o n o f u s e , c o m b i n a t i o n s o f p r o t e c t a n t s and e r a d i c a n t s t h a t have n o n - s p e c i f i c and s p e c i f i c modes o f a c t i o n , and c o m b i n a t i o n s of e r a d i c a n t s w i t h d i f f e r e n t s p e c i f i c modes o f a c t i o n . Knowledge on modes o f a c t i o n and r e s i s t a n c e mechanisms o f f e r some approaches t h a t can be d e v e l o p e d t o c i r c u m v e n t r e s i s t a n c e p r o b l e m s . Many o f t h e s e i d e a s were d i s c u s s e d a t a r e c e n t symposium a d d r e s s i n g novel approaches f o r r e s e a r c h on a g r i c u l t u r a l c h e m i c a l s ( 3 1 ) . S e l e c t i v e A c t i o n . E v i d e n c e s u g g e s t s t h a t a t t e n t i o n s h o u l d be g i v e n to t h e development and use o f c h e m i c a l s w i t h modes o f a c t i o n t h a t s e l e c t f o r r e s i s t a n t s t r a i n s which do n o t cause d i s e a s e p r o b l e m s . I n the s e c t i o n on E B I f u n g i c i d e s , i t was p o i n t e d o u t t h a t r e s i s t a n t

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mutants have been d e v e l o p e d i n the l a b o r a t o r y , but t h a t t h e r e have been no c a s e s of d i s e a s e c o n t r o l f a i l u r e a t t r i b u t e d to r e s i s t a n c e i n the f i e l d . Perhaps t h i s p a r t i c u l a r mechanism i s s u c h t h a t m u t a t i o n s to overcome i t impart c h a r a c t e r i s t i c s i n a fungus t h a t reduce pathogenicity (18). C i r c u m v e n t i o n o f S i t e M o d i f i c a t i o n . As i n d i c a t e d e a r l i e r , c a r b o x i n i n h i b i t s s u c c i n i c dehydrogenase a c t i v i t y . In s t u d i e s u s i n g r e s i s t a n t l a b o r a t o r y mutants and a l a r g e number of a n a l o g u e s o f the p a r e n t compound, d a t a i n d i c a t e d t h a t f o r any m u t a t i o n i n the l o c i a f f e c t i n g c a r b o x i n s e n s i t i v i t y , a s p e c i f i c s t r u c t u r a l change i n the p a r e n t compound c o u l d a l l e v i a t e or r e v e r s e the f a i l u r e to c o n t r o l the fungus (32). In s c r e e n i n g s t u d i e s , a number of a n a l o g u e s were i d e n t i f i e d t h a t showed i n v i t r o b i n d i n g a f f i n i t y f o r s u c c i n i c d e h y d r o g e n a s e - m i t o c h o n d r i a l complex I I . Some a n a l o g u e s were e f f e c t i v e a g a i n s t w i l d type and some a g a i n s t r e s i s t a n t mutants. A l o g i c a l s t r a t e g y would be to use m i x t u r e s of t h e s e c h e m i c a l s o r t o use them s e p a r a t e l y i n a scheme would not work i a c t i v e a n a l o g u e s were not s y s t e m i c . However, m o d i f i c a t i o n of the c h e m i c a l s t r u c t u r e i s one a p p r o a c h to c o n t r o l the development o f resistance. C o n s i d e r a t i o n of A c t i v a t i o n and S t e r e o s e l e c t i v i t y . S t u d i e s on t r i a d i m e f o n , one of the EBI compounds, p r o v i d e thoughts to c o n s i d e r i n d e v e l o p i n g new compounds as w e l l as a d d r e s s i n g r e s i s t a n c e problems. In e v a l u a t i n g the e f f e c t i v e n e s s of t r i a d i m e f o n a p p l i e d t o p l a n t s f o r p e s t c o n t r o l , the f o l l o w i n g must be t a k e n i n t o c o n s i d e r a t i o n (33): E x t e n t of c o n v e r s i o n to t r i a d i m e n o l by h o s t and pathogen I n h e r e n t a c t i v i t i e s of the c h e m i c a l p r e s e n t (stereoselectivity) * Time r e q u i r e d f o r an e f f e c t i v e dose to r e a c h the s p e c i f i c i n t r a c e l l u l a r s i t e of a c t i o n * F u r t h e r m e t a b o l i s m of the c h e m i c a l by the pathogen or h o s t p l a n t to l e s s t o x i c compounds W h i l e a l l o f t h e s e f a c t o r s must be t a k e n i n t o a c c o u n t , the l a t t e r two a r e dependent on the f i r s t two, and we w i l l f o c u s on the f i r s t two as p o i n t s of c o n s i d e r a t i o n f o r r e s i s t a n c e as w e l l as s t r a t e g i e s f o r the f u t u r e e f f e c t i v e use of t h i s compound. F u n g i s e n s i t i v e to t r i a d i m e f o n c o n v e r t the p a r e n t compound t h r o u g h b i o c h e m i c a l r e d u c t i o n to t r i a d i m e n o l a t a h i g h r a t e , whereas r e s i s t a n t s p e c i e s d e m o n s t r a t e l i t t l e or no c o n v e r s i o n ( 3 4 ) . This i n v e s t i g a t i o n a l s o demonstrated t h a t p l a n t s enhance f u n g i t o x i c i t y by this conversion. Thus an a c t i v a t i o n p r o c e s s i n b o t h pathogen and h o s t c o u l d p o s s i b l y be a l t e r e d to p o t e n t i a t e e f f e c t i v e n e s s . To add t o t h i s somewhat complex s i t u a t i o n , two d i a s t e r e o i s o m e r s of t r i a d i m e n o l a r e formed i n the r e d u c t i o n p r o c e s s , and one i s more a c t i v e than the o t h e r ( 3 5 ) . A h i g h r a t e of c o n v e r s i o n to t r i a d i m e n o l i n a d d i t i o n to a h i g h r a t i o of the more a c t i v e d i a s t e r e o m e r to the l e s s a c t i v e can be assumed t o be p r e s e n t i n t r i a d i m e f o n - s e n s i t i v e f u n g i whereas the o p p o s i t e s i t u a t i o n may be found i n r e l a t i v e l y i n s e n s i t i v e f u n g i . The c o n v e r s i o n p r o c e s s i n p l a n t s a f f o r d s the o p p o r t u n i t y to c o n t r o l i n s e n s i t i v e f u n g i which l a c k the c o n v e r s i o n mechanism· β

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A number of f a c t o r s must be c o n s i d e r e d i n the a r e a of s t e r e o s e l e c t i v i t y (31). D i s t i n c t d i f f e r e n c e s a r e n o t e d between s t e r e o i s o m e r s i n a b s o r p t i o n / uptake, a c t i v i t y a t the t a r g e t or r e c e p t o r s i t e , pathways and r a t e of b i o c o n v e r s i o n , and p h a r m a c o k i n e t i c s . A l l o f t h e s e f a c t o r s a r e a p p l i c a b l e i n the c a s e of t r i a d i m e f o n and a r e examples of p r o c e s s e s t h a t may be m a n i p u l a t e d to enhance d i s e a s e c o n t r o l e f f e c t i v e n e s s . I n h i b i t i o n o f a R e s i s t a n c e Mechanism. Another i n t e r e s t i n g c a s e s e r v e s as an example of the importance of u n d e r s t a n d i n g r e s i s t a n c e mechanisms. I n t h i s s i t u a t i o n r e s i s t a n c e i s not r e l a t e d to the mode of a c t i o n . O b s e r v a t i o n s i n d i c a t e t h a t i n a w i l d type s t r a i n o f A s p e r g i l l u s n i d u l a n s , u p t a k e of the EBI f u n g i c i d e , f e n a r i m o l , i s a r a p i d , p a s s i v e i n f l u x w h i c h i n d u c e s an energy dependent e f f l u x r e s u l t i n g i n an energy dependent s t a t e of e q u i l i b r i u m ( 3 6 ) . Toxic a c t i o n o c c u r s p r i o r to s u f f i c i e n t e f f l u x to r e d u c e l e v e l s below toxic concentrations. T h i s same s t u d y found t h a t the e f f l u x system in resistant strains is uptake of the f u n g i c i d e mechanism i s i n v o l v e d i n n i d u l a n s s t r a i n s r e s i s t a n t to i m a z a l i l , a n o t h e r EBI f u n g i c i d e ( 3 7 ) . E f f l u x i s b l o c k e d by compounds such as r e s p i r a t o r y i n h i b i t o r s t h a t d e c r e a s e l e v e l s of ATP. A p p a r e n t l y the i n f l u x - e f f l u x mechanism i s dependent on i n t r a c e l l u l a r ATP, which i s u t i l i z e d by the plasma membrane ATPase and thus d r i v e s the e f f l u x p r o c e s s ( 3 6 ) . The s y n e r g i s t i c e f f e c t s of r e s p i r a t o r y i n h i b i t o r s c o u l d s e r v e as the b a s i s f o r u s i n g a c o m b i n a t i o n of i n h i b i t o r s . T h i s a l s o b r i n g s up the p o s s i b i l i t y t h a t t h r o u g h the use of r e s p i r a t o r y i n h i b i t o r s , f u n g i t h a t a r e n o r m a l l y i n s e n s i t i v e or o n l y s l i g h t l y s e n s i t i v e to f e n a r i m o l o r i m a z a l i l due t o e f f l u x systems c o u l d be c o n t r o l l e d . Conclusions New approaches f o r the c h e m i c a l c o n t r o l o f p l a n t pathogens emphasize the need to u n d e r s t a n d the p h y s i o l o g i c a l , b i o c h e m i c a l , and m o l e c u l a r l e v e l s of the modes o f a c t i o n and the mechanisms o f pathogen resistance. These approaches i n v o l v e the use of c h e m i c a l s i n a way t h a t w i l l r e d u c e or p r e v e n t the development o f r e s i s t a n t p a t h o g e n s . They a l s o take advantage of compounds t h a t s e r v e as antipathogenic a g e n t s to p r e v e n t d i s e a s e development, an a p p r o a c h w h i c h r e q u i r e s an u n d e r s t a n d i n g of h o s t - p a r a s i t e p h y s i o l o g y and p a t h o g e n i c p r o c e s s e s . The a n t i p a t h o g e n i c a g e n t s are h i g h l y s p e c i f i c and u s u a l l y t a r g e t e d f o r a d e s i g n a t e d pathogen on a g i v e n p l a n t . In l o o k i n g t o the f u t u r e , one must be p r a c t i c a l and take i n t o a c c o u n t the f a c t t h a t s u c h c h e m i c a l s a r e l i k e l y to be c o m m e r c i a l l y a v a i l a b l e o n l y f o r major d i s e a s e s on major c r o p s . The c u r r e n t c o s t s of development, r e g i s t r a t i o n , and m a r k e t i n g put r e s t r i c t i o n s on w i d e s p r e a d use o f t h e s e t h i r d g e n e r a t i o n f u n g i c i d e s . Thus, one must take advantage o f f i r s t and second g e n e r a t i o n type compounds. The i n f o r m a t i o n we have p r e s e n t e d on t h e s e m a t e r i a l s i n d i c a t e s t h a t a r e a s o n a b l e l e v e l o f knowledge e x i s t s on how they work. We have seen examples of the b r o a d range o f c e l l u l a r p r o c e s s e s t h a t second g e n e r a t i o n f u n g i c i d e s affect. A d d i t i o n a l d a t a w i l l u n d o u b t e d l y enhance t h i s i n f o r m a t i o n b a s e . New approaches w i l l take advantage of e x i s t i n g knowledge and

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emphasize the Importance of additional basic information on the mechanisms of action and resistance as well as host-pathogen relationships. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Sisler, H. D.; Ragsdale, N.N. in "Handbook of Pest Management in Agriculture"; Pimental, D., Ed.; CRC Press: Boca Raton, Florida, 1981; pp. 17-47. Siegel, M. R. in "Pesticide Selectivity"; Street, J . C . , E d . ; Marcel Dekker: New York, 1975; pp. 21-46. Edgington, L. V . ; Martin, R. Α.; Bruin, G. C . ; Parsons, I.M. Plant D i s . 1980, 64, 19-23. Sisler, H. D.; Ragsdale, Ν. N. in "Agricultural Chemicals of the Future"; Hilton, J . L., Ed.; Rowman and Allanheld Publishers: Totown, New Jersey, in press. Sisler, H. D. Annu Rev Phytopathology 1969 7 311-30 Dekker, J. in "Fungicides" Academic Press: Ne , ; pp Siegel, M. R. in "Antifungal Compounds", Vol. 2; Siegel, M. R.; Sisler, H. D. , Eds.; Marcel Dekker: New York, 1977; pp. 399-438. Davidse, L. C. in "Fungicide Resistance in Crop Protection"; Dekker, J.; Georgopoulos, S. G . , Eds.; Pudoc: Wageningen, The Netherlands, 1982; pp. 60-70. Hammerschlag, R. S.; Sisler, H. D. Pestic. Biochem. Physiol. 1973, 3, 42-54. Howard, R. J.; Aist, J. R. Protoplasma 1977, 92, 195-210. Davidse, L. C.; deWaard, M. A. Adv. Plant Pathology. 1984, 2, 191-257. Mathre, D. E . Pestic. Biochem. Physiol. 1971, 1, 216-24. White, G. A. Biochem. Biophys. Res. Commun. 1971, 44, 1210-12. Misato, T . ; Kakiki, K. in "Antifungal Compounds", Vol. 2; Siegel, M. R.; Sisler, H. D., Eds.; Marcel Dekker: New York, 1977; pp. 277-300. Ragsdale, Ν. N. in "Antifungal Compounds, Vol. 2; Siegel, M. R.; Sisler, H. D., Eds.; Marcel Dekker; New York, 1977; pp. 333-63. Kaars Sypesteijn, A. in "Fungicide Resistance in Crop Protection"; Dekker, J.; Georgopoulos, S. G . , Eds.; Pudoc: Wageningen, The Netherlands, 1982; pp. 32-45. Ragsdale, Ν. N.; Sisler, H. D. Biochem. Biophys. Res. Commun. 1972, 46, 2048-53. Sisler, H. D.; Ragsdale, Ν. N. in "Mode of Action of Antifungal Agents"; Trinci, A. P . J . ; Ryley, J . F . , Eds.; Cambridge University Press: London, 1984; pp. 257-82. Petranyi, G.; Ryder, N. S.; Stütz, A. Science 1984, 224, 1239-41. Kato, T . ; Shoami, M.; Kawase, Y. J. Pestic. Sci. 1980, 5, 69-79. Kerkenaar, Α.; Uchiyama, M.; Versluis, G. G. Pestic. Biochem. Physiol. 1981, 16, 97-104. Henry, M. J.; Sisler, H. D. Pestic. Biochem. Physiol. 1984, 22, 262-75. Gadher, P.; Mercer, Ε. I . ; Baldwin, B. C . ; Wiggins, T. E . Pestic. Biochem. Physiol. 1983, 19, 1-10.

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Coolbaugh, R. C.; Hirano, S. S.; West, C. A. Plant Physiol. 1978, 62, 571-6. Kolata, G. Science 1984, 225. 913-4. Wolfe, M. S. personal communication. Chrysayi Tokousbalides, M.; Sisler, H. D. Pestic. Biochem. Physiol. 1978, 8, 26-32. Chrysayi Tokousbalides, M.; Sisler, H. D. Pestic. Biochem. Physiol. 1979, 11, 64-73. Woloshuk, C. P.; Sisler, H. D. J . Pestic. Sci. 1982, 7, 161-6. Georgopoulos, S. G. in "Antifungal Compounds", Vol. 2; Siegel, M. R.; Sisler, H. D., Eds.; Marcel Dekker: New York, 1977; pp. 439-84. Fuchs, Α.; Davidse, L . C . ; deWaard, Μ. Α.; deWit, P. J . G. M. Pestic. Sci. 1983, 14, 272-93. White, G. Α.; Thorn, G. D. Pestic. Biochem. Physiol. 1980, 14, 26-40. Deas, Α. H.B.; Clifford, D. R. Pestic. Biochem. Physiol. 1982, 17, 120-33. Gasztonyi, M.; Josepovits Buchenauer, H. in "Abstracts of Papers 1X International Congress of Plant Protection", 1979, No. 939. DeWaard, Μ. Α.; van Nistelrooy, J . G. M. Pestic. Biochem. Physiol. 1980, 13, 255-66 Siegel, M. R.; Solel, J . Pestic. Biochem. Physiol. 1981, 15, 222-33.

RECEIVED January 15, 1985

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

4 Elicitation of Disease Resistance in Plants by the Expression of Latent Genetic Information STEVEN D. SALT and JOSEPH KUĆ Department of Plant Pathology, University of Kentucky, Lexington, KY 40546-0091 Cucumber, melon, tobacco, and bean varieties, supposedly l a c k i n g s i g n i f i c a n t resistance to particular diseases, can be made highly resistant systemically b exposure to pathogens stress do not e l i c i t persistent systemic resistance. These findings suggest that plants generally possess genetic information for disease resistance mechanisms and that resistance is generally determined by the speed and magnitude of response to pathogens. We s h a l l present aspects of our investigations into immunization of tobacco, c u c u r b i t s , and beans (Phaseolus vulgaris L.) and b r i e f l y review other workers' i n v e s t i g a t i o n s into bioticallyand chemically-induced disease resistance of plants. Enhanced resistance to disease in plants after an i n i t i a l infection has fascinated observers for over 100 years. A review of the subject by Chester in 1933 contains 201 references (1). "Immunization", "acquired systemic resistance", or "induced resistance" of plants have been reviewed in recent years (2-11). We shall not exhaustively review the literature, but shall focus on general principles and phenomena of particular relevance to the use of "plant immunization" for the practical control of disease. This paper w i l l stress examples from our own research program, but will also include literature citations to provide the reader with an appreciation of important research c o n t r i b u t i o n s of others previously and presently active in the f i e l d . Most examples presented w i l l deal with fungal, bacterial, or v i r a l diseases of crop plants, but similar principles may apply to infestations by nematodes and, possibly, insects. Protection of Plants Against Pests Immunization of plants via priming for expression of latent genetic information encoding disease resistance mechanisms may be

0097-6156/85/0276-0047$06.50/0 © 1985 American Chemical Society

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i n t r o d u c e d by c o n t r a s t i n g i t w i t h o t h e r s t r a t e g i e s u t i l i z e d f o r p r o t e c t i n g p l a n t s from d i s e a s e s The s t r a t e g i e s c o n s i d e r e d can be c a t e g o r i z e d as: e c o l o g i c a l , p e s t i c i d e s , a n t i t o x i n s , a l t e r a t i o n s of p l a n t p h y s i o l o g y , n o n s p e c i f i c p h y t o a l e x i n i n d u c t i o n , and plant s e n s i t i z a t i o n (immunization). Ecological. This h i s t o r i c a l l y venerable strategy i s characterized by t a c t i c s d e s i g n e d to m i n i m i z e exposure o f p l a n t s to pathogens and make e n v i r o n m e n t a l c o n d i t i o n s u n f a v o r a b l e f o r d i s e a s e development. Measures i n c l u d e c r o p r o t a t i o n , s a n i t a t i o n , and q u a r a n t i n e . Such p r o c e d u r e s are v a l u a b l e and worthy of f u r t h e r d e v e l o p m e n t , but they are g e n e r a l l y of l i m i t e d e f f e c t i v e n e s s i n p r o v i d i n g r e l i a b l y h i g h y i e l d and q u a l i t y o f c r o p s i n a r e a s w i t h s e v e r e o r p e r s i s t e n t d i s e a s e problems. Pesticides. The a p p l i c a t i o n o f exogenous c h e m i c a l s t o x i c to p l a n t pathogens and p e s t s has in t e c h n o l o g i c a l l y develope strategy i s w e l l suited and has v e r y s u c c e s s f u l l y enhanced c r o p p r o d u c t i o n . Numerous and s e r i o u s l o n g - t e r m problems have, however, become e v i d e n t . Heavy r e l i a n c e on, and c o n f i d e n c e i n , p e s t i c i d e s has caused other strategies t o become r e l a t i v e l y neglected. The expense of p e s t i c i d e s and n e c e s s a r y a n c i l l a r y e q u i p m e n t , as w e l l as t e c h n o l o g i c a l e x p e r t i s e r e q u i r e d f o r t h e i r e f f e c t i v e and s a f e use, are burdensome and o f t e n beyond the r e a c h o f farmers i n d e v e l o p i n g nations. H e a v y and i m p r o p e r use o f p e s t i c i d e s has l e d to the a p p e a r a n c e o f r e s i s t a n t p e s t s and c o n s e q u e n t l o s s o f p e s t i c i d e efficacy. M o s t s e r i o u s l y , t h e d a n g e r o f i n j u r y to n o n - t a r g e t organisms, e s p e c i a l l y t o x i c i t y , t e r a t o g e n i c i t y , and carcinogenicity to humans, has l e d to cumbersome and e x p e n s i v e r e s t r i c t i o n s on the development, s a l e , and use o f p e s t i c i d e s . Antitoxins. This i s a s t r a t e g y of disarmament i n which a p p l i e d c h e m i c a l s are not d i r e c t l y t o x i c to pathogens, but i n t e r f e r e w i t h t h e i r mechanisms f o r p a t h o g e n e s i s , e.g., p e n e t r a t i o n , m a c e r a t i o n of t i s s u e s , or i n d u c t i o n of w i l t i n g or a b n o r m a l growth. Few s u c h compounds have been d e v e l o p e d f o r commercial use. Tricyclazoles a p p e a r t o f u n c t i o n by i n h i b i t i n g t h e m e l a n i z a t i o n o f fungal a p p r e s s o r i a and thus reduce p e n e t r a t i o n i n t o h o s t p l a n t s ( 12-14). Other r e p o r t e d examples i n c l u d e i n a c t i v a t i o n of p i r i c u l a r i n , t o x i n o f the r i c e b l a s t f u n g u s P i r i c u l a r i a o r y z a e , by f e r u l i c and c h l o r o g e n i c a c i d s (15), and i n h i b i t i o n o f Fusarium w i l t symptoms i n tomato by c a t e c h o l , which at e f f e c t i v e c o n c e n t r a t i o n s appears non­ t o x i c t o t h e f u n g u s and n e i t h e r p r e v e n t s nor r e d u c e s i n f e c t i o n (16). The b i o c h e m i s t r y of p a t h o g e n e s i s i n p l a n t d i s e a s e , however, i s i n an e a r l y s t a g e o f i n v e s t i g a t i o n and the r a t i o n a l d e s i g n o f t o x i n i n h i b i t o r s or o t h e r a n t i p a t h o g e n e t i c compounds may be e v e n more d i f f i c u l t t h a n t h a t o f p e s t i c i d e s . Random s c r e e n i n g o f compounds f o r a n t i t o x i n a c t i v i t y has i n h e r e n t d i f f i c u l t i e s f o r b i o a s s a y , and the a p p l i c a t i o n o f a n t i t o x i n s s u f f e r s f r o m t h e d i f f i c u l t i e s encountered i n the e n v i r o n m e n t a l r e l e a s e of c h e m i c a l s as c i t e d f o r p e s t i c i d e s .

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A l t e r a t i o n s o f P h y s i o l o g y . S u s c e p t i b l e p l a n t s p e c i e s or c u l t i v a r s may be r e n d e r e d resistant t o d i s e a s e by so a l t e r i n g their p h y s i o l o g y , e.g., h o r m o n a l b a l a n c e s , i o n f l u x e s , c o n s t i t u t i v e secondary m e t a b o l i s m , or r e s p i r a t o r y r a t e s , as to render them u n f i t o r h o s t i l e e n v i r o n m e n t s f o r p a t h o g e n d e v e l o p m e n t , i . e . , t o make them n o n - h o s t s . T h i s c o u l d be a c c o m p l i s h e d , f o r e x a m p l e , by the use o f growth r e g u l a t o r s or b r e e d i n g . T h i s approach may not p r o v e g e n e r a l l y p r a c t i c a b l e , however, because crop p l a n t s , o v e r the ages, have been s e l e c t e d p r e c i s e l y f o r t h o s e p h y s i o l o g i c a l c h a r a c t e r i s t i c s w h i c h make them more agronomica1 l y d e s i r a b l e than f r e q u e n t l y hardier w i l d r e l a t i v e s . D r a s t i c c h a n g e s i n c o n s t i t u t i v e p h y s i o l o g y may r e d u c e a g r o n o m i c f i t n e s s e v e n i f d i s e a s e r e s i s t a n c e i s enhanced. Breeders are o f t e n f a c e d w i t h the p r o b l e m o f r e i n c o r p o r a t i n g y i e l d and q u a l i t y f a c t o r s back i n t o d i s e a s e - a n d i n s e c t - r e s i s t a n t p l a n t s . N o n - s p e c i f i c or C o n s t i t u t i v e P h y t o a l e x i n I n d u c t i o n Many p l a n t s are r e p o r t e d to produc a n t i m i c r o b i a l compounds These compounds r a p i d l y a c c u m u l a t e to h i g h c o n c e n t r a t i o n s i n p l a n t t i s s u e s i m m e d i a t e l y a d j a c e n t to s i t e s of i n f e c t i o n i n r e s i s t a n t c u l t i v a r s , and they f u n c t i o n to r e s t r i c t d e v e l o p m e n t of f u n g a l and b a c t e r i a l pathogens o f p l a n t s (17,18). Susceptible c u l t i v a r s may u l t i m a t e l y a c c u m u l a t e as much o r more t o t a l p h y t o a l e x i n s d u r i n g d i s e a s e development, but a c c u m u l a t i o n i s s l o w e r and d i f f u s e , and l o c a l i z e d c o n c e n t r a t i o n s i n advance of the pathogen appear to be i n s u f f i c i e n t to i n h i b i t pathogen development. Many a b i o t i c agents, i n c l u d i n g heavy m e t a l s a l t s (19), heat (20), c h l o r o f o r m v a p o r s ( 2 1 ) , u l t r a v i o l e t i r r a d i a t i o n ( 2 2 ) , f u n g i c i d e s s u c h as maneb and b e n o m y l ( 2 3 ) , and e v e n i n n o c u o u s s u b s t a n c e s s u c h as s u c r o s e (24) e l i c i t p h y t o a l e x i n a c c u m u l a t i o n i n some p l a n t s . P r o p o s a l s have b e e n made t h a t g e n e r a l n o n - s p e c i f i c i n d u c t i o n o f p h y t o a l e x i n a c c u m u l a t i o n i n p l a n t s may be an effective and " n a t u r a l " method o f p l a n t p r o t e c t i o n from p e s t s . U n f o r t u n a t e l y , p h y t o a l e x i n s , and the e f f e c t i v e c o n c e n t r a t i o n s o f p h y t o a l e x i n e l i c i t o r s , a r e o f t e n t o x i c a n t s which i n j u r e or k i l l p l a n t c e l l s (25). In d i s e a s e r e s i s t a n c e i n v o l v i n g p h y t o a l e x i n a c c u m u l a t i o n , the p l a n t s a c r i f i c e s a few o f i t s own c e l l s i m m e d i a t e l y a d j a c e n t to i n v a d i n g pathogens i n o r d e r t o s a v e the e n t i r e p l a n t . General or c o n s t i t u t i v e i n d u c t i o n o f p h y t o a l e x i n a c c u m u l a t i o n may p r o t e c t p l a n t s a g a i n s t i n f e c t i o u s d i s e a s e , but i t would l i k e l y be d i s a s t r o u s l y c o u n t e r p r o d u c t i v e to o v e r a l l p l a n t h e a l t h because o f a u t o t o x i c i t y and t h e need f o r c o n t i n u o u s d i v e r s i o n o f m e t a b o l i c e n e r g y and primary m e t a b o l i t e s into secondary metabolism. In a d d i t i o n , phytoalexins require higher c o n c e n t r a t i o n s i n p l a n t t i s s u e s than commercial synthetic p e s t i c i d e s to achieve comparable effectiveness. They a l s o are not t r a n s l o c a t e d w i t h i n p l a n t s , and a r e r a p i d l y d e g r a d e d by p l a n t s and many p a t h o g e n s . To m a i n t a i n e f f e c t i v e l y h i g h s y s t e m i c l e v e l s o f p h y t o a l e x i n s , t h e r e f o r e , would l i k e l y r e q u i r e repeated a p p l i c a t i o n s of p h y t o t o x i c elicitor s u b s t a n c e s or the b r e e d i n g o f p l a n t s w i t h c o n s t i t u t i v e l y h i g h l e v e l s of p h y t o a l e x i n s . F u r t h e r m o r e , many p h y t o a l e x i n s may be t o x i c t o mammals, and p l a n t t i s s u e c o n t a i n i n g h i g h total c o n c e n t r a t i o n s may be u n p a l a t a b l e o r h a z a r d o u s f o r c o n s u m p t i o n (25).

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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S e n s i t i z a t i o n of Plants. This strategy f o rplant p r o t e c t i o n i s c o n c e p t u a l l y , i f not m e c h a n i s t i c a l l y , a n a l o g o u s t o immunization v i a v a c c i n a t i o n o f mammals. An i n i t i a l s t i m u l u s e l i c i t s a l o n g - t e r m s u b t l e a l t e r a t i o n i n a p l a n t such that subsequent exposure to a pathogen o r pest r e s u l t s i n a v i g o r o u s and r a p i d a c t i v a t i o n o f the p l a n t ' s endogenous g e n e t i c a l l y e n c o d e d d e f e n s e m e c h a n i s m s . However, a s i d e f r o m a r e l a t i v e l y b r i e f i n i t i a l s e n s i t i z a t i o n p e r i o d , d i s e a s e r e s i s t a n c e mechanisms remain l a t e n t u n t i l a f t e r i n f e c t i o n and a r e g e n e r a l l y l o c a l i z e d a t the s i t e s o f i n f e c t i o n . T h e r e i s no g r o s s d e r a n g e m e n t o f p l a n t c o n s t i t u t i v e m e t a b o l i s m . There i s l i t t l e consumption o f energy or m e t a b o l i t e s and t h e e x p e n d i t u r e o c c u r s when and where needed. A u t o t o x i c i t y from non­ s p e c i f i c or c o n s t i t u t i v e defense r e a c t i o n s i n the absence of i n f e c t i o n i s avoided. A " s e n s i t i z e d " p l a n t may be s a i d t o p o s s e s s t o some d e g r e e a s t a t e o f " i m m u n i t y " o r " a c q u i r e d / i n d u c e d resistance"· The remainder o f t h i s paper s h a l l p r e s e n t g e n e r a l p r i n c i p l e s , examples of a p p l i c a t i o n s l i m i t a t i o n s of s e n s i t i z a t i o a g a i n s t i n f e c t i o u s d i s e a s e s and damage from o t h e r p e s t s .

Statement o f T h e s i s V i r t u a l l y a l l p l a n t s , even those considered " s u s c e p t i b l e " to particular diseases, possess genetic information f o r the biochemical pathways r e s p o n s i b l e f o r e f f e c t i v e d i s e a s e r e s i s t a n c e mechanisms. S u s c e p t i b i l i t y i s due t o a f a i l u r e o r d e l a y i n recognizing the pathogen, suppression of a c t i v a t i o n of host d e f e n s e s by t h e p a t h o g e n , inactivity o f gene p r o d u c t s , or m o d i f i c a t i o n o f c o m p o n e n t s o f t h e r e s i s t a n c e r e s p o n s e . The d a t a presented i n t h i s paper support the t h e s i s t h a t , given an a p p r o p r i a t e i n d u c i n g s t i m u l u s , s u s c e p t i b l e p l a n t s can be s e n s i t i z e d t o a c t i v a t e d e f e n s e m e c h a n i s m s a g a i n s t p a t h o g e n s so as t o become r e s i s t a n t to d i s e a s e . However, though enhancement o f e x p r e s s i o n o f g e n e t i c a l l y e n c o d e d p l a n t d i s e a s e r e s i s t a n c e m e c h a n i s m s may be a g e n e r a l l y e f f e c t i v e means o f i m m u n i z i n g p l a n t s , we do n o t c l a i m u n i v e r s a l efficacy. In some e n v i r o n m e n t s , gene p r o d u c t s may be i n e f f e c t u a l in restricting or c o n d i t i o n i n g the r e s t r i c t i o n o f pathogen d e v e l o p m e n t , e.g., a c t i v i t y o f gene p r o d u c t s f o r r e s i s t a n c e may have s p e c i f i c temperature or l i g h t requirements. In p l a n t s s e v e r e l y weakened by i n j u r y o r d i s e a s e , f a c u l t a t i v e p a r a s i t e s or n o r m a l l y i n c o m p a t i b l e pathogens may be a b l e to p a r a s i t i z e n o r m a l l y nonhost o r r e s i s t a n t p l a n t s due t o the p l a n t ' s i n a b i l i t y t o d e v e l o p an e f f e c t i v e m e t a b o l i c response. Some pathogens may a c t i v a t e host d e f e n s e mechanisms y e t d e v e l o p i n host t i s s u e s and cause d i s e a s e by inactivating c o m p o n e n t s o f t h e r e s i s t a n c e m e c h a n i s m s , e.g., metabolism of kievetone, a p h y t o a l e x i n o f b e a n s , by F u s a r i u m (26,27) and the d e t o x i f i c a t i o n o f p i s a t i n , a p h y t o a l e x i n o f pea, by N e c t r i a hematococca ( 2 8 ) .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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KUC

Disease Resistance in Plants

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Methods of S e n s i t i z a t i o n Gene t i c . The d i f f e r e n c e s b e t w e e n many s u s c e p t i b l e and r e s i s t a n t c u l t i v a r s are the a b i l i t i e s o f the l a t t e r to a p p r o p r i a t e l y respond q u a n t i t a t i v e l y i n t i m e and i n s p a c e t o i n f e c t i o n , r a t h e r t h a n q u a l i t a t i v e d i f f e r e n c e s i n b i o c h e m i c a l pathways. T h i s type of g e n e t i c r e s i s t a n c e may be c o n s i d e r e d a f o r m o f an e n d o g e n o u s c o n s t i t u t i v e ly sensitized state. Genetic s e n s i t i z a t i o n of a c u l t i v a r r e q u i r e s b o t h a s o u r c e o f a p p r o p r i a t e g e r m p l a s m and a m e t h o d o f e f f e c t i v e t r a n s f e r and i n c o r p o r a t i o n o f t h e g e n e t i c m a t e r i a l i n t o the r e c i p i e n t c u l t i v a r . Both r e q u i r e m e n t s cannot a l w a y s be met. F u r t h e r m o r e , u n d e s i r a b l e t r a i t s o f t e n accompany desired resistance traits. This v e r y important area of p l a n t s e n s i t i z a t i o n , b r o a d l y c o n s t r u e d , l i e s beyond the scope of t h i s paper. Physical. A n e c d o t a l a c c o u n t s abound of enhanced p l a n t r e s i s t a n c e to d i s e a s e a c h i e v e d b physical stimuli, e.g. electromagnetic r a d i a t i o n , e l e c t r i c c u r r e n t , s o u n d w a v e s , and vibration. In our own l a b o r a t o r y , we have made cucumbers r e s i s t a n t t o a n t h r a c n o s e by v i b r a t i o n ( S t r o m b e r g and Kuc, unpublished). H o w e v e r , t h e s e phenomena a r e p o o r l y u n d e r s t o o d and may include enhanced r e s i s t a n c e r e s u l t i n g f r o m n o n - s p e c i f i c a l t e r e d ( s t r e s s ) p h y s i o l o g y , n o n s p e c i f i c p h y t o a l e x i n e l i c i t a t i o n , m o d i f i c a t i o n of t h e a c t i o n o f gene p r o d u c t s , o r s e n s i t i z a t i o n . T h i s i n t e r e s t i n g b u t l i t t l e e x p l o r e d a r e a w i l l n o t be f u r t h e r d i s c u s s e d i n t h i s paper. Biotic. Enhanced r e s i s t a n c e to d i s e a s e which r e s u l t s from p r i o r exposure to a v i r u l e n t pathogens o r n o n p a t h o g e n i c o r g a n i s m s o r t o v i r u l e n t pathogens under c o n d i t i o n s u n f a v o r a b l e f o r disease d e v e l o p m e n t , i s the major focus of t h i s paper and w i l l be d i s c u s s e d at l e n g t h . C h e m i c a 1 . T h e r e a r e a number o f r e p o r t s o f e x o g e n o u s c h e m i c a l s , b o t h s y n t h e t i c and n a t u r a l , which enhance endogenous p l a n t d e f e n s e mechanisms. We s h a l l r e v i e w some o f these r e p o r t s a t a l a t e r p o i n t i n t h i s paper. In some c a s e s , n e i t h e r t h e compounds n o r their m e t a b o l i c p r o d u c t s are d i r e c t l y t o x i c to the pathogens under study. In o t h e r cases, enhanced p l a n t d e f e n s e r e s p o n s e s appear c o e x i s t e n t with d i r e c t p e s t i c i d a l activity of the compounds o r their metabolites. Few o f these c a s e s have been t h o r o u g h l y examined at the b i o c h e m i c a l l e v e l . However, the p o s s i b i l i t y t h a t s e n s i t i z a t i o n of p l a n t s towards pathogens may be a c h i e v e d by exogenous c h e m i c a l s has g r e a t p o t e n t i a l f o r p r a c t i c a l a p p l i c a t i o n s . Of c o u r s e , a l l difficulties inherent i n r e l e a s i n g a l i e n c h e m i c a l s i n t o the e n v i r o n m e n t may be e n c o u n t e r e d i n t h i s a p p r o a c h . R e g a r d l e s s of the n a t u r e o f the o r i g i n a l s e n s i t i z i n g s t i m u l u s , however, induced r e s i s t a n c e i s mediated and e x p r e s s e d through the endogenous g e n e t i c a l l y encoded b i o c h e m i c a l mechanisms of the p l a n t .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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G e n e r a l Occurrence

o f R e s i s t a n c e Induced

by B i o t i c

Agents

Numerous r e p o r t s o f " a c q u i r e d r e s i s t a n c e " o r " i n d u c e d r e s i s t a n c e " o r " i m m u n i z a t i o n " o f p l a n t s , e s p e c i a l l y t o d i s e a s e s c a u s e d by v i r u s e s , date back as f a r as a c e n t u r y (_1). Only a s m a l l sampling of e x a m p l e s i s p r e s e n t e d i n T a b l e I to i l l u s t r a t e the v a r i e t y o f host-disease combinations f o r w h i c h t h i s phenomenon has b e e n reported. Many examples have not been i n d e p e n d e n t l y v e r i f i e d or extensively investigated. D i r e c t a n t i b i o s i s or changes i n host p h y s i o l o g y or pathogen v i r u l e n c e may be c o n f u s e d w i t h , or obscure, s e n s i t i z a t i o n towards subsequent r e i n f e c t i o n by the same pathogen. However, enough c a s e s h a v e been c l o s e l y examined to s u g g e s t the g e n e r a l i t y of b i o t i c a l l y induced r e s i s t a n c e i n p l a n t s (2-11). S i m i l a r phenomena h a v e a l s o b e e n r e p o r t e d f o r n e m a t o d e s (29) and f o r i n s e c t s (30). N o n - s p e c i f i c i t y of

Protection

One o f t h e most f a s c i n a t i n r e s i s t a n c e i n d u c e d i n p l a n t s by b i o t i c a g e n t s i s t h e f r e q u e n t l y o b s e r v e d a b i l i t y of i n f e c t i o n by one organism to induce r e s i s t a n c e a g a i n s t d i s e a s e caused by o t h e r organisms d i s t a n t l y r e l a t e d both to the i n d u c i n g organism and to one another. Limited foliar infection o f cucumber, watermelon, and muskmelon by C o l l e t o t r i c h u m l a g e n a r i u m , a pathogen o f l e a v e s and f r u i t s , o r by t h e n o n - p a t h o g e n t o b a c c o n e c r o s i s v i r u s (TNV), s y s t e m i c a l l y p r o t e c t e d a g a i n s t d i s e a s e c a u s e d by C. l a g e n a r i u m , Cladosporium cucumerinum, Mycosphaerelia melonis, Fusarium o x y s p o r u m f . s p . c u e urne r i n u m , P s e u d o p e r o n o s p o r a c u b e n s i s, Pseudomonas lachrymans, E r w i n i a t r a c h e i p h i l a , TNV ( l o c a l n e c r o s i s ) , or P h y t o p h t h o r a i n f e s t a n s (9,49-55). Thus p r o t e c t i o n was a c h i e v e d a g a i n s t n e c r o t r o p h i c and b i o t r o p h i c pathogens; f u n g i , v i r u s e s , and b a c t e r i a ; f o l i a r , stem, f r u i t , and r o o t p a t h o g e n s ; and a g a i n s t v a r i o u s d i s e a s e types - s y s t e m i c and l o c a l ; w i l t s , r o t s , b l i g h t s , l e a f s p o t s , mildews, scabs, and l o c a l n e c r o s i s . The o n l y d i s e a s e a g a i n s t w h i c h we were u n a b l e t o i n d u c e r e s i s t a n c e w i t h TNV o r C. l a g e n a r i u m was p o w d e r y m i l d e w c a u s e d by S p h a e r o t h e c a fulginea. However, B a s h a n and Cohen (56) h a v e more r e c e n t l y r e p o r t e d i n d u c t i o n of r e s i s t a n c e a g a i n s t powdery m i l d e w o f cucumbers by TNV. G r e e n b e a n s ( P h a s e o l u s v u l g a r i s L.) c a n be systemically p r o t e c t e d a g a i n s t c u 1 t i v a r - p a t h o g e n i c r a c e s of the a n t h r a c n o s e fungus, C o l l e t o t r i c h u m 1indemuthianum, by p r i o r i n o c u l a t i o n e i t h e r w i t h c u l t i v a r - i n c o m p a t i b l e r a c e s o f the same fungus (57-59) or by c u l t i v a r - p a t h o g e n i c r a c e s i f the i n f e c t i o n was a t t e n u a t e d i n s i t u by h e a t t r e a t m e n t p r i o r t o a p p e a r a n c e o f symptoms ( 6 0 , 6 1 ) . Bean c u l t i v a r s s u s c e p t i b l e to a l l known r a c e s o f C^ 1indemuthianum and a l s o t h o s e p o s s e s s i n g " g e n e t i c " r e s i s t a n c e t o some f u n g a l r a c e s were r e n d e r e d r e s i s t a n t t o a l l r a c e s o f t h e p a t h o g e n by p r i o r i n f e c t i o n w i t h the pathogen o f c u c u r b i t s , Co 1 l e t o t r i c h u m l a g e n a r i u m ( 57_, 62 ). T o b a c c o was r e c i p r o c a l l y p r o t e c t e d a g a i n s t TMV, TNV, or T h i e l a v i o p s i s b a s i c o l a (34) ; a g a i n s t P h y t o p h t h o r a p a r a s i t i c a v a r . nicot ianae with TNV ( 63); and a g a i n s t TMV and Erys iphe c i c h o r a c e a r u m (powdery mildew) w i t h P e r o n o s p o r a t a b a c i n a ( 6 4 - 6 6 ) .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

4.

SALT AND KUC Table

I.

Disease Resistance in Plants

Some Reports of B i o t i c a l l y

Plant

Disease

b l u e mold tobacco mosaic tobacco r i n g s p o t b l a c k shank black root r o t

Tobacco

Cabbage, carnation, tomato, watermelon, flax, s i l k tree, cotton

wilt

53

Induced

R e s i s t a n c e In P l a n t s

Pathogen

Reference(s)

Peronospora t a b a c i n a virus virus Phytophthora p a r a s i t i c a Thielaviopsis basicola Fusarium

5,31,32 1,3 1,3 33 34

spp.

35

Sugar cane

mosaic corn streak

Coffee

tree

rust

Hemileia

vastatrix

36,37

Euphorbia cyparissias

rust

Uromyces

pisi

38

Daisies

gall

Agrobacterium

Cedars, apples

rust

Gymnosporangium macropus

40

Carrots

root rot

Botrytis

41

Cucurbits (cucumbers, melons)

anthracnose mosaic a n g u l a r l e a f spot

C o l l e t o t r i c h u m lagenarium virus Pseudomonas lachrymans

Peaches, plums

canker

Cytospora

Green beans

anthracnose

Colletotrichum 1indemuthianum

45

A p p l e s , pears

fire

Erwinia

46-48

blight

virus virus

1 1

tumefasciens

cinerea

cincta

amylovora

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

39

42,43,54 1 10 44

54

BIOREGULATORS FOR PEST CONTROL

However, i n o c u l a t i o n w i t h c i c h o r a c e a r u m , TMV, cucumber mosaic virus, A l t e r n a r i a so 1 a n i , Helminthosporium turc icum,or P s e u d o p e r o n o s p o r a c u b e n s i s f a i l e d to p r o t e c t t o b a c c o a g a i n s t Peronospora t a b a c i n a or c i c h o r a c e a r u m (65)» Thus, w h i l e b i o t i c agents often protect against c l o s e l y or d i s t a n t l y r e l a t e d organisms, the e f f e c t i v e c o m b i n a t i o n s o f p l a n t , b i o t i c i n d u c e r , and c h a l l e n g e pathogen e x h i b i t some s p e c i f i c i t y . It i s p u z z l i n g that r e c i p r o c a l c o m b i n a t i o n s may be i n e f f e c t i v e , and t h a t some pathogens seem i n e f f e c t i v e i n e l i c i t i n g r e s i s t a n c e a g a i n s t t h e m s e l v e s o r c l o s e l y r e l a t e d organisms, but h i g h l y e f f e c t i v e a g a i n s t d i s t a n t l y r e l a t e d pathogens! Some more u n u s u a l e f f e c t i v e i n d u c e r / p a t h o g e n c o m b i n a t i o n s i n c l u d e t h e use o f t h e n e m a t o d e s P r a t y l e n c h u s p e n e t r a n s o r P. b r a c h y u r r u s a g a i n s t the b l a c k shank fungus P. p a r a s i t i c a i n t o b a c c o (33,67,68), and use of TMV a g a i n s t the a p h i d Myzus p e r s i c a e as w e l l as a g a i n s t TMV, j \ _ p a r a s i t i c a v a r . n i c o t i a n a e , Pseudomonas t a b a c i ( w i l d f i r e b a c t e r i u m ) , and Peronospora t a b a c i n a (69). Dynamics of Induced

Resistanc

Initial reaction. In a l l known c a s e s of e f f e c t i v e b i o t i c s e n s i t i z a t i o n of p l a n t s r e p o r t e d to date, a c r i t i c a l f a c t o r appears to be the n e c r o s i s o f h o s t c e l l s i n the zone of i n i t i a l i n f e c t i o n . However, w h i l e n o n - n e c r o t i c i n f e c t i o n s a r e i n e f f e c t i v e i n d u c e r s , n e c r o s i s per se i s not e f f e c t i v e i n i n d u c i n g r e s i s t a n c e . I n j u r y by a b i o t i c a g e n t s s u c h as h e a t , c h e m i c a l s , d r y i c e , o r various e x t r a c t s f r o m p l a n t s and m i c r o b e s d o e s n o t p r o t e c t c u c u m b e r s against l a g e n a r i u m (8-10). I n f e c t i o n o f t o b a c c o by a w i d e v a r i e t y of P e r o n o s p o r a l e s f u n g i o t h e r than P^_ t a b a c i n a frequently c a u s e s s e v e r e n e c r o s i s , but does not i n d u c e s y s t e m i c r e s i s t a n c e a g a i n s t b l u e mold (Tuziin and Kuc, unpublished). The e f f e c t i v e n e s s o f immunization i s d i r e c t l y r e l a t e d to the e x t e n t of i n f e c t i o n by, or i n o c u l u m c o n c e n t r a t i o n o f , the inducer o r g a n i s m up t o a p o i n t o f m a x i m a l r e s p o n s e o r saturation (49,53, 70). H o w e v e r , one l e s i o n c a u s e d by C. l a g e n a r i u m o r e i g h t l e s i o n s caused by TNV on one i n d u c e r l e a f can s i g n i f i c a n t l y induce s y s t e m i c d i s e a s e r e s i s t a n c e i n cucumbers. S p a t i a l and Temporal r e l a t i o n s h i p s . A common c h a r a c t e r i s t i c o f b i o t i c s e n s i t i z a t i o n of p l a n t s i s a l a t e n t or l a g p e r i o d between i n i t i a t i o n o f the i n d u c e r i n f e c t i o n and m a n i f e s t a t i o n o f d i s e a s e resistance. In the cucumber/TNV or l a g e n a r i u m system, enhanced r e s i s t a n c e a g a i n s t pathogens i s f i r s t m a n i f e s t about 48-72 hr a f t e r i n i t i a l i n f e c t i o n and maximal r e s i s t a n c e i s a c h i e v e d by 120-144 hr (49,53,70). In tobacco, r e s i s t a n c e a g a i n s t Ρ^_ t a b a c i n a a c h i e v e d by l i m i t e d stem i n f e c t i o n by the same organism i s not e v i d e n t b e f o r e 9 d a y s and i n c r e a s e s u n t i l a b o u t 21 d a y s a f t e r i n d u c t i o n ( 5 ) . An induction period o f 3-6 days i s r e q u i r e d for expression of r e s i s t a n c e t o TMV o r TNV i n d u c e d i n t o b a c c o by T h i e l a v i o p s i s b a s i c o l a ( 34). R e m o v a l o f the i n d u c e r l e a f 72-96 h r a f t e r i n i t i a t i o n o f the i n d u c i n g i n f e c t i o n i n cucumbers d i d not r e s u l t i n the l o s s o f s y s t e m i c r e s i s t a n c e i n the r e s t o f the p l a n t (53). Likewise, l e a v e s d i s t a l from the i n d u c e r l e a f r e t a i n e d r e s i s t a n c e a f t e r detachment from the p l a n t once immunization was e s t a b l i s h e d .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

4.

SALT AND

KUC

Disease Resistance in Plants

55

D u r a b i l i t y o f b i o t i c a l l y i n d u c e d r e s i s t a n c e v a r i e s w i t h the ρlant/inducer/pathogen combination. In c u c u m b e r s , r e s i s t a n c e i n d u c e d by l a g e n a r i u m o r TNV gradually declines unless a " b o o s t e r " i n o c u l a t i o n i s g i v e n 3-6 weeks a f t e r i n d u c t i o n ( 7 0 ) . With a b o o s t e r i n o c u l a t i o n , i m m u n i z a t i o n of cucumbers appears s u f f i c i e n t l y d u r a b l e to p r o t e c t the p l a n t t h r o u g h f l o w e r i n g and fruiting. However, cucumbers cannot be immunized a f t e r i n i t i a t i o n of f l o w e r i n g (7_1_)· T h i s s u g g e s t s a h o r m o n a l e f f e c t upon immunization. In t o b a c c o , i m m u n i z a t i o n a g a i n s t t a b a c i n a by v i r u l e n t P. t a b a c i n a i s hazardous b e f o r e the p l a n t s are at l e a s t 20 cm t a l l due t o f r e q u e n t s y s t e m i c s p r e a d o f t h e f u n g u s i n y o u n g p l a n t s , but a s i n g l e i n o c u l a t i o n o f p l a n t s o v e r 20 cm i n h e i g h t i s e f f e c t i v e t h r o u g h o u t the p l a n t ' s l i f e (5,72). Protection of t o b a c c o a g a i n s t P h y t o p h t h o r a p a r a s i t i c a v a r . n i c o t i a n a e by TMV seems to r e q u i r e r e p e a t e d a d m i n i s t r a t i o n o f the v i r u s (69). W h i l e b i o t i c s e n s i t i z a t i o n of p l a n t s a g a i n s t pathogens may a c t s y s t e m i c a l l y , not a l l organs or t i s s u e s are n e c e s s a r i l y p r o t e c t e d equally. Recent work i of r e s i s t a n c e a g a i n s t a n t h r a c n o s d i f f e r e n t p o s i t i o n s on i m m u n i z e d c u c u m b e r p l a n t s v a r i e s i n a complex manner not n e c e s s a r i l y d i r e c t l y p r o p o r t i o n a l to p r o x i m i t y o f the i n d u c e r l e s i o n s (73). Removal of e p i d e r m a l l a y e r s from immunized cucumber l e a v e s reduces r e s i s t a n c e to a n t h r a c n o s e (74). Mechanisms o f R e s i s t a n c e . The broad s p e c t r u m o f e f f e c t i v e n e s s o f induced s y s t e m i c r e s i s t a n c e a g a i n s t b a c t e r i a , v i r u s e s , f u n g i , and n e m a t o d e s , makes i t seem u n l i k e l y t h a t o n l y a s i n g l e r e s i s t a n c e mechanism i s a c t i v a t e d . Phytoalexins. Many, but not a l l , p l a n t f a m i l i e s are r e p o r t e d to u t i l i z e p h y t o a l e x i n a c c u m u l a t i o n as a means of d e f e n s e a g a i n s t pathogens. A major p h y t o a l e x i n i n green beans, P h a s e o l u s v u l g a r i s , i s t h e i s o f l a v o n o i d p h a s e o l l i n (18). R e a c t i o n s of " g e n e t i c a l l y r e s i s t a n t " bean v a r i e t i e s a g a i n s t incompatible r a c e s of C. 1 indemuthianum or of immunized " s u s c e p t i b l e " b e a n p l a n t s against v i r u l e n t r a c e s are marked by r a p i d a c c u m u l a t i o n of h i g h l e v e l s o f p h a s e o l l i n i n t i s s u e s a d j a c e n t to the i n v a d i n g fungus, r e s u l t i n g i n c o n t a i n m e n t o f the f u n g a l h y p h a e ( 62,75 ; F i g u r e 1). On t h e o t h e r hand, w h i l e u l t i m a t e p h y t o a l e x i n a c c u m u l a t i o n on a t o t a l p l a n t t i s s u e b a s i s c a n be g r e a t e r i n nonimmunized i n f e c t e d s u s c e p t i b l e p l a n t s , t h e o n s e t o f a c c u m u l a t i o n i s d e l a y e d ( F i g u r e 1); l o c a l concentrations a r e i n s u f f i c i e n t t o c o n t a i n t h e f u n g u s , and the p l a n t i s s u c c e s s f u l l y c o l o n i z e d by the pathogen. S i g n i f i c a n t l y , as shown i n F i g u r e 1, p h y t o a l e x i n s do not a c c u m u l a t e i n the immunized " s u s c e p t i b l e " bean p l a n t d i s t a n t from the s i t e of i n d u c t i o n p r i o r to exposure to the pathogen. That i s , s e n s i t i z e d r e s i s t a n c e i s not due to h i g h constitutive l e v e l s of p h y t o a l e x i n s . Induced r e s i s t a n c e through p r i o r b i o t i c i n f e c t i o n , t h e r e f o r e , appears to be at l e a s t a two-step phenomenon: s e n s i t i z a t i o n f o l l o w e d by a r a p i d e x p r e s s i o n of r e s i s t a n c e mechanisms a f t e r subsequent i n f e c t i o n . I n h i b i t i o n o f p e n e t r a t i o n and s p r e a d w i t h i n t i s s u e s . In c u c u r b i t s , c l a s s i c a l p h y t o a l e x i n s h a v e n o t b e e n i d e n t i f i e d and d i s e a s e r e s i s t a n c e appears due to o t h e r mechanisms. Immunization

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST C O N T R O L

150

125

£ 100

a* ~ 75 Ο

S

50


100 >100

Abyssinol A (II), Β (III) and C (IV), without the acetoxyl moiety, were less active than abyssini the acetoxyl moiety. W antifeedant a c t i v i t y due to the removal of the acetoxyl group when we investigated antifeedant a c t i v i t i e s o f the ajugarins (11). Tetrahydroabyssinin (IX) and i t s epoxy-hydrolyzed derivative (XVI) were found to be inactive even at 100 ug/disk. This suggests both the α-pyrone ring and the epoxide group are required f o r the strong antifeedant a c t i v i t y of abyssinin (I). We also conducted a 'no-choice a r t i f i c i a l d i e t feeding assay (12), since compounds not showing antifeedant a c t i v i t y but having growth inhibitory a c t i v i t y would be overlooked i n the previous 'choice' s i t u a t i o n . As a r e s u l t , we isolated only abyssinin as an insect growth i n h i b i t o r while monitoring by an a r t i f i c i a l d i e t feeding assay. In f a c t , abyssinol A (II), Β (III) and C (IV), isolated monitoring by the l e a f disk assay, d i d not show any insect growth inhibitory a c t i v i t y , as i s shown i n Table III. 1

Table III.

Growth inhibitory a c t i v i t y (ED50 i n ppm) of bufadienolides against Pectinophora g o s s y p i e l l a .

Compounds

EDso(ppm)

(I)

10

(ID

>50

(III)

>100

(IV)

>100

(IX)

>150

(XVI)

>150

G a l l i c acid

>500

Methyl

>500

gallate

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

200

BO IREGULATORS FOR PEST CONTROL

Thus, the growth inhibitory a c t i v i t y of abyssinin (I) compared to abyssinols (II-IY) could be attributed to a 'potent' antifeedant e f f e c t which can cause starvation. Accordingly, the antifeedant a c t i v i t i e s of the abyssinols (II-IY) appear not to be strong enough as to force starvation. G a l l i c acid and methyl gal l a t e , isolated monitoring by antimicrobial a c t i v i t y against subtil i s , were inactive against P. gossypiella even a t a concentration of 500 ppm. On the other Fana, abyssinin (I) did not show microbial a c t i v i t y against EL subtil i s even at a high concentration. This shows another example where d i f f e r e n t type of secondary compounds a c t i n a variety of ways as defense adaptations of a plant. Acknowledgments Insects f o r the bioassay were kindly supplied by the agencies of the USDA i n Phoenix, AZ; T i f t o n , GA; and Brownsville, TX. The authors are grateful to Dr. H. Naoki and Professor A. S . Kende f o r NMR measurements, Professo J . A. Klocke f o r antifeedant bioassay. Literature

Cited

1.

J. O. Kokwaro, "Medicinal Plants of East Africa"; East African Literature Bureau, Nairobi, 1976; p. 159. 2. I. Kubo and K. Nakanishi, "Host Plant Resistance to Pests"; ed. by P. A. Hedin, ACS Symposium Series 62, American Chemical Society: Washington, D.C., 1977; pp. 165-178. 3. S. M. Kupchan and I. Ognyanov, Tetrahedron Lett., 1709 (1969), S. M. Kupchan, R. J. Hemingway and J. C. Hemingway, J. Org. Chem., 34, 3894 (1969). 4. P. Zanno, I. Miura, K. Nakanishi and D. Elder, J. Am. Chem. Soc., 97, 1975 (1975). 5. P. J. May, Terpenoidas and Steroids, 1, 527 (1971). 6. L. Gsell and Ch. Tamm, Helv. Chim. Acta., 52, 551 (1969). 7. K. Nakanishi, T. Goto, S. Ito, S. Natori and S. Nozoe, "Natural Products Chemistry"; Academic Press: New York, 1974; p. 469-476. 8. J. Meinwald, D. F. Wiemer and T. Eisner, J. Am. Chem. Soc., 101, 3055 (1979). 9. J. Goto, N. Goto, F. Shamsa, M. Sato, S. Komatsu, K. Suzaki and T. Nambara, Anal. Chim. Acta., 147, 397 (1983). 10. I. Kubo and J. A. Klocke, l'Colloques de l'I.N.R.A., 7, 117 (1981). 11. B. G. Chan, A. C. Waiss, Jr., W. L. Stanley and A. E. Goodban, J. Econ. Ent., 71, 366 (1978). RECEIVED

November 8, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

13 Cockroach Control with Juvenoids G E R A R D U S B. STAAL, CLIVE A. HENRICK, DAVID L. GRANT, DAVID W. MOSS, M I C H A E L C. JOHNSTON, ROBIN R. R U D O L P H , and WILLIAM A. D O N A H U E Zoecon Corporation, Palo Alto, C A 94304

The potential of juvenoids to control cockroach populations depends on their ability to stop reproduction. The therefore relate to property with short term responses such as morphogenetic abnormalities. In this study, several dienoates with high activity on various other insect targets and a few other prominent juvenoids were compared in a feeding assay for activity on the German cockroach. Of the compounds tested, hydroprene was the most potent, whereas methoprene was considerably less active against this target species. Further studies on the persistence of residues on various substrates of technical hydroprene confirmed the volatility of hydroprene. Through formulation, the volatility could be greatly reduced with retention of full activity. However, volatility may well play a contributory role in the efficacy of practical household applications. The coapplication of conventional insecticides contributes term reduction in population and consumer acceptance of juvenoids.

to the short thus enhances

S t u d i e s i n the e a r l y and mid s e v e n t i e s on t h e e f f e c t of s e v e r a l j u v e n o i d s on cockroaches have i n d i c a t e d t h e i r a c t i v i t y i n terms of i n h i b i t i o n of metamorphosis, i n c r e a s e d m e l a n i z a t i o n , development o f o v a r i e s , m o r t a l i t y of a d u l t s and progeny, e f f e c t s on r e g e n e r a t i o n , d e f i c i e n c i e s i n mating b e h a v i o r , d e l a y of the f i n a l molt and, l a s t but not l e a s t , the i n h i b i t i o n of r e p r o d u c t i o n (1-11). The p o t e n t i a l of c e r t a i n j u v e n o i d s , p a r t i c u l a r l y t h a t of hydroprene, t o control cockroach populations by i n h i b i t i n g the reproduction through exposure t o t h e j u v e n o i d s i n a c r i t i c a l period before metamorphosis was c l e a r l y suggested by t h i s e a r l i e r work. However, t h i s was not f o l l o w e d up a t t h a t time because measures r e q u i r i n g several months t o a c h i e v e c o n t r o l were not c o n s i d e r e d t o be commercially a t t r a c t i v e . W i t h j u v e n o i d s alone i t would i n d e e d take s e v e r a l months f o r t h e t o t a l d i s a p p e a r a n c e by a t t r i t i o n of the l a s t normal and s t e r i l e a d u l t s , s i n c e the average l o n g e v i t y of female a d u l t s i s about 5 months.

0097-6156/ 85/ 0276-0201 $06.00/ 0 © 1985 American Chemical Society In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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A t l e a s t two events were r e s p o n s i b l e f o r a change i n t h i s a t t i t u d e and provided the i n c e n t i v e t o s e l e c t and develop an e f f e c t i v e j u v e n o i d f o r cockroach c o n t r o l : a) the s u c c e s s a c h i e v e d with the j u v e n o i d methoprene i n the c o n t r o l of b r e e d i n g flea populations i n household carpets and upholstery (17, 18) b) subjective observations by tenants of apartments treated with methoprene f o g g e r s d u r i n g " f i e l d " e x p e r i m e n t s conducted i n 1981 d e s i g n e d to e s t a b l i s h the e f f i c a c y on f l e a p o p u l a t i o n s i n d i c a t i n g t h a t c o c k r o a c h p o p u l a t i o n s a l s o appeared t o be a f f e c t e d . Over the p a s t decade, the o u t l o o k f o r c o c k r o a c h c o n t r o l had undergone changes due to the r e a l i z a t i o n t h a t the most e f f e c t i v e p e s t i c i d e t r e a t m e n t s , even i f a p p l i e d by e x p e r i e n c e d p e s t c o n t r o l operators (P.C.O.'s) a l t h o u g h seemingly effective f o r a short while, never appeared t o y i e l d l o n g l a s t i n g c o n t r o l . This i s p r o b a b l y r e l a t e d i n p a r t to c o c k r o a c h b e h a v i o r a l a d a p t a t i o n s such as h i d i n g i n c r a c k s and c r e v i c e s when i n s e c t i c i d e p r e s e n c e i s sensed or to temporary r e l o c a t i o n t o u n t r e a t e d a d j a c e n t h a b i t a t s u n t i l the r e s i d u e s hav of the most e f f i c a c i o u and r e s i s t a n c e a g a i n s t the r e m a i n i n g ones have become w i d e s p r e a d (19). Hence, an i n c r e a s i n g i n t e r e s t developed f o r the i n t r o d u c t i o n of l e s s t o x i c p r o d u c t s w i t h a n o v e l mode of a c t i o n . I n a f u r t h e r development of the j u v e n o i d c o n t r o l approach, t h e r e appeared t o be a very good promise a l s o f o r combinations of conventional insecticides (whether really effective or only perceived as such) with effective long term j u v e n o i d control agents. T h e r e f o r e , we undertook t o c o n f i r m whether hydroprene (as y e t u n r e g i s t e r e d f o r any insect c o n t r o l use) was indeed the most e f f i c a c i o u s of the j u v e n o i d s p r o p r i e t a r y to Zoecon and whether the use of methoprene, another d i e n o a t e which was a l r e a d y r e g i s t e r e d f o r h o u s e h o l d i n s e c t c o n t r o l t a r g e t s ( i . e . f l e a s ) , c o u l d be e q u a l l y e f f i c a c i o u s i f a p p l i e d at h i g h e r r a t e s . We a l s o wanted to compare our most e f f e c t i v e d i e n o a t e s w i t h j u v e n o i d s under c o n s i d e r a t i o n f o r commercial development by o t h e r p a r t i e s . The a c t i v i t y of v a r i o u s j u v e n o i d s on a spectrum of i n s e c t t a r g e t s has been d e s c r i b e d i n s e v e r a l review papers (20-22). F u r t h e r m o r e , i f the study would suggest that a dienoate juvenoid could indeed be s u c c e s s f u l l y a p p l i e d a g a i n s t cockroaches, f u r t h e r s t u d i e s on the most e f f e c t i v e mode of a p p l i c a t i o n , l o n g e v i t y of r e s i d u e s , o p t i m i z e d f o r m u l a t i o n s , c o m b i n a t i o n s w i t h a d u l t i c i d e s , e t c . would be u n d e r t a k e n . Our p r i n c i p a l t a r g e t was the German cockroach, (Blattella germanica) as the most w i d e s p r e a d d o m e s t i c c o c k r o a c h i n the USA, but a l i m i t e d number of c o n f i r m a t o r y s t u d i e s were done on the American cockroach (Periplaneta americana) and the oriental cockroach ( B l a t t a o r i e n t a l i s ) . A s s a y f o r Comparative

Activity

To study the r e l a t i v e i n t r i n s i c a c t i v i t y of a s e r i e s of d i e n o a t e juvenoids, a forced feeding test was s e l e c t e d i n which the m a t e r i a l s were i n c o r p o r a t e d i n the o n l y f o o d a v a i l a b l e t o B l a t f c e l l a germanica p o p u l a t i o n s throughout the d u r a t i o n of the e x p e r i m e n t . T h i s c h o i c e was made because of the d i f f i c u l t y i n s y n c h r o n i z i n g the peak of s e n s i t i v i t y i n the l a s t nymphal i n s t a r due t o the l o n g

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

13.

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203

d u r a t i o n of t h a t (and o t h e r ) s t a g e s i n c o c k r o a c h e s . The absence of d e f i n i t e developmental markers o t h e r than the m o l t i n g p r o c e s s and variations i n the d u r a t i o n of the last nymphal instar make determination of the sensitive period unreliable. Food a d m i n i s t r a t i o n would at l e a s t secure the presence of the compound throughout the f e e d i n g p e r i o d of the l a s t nymphal s t a g e , c o v e r i n g the window of s e n s i t i v i t y . S i n c e many y e a r s of j u v e n o i d t e s t i n g have c o n f i r m e d t h a t the r e s u l t s of f e e d i n g t e s t s p a r a l l e l those of t o p i c a l a p p l i c a t i o n s i n s e v e r a l i n s e c t s p e c i e s ( p r i m a r i l y phytophagous L e p i d o p t e r a ) w i t h a b e t t e r s y n c h r o n i e a b l e s e n s i t i v e p e r i o d , we f e l t r e a s o n a b l y secure w i t h a food t e s t . However, i t was known a l r e a d y at t h a t time t h a t a f o o d b a i t i s an i n e f f e c t i v e way of a d m i n i s t e r i n g j u v e n o i d s when the b a i t i s i n c o m p e t i t i o n w i t h a c h o i c e of o t h e r h o u s e h o l d f o o d sources (unpublished s t u d i e s ) . Other t e s t p o s s i b l i t i e s such as continuous s u b s t r a t e c o n t a c t , seemed more r e l e v a n t but were not pursued because t h e i r a c c u r a c y and r e p r o d u c i b i l i t y was unknown a t the time, a t l e a s t f o Cruickshank & Palmere c a l l e d " b a i t " o r "bedding") d i d not a p p e a l t o us because, i n our e x p e r i e n c e , s t a r c h c o u l d a c t as a s o r p t i v e dust c o n t r i b u t i n g t o the cockroaches demise by causing dehydration. As w i l l become 1

apparent l a t e r , s u b s t r a t e r e s i d u e exposure has many parameters t h a t would i n d e e d have i n t e r f e r e d w i t h a comparative evaluation. We c o n s i d e r e d t h a t the use of a f o o d s u b s t r a t e t o a d m i n i s t e r the juvenoids would probably minimize the uncertainties i n the persistence of r e s i d u e s i n the bedding m a t e r i a l or on other s u r f a c e s i n the h a b i t a t . However, the chosen method of food incorporation could have one potential flaw: i f any of the i n c o r p o r a t e d m a t e r i a l s were r e p e l l a n t at the a p p l i e d c o n c e n t r a t i o n , death by s t a r v a t i o n could result. Careful observation could p r o b a b l y r u l e t h i s out. H a v i n g no r e a l e s t i m a t e of the e f f e c t i v e c o n c e n t r a t i o n s under our c o n d i t i o n s , the i n i t i a l phase of this experiment was carried out at 1000 ppm for a l l materials investigated. In f o l l o w i n g t e s t c y c l e s , the most a c t i v e m a t e r i a l s were a p p l i e d a t lower doses f o r f u r t h e r d i f f e r e n t i a t i o n . The r e s u l t s of these s u c c e s s i v e c y c l e s a r e here d e s c r i b e d t o g e t h e r as one e x p e r i m e n t . M a t e r i a l s & Methods. Batches of f i f t y 4th i n s t a r mixed sex B. germanica were p l a c e d i n s t a n d a r d 28x17x12 cm d i s p o s a b l e mouse cages. The i n s i d e w a l l s of the cages were t r e a t e d w i t h F l u o n ( F l u o n AD-1 l i q u i d t e f l o n s u s p e n s i o n . N o r t h e a s t C h e m i c a l Co., I n c . 153 Hamlet Avenue, P.O. Box 1175, Woonsocket, RI 02895) t o p r e v e n t the cockroaches f r o m e s c a p i n g . Water and harborage was p r o v i d e d , and cages were f i t t e d w i t h f i n e mesh s c r e e n tops (white p o l y e s t e r sheer c u r t a i n f a b r i c . C h a n d l e r E n t e r p r i s e s , P.O. Box 4934 F o s t e r City, CA 94404) t o prevent i n g r e s s or e g r e s s . Cages were m a i n t a i n e d at 27°C i n a 16 hr phot ope r i o d and a p p r o x i m a t e l y 50% RH i n an a i r c o n d i t i o n e d room (no a i r r e c y c l e d . ) These t e s t s were a l l performed i n t r i p l i c a t e .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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FOR PEST C O N T R O L

The f o o d was p r e p a r e d by p u l v e r i z i n g Gaines burger dog f o o d i n a Waring b l e n d e r , then m i x i n g t h i s w i t h the a c t i v e ingredient d i s s o l v e d i n acetone. A f t e r m i x i n g , the f o o d was l e f t i n an uncovered s t o r a g e j a r f o r 24 hours t o a l l o w f o r e v a p o r a t i o n of t h e solvent. A p p r o x i m a t e l y 10 g of f o o d was s u p p l i e d t o each cage, and the r e s t (40 grams) was s t o r e d a t 5°C i n s e a l e d j a r s f o r use as needed t o r e s u p p l y consumed f o o d i n the t e s t cages. The d i e n o a t e s #1, #2, #4-15, #17, #18, i n c l u d i n g methoprene (#9) and hydroprene (#1; 25) have been d e s c r i b e d by H e n r i c k e t a l i n c l u d i n g t h e i r s y n t h e s i s and b i o l o g i c a l a c t i v i t y a g a i n s t various i n s e c t t a r g e t s , and were s y n t h e s i z e d i n Zoecon C o r p o r a t i o n (24, 25). Compound #3 (R-20458) was s u p p l i e d by S t a u f f e r C h e m i c a l Company ( 2 6 ) ; compound #16 was s u p p l i e d by M. Schwartz (USDA) ( 2 7 ) . A l l p o p u l a t i o n s were r e g u l a r l y s c o r e d f o r e i t h e r p r e s e n c e o r absence of unambiguous morphogenetic a b n o r m a l i t i e s ( r a n g i n g f r o m slightly deformed or crumpled wings to completely nymphal supernumerary i n s t a r s ) as w e l l as f o r the number of F^ progeny. It was deemed e s s e n t i a l t l o n g p e r i o d a f t e r the mol of most j u v e n o i d s t o d e l a y the metamorphic m o l t . In a l l c a s e s , the f i n a l r e p r o d u c t i v e s c o r i n g was done a f t e r the f i r s t o v a r i a n c y c l e i n a l l t r e a t m e n t s was completed and the f i r s t round of egg c a p s u l e s ( v i a b l e or u n v i a b l e ) were dropped. We have o b s e r v e d t h a t non r e p r o d u c t i v e f e m a l e s would remain s t e r i l e i n subsequent ovarian c y c l e s under c o n t i n u o u s exposure t o the j u v e n o i d . The p o s s i b i l i t y of a r e v e r s a l a f t e r removal f r o m exposure i s the s u b j e c t of o n g o i n g studies. However, e a r l i e r s t u d i e s (29) have i n d i c a t e d t h a t t h i s would not o c c u r . Results. I n n e a r l y a l l cases, the t r i p l i c a t e cages f o r each dose yielded identical results. The o r d e r of a c t i v i t y on r e p r o d u c t i o n o b t a i n e d i n these f o o d experiments i s i n d i c a t e d by t h e i r r a n k i n g order i n T a b l e I. At 10 ppm, hydroprene (#1) appears to be somewhat more e f f e c t i v e than the o t h e r d i e n o a t e s and the S t a u f f e r cpd (#3). Methoprene (#9) f a i l e d even at 1000 ppm, i n d i c a t i n g a l e s s e r e f f i c a c y as compared t o hydroprene by a f a c t o r between 10 and hundred. S e v e r a l o t h e r d i e n o a t e s and Schwartz's compound (#16), a l t h o u g h e x h i b i t i n g at l e a s t f a i r t o good a c t i v i t y on o t h e r i n s e c t t a r g e t s (22, 24), f a i l e d i n t h i s t e s t even at 1000 ppm. For the 2,4-dienoate e s t e r s t e s t e d , the p r e s e n c e of an 11-methoxy or an 11-hydroxy group produces a sharp drop i n a c t i v i t y (#1 vs_ #13 and #7 vs_ #9). A s i m i l a r r e s u l t i s o b t a i n e d i n T a b l e I f o r the Si s o p r o p y l e s t e r s (#5 vs_ #11). The ketone #2, which has a shape s i m i l a r t o t h a t of the e t h y l e s t e r #1 has an a c t i v i t y v e r y s i m i l a r to #1. F o r the dienamides, the Nj-ethyl a n a l o g s are more a c t i v e t h a n the N , ^ - d i e t h y l compounds (#4 vs_ #8 and #6 vs_ #10). The s e c butyl ester #12 i s much l e s s active than the c o r r e s p o n d i n g i s o p r o p y l e s t e r #7. I t s h o u l d be noted t h a t these r e s u l t s d i f f e r f r o m those o b t a i n e d by Radwan & Sehnal ( 1 2 ) , who f o u n d methoprene t o be more a c t i v e than hydroprene i n topical applications to Nauphoeta c i n e r e a . They found t h a t methoprene, hydroprene and the i s o p r o p y l e s t e r #7 were a l l very a c t i v e a t i n h i b i t i n g metamorphosis of N. c i n e r e a , w i t h methoprene b e i n g the most a c t i v e . The a r o m a t i c e t h e r #3, and s e v e r a l r e l a t e d a n a l o g s , showed much lower a c t i v i t y

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

100 100 100 100 100 100 100 100 24 98 100 100 0 0 0 0 0 0 0

(0) (0) (0) (0) (0) (0) (0) (0) (266) (13) (0) (10) (116) (194) (215) (148) (400+) (150) (400+)

1000 ppm

Note: - = not t e s t e d a t t h i s

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 ( u n t r e a t e d )

Compound No.

(0) (0) (0) (0) (0) (0) (0) (0) (400+) (400+) (400+) (400+)

level.

--

100 100 100 100 100 100 100 100 11 94 99 98 -

500 ppm

100 100 100 100 98 100 100 99 9 76 92 47 -

(0) (0) (0) (0) (0) (0) (0) (0) (400+) (400+) (400+) (400+)

250 ppm

--

-

-

-

-

--

--

11 12 13 11 4 13 0 8 -

--

(0) (0) (0) (0) (0) (0) (0) (188)

(98) (253) (260) (391) (397) (421) (448) (350)

10 ppm

100 100 100 100 54 100 87 31 -

100 ppm

-

--

-

--

10 6 11 0 2 8 0 4 (313) (469) (322) (402) (493) (494) (438) (315)

5 ppm

Table I . P e r c e n t Abnormal and Number o f F i r s t C y c l e Progeny ( i n P a r e n t h e s e s ) R e s u l t i n g from "No C h o i c e " Feeding o f B l a t t e l l a germanica Nymphs on V a r i o u s J u v e n o i d s I n c o r p o r a t e d i n Food

206

BIOREGULATORS FOR PEST CONTROL

3 .

4.

7.

^ ° γ γ ν γ ^ ^

1 .

2 .

s.

ν γ γ ν γ \ / γ

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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207

a g a i n s t t h i s s p e c i e s (12)· R i d d i f o r d e t a l had r e p o r t e d p r e v i o u s l y t h a t under t h e i r t e s t i n g c o n d i t i o n s (constant contact with the compound d e p o s i t e d on c o r n s t a r c h bedding m a t e r i a l ) hydroprene (//l) was s u b s t a n t i a l l y more a c t i v e than methoprene (#9), k i n o p r e n e (22) and #3 a g a i n s t B l a t t e l l a germanica (14)· T r i p r e n e (22) was found to be r e a s o n a b l e a c t i v e under t h e i r c o n d i t i o n s . The low a c t i v i t y they observed f o r #3 ( c f . T a b l e I ) c o u l d be due t o i t s r e l a t i v e l y low v o l a t i l i t y and/or i n c r e a s e d b i n d i n g t o the c o r n s t a r c h . This experiment also allowed us to draw an important c o n c l u s i o n t h a t c o u l d be s u b s t a n t i a t e d i n a l l f u r t h e r work on hydroprene: morphogenetic abnormality and full reproductive i n h i b i t i o n were c o r r e l a t e d w i t h each o t h e r at a l l dose l e v e l s i n the sense t h a t m o r p h o g e n e t i c a l l y a f f e c t e d a d u l t s always f a i l e d t o reproduce (compare T a b l e I ) . The r e v e r s e was not always t r u e ; a t m a r g i n a l doses of j u v e n o i d s , m o r p h o g e n e t i c a l l y normal a d u l t s were found o c c a s i o n a l l y t h a t were unable t o r e p r o d u c e . However, the o b s e r v e d c o r r e l a t i o n may not h o l d t r u e f o r s t i l l o t h e r types of juvenoids. We found n j u v e n o i d s at the c o n c e n t r a t i o n methoprene had shown t h i s i n p r e l i m i n a r y e x p e r i m e n t s at 10,000 ppm. Residue t e s t

on

latex painted

surface

In this test s e r i e s t e c h n i c a l hydroprene compared w i t h a r e s i d u a l dust f o r m u l a t i o n (RF substrate.

and 10%)

methoprene were on a l a t e x p a i n t

M a t e r i a l s & Methods. The bottoms of s t a n d a r d d i s p o s a b l e mouse cages were f i r s t p a i n t e d w i t h a f l a t l a t e x i n t e r i o r p a i n t . After l e t t i n g the p a i n t dry f o r one t o t h r e e days, the t e s t m a t e r i a l s were a p p l i e d by s p r e a d i n g e i t h e r acetone d i l u t i o n s d r i b b l e d on f r o m a s y r i n g e or by s p r e a d i n g the dust f o r m u l a t i o n w i t h a s p a t u l a over the p a i n t e d s u r f a c e s . One day l a t e r , these cages were i n f e s t e d w i t h f i f t y 4th i n s t a r nymphs of B l a t t e l l a germanica and f u r t h e r t r e a t e d and s c o r e d as d e s c r i b e d i n Experiment 1. A l l t e s t doses were s e t up i n d u p l i c a t e . F i n a l s c o r i n g was performed 100 days a f t e r i n i t i a t i o n of the e x p e r i m e n t . Results. T e c h n i c a l hydroprene as w e l l as the RF f o r m u l a t i o n were both f u l l y e f f e c t i v e i n i n h i b i t i n g r e p r o d u c t i o n and p r o d u c i n g a h i g h or complete of morphogenetic a b n o r m a l i t i e s at a r a t e as low as 1 ug/cm (Table I I ) . T e c h n i c a l methoprene r e q u i r e d 1000 pg/cm , and thus was 100 to 1000 times less effective than hydroprene t e c h n i c a l . Methoprene RF was at l e a s t 10 times more e f f e c t i v e t h a n t e c h n i c a l methoprene and t h e r e f o r e a p p r o x i m a t e l y 100 times l e s s e f f e c t i v e than hydroprene i n a s i m i l a r formulation. H i g h c o n c e n t r a t i o n s of the i n e r t s of RF f o r m u l a t i o n s appeared t o be t o x i c by themselves, at l e a s t when f o r m u l a t e d w i t h d i e n o a t e s . A f o r m u l a t i o n b l a n k was not i n c l u d e d because the blank RF f o r m u l a t i o n was found t o be p h y s i c a l l y not comparable ( o n l y a f o r m u l a t i o n w i t h an inert look alike dienoate would have made a relevant comparison).

incidence

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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T a b l e I I . The i n f l u e n c e of f o r m u l a t i o n on the e f f i c a c y of hydroprene and methoprene on B l a t t e l l a germanica nymphs when a p p l i e d t o a l a t e x p a i n t s u b s t r a t e . % abnormal morphogenesis 0.1

1

ug/cm 1000

100

10

4

Hydroprene Tech

3 r

87 0

100 0

100 ο

100 ο

Hydroprene RF

6 r

100 0

100 0

_a

-

10%

-

Methoprene Tech. Methoprene RF

10%

r • reproduction -

a

no

survival;

observed; [] few

7 V

4 V

3 r

6 V

64 V

100 ο

ο = no reproduction;

75 ο [100 o]

- = not done.

survivor

Discussion. The i n c r e a s e i n a c t i v i t y f o r the RF f o r m u l a t i o n s of methoprene and the r e t e n t i o n of a c t i v i t y f o r hydroprene was a s u r p r i s e s i n c e f o r m u l a t i o n s w i t h a l o n g r e s i d u a l g e n e r a l l y tend t o d e c r e a s e acute a c t i v i t y . However, the i n c r e a s e d s t a b i l i t y , the decrease in volatility and the intense contact with a dust f o r m u l a t i o n by grooming cockroaches may have c o n t r i b u t e d t o t h i s result. I t a l s o appeared t h a t methoprene (RF 10%) at 100 ug/cm was an e f f e c t i v e c o n t r o l dose. Persistence substrates

of

hydroprene

The e f f e c t of t h r e e hydroprene f o r a g i n g d i f f e r e n t substrates

residues

on

various

household

surface

d i f f e r e n t f o r m u l a t i o n s on the p e r s i s t e n c e of p e r i o d s of up to 32 weeks was s t u d i e d on f o u r under average h o u s e h o l d exposure c o n d i t i o n s .

Preliminary observations had i n d i c a t e d that residues of t e c h n i c a l hydroprene of 0.5 ug/cm on glass (after overnight exposure t o ambient room c o n d i t i o n s ) y i e l d e d o n l y m a r g i n a l r e s u l t s p r o b a b l y due t o the v o l a t i l i t y of t h i s d i e n o a t e . T h i s l e d us t o investigate two existing commercial formulations and one experimental f o r m u l a t i o n at higher rates for t h e i r persistence throughout extended p e r i o d s of exposure t o ambient c o n d i t i o n s . M a t e r i a l s & Methods. R e c t a n g l e s (14x23 cm) of v i n y l f l o o r t i l e , u n f i n i s h e d plywood, g l a s s , and v i n y l t i l e s p a i n t e d w i t h enamel p a i n t (U.S. Navy) were t r e a t e d w i t h the f o l l o w i n g f o r m u l a t i o n s : a) e m u l s i f i a b l e concentrate (5E) d i l u t e d i n water s p r a y e d on w i t h a De V i l b i s s s p r a y e r at 5 p . s . i . at r a t e s of 1 and 2 ug/cm b) A 10% m i c r o e n c a p s u l a t e d s u s p e n s i o n (M 10%) equivalent in manufacture t o ALT0SID SR10 was a p p l i e d i n the same way d i l u t e d i n water, y i e l d i n g the same r a t e s of A.I. c) The RF dust f o r m u l a t i o n (9-10% A.I.) was a p p l i e d as dry powder and s p r e a d w i t h a spatula. T h i s f o r m u l a t i o n was o n l y a p p l i e d t o plywood and glass. A l l p l a t e s were then kept f o r s t a t e d i n t e r v a l s at ambient room c o n d i t i o n s s i m i l a r t o those i n experiment 1, w i t h ambient

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

13.

Cockroach Control with Juvenoids

STAALETAL.

209

l i g h t from f l u o r e s c e n t f i x t u r e s (equipped w i t h i n f r a r e d a b s o r b i n g shields). P l a t e s t r e a t e d w i t h a) and b) were s t o r e d u p r i g h t , p l a t e s w i t h c ) were k e p t h o r i z o n t a l l y t o p r e v e n t the l o s s of powdery r e s i d u e s . A f t e r the exposure p e r i o d ( a c t u a l l y 24 h r s f o r the 0 weeks of exposure), the s u b s t r a t e p l a t e s were t h e n i n s e r t e d i n s t a n d a r d mouse cages, c o v e r i n g most of the bottom. I n each cage twentyfive 4th i n s t a r B l a t t e l l a germanica nymphs were i n t r o d u c e d , a f t e r w h i c h the r e a r i n g c o n d i t i o n s and s c o r i n g t e c h n i q u e s described under experiment 1 were a p p l i e d . Results. T a b l e I I I i n d i c a t e s t h a t the r e s i d u a l p e r s i s t e n c e v a r i e d w i t h the f o r m u l a t i o n and the s u b s t r a t e . V i n y l t i l e and p a i n t e d s u r f a c e s r e t a i n e d residues b e t t e r than g l a s s . Bare plywood was b e t t e r f o r M than f o r the EC. The RF f o r m u l a t i o n was a c l e a r

Table I I I . Residua on various househol active ingredient

Substrate Vinyl

tile

Enamel P a i n t

Plywood

Formulation

0

1 ug/cm 2 4 8 16 32

EC 60%

0

0

M

10 %

0

0

r

r

Untreated

r

r

r

EC 60 %

0

r

M

0

Untreated

EC 60 %

r

0

0

0

0

r

r

r

r

0

0

0

r

r

r

r

r

r

r

r

r

r

r

r

r

r

r

0

0

0

0

r

0

r

r

r

0

0

0

r

r

r

r

r

r

r

r

r

r

r

r

0

r

r

r

r

0

r

0

r

r

0

r

r

r

-

r

-

10 %

0

0

r

r

r

0

0

0

r

r

R F 10 %

0

0

0

0

0

r

0

0

0

0

0

0

Untreated

r

r

r

r

r

r

r

r

r

r

r

r

EC 60 %

0

r

r

r

r

0

r

r

r

r

M

10 %

0

r

r

r

r

0

r

r

r

r

RF 10% Untreated

0 r

0 r

0 r

0 r

0 r

0 r

0 r

0 r

0 r

0 r

M

Glass

10%

2 ug/cm Weeks of a g i n g 0 2 4 8 16 32

0 r

0 r

0 « no r e p r o d u c t i o n ; r = r e p r o d u c t i o n ; - = not t e s t e d . EC = e m u l s i f i a b l e c o n c e n t r a t e , M - m i c r o e n c a p s u l a t e d formulation (suspension), R F = r e s i d u a l f o r m u l a t i o n ( d r y powder).

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

210

BIOREGULATORS FOR PEST CONTROL

w i n n e r . Only at 32 weeks and a t the 1 ug dose i s t h e r e a b e g i n n i n g of breakdown of r e p r o d u c t i v e c o n t r o l . We concluded t h a t the RF f o r m u l a t i o n a f f o r d e d a s u p e r i o r p r o t e c t i o n a g a i n s t e v a p o r a t i o n and possibly also against photo-instability without decreasing biological availability. I n t h i s t a b l e o n l y the complete i n h i b i t i o n of r e p r o d u c t i o n i s r e p r e s e n t e d s i n c e we concluded t h a t p a r t i a l f a i l u r e of reproduc­ t i o n c o u l d not be a s s e s s e d q u a n t i t a t i v e l y ( i t was not known, f o r i n s t a n c e , from how many s u r v i v i n g a d u l t s a low number of o f f s p r i n g c o u l d have o r i g i n a t e d ) . Vapor

test

I n o r d e r t o t e s t the v o l a t i l i t y and the containment of such vapors i n the u s u a l mouse cages w i t h s c r e e n t o p s , 50 mg of t e c h n i c a l h y d r o p r e n e or 50 mg A . I . of hydroprene RF 10% were p l a c e d i n aluminum boats w i t h 1/2 cm u p t u r n e d edges i n the top 3 cm of the cages i n such a way t h a c o c k r o a c h e s c o u l d take p l a c e t o those d e s c r i b e d i n the o t h e r e x p e r i m e n t s . The t e s t was s c o r e d for morphological as w e l l as r e p r o d u c t i v e effects well after c o m p l e t i o n of the f i r s t r e p r o d u c t i o n c y c l e i n the c o n t r o l s (Table IV). Results. The r e s u l t s c o n v i n c i n g l y demonstrate the vapor a c t i v i t y of t e c h n i c a l hydroprene i n both types of s c o r i n g and the absence of i t i n the RF f o r m u l a t i o n . A l t h o u g h one of the RF r e p l i c a t e s produced a sizable number of morphogenetically affected i n d i v i d u a l s , the r e p r o d u c t i o n was not n o t i c e a b l y a f f e c t e d . Even though the doses of hydroprene ( e q u i v a l e n t t o 100 vg/cm ) were on T a b l e IV. M o r p h o g e n e t i c i n h i b i t i o n and e f f e c t on r e p r o d u c t i o n as a r e s u l t of exposure t o h y d r o p r e n e as vapor o n l y a t 50 mg/cage ( e q u i v a l e n t t o 100 pg/cm ).

Cage 1

Compound

Abnormal

Reproduction

2

27

R

2

3

18

R

3

2

18

R

4

untreated

Normal

23

0

0

5

18

0

0

6

21

0

0

7

hydroprene

hydroprene RF

10%

18

a )

4

R

8

2

33

R

9

0

30

R

Possible false positive,

see

text.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

13.

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Cockroach Control with Juvenoids

the h i g h s i d e , the RF f o r m u l a t i o n f a i l e d t o produce any vapor effect. The f a l s e p o s i t i v e was p r o b a b l y caused by a v e r y m a r g i n a l spill of powder f o r m u l a t i o n or a vapor c o n t a m i n a t i o n f r o m an a d j a c e n t cage. Aquarium

and chamber experiments

3 d i f f e r e n t and independent l o n g term t e s t s w i t h hydroprene were run e i t h e r i n e x p e r i m e n t a l chambers s i m u l a t i n g h o u s e h o l d c o c k r o a c h h a b i t a t s or i n c o n t a i n e r s (aquariums) t h a t had been exposed t o methoprene or hydroprene under s i m u l a t e d f i e l d c o n d i t i o n s . D a l l a s Aquarium T e s t s . I n October of 1981, t e s t s were i n i t i a t e d a t Zoecon I n d u s t r i e s ( D a l l a s , T e x a s ) , u s i n g t h r e e 10 g a l l o n aquariums, each i n f e s t e d w i t h 5 a d u l t male and 5 a d u l t female B»_ g e r m a n i c a . One aquarium, i n c l u d i n g a harborage, was p l a c e d i n a 3000 cu f t room which was then f o g g e d w i t h 3 oz of 0.15% hydroprene or a p p r o x i m a t e l y 0.23 pg/c s i m i l a r fashion with 3 o aquarium was l e f t u n t r e a t e d . A f t e r f o g g i n g , the aquariums w i t h the f o g g i n g r e s i d u e s were removed t o a c l e a n room and m a i n t a i n e d at ambient temperature (18-24°C) and 30-60% RH. Food and water were s u p p l i e d r o u t i n e l y t o a l l t h r e e chambers. S i n c e r e p r o d u c t i o n of a d u l t c o c k r o a c h e s was a l r e a d y known to be u n a f f e c t e d by j u v e n o i d type compounds a p p l i e d i n t h a t s t a g e , the original 10 a d u l t s were removed f r o m each aquarium on day 65, l e a v i n g an e s t a b l i s h e d F^ t a r g e t p o p u l a t i o n . Total population counts were made p e r i o d i c a l l y over a s i x month p e r i o d . F i g u r e 1 shows the changes i n the t o t a l p o p u l a t i o n s i z e w i t h time. By the end of s i x months the hydroprene p o p u l a t i o n was r e d u c e d t o n e a r z e r o ; the few r e m a i n i n g c o c k r o a c h e s were non reproductive adults. The c o n t r o l p o p u l a t i o n i n c r e a s e d from 10 t o a p p r o x i m a t e l y 1000 d u r i n g the t e s t p e r i o d . 3

D a l l a s Chamber T e s t . I n F e b r u a r y of 1982, two 1000 f t chambers were s e t up as s i m u l a t e d k i t c h e n s i n Zoecon I n d u s t r i e s (Dallas, Texas). These chambers were s u p p l i e d w i t h ready made k i t c h e n c a b i n e t s w i t h u n f i n i s h e d plywood t o p s . The temperature was k e p t n e a r 27°C u s i n g a s m a l l space h e a t e r and a b u i l t i n a i r c o n d i t i o n e r as n e c e s s a r y and h u m i d i t y was m a i n t a i n e d a t 65%. The d i u r n a l c y c l e was s e t at 16 hrs l i g h t , 8 hrs d a r k . Food (Waynes Pro M i x dry dog f o o d ) , water and p a r t i t i o n e d h a r b o r a g e boxes were p l a c e d i n 6 l o c a t i o n s throughout the chambers. The chambers were i n f e s t e d w i t h a p p r o x i m a t e l y 500 Blattella germanica (mixed p o p u l a t i o n ) e a c h . These p o p u l a t i o n s were a l l o w e d to a c c l i m a t i z e and b u i l d up f o r about a month. Chamber A was t r e a t e d w i t h a 1.2% hydroprene f o g g e r a t the r a t e of 2 oz p e r 1000 ft. Chamber Β was t r e a t e d w i t h a b l a n k f o g g e r (no A . I . ) a t the r a t e of 2 oz per 1000 f t . or a p p r o x i m a t e l y 0.62 pg/cm . Both chambers were m o n i t o r e d monthly by means of counts of cockroaches p r e s e n t i n the s i x h a r b o r a g e s . These c o c k r o a c h e s were r e t u r n e d t o the chamber a f t e r c o u n t i n g .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

212

BIOREGULATORS FOR PEST CONTROL

F i g u r e 2 shows t h a t the hydroprene t r e a t e d p o p u l a t i o n remained n e a r l y c o n s t a n t at about 1000 over the e n t i r e one y e a r d u r a t i o n of the t e s t whereas the u n t r e a t e d p o p u l a t i o n i n c r e a s e d t o over 60,000 c o c k r o a c h e s , which c o n s t i t u t e s 98.5% c o n t r o l by comparison. The s t a b l e p o p u l a t i o n i n the t r e a t e d chamber s u g g e s t s t h a t progeny of the original hydroprene population were indeed unable to reproduce. I n c o m p l e t e l y i s o l a t e d chambers the p o p u l a t i o n c o u l d have been e x p e c t e d t o d i s a p p e a r by a t t r i t i o n i n a p p r o x i m a t e l y 6 months. The population r e m a i n i n g i n the hydroprene chamber p r o b a b l y r e f l e c t e d a c o n t i n u i n g low l e v e l of r e i n f e s t a t i p n w i t h u n t r e a t e d c o c k r o a c h e s because the chambers appeared t o be not f u l l y cockroachproof· Purdue University Kitchen Test. I n the f a l l of 1982, E.S. Runstrom, a r e s e a r c h a s s o c i a t e working w i t h Dr. G.W. Bennett of Purdue U n i v e r s i t y (West L a f a y e t t e , I n d i a n a ) , i n i t i a t e d t e s t s of hydroprene a g a i n s t B l a t t e l l a germanica i n f o u r i m i t a t i o n k i t c h e n s s e t up i n a vacant campu s i m u l a t e the h o u s e h o l d b u i l t out of wood, and measured ΙΟ'χβ'χό'. Each chamber was made escape p r o o f and c o n t a i n e d a base c a b i n e t w i t h two doors under the s i n k , two s i d e u n i t s , each w i t h one drawer and one door, and two w a l l mounted c a b i n e t s above the base u n i t s . L i g h t s were i n s t a l l e d i n each chamber and were programmed f o r a 12:12 photophase. Two chambers s e r v e d as u n t r e a t e d c o n t r o l s , w h i l e two were t r e a t e d w i t h hydroprene at the r a t e of 5.95 ml of hydroprene 5E ( 6 5 % A . I . ) p e r g a l l o n of water a p p l i e d at 1 g a l l o n p e r 1000 f t . amounting t o approximately 4.4 pg/cm · Food and water were supplied continuously. Each chamber was i n f e s t e d b e f o r e t r e a t m e n t w i t h a p o p u l a t i o n of 750 B l a t t e l l a germanica c o n s i s t i n g of 250 nymphs of mixed age, 250 a d u l t males, and 250 a d u l t f e m a l e s . P o p u l a t i o n s were sampled monthly w i t h m o n i t o r i n g t r a p s made f r o m baby f o o d j a r s b a i t e d w i t h b r e a d soaked i n beer. These escape p r o o f j a r s were p l a c e d i n the chambers f o r a p e r i o d of 24 hours once per month. Trapped c o c k r o a c h e s were counted as male, female, g r a v i d female, a d u l t s showing j u v e n o i d e f f e c t s , l a r g e and s m a l l nymphs. S i n c e the u l t i m a t e g o a l was t o reduce the p o p u l a t i o n t o z e r o t h r o u g h the s t e r i l i z i n g e f f e c t of hydroprene, o n l y the monthly t o t a l t r a p counts are shown. The d a t a p o i n t s i n f i g u r e 3 r e p r e s e n t the combined t o t a l t r a p counts of two hydroprene t r e a t e d chambers and of two u n t r e a t e d ones. The s t r i k i n g d i v e r g e n c e i n p o p u l a t i o n l e v e l s began a t the s i x t h month, which i s the time when u n a f f e c t e d c o c k r o a c h e s , which were a d u l t s at the time of t r e a t m e n t , s t a r t e d t o die out. By month 10 the t r e a t e d p o p u l a t i o n s were near z e r o and t h o s e r e m a i n i n g were o n l y s t e r i l e a d u l t s . The r e s u l t s o b t a i n e d i n t h e s e chamber and aquarium t e s t s clearly c o n f i r m e d the p o t e n t i a l of hydroprene as a c o c k r o a c h c o n t r o l agent w i t h consumer o r i e n t e d a p p l i c a t i o n methods. Field

Experiments

D u r i n g 1981-83 f i e l d experiments were conducted i n s i n g l e f a m i l y r e s i d e n c e s and i n apartment complexes i n s e v e r a l l o c a t i o n s i n the USA i n compliance w i t h E x p e r i m e n t a l Use P e r m i t s (E.U.P.'s) f r o m the

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

STAAL ET AL.

213

Cockroach Control with Juvenoids

# ROACHES

10000 τ

• UNTREATED Ο HETHOPRENE Δ HYDROPRENE 6 MONTHS

1 F i g u r e 1. Aquarium t e s t s D a l l a s (1981) Population development over 6 months i n a q u a r i a exposed t o f o g g i n g h y d r o p r e n e and methoprene.

with

# ROACHES • UNTREATED Ο HYDROPRENE

10000 h

1000 h

100 h

MONTHS

F i g u r e 2. Chamber t e s t s D a l l a s ( 1 9 8 2 ) . Population development over 12 months a f t e r hydroprene a p p l i c a t i o n .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

214

BIOREGULATORS FOR PEST CONTROL

US E n v i r o n m e n t a l P r o t e c t i o n Agency (E.P.A.)· In a l l cases, apartment complexes were selected that harbored a sizable p o p u l a t i o n of B l a t t e l l a germanica and i n which f u l l c o o p e r a t i o n of owner and t e n a n t s f o r a l e n g t h y t r e a t m e n t and o b s e r v a t i o n s c h e d u l e c o u l d be s e c u r e d . U n i f o r m treatments were a p p l i e d i n a l l u n i t s of a complex (4-6 u n i t s ) . The treatments were g e n e r a l l y 0.6 % or 1.2% hydroprene total release aerosol foggers, o f t e n combined with simultaneous spray treatment of cracks and c r e v i c e s w i t h a conventional i n s e c t i c i d e . The g r e a t v a r i e t y of treatment s c h e d u l e s and v a r i a t i o n s between l o c a t i o n s e t c make a comprehensive d i s c u s s i o n of the r e s u l t s w i t h i n the scope of t h i s paper i m p o s s i b l e . However, the f o l l o w i n g g e n e r a l c o n c l u s i o n s c o u l d be drawn: 1.

2.

3.

4.

F u l l e r a d i c a t i o n of c o c k r o a c h p o p u l a t i o n s i n s i n g l e f a m i l y r e s i d e n c e s w i t h hydroprene f o g g e r s a l o n e o r i n c o m b i n a t i o n w i t h conventional insecticides has been consistently achieved (Figure 4). Simultaneous insecticid substantially t o t h e e a r l y demise o f c o c k r o a c h populations (29). F u l l s u c c e s s i n apartment complexes i s e q u a l l y f e a s i b l e but i s o f t e n d e l a y e d by compliance f a i l u r e s , t e n a n t s moving i n and out, e t c . Retreatment s c h e d u l e s a t 4-6 months i n t e r v a l s u s u a l l y y i e l d t h e best r e s u l t s .

As an i l l u s t r a t i o n , population curves obtained i n single residences i n various locations (Dallas, Denver, C h i c a g o ) were depicted i n figure 4. The t r e a t m e n t s i n t h i s group of f i e l d experiments were a ) hydroprene 0.6 % f o g g e r s b) hydroprene 1.2% foggers plus dichlorvos/propoxur foggers c) dichlorvos/propoxur foggers only. The d i c h l o r v o s / p r o p o x u r t r e a t m e n t s were a p p l i e d a t t w i c e the u s u a l treatment r a t e i n both b) and c ) . The r e s u l t s c l e a r l y i n d i c a t e d t h a t hydroprene e i t h e r a l o n e or i n c o m b i n a t i o n w i t h i n s e c t i c i d e s p r o v i d e d a more e f f e c t i v e and l o n g e r l a s t i n g c o n t r o l than the c o n v e n t i o n a l i n s e c t i c i d e s by t h e m s e l v e s . The c o m b i n a t i o n treatment appears t o y i e l d s u p e r i o r c o n t r o l a t l e a s t initially. Discussion Hydroprene has proven t o be the j u v e n o i d of c h o i c e f o r the c o n t r o l of the major domestic c o c k r o a c h s p e c i e s . I t s s u c c e s s appears t o be due t o i t s very h i g h i n t r i n s i c a c t i v i t y as compared w i t h most o t h e r j u v e n o i d s and p r o b a b l y a l s o t o i t s v o l a t i l i t y which may a l l o w f o r a p e n e t r a t i o n of vapor i n i n a c c e s s i b l e c o c k r o a c h h a r b o r a g e s . It i s likely t h a t hydroprene vapors readily t r a n s l o c a t e between many h o u s e h o l d s u r f a c e m a t e r i a l s and thus remain a c c e s s i b l e t o r e s i d e n t c o c k r o a c h p o p u l a t i o n s . T h i s v o l a t i l i t y of hydroprene c o u l d a l s o be a l i a b i l i t y f o r p e r s i s t e n c e i n places with high a i r displacement. In t h i s case, hydroprene f o r m u l a t e d f o r slow r e l e a s e i n a dust (RF 10%) c o u l d p r o v i d e a very p e r s i s t e n t r e s i d u e , a l b e i t w i t h o u t the vapor b e n e f i t s .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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F i g u r e 3. Purdue U n i v e r s i t y k i t c h e n t e s t ( E . S . Runstrom and G.W. B e n n e t t 1982). P o p u l a t i o n development over 11 months a f t e r hydroprene a p p l i c a t i o n . PERCENT CONTROL

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F i g u r e 4. F i e l d t e s t s i n s i n g l e family residences with h y d r o p r e n e and d i c h l o r v o s / p r o p o x u r f o g g e r s . Population development over 7 months a f t e r a p p l i c a t i o n .

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Methoprene, a compound already registered for many a p p l i c a t i o n s , can be used f o r c o c k r o a c h c o n t r o l but at h i g h e r r a t e s than for hydroprene and only if formulated for optimal e f f e c t i v e n e s s , such as i n the RF 10% f o r m u l a t i o n . Many d i f f e r e n t e f f e c t s have been d e s c r i b e d as a r e s u l t of j u v e n o i d treatment on c o c k r o a c h e s , p r i m a r i l y B l a t t e l l a g e r m a n i c a . I n none of the t e s t s we undertook have we seen a s i g n i f i c a n t m o r t a l i t y a t t r i b u t a b l e to e i t h e r a d i r e c t e f f e c t of the compounds t e s t e d , (13, 14, 23), an i n a b i l i t y t o molt p r o p e r l y , or a f a i l u r e t o molt (5. 8, 11). On the c o n t r a r y , many of the m o r p h o g e n e t i c a l l y a f f e c t e d a d u l t s or a d u l t o i d s appeared t o s u f f e r no i l l e f f e c t f r o m the i n d i g n i t i e s i n f l i c t e d by the j u v e n o i d s , o t h e r t h a n f a i l u r e t o reproduce. Although the treatment methods may be partly responsible for differences between the results of d i f f e r e n t a u t h o r s , i t i s more l i k e l y t h a t q u a l i t a t i v e d i f f e r e n c e s between types of j u v e n o i d s are p a r t l y r e s p o n s i b l e . Delays i n the final molt were f r e q u e n t l y o b s e r v e d i n batches a f f e c t e d by juvenoids. Most, i f not a l l , of thes non r e p r o d u c i n g , adultoids The s e n s i t i v e p e r i o d f o r morphogenetic and s t e r i l i t y i n d u c t i o n was c l e a r l y l i m i t e d t o the l a s t nymphal i n s t a r , e x c l u d i n g the l a s t 10 days b e f o r e the molt. ( I f batches of l a s t i n s t a r nymphs of mixed age are exposed t o hydroprene, a d u l t s m o l t i n g i n the f i r s t t e n days a f t e r the treatment appear t o be m o r p h o g e n e t i c a l l y as w e l l as reproductively unaffected). Treatment of a d u l t s themselves was a l s o without consequences, a l t h o u g h a temporary r e d u c t i o n in r e p r o d u c t i o n r a t e was observed on s e v e r a l o c c a s i o n s . However, i t remains possible that such treatment does not affect total r e p r o d u c t i v e c a p a c i t y of the f e m a l e s . The l i t e r a t u r e on e f f e c t s on the males i s very s c a n t . However, t h e r e are i n d i c a t i o n s t h a t males may a l s o be a f f e c t e d and r e p r o d u c t i v e l y i n c a p a c i t a t e d . Whether t h i s would take p l a c e at the same dose l e v e l as t h a t f o r the f e m a l e s , remains t o be e s t a b l i s h e d . The t r u e n a t u r e of the reduced fertility effect has never been established with certainty. However, s i n c e o n l y exposure d u r i n g the s e n s i t i v e p e r i o d i n the l a s t nymphal i n s t a r i n d u c e s permanent s t e r i l i t y , t h e r e must be a l i n k w i t h metamorphosis. In our t r i a l s we established a link between external metamorphic inhibition and sterility for hydroprene i n the sense t h a t a d u l t o i d s ( c h a r a c t e r i z e d by deformed or crumpled w i n g s ) were always s t e r i l e . However, females c o u l d sometimes be non reproductive w h i l e not visibly affected. Non f e r t i l e females d i d produce oothecae c o n t a i n i n g no eggs and h a v i n g a s h r i v e l e d appearance. Yet, on d i s s e c t i o n , h y p e r t r o p h i e d ovaria were o f t e n f o u n d . The opposite effect, atrophied ovaria, was d e s c r i b e d by Das & Gupta ( 3 ) . In one of our p r e l i m i n a r y trials, f e m a l e s kept s e p a r a t e d f r o m males as a d u l t s , d i d produce e q u a l l y a f f e c t e d and s t e r i l e o o t h e c a e . I t i s t h e r e f o r e not p o s s i b l e t o s t a t e t h a t the o b s e r v e d i n f e r t i l i t y c o u l d not r e s u l t from a f a i l u r e to mate, e i t h e r because of m o r p h o l o g i c a l or b e h a v i o r a l inadequacy or the absence of c h e m i c a l s i g n a l s . C r u i c k s h a n k & Palme re (23). observed that exposure t o c e r t a i n amide j u v e n o i d s caused the premature dropping of oothecae. This is likely t o be an i r r i t a t i o n response, s i n c e i t i s a l s o o b s e r v e d as a r e s u l t f r o m exposure t o many o t h e r c o n v e n t i o n a l i n s e c t i c i d e s .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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We have been unable t o i n t e r p r e t Edwards f i n d i n g t h a t exposure t o e a r l i e r l a r v a l i n s t a r s i n t e n s i f i e s the response t o methoprene (28). Other a u t h o r s , working w i t h more a c t i v e j u v e n o i d s u s u a l l y r e p o r t e d i n c r e a s i n g s e n s i t i v i t y d u r i n g the l a s t nymphal i n s t a r s (10). S i n c e methoprene i s only m a r g i n a l l y a c t i v e at 1000 ppm and m o r t a l i t y was p r e v a l e n t i n Edwards' t r e a t m e n t s , a c o n f i r m a t i o n i s much needed. I t i s very obvious t h a t more r e s e a r c h i n t o the n a t u r e of the i n f e r t i l i t y i n d u c e d by j u v e n o i d s i n g e n e r a l i s much overdue. The v o l a t i l i t y of hydroprene was e a s i l y e s t a b l i s h e d , but i t was a s u r p r i s e t h a t a c t i v i t y c o u l d be e a s i l y c o n t a i n e d f o r l o n g p e r i o d s i n cages w i t h a s c r e e n t o p . Our r e s u l t s suggest t h a t hydroprene i s m i g r a t i n g between the v a r i o u s s u b s t r a t e s i n these cages without significant loss. Cockroaches appear t o be e a s i l y a f f e c t e d by such r e s i d u e s e i t h e r by c o n t a c t or perhaps a l s o by vapor. Volatile residues may also increase the efficacy by penetrating into i n a c c e s s i b l e h a r b o r a g e s , thus i n c r e a s i n g the c o c k r o a c h e s exposure d u r i n g the i n a c t i v e ( d a y t i m e ) p e r i o d s The v o l a t i l i t y was almost completely repressed i w h i c h proved t o be als r e l a t i v e e f f i c a c y of v a r i o u s f o r m u l a t i o n s i n h o u s e h o l d h a b i t a t s has not y e t been e s t a b l i s h e d , but i t appears t h a t compounds such as hydroprene can be t a i l o r e d t o the r e q u i r e m e n t s of p r a c t i c a l l y any cockroach h a b i t a t . 1

Literature 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17.

cited

Bell, W.J.; Barth, R.H. J. Insect Physiol. 1970, 16, 2303-13. Das, Y.T. PH.D. Thesis, Rutgers University, 1975. Das, Y.T.; Gupta, A.P. Experientia 1974, 30, 1093-5. Das, Y.T.: Gupta, A.P. Experientia 1977, 33, 968-70. Hangartner, W.; Masner P. Experientia 1973, 19, 1358-9. Kunkel, J.P. J. Insect Physiol. 1973, 19, 1285-97. Masner, P.; Hangartner, W.; Suchy, M. J. Insect Physiol. 1975, 21, 1755-62. Masner, P.; Dorn, S.; Vogel, W., Kahn, M.; Graf, O.; Gunthart, E. In: Scientific Papers of the Institute of Organic and Physical Chemistry of Wroclaw Technical University, 1981, 22, 809-818. Pincus, D.S. J. Insect Physiol. 1977, 23, 73-7. Roberts, B. Austr. J. Zool., 1977, 25, 233-41. Vogel, W.; Masner, P.; Graf, O.; Dorn, S. Experientia 1979, 35, 1254-6. Radwan, W.; Sehnal, F. Experientia 1974, 30, 615-8. Cruickshank, P.Α.; Mitt. Schweiz. Entomol. Ges. 1971, 44, 98-133. Riddiford, L.M.; Ajami, A.M; Boake, Chr. J. Econ. Ent. 1975, 68, 46-8. Cristodorescu, G.; Nosec, I.; Tacu, V. Arch. Roum. Path. Exp. Microbiol. 1978, 37, 47-53. Cornwell P.B. The Cockroach, vol I, Hutchinson, London 1968, p. 48. Chamberlain, W.F.; Becker, J.D. The Southwestern Entomologist 1977, 2, 179-182.

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Clark, G.N. Chemical Times and Trends 1982, 5, 52-5. Cochran, D.G. Pest Control, 1982, 50, 16-20. Staal, G.B. In: Annual Review of Entomology 1975, 20, 417-460. Edwards, J.P.; Menn, J.J. In: Chemie der Pflanzenschutz und Schadlingsbekampfungmittel. Springer Verlag New York 1981, 185-214. Henrick, C.A. In Insecticide Mode of Action; Academic Press Inc., New York, 1982, Chapt. 11, pp 315-401. Cruickshank, Ph.A.; Palmere R.M. Nature 1971, 233-488-g. Henrick, C.A.; Willy, W.E.; Staal, G.B. Agr. Food Chem. 1976, 24, 207-18. Henrick, C.A. US Patent 4,021,461, 1977. Pallos, F.M.; Menn, J.J.; Letchworth, P.E.; Miaullis, J.B. Nature (London) 1971, 232, 486Schwartz, M.; Miller, R.W.; Wright, J.E.; Chamberlain, W.F.; Hopkins, D.E. J. Econ. Entomol. 1974, 67, 598 Edwards, J.P. Proc 267-75. Bilbie, I.; Nicolescu, G. Arch. Roum. Path. Exp. Microbiol. 1981, 40, 173-5.

RECEIVED November 23, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

14 Some Chemical Ecological Approaches to the Control of Stored-Product Insects and Mites IZURU YAMAMOTO Tokyo University of Agriculture, Setaga-ku, Tokyo 156, Japan

Three novel approaches to the control of insects are described and evaluated. of behavior. Control of mating. type pheromone found present in Callosobruchus chinensis, which is not a conventional sex attractant, but rather induces the male to extrude his genital organ and to effect copulation. A female dummy bearing the pheromone mimic, elicits copulation and ejaculation by the male. Control of oviposition. C. chinensis and C. maculatus develop a strategy to reduce competition among larvae by first marking the beans and then elimi­ nating the excess eggs, using the same marker at accumulated doses. The marking pheromone is a mix­ ture of lipids, and precoating the beans with certain edible oils kills the deposited eggs and prevents the injury. Control of feeding. Many bean weevils Callosobruchus spp. deposit eggs on kidney beans, but the penetrated larvae do not develop. An unidentified fraction of the bean was found as the growth inhibitor, but it is not lectin. I f a crop i s c o m p l e t e l y p r o t e c t e d from p e s t s , i t s y i e l d may be expected t o double. T h i s g o a l has been e a r n e s t l y pursued i n t h e f i e l d t o improve crop p r o d u c t i o n . L a t e l y , t h e need t o p l a c e more emphasis on p r o t e c t i o n from i n s e c t s d u r i n g p o s t - h a r v e s t f o o d s t o r a g e has been r e c o g n i z e d . There a r e s e v e r a l reasons why t h i s a r e a has been n e g l e c t e d : f i r s t , i n s u f f i c i e n t r e s e a r c h has been conducted; second, because l o s s e v a l u a t i o n i s d i f f i c u l t and s e e m i n g l y no problem e x i s t s w i t h the modern p r o d u c t i o n t e c h n o l o g y , t h e r e i s l i t t l e i n c e n t i v e ; t h i r d , t h e r e i s an assumption t h a t food s t o r a g e i s u n n e c e s s a r y under c o n d i t i o n s where t h e r e i s no f o o d surplus. We a r e more o r l e s s accustomed t o handle s t o r a g e problems w i t h our advanced t e c h n o l o g y , b u t i n d e v e l o p i n g c o u n t r i e s , p o s t -

0097-6156/ 85/0276-0219$06.00/ 0 © 1985 American Chemical Society

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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h a r v e s t l o s s e s a r e about 30%, thus n u l l i f y i n g much o f the e f f o r t invested i n f i e l d production strategies. The major r e a s o n f o r t h e s e l o s s e s i s i n s e c t p e s t s . U n t i l the p r e s e n t , p e s t i c i d e s have p l a y e d a major r o l e i n p e s t c o n t r o l . However, t h e i r n e g a t i v e e n v i r o n m e n t a l impact i s b e i n g s e v e r e l y c r i t i c i z e d , making the con­ c e p t o f i n t e g r a t e d p e s t management r e l a t i v e l y more a t t r a c t i v e . D i f f e r e n t c o n t r o l methods have been e x p l o r e d i n s t o r a g e i n s e c t management, but examples of a t r u l y i n t e g r a t e d approach a r e few and t h e assessment o f the p r a c t i c a l i t y of any of t h e s e new approaches has not been completed. Considering p e s t i c i d e s for storage insect c o n t r o l , p a r t i c u l a r l y f u m i g a n t s , the use of some of the e x i s t i n g ones i s d e c r e a s i n g because of problems of t o x i c i t y , r e s i d u e s and resistance. The development of new p e s t c o n t r o l c h e m i c a l s based on new c o n c e p t s i s a d e s i r a b l e g o a l and may be r e w a r d i n g . The emerging c o u n t r i e s i n the t r o p i c s , h a v i n g a f a v o r a b l e c l i m a t i c con­ d i t i o n f o r p e s t emergence, do not have t h e economic r e s o u r c e s t o p r o v i d e f o r p r o p e r s t o r a g e space and/or f u m i g a t i o n . T h e r e f o r e , a simp1er t e c h n o l o g y w h i c needed. P e s t Management w i t h

Ecochemicals

The p r e s e n t c o n c e p t s o f p e s t management have e v o l v e d from b a s i c knowledge o f the c h e m i c a l i n t e r a c t i o n s between organisms. C h e m i c a l s d i r e c t l y i n v o l v e d i n the e c o l o g i c a l r e l a t i o n s h i p between organisms a r e c a l l e d e c o c h e m i c a l s , ecomones, o r s e m i o c h e m i c a l s ; t h o s e a c t i n g between d i f f e r e n t s p e c i e s a r e c a l l e d kairomones, a l l o mones, o r a l l e l o c h e m i c s , w h i l e t h o s e a c t i n g w i t h i n the same s p e c i e s a r e c a l l e d pheromones. R e s e a r c h on e c o c h e m i c a l s i s most advanced i n the case of i n s e c t s , thus p r o v i d i n g the o p p o r t u n i t y f o r i t s a p p l i c a t i o n t h r o u g h p e s t management. Through e v o l u t i o n a r y p r o c e s s e s , i n s e c t s have a c q u i r e d h a b i t s w h i c h a r e c o n d i t i o n e d by pheromones and/or a l l o m o nes. The r e a c t i o n s t o t h e s e s u b s t a n c e s by i n s e c t s d i f f e r from t h a t t o p o i s o n o u s i n s e c t i c i d e s i n t h a t i t i n v o l v e s a normal and s p e c i f i c response. In g e n e r a l , e c o c h e m i c a l s have been found t o be e f f e c t i v e a t v e r y low l e v e l s because t h e i r d i r e c t e f f e c t i s m a g n i f i e d through behavioral responses. Moreover, t h e s e e c o c h e m i c a l s g e n e r a l l y have a s i m p l e c h e m i c a l s t r u c t u r e and o f t e n t h e i r a n a l o g s a r e a v a i l a b l e . The p r e s e n t emphasis o f u t i l i z i n g t h e s e c h e m i c a l s i s t o r e g u l a t e p e s t a c t i v i t i e s i n o r d e r t o p r e v e n t c r o p and f o o d i n j u r y r a t h e r t h a n t o k i l l p e s t s . In the remainder o f t h i s r e p o r t , r e s e a r c h con­ d u c t e d a t our l a b o r a t o r i e s w i l l be d e s c r i b e d and d i s c u s s e d . Control of Feeding

Behavior

I n s e c t p r e f e r e n c e s f o r c e r t a i n t y p e s of f o o d can be c o n s i d e r e d from a c h e m i c a l e c o l o g i c a l p o i n t o f view as f o l l o w s : p r e s e n c e of a t t r a c t a n t , f i x i n g f a c t o r o v i p o s i t i o n - s t i m u l a n t , and f e e d i n g s t i m u ­ l a n t ; absence of r e p e l l e n t , o v i p o s i t i o n d e t e r r e n t , f e e d i n g d e t e r r e n t , n u t r i t i o n a l d e f e c t , and g r o w t h - d e t e r r e n t . Conversely, t h e o p p o s i t e i s t r u e f o r c e r t a i n f o o d t y p e s u n d i s t u r b e d by i n s e c t s .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

14. YAM AMOTO

Control of Stored-Product Insects and Mites

A t t r a c t a n t s i n c e r e a l g r a i n s o f S i t o p h i l u s zeamais. J5. zeamais i s known as the r i c e w e e v i l i n Japan, but as the maize w e e v i l i n the U.S., w h i l e S^. o r y z a e i s the s m a l l r i c e w e e v i l i n Japan, but the r i c e w e e v i l i n the U.S. Both i n s e c t s cause i n j u r y to r i c e , maize and wheat, or t o t h e i r p r o c e s s e d p r o d u c t s , thus b e i n g the most i m p o r t a n t i n j u r i o u s i n s e c t s to c e r e a l s . T h e r e i s e v i d e n c e t h a t o l f a c t o r y s t i m u l u s i s i n v o l v e d i n the h o s t s e l e c t i o n . In warm a r e a s , the i n s e c t s m i g r a t e d u r i n g the h a r v e s t o f r i c e , maize or wheat i n t o the f i e l d s from the s t o r e - h o u s e s , t h e r e b y c a u s i n g p o s t harvest injury a f t e r harvest. We began t h i s s t u d y w i t h the o b j e c ­ t i v e of e l u c i d a t i n g chemical f a c t o r s i n g r a i n s i n v o l v e d i n h o s t - f i n d i n g b e h a v i o r of JS. zeamais. U s i n g o l f a c t o m e t e r s , the f o l l o w i n g samples, f r a c t i o n s , e x t r a c t s , or compounds were shown t o p o s s e s s a t t r a c t a n c y : 1. p o l i s h e d r i c e , brown r i c e , maize and wheat; 2. e t h e r , methanol, a c e t o n e or hexane e x t r a c t s o f the above samples, among which the e t h e r e x t r a c t showed the h i g h e s t a c t i v i t y ; 3 a c i d i c and n e u t r a l f r a c t i o n s of the e t h e r e x t r a c t t h e c o m p o s i t i o n o f the a t t r a c t i v r a t h e r than a s i m p l e n a t u r e . The a t t r a c t a n t s i n r i c e and maize g r a i n s were found t o be a m i x t u r e of C$ and Cj c a r b o x y l i c a c i d s , γl a c t o n e s of C3 h y d r o x y c a r b o x y l i c a c i d s , and p h e n y l e t h a n o l . Of t h e s e , h e x a n o i c a c i d and 2 - n o n e n - 4 - o l i d e p l a y the major r o l e . S i m i l a r a t t r a c t a n t s were a l s o found i n wheat. The a t t r a c t a n c y o f r e l a t e d c a r b o x y l i c a c i d s was i n v e s t i g a t e d e x t e n s i v e l y , however no o t h e r s were found t o be a t t r a c t i v e beyond the above-mentioned com­ pounds. As f o r the l a c t o n e s , s e v e r a l s y n t h e s i z e d compounds were found t o be a t t r a c t i v e . The above f o o d a t t r a c t a n t appears t o be i m p o r t a n t i n u n d e r s t a n d i n g the h o s t - f i n d i n g b e h a v i o r o f the r i c e weevil. S t u d i e s s h o u l d be conducted to d e t e r m i n e whether t h e r e i s a c o r r e l a t i o n between the c o n c e n t r a t i o n of a t t r a c t a n t s i n g r a i n s and o b s e r v e d s u s c e p t i b i l i t y . Then, b r e e d i n g f o r r e s i s t a n t v a r i e t i e s c o u l d be conducted on a r a t i o n a l b a s i s . A t t r a c t a n t i n Cheese o f Tyrophagus p u t r e s c e n t i a e . T. p u t r e s c e n t i a e ( f o r m e r l y T\ d i m i d i a t u s ) i s a g r a i n mite t h a t f e e d s on s t o r e d f o o d s t u f f s s u c h as d a i r y p r o d u c t s , g r a i n powders, c h o c o l a t e , s p i ­ c e s , soybean p a s t e and d r i e d f i s h . The m i t e has been found to p r e f e r cheese, and i s thus commonly c a l l e d the cheese m i t e . A q u e s t i o n a r i s e s as t o whether these f o o d s t u f f s have the same or d i f f e r e n t f a c t o r s t h a t g o v e r n h o s t - f i n d i n g by the m i t e . For t h i s , an o l f a c t o m e t e r b i o a s s a y was d e s i g n e d u s i n g cheddar cheese w h i c h i s e a s i l y a v a i l a b l e and p o s s e s s e s a s t r o n g a t t r a c t a n c y as the s o u r c e material. Upon f r e e z e d r y i n g the cheese, the a t t r a c t a n c y was condensed i n t h e c o l d t r a p w i t h the v o l a t i l e s and f u r t h e r c o n c e n t r a t e d i n t o a n e u t r a l f r a c t i o n , which was s e p a r a t e d i n t o 3 f r a c t i o n s by chroma­ t o g r a p h y . E a c h f r a c t i o n was assayed to d e t e r m i n e i t s a t t r a c t a n c y . F r a c t i o n I gave no a c t i v i t y ; f r . I I gave 1/6 of the s t a r t i n g a c t i ­ v i t y , and f r . I l l gave f a i n t a c t i v i t y , but when f r . I I was combined w i t h f r . I or I I I , the a t t r a c t a n c y i n c r e a s e d t o 1/2; e v i d e n c e o f s y n e r g i s m between f r a c t i o n s . By combining a l l o f the f r a c t i o n s , t h e o r i g i n a l a t t r a c t a n c y was r e s t o r e d . 8-Nonen-2-one o f f r . I I showed some a c t i v i t y , but o t h e r m e t h y l k e t o n e s i n the same f r a c t i o n

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were not a c t i v e . However, a m i x t u r e of 8-nonen-2-one w i t h heptan-2-one, octan-2-one and nonan-2-one showed s t r o n g a c t i v i t y , a s y n e r g i s t i c e f f e c t among m e t h y l k e t o n e s . F r . I l l c o n t a i n e d lower a l c o h o l s , but the a t t r a c t a n c y of f r . I l l appeared t o be due t o 3 - m e t h y l b u t a n o l a l o n e which f u r t h e r s y n e r g i z e d the a c t i v i t y o f the m i x t u r e of the above-mentioned m e t h y l k e t o n e s . M e t h y l k e t o n e s a r e p r e s e n t i n d a i r y p r o d u c t s , formed from t r i g l y c e r i d e s t h r o u g h $ - k e t o a c i d s . 3 - M e t h y l b u t a n o l o r i g i n a t e s from L - l e u c i n e and i s p r e s e n t i n v a r i o u s fermented p r o d u c t s . The p r e ­ sence of such common c o n s t i t u e n t s i s thought t o be i n v o l v e d i n h o s t f i n d i n g f o r d i f f e r e n t k i n d s of f o o d s t u f f s by the m i t e . Much l e s s r e s e a r c h has been done on f o o d a t t r a c t a n t s than on sex pheromones. More emphasis on the u n d e r s t a n d i n g of such a l l o m o nes and t h e i r p o t e n t i a l use f o r i n s e c t c o n t r o l i s needed. Growth i n h i b i t o r i n k i d n e y bean o f bean w e e v i l s . A l a r v a o f the a z u k i bean w e e v i l , C a l l o s o b r u c h u s c h i n e n s i s , grows i n s i d e the a z u k i bean and o t h e r beans, bu ney bean. I s h i i (1) ha growth i n h i b i t o r i n the bean, and o t h e r workers had attempted t o grow r e s i s t a n t bean p l a n t s by g r a f t i n g . J a n z e n e t a l . (2) c l a i m e d t h a t the cowpea w e e v i l , (Σ. macula t u s , i s not a b l e t o grow i n s i d e t h e k i d n e y bean due t o the a c t i o n of l e c t i n . We have attempted t o show t h e s e same r e l a t i o n s h i p s w i t h chinensis. The l e c t i n f r a c ­ t i o n , p u r i f i e d by a f f i n i t y chromatography, d i d not show any growth i n h i b i t o r y a c t i v i t y , however. A p o s s i b i l i t y t h a t a t r y p s i n i n h i b i ­ t o r i s r e s p o n s i b l e f o r the growth i n h i b i t i o n remains. Such a s t u d y i n c o m b i n a t i o n w i t h e f f o r t s t o breed beans f o r r e s i s t a n c e c o u l d c o n t r i b u t e t o the a t t a i n m e n t of a w e e v i l - f r e e bean. C o n t r o l of M a t i n g

Behavior

The use o f sex a t t r a c t a n t s seems p r o m i s i n g p a r t i c u l a r l y w i t h s t o r e d p r o d u c t i n s e c t s because t h e y must l i v e i n a r e s t r i c t e d space; t h i s has been w e l l documented by B u r k h o l d e r ( 4 ) . R e c e n t l y , a new type of sex pheromone was found from C^. c h i n e n s i s by u s . M a t i n g pheromones of C. c h i n e n s i s . Two sex pheromones a r e i n v o l v e d i n t h e m a t i n g b e h a v i o r o f C^. c h i n e n s i s : a female sex a t t r a c t a n t , and a c o p u l a t i o n r e l e a s e pheromone. The former a t t r a c t s the male t o the female, but does not i n d u c e f u r t h e r responses. The l a t t e r i n d u c e s the male t o e x t r u d e h i s g e n i t a l o r g a n and t o attempt c o p u l a t i o n , and thus i s named " e r e c t i n " ( 3 ) . E r e c t i n i s r e l e a s e d by b o t h the male and the female (more by the f e m a l e ) , but o n l y the male responds t o i t . The e v i d e n c e f o r c o p u l a t i o n r e l e a s e a c t i v i t y has been demonstrated w i t h s e v e r a l i n s e c t s : Costelytra zealandica; Trogoderma glabrum; L i m o n i u s canus; T r i b o l i u m confusum; T e n e b r i o m o l i t o r , and C a l l o s o b r u c h u s m a c u l a t u s ( F . ) ( 4 ) . However, e r e c t i n of c h i n e n s i s was the f i r s t to be i d e n t i f i e d , and i t i s i n v o l v e d only i n copulation release a c t i v i t y . C h e m i c a l l y , e r e c t i n c o n s i s t s of two s y n e r g i s t i c a l l y a c t i n g f r a c t i o n s , n e i t h e r h a v i n g any a c t i v i t y . One i s a m i x t u r e of C26-C35 h y d r o c a r b o n s and the o t h e r i s a d i c a r b o x y l i c a c i d , named

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YAMAMOTO

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"callosobruchusic a c i d " , (E)-3,7-dimethyl-2-octene-l,8-dioic acid. (5). I f e r e c t i n can be made a v a i l a b l e i n s u f f i c i e n t q u a n t i t i e s f o r p r a c t i c a l use, and i f the c o p u l a t i o n of males w i t h female dummies b e a r i n g e r e c t i n can e f f e c t the l o w e r i n g of the p o p u l a t i o n d e n s i t y , t h i s a p p a r e n t r e v e r s a l of the s t e r i l i z e d male t e c h n i q u e c o u l d become a new c o n t r o l a p p r o a c h . The f i r s t r e q u i s i t e f o r p r a c t i c a l use was a c h i e v e d by the s y n t h e s i s of c a l l o s o b r u c h u s i c a c i d , and the s u b s t i t u t i o n of a complex h y d r o c a r b o n m i x t u r e by o c t a d e c a n e . Only the (E) form was active. S y n t h e s i s of the two o p t i c a l forms was a l s o a c h i e v e d , but b o t h forms showed the same a c t i v i t y as the n a t u r a l c a l l o s o b r u c h u s i c a c i d , thus making i t i m p o s s i b l e t o a s s i g n the a b s o l u t e con­ f i g u r a t i o n ( 5 ) . The second r e q u i r e m e n t has been pursued by s e l e c t i n g d i f f e r e n t dummies. When an amount of e r e c t i n e q u i v a l e n t t o t h a t of one female was a p p l i e d t o a g l a s s r o d , the male a t t e m p t e d c o p u l a t i o n , but d i d not e j a c u l a t e However the male d i d show the i n s e r t i o n and e j a c u l a t i o num f o i l tube b e a r i n g e r e c t i n C o n t r o l of O v i p o s i t i o n B e h a v i o r Examples of the c o n t r o l o f o v i p o s i t i o n b e h a v i o r w i t h c h e m i c a l s have been demonstrated i n t h i s l a b o r a t o r y w i t h an o v i p o s i t i o n s t i m u l a n t for c h i n e n s i s and m a c u l a t u s and an o v i p o s i t i o n r e g u l a t o r . O v i p o s i t i o n s t i m u l a n t from bean seed c o a t . O v i p o s i t i o n on k i d n e y , cowpea and a z u k i beans by JC. c h i n e n s i s and _C. m a c u l a t u s i s stimu­ l a t e d by a t l e a s t two f a c t o r s . T h i s was shown u s i n g g l a s s beads of d i f f e r e n t s i z e s t r e a t e d w i t h the e x t r a c t of the bean seed c o a t s as the o v i p o s i t i o n s u b s t r a t e . jC. m a c u l a t u s o v i p o s i t e d o n l y when a c h e m i c a l s t i m u l a n t was p r o v i d e d , whereas c h i n e n s i s r e q u i r e d ade­ q u a t e p h y s i c a l s t i m u l i s u c h as s i z e , and the c h e m i c a l s t i m u l a n t played only a secondary r o l e . I s o l a t i o n o f the c h e m i c a l (5) i s i n progress. Such an o v i p o s i t i o n s t i m u l a n t w i t h a s u i t a b l e s u b s t r a t e c o u l d m o d i f y the o v i p o s i t i o n b e h a v i o r of t h e s e p e s t i n s e c t s . O v i p o s i t i o n r e g u l a t o r of C. c h i n e n s i s and C. m a c u l a t u s . There a r e two prominent phenomena t h a t govern the o v i p o s i t i o n b e h a v i o r o f the two w e e v i l s . F i r s t , under low d e n s i t y c o n d i t i o n s , f e m a l e s o v i p o s i t e v e n l y among beans. Second, under h i g h d e n s i t y c o n d i t i o n s where e a c h bean h o l d s many eggs, the egg d i s t r i b u t i o n becomes random and o n l y a few eggs h a t c h , l e a v i n g the r e s t t o d i e . The bean s u r f a c e becomes more s h i n y as o v i p o s i t i o n p r o g r e s s e s . An e t h e r - s o l u b l e s u b s t a n c e which i m p a r t s t h i s s h i n i n e s s was shown t o have marking activity. The w e e v i l p r e f e r s l e s s e r marked beans f o r f u r t h e r o v i ­ p o s i t i o n , thus r e s u l t i n g i n an even d i s t r i b u t i o n of the eggs. Moreover, as t h i s s u b s t a n c e i n c r e a s e s t o a l e v e l of 100 pg/bean, c a u s i n g many eggs t o be d e p o s i t e d , the s u b s t a n c e shows o v i c i d a l a c t i o n , thus e l i m i n a t i n g most of the eggs e x c e p t t h o s e o v i p o s i t e d i n e a r l i e r s t a g e s t h a t had a l r e a d y h a t c h e d and p e n e t r a t e d i n t o the beans. These i n s e c t s thus have d e v e l o p e d a s t r a t e g y t o reduce com­ p e t i t i o n among l a r v a e , and t o m a x i m a l l y u t i l i z e the h o s t beans by u s i n g the same s u b s t a n c e a t d i f f e r e n t l e v e l s .

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This pheromone consists of t r i g l y c e r i d e s , f a t t y acids, and hydrocarbons, and each has some marking and o v i c i d a l a c t i v i t y . The f a t t y acid composition of the t r i g l y c e r i d e s i s similar to that of edible o i l s , and treatment of the azuki bean with certain edible o i l s (200 pg/bean) could protect the beans from injury by maculatus, chinensis, and Zabrotes subfaciatus. Conclusion As i l l u s t r a t e d , new approaches to pest control are being developed that have the p o t e n t i a l i t y to lessen i n j u r i e s by lowering the insect population without direct k i l l . One future d i r e c t i o n may well be that of i d e n t i f y i n g ecochemicals of natural o r i g i n as a forerunner to the development of synthetic " p e s t i s t a t i c s " which mimic the function of the former. The methods of crop and food protection w i l l c e r t a i n l y change with the transformation of s o c i a l consciousness and with progress of science and technology. Along with this trend, the for inevitably be altered. Literature 1. 2. 3. 4. 5.

Cited

Ishii, S. Bull. Natl. Inst. Agric. Sci. Ser. C., No. 1. 1952, 185. Janzen, D. H.; Juster, H. B.; Liener, I. E. Science 1976, 192, 795. Tanaka, K.; Ohsawa, K.; Honda, H.; Yamamota, I. J. Pesticide Sci. 1981, 6, 75. Yun-Tai Qi; Burkholder, W. E. J. Chem. Ecol. 1982, 8, 527. Mori, K.; Ito, T.; Tanaka, K.; Honda, H.; Yamamoto, I. Tetrahedron 1983, 39, 2303.

RECEIVED

December 14, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

15 Phytochemical Disruption of Insect Development and Behavior WILLIAM S. BOWERS Department of Entomology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456

Plants deploy attack by insects. toxic actions against insects are well known and have often served as prototypic models for the development of optimized analogs subsequently reduced to commercial practice. Less well understood and investigated are those secondary chemicals targeted to more subtle disruption of pest specific biology. These include mimics of important developmental hormones and chemicals of communicative significance.

C o m p e t i t i v e s p e c i e s l i v i n g t o g e t h e r i n a r e s t r i c t e d space even­ t u a l l y seek t o e x c l u d e o r prey upon each o t h e r . P l a n t s and i n s e c t s a r e prime examples o f c o e x i s t i n g s p e c i e s l o c k e d i n a n e t e r n a l s t r u g g l e f o r space and s u s t e n a n c e . P l a n t s , a s the u l t i m a t e c o l l e c t o r s o f s o l a r energy and t h e v e g e t a t i v e dominants, a r e w e l l matched a g a i n s t i n s e c t s , which a r e the p r i n c i p a l animate l i f e forms. I n s e c t s a r e so s u c c e s s f u l because o f t h e i r m o b i l i t y , h i g h reproductive p o t e n t i a l , a b i l i t y t o e x p l o i t plants a s a food r e s o u r c e , and t o occupy so many e c o l o g i c a l n i c h e s . Plants are e s s e n t i a l l y s e s s i l e and can be seen t o produce f l o w e r s , n e c t o r , p o l l e n , and a v a r i e t y o f c h e m i c a l a t t r a c t a n t s t o i n d u c e i n s e c t cooperation i n c r o s s - p o l l i n a t i o n . However, i n o r d e r t o reduce t h e e f f i c i e n c y o f i n s e c t prédation upon them, p l a n t s a l s o produce a h o s t o f s t r u c t u r a l , m e c h a n i c a l , and c h e m i c a l d e f e n s i v e a r t i f i c e s . The most v i s i b l e c h e m i c a l d e f e n s e s a r e p o i s o n s , b u t c e r t a i n chemi­ c a l s , not i n t r i n s i c a l l y t o x i c , a r e t a r g e t e d t o d i s r u p t s p e c i f i c c o n t r o l systems i n i n s e c t s t h a t r e g u l a t e d i s c r e t e a s p e c t s o f i n s e c t p h y s i o l o g y , b i o c h e m i s t r y , and b e h a v i o r . Hormones and pheromones a r e unique r e g u l a t o r s o f i n s e c t growth, development, r e p r o d u c t i o n , d i a p a u s e , and b e h a v i o r . P l a n t secondary c h e m i c a l s f o c u s e d o n t h e d i s r u p t i o n o f i n s e c t e n d o c r i n e and pheromone mediated p r o c e s s e s c a n be v i s u a l i z e d a s i m p o r t a n t components o f p l a n t d e f e n s i v e mechanisms.

0097-6156/85/0276-0225$06.00/0 © 1985 American Chemical Society

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Notwithstanding the continuing dynamics of evolutionary processes, certain plant secondary chemicals employed as defensive strategies against pests and pathogens can serve as prototypic models leading to new chemical control agents and/or resources f o r the genetic engineering of pest r e s i s t a n t crop c u l t i v a r s . Various plant derived toxicants have provided the i n i t i a l chemistry leading to successful commercial i n s e c t i c i d e s including the pyrethroids and carbamates. Less well explored are those plant chemical defenses targeted to more subtle interruption of i n s e c t - s p e c i f i c aspects of development and behavior.

Insect Phytohormpnes Phvtojuvenoids. Wigglesworth (JL) demonstrated that a hormone secreted by the insect corpora a l l a t a was responsible f o r the control of d i f f e r e n t i a t i o n i n immature insects and reproduction i n adult female insects. Williams (£) prepared an active extract of t h i s hormone from adult hormone". We were able chemistry of the juvenile hormone from the study of the active cecropia extract to synthesize JH I I I (3.). Seven years l a t e r i t s presence as a natural hormone i n the tobacco hornworm was confirmed (JL). Three other analogous juvenile hormones (JH 0, I, II) have been found to occur only i n lepidoptera (ϋ, L, X) (Figure 1). Juvenile hormone III i s the p r i n c i p a l juvenile hormone of insects and has been demonstrated i n a l l of the insect taxa investigated. In 1965 Slama and Williams (£) observed that contact with certain paper products disrupted the metamorphosis of the linden bug. The b i o l o g i c a l a c t i v i t y was i d e n t i c a l i n every respect to that produced by the insect juvenile hormone. Thus the ultimate nymphal stages were induced to molt into nymphal-adult i n t e r ­ mediates or supernumerary nymphs which eventually died without completing metamorphosis. They demonstrated that the active agent(s) was present i n the balsam f i r tree from which the paper had been manufactured. Bowers et a l . ( and t h e s t r u c t u r e proposed by K a r l s o n e t a l . (2k) was c o n f i r m e d w i t h a f u l l e l u c i d a t i o n o f s t r u c t u r e and a b s o l u t e c o n f i g u r a t i o n by Huber and Hoppe (25.) t h r o u g h X-ray a n a l y s i s . A second hormone was c h a r a c t e r i z e d a s 20-hydroxyecdysone by s e v e r a l l a b o r a t o r i e s i n c l u d i n g Hampshire and Horn (2L) > Hocks and W i e c h e r t ( 2 1 ) , and K a p l a n i s e t a l metabolites with s i g n i f i c a n subsequently i d e n t i f i e d (Figure 2 ) . In 1966 N a k a n i s h i i e t a l . (21) i d e n t i f i e d p o n a s t e r o n e A, a p o l y h y d r o x y s t e r o i d a l component o f t h e p l a n t Podocarpus n a k a i i ( F i g u r e 3 ) . R e c o g n i z i n g i t s s i m i l a r i t y t o t h e i n s e c t ecdysones he was a b l e t o c o n f i r m t h a t i t p o s s e s s e d a u t h e n t i c m o l t i n g hormone activity. L a t e r , t h i s p h y t o e c d y s t e r o i d was d i s c o v e r e d t o e x i s t a s a n a t u r a l ecdysone o f s e v e r a l C r u s t a c e a (30.)· S i m i l a r l y the p h y t o e c d y s t e r o i d i n o k o s t e r o n e ( F i g u r e 3) was shown t o o c c u r i n t h e c r u s t a c e a n C a l l i n e o t e s s a p i d u s ( 3 1 ) . The d i s c o v e r y o f p l a n t s t e r o i d s w i t h m o l t i n g hormone a c t i v i t y s t i m u l a t e d a w i d e s p r e a d s e a r c h among p l a n t s f o r s i m i l a r hormone mimics. R e c e n t l y , 111 p l a n t f a m i l i e s have been shown t o c o n t a i n a t l e a s t 69 p h y t o e c d y s t e r o i d s w i t h m o l t i n g hormone a c t i v i t y (32)· I t i s e s p e c i a l l y noteworthy t h a t t h e n a t u r a l i n s e c t m o l t i n g hormone, 20hydroxy ecdysone, i s t h e most commonly e n c o u n t e r e d hormone i n t h e major p l a n t d i v i s i o n s P t e r i d o p h y t a , Gymnospermae, and Angiospermae. I n d e s c e n d i n g o r d e r the most abundant e c d y s t e r o i d s a r e ponasterone A, p o l y p o d i n e B, ecdysone, and p t e r o s t e r o n e (32)· Ponasterone A, a n a t u r a l phytoecdysone, i s r e p o r t e d t o have the h i g h e s t hormonal a c t i v i t y i n i n s e c t s and t o show t h e g r e a t e s t a f f i n i t y f o r e c d y s t e r o i d r e c e p t o r s (33.). E c d y s t e r o i d s and a n a l o g s i n c o r p o r a t e d i n t o a r t i f i c i a l d i e t s a r e shown t o d e t e r f e e d i n g i n many i n s e c t s (3k) a s w e l l a s t o i n h i b i t growth and r e p r o d u c t i o n (21=30.)· G a l b r a i t h and Horn (39.) were t h e f i r s t t o suggest t h a t the p h y t o e c d y s t e r o i d s m i g h t c o n s t i t u t e a p l a n t d e f e n s i v e s t r a t e g y . T h i s t h e s i s has been echoed by numerous i n v e s t i g a t o r s and i s r e i n ­ f o r c e d by t h e i r c l e a r b i o l o g i c a l e f f e c t s on a wide v a r i e t y o f insects. I n view o f t h e w i d e s p r e a d o c c u r r e n c e o f p h y t o e c d y s o n e s and t h e i r acknowledged i n t e r f e r e n c e w i t h i n s e c t m o l t i n g and metomorphosis, t h e i r r o l e a s p l a n t d e f e n s i v e components seems assured. A n t i - J u v e n i l e Hormones. A p l a n t d e f e n s i v e s t r a t e g y t a r g e t e d t o d i s r u p t i o n o f the endocrine r e g u l a t i o n o f the e a r l y l a r v a l stages o f metamorphosis was r e v e a l e d w i t h t h e d i s c o v e r y o f a n t i - j u v e n i l e

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST CONTROL

JH

III

JH

/kj/N/^/N/^s/^o'

PRINCIPLE JH OF INSECTS

II

^SPECIALTY J H ' S OF JH I

LEPIDOPTERA

JH 0

F i g u r e 1.

The j u v e n i l e hormones o f i n s e c t s .

ECDYSONE

20-HYDROXYECDYSONE

20,26-DiHYDROXYECDYSONE

24-METHYL-20-HYDROXYECDYSONE

Η Ο 2-DEOXYECDYSONE

F i g u r e 2.

The i n s e c t m o l t i n g hormones, "ecdysones".

OH

OH

INOKOSTERONE

F i g u r e 3. R e p r e s e n t a t i v e phytoecdysones a l s o found i n invertebrates.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Phytochemical Disruption of Insects

15. BOWERS

Table I.

229

P h y t o c h e m i c a l s w i t h J u v e n i l e Hormone A c t i v i t y

PHYTOJUVENOID

OCCURRENCE

A/\A/SA/\)H

REFERENCE

Ubiquitous

Schmialek (10)

Farnesol

Ο Juvabione

/

0

?

s l a m a

Abie* bonanza

&

w

i

l

l

i

a

m

()

s

8

Bowers, et a l . (9) Cerny, et a l . (11)

Dehydrojuvabione

Cjv^O

Sesamin

Bowers (12, 13)

Snàamum Indicim

Sesamolin

L_>^

Ç^

Thujic acid

0H

ΝΛΑΛΑΛΛΛΛ,Ό η Sterculic

Barton, et a l . (14)

Tkuja ptiavta

StzKojuJUùx. faitiAa Thompson & Barlow (15) acid

0 /k/^s/S^s^

/

^

/ Λ ν /

^

/ Ν

|Γ^^

Tagetone

Tagvtu minuta

Ostruthin

JmpeAatoUa.

Saxena & Srivastava (16)

c

_

.

.

OH Abscisic

ΛΑ^^Α^

acid

E c h i nolone

Ubiquitous

EchuidCZaz

angu&Uéolia Bakuchiol

Juvocimene

PàonalzA

I

OcAMum ba&UÀam Juvicmene

Slama (18)

.

.

.

,

^cobson, et a l . (19) „.

.

,

/ x 9n

Bowers & Nishida (22)

II

UacAûpipeA zxczJUm Nishida, et a l . (21)

\y^\y^y\y& Me = Macrosiphum e u p h o r b i a e (Thomas), Mp » Myzus p e r s i c a e ( S u l z e r ) , Oa = Oncophanes americanus (Weed), Of = Oncopeltus f a s c i a t u s ( D a l l a s ) , Pf • P e d i o b i u s f o v e o l a t u s (Crawford), Pr = P i e r i s rapae ( L i n n . ) , Pv = P l a g i o d e r a v e r s i c o l o r a ( L a i c h a r t i n g ) , Sf = Spodoptera f r u g i p e r d a ( J . E . S m i t h ) , Tu = T e t r a n y c h u s u r t i c a e Koch, and Tv = T r i r h a b d a v i r g a t a ( L e C o n t e ) . Adapted w i t h p e r m i s s i o n from Ref 21. C o p y r i g h t 1984, E x p e r i e n t i a .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

274

BIOREGULATORS FOR PEST CONTROL

t e r i n g o f chewing h e r b i v o r e s i n the r e g i o n of b o t h h i g h t r a n s - E H and h i g h t r a n s / c i s r a t i o s , whereas entomophagous, s u c k i n g , and more s p e c i a l i z e d f e e d e r s group a t low t r a n s - E H and a t low r a t i o s of activities. Indeed, the 20 chewing h e r b i v o r o u s p e s t s of economic importance s u r v e y e d here had on the average a 3 0 - f o l d h i g h e r t r a n s EH and a 1 3 - f o l d h i g h e r EH r a t i o than the f o u r s u c k i n g h e r b i v o r e s ( T a b l e I I I ) , whereas the l a t t e r group and the b e n e f i c i a l a r t h r o p o d s had s i m i l a r EH p r o f i l e s . The spectrum of EH a c t i v i t i e s and r a t i o s were much l e s s v a r i a ­ b l e i n entomphagous r e l a t i v e t o phytophagous a r t h r o p o d s ( T a b l e I I , F i g u r e 2 ) , p r o b a b l y s i n c e d i e t a r y and endogenously formed e p o x i d e s would be more e q u i v a l e n t i n the f o o d of the l a t t e r . To f u r t h e r e x p l o r e the s i t u a t i o n of the h e r b i v o r e , gut EH l e v e l s from a d u l t l e a f f e e d i n g b e e t l e s of the Chrysomelidae were examined r e l a t i v e t o encounter w i t h p l a n t a l l e l o c h e m i c a l s as e s t i m a t e d by host p l a n t range ( F i g u r e 3 ) . R a t i o s of t r a n s / c i s EH c o r r e l a t e d w e l l (r>0.92) w i t h e i t h e r number o f p l a n t f a m i l i e s o r p l a n t g e n e r a consumed. This strongly implicate derived epoxides. S y n e r g i s t s of P l a n t T o x i c a n t s as S e l e c t i v e P e s t B i o r e g u l a t o r s The c l e a r a s s o c i a t i o n of a cytochrome P-450 e p o x i d a s e and t r a n s - E H w i t h h e r b i v o r y may have i m p o r t a n t consequences f o r i n t e g r a t e d p e s t management (IPM), s i n c e t h e s e enzymes w i l l d e t e r m i n e , i n p a r t , the a b i l i t y o f phytophagous a r t h r o p o d s t o be p e s t s . Epoxidase a c t i o n w i l l a c t i v a t e many p h y t o c h e m i c a l d e f e n s e s and thus be d i s a d v a n t a ­ geous t o the h e r b i v o r e u n l e s s a c o n c u r r e n t d e t o x i f i c a t i o n event such as EH i s a v a i l a b l e . Hence, the w e l l - d e v e l o p e d o l e f i n t o d i o l pathway of chewing crop p e s t s may be c o u n t e r e d by s e l e c t i v e i n h i b i ­ t i o n of t r a n s - E H . Potent i n v i t r o i n h i b i t o r s of t h i s enzyme a r e known f o r mammals a l t h o u g h they appear l e s s e f f i c a c i o u s f o r a r t h r o ­ pods ( 1 5 , 3 0 ) . The a p p r o p r i a t e s t r u c t u r a l m o d i f i c a t i o n of c h a l c o n e o r f l a v o n o i d d e r i v a t i v e s may l e a d to s e l e c t i v e h e r b i v o r e b i o r e g u l a ­ t o r s t h a t have l i t t l e impact on entomophagous a r t h r o p o d s important i n IPM. Perhaps the s i m i l a r e n z y m o l o g i e s of phloem-feeding aphids and c a r n i v o r e s ( T a b l e s I I , I I I ) may l i m i t use of i d e n t i c a l c h e m i c a l s t r a t e g i e s f o r aphid c o n t r o l unless e c o l o g i c a l s e l e c t i v i t y v i a s y s t e m i c a p p l i c a t i o n s i s implemented. I n h i b i t o r s of a h e r b i v o r e ' s a b i l i t y t o d e t o x i f y p l a n t t o x i ­ c a n t s may e x p e c t e d l y be more s l o w - a c t i n g than n e u r o t o x i c a n t s such as o r g a n o p h o s p h a t e s . N e v e r t h e l e s s , securance o f s y n e r g i s t s of d i e t a r y t o x i c a n t s t h a t complement a c t i v a t i o n r e a c t i o n s and i n h i b i t d e t o x i f i c a t i o n r e a c t i o n s i n the t a r g e t e d p e s t s w i l l have p o t e n t i a l f o r IPM, s i n c e they s h o u l d be c o m p a t i b l e or moreover, augment p l a n t a n t i b i o s i s , n a t u r a l enemies and c o n c u r r e n t l y used p e s t i c i d e s . R e l e v a n c e of D e t o x i f i c a t i o n D i s s i m i l a r i t i e s

to In V i v o T o x i c o s i s

R e c e n t l y much work has been devoted i n understanding the a n t i ­ j u v e n i l e hormone a c t i o n o f p l a n t chromenes from Ageratum spp. (10, 31,32). The a l l a t o c i d a l a c t i v i t y o f precocenes i s a p p a r e n t l y due t o a b a l a n c e of d e c r e a s e d d e t o x i f i c a t i o n i n p e r i p h e r a l t i s s u e s and

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

19.

MULLIN

0

Detoxification Enzymes in Arthropods

2

4

6

8

10

275

12

14

TRANS/CIS RATIO OF EPOXIDE HYDROLASE ACTIVITIES

F i g u r e 3. A s s o c i a t i o n o f t r a n s - e p o x i d e h y d r o l a s e herbivory. T r a n s / c i s r a t i o s of epoxide h y d r o l a s e adult chrysomelid beetles are p l o t t e d r e l a t i v e to p l a n t f a m i l i e s ( φ ) o r g e n e r a ( Δ ) found w i t h i n o f each s p e c i e s . See F i g u r e 2 f o r d e t a i l s .

with arthropod i n midguts o f number o f t h e h o s t range

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

276

BIOREGULATORS FOR PEST CONTROL

TABLE I I I .

Epoxide H y d r o l a s e i n P e s t R e l a t i v e t o A r t h r o p o d s o f Economic Importance

Beneficial

Trans cis

Economic Group

Trans

Cis

Chewing H e r b i v o r e s η = 20

20.7

a

3.0

a

10.2

a

Sucking H e r b i v o r e s η = 4

0.7

0.8

b

0.8

b

Bénéficiais η = 7

l

b

b

l

b

l

b

Measured i n gut p r e p a r a t i o n s except f o r a c a r i n e s , a p h i d s p a r a s i t o i d s , where whole body p r e p a r a t i o n s u s e d . Groups same l e t t e r a r e not s i g n i f i c a n t l

and with

enhanced o x i d a t i v e b i o a c t i v a t i o n t o a c y t o t o x i c e p o x i d e i n the corpora a l l a t a (CA). I t i s i n t e r e s t i n g t h a t most of the i n s e c t s s e n s i t i v e t o p r e c o c e n e , and thus expected t o e x h i b i t enhanced epox­ i d a s e i n the CA but d e c r e a s e d p e r i p h e r a l d e t o x i f i c a t i o n t o d i o l s , g l u c o s i d e s , e t c . , a r e s u c k i n g s p e c i e s and n o t h o l o m e t a b o l o u s chew­ ing herbivores (10,32). I n h i b i t o r s of a p p r o p r i a t e d e t o x i f i c a t i o n enzymes s h o u l d s y n e r g i z e p r e c o c e n e a c t i o n . A CA e p o x i d a s e perhaps i d e n t i c a l t o the p r e c o c e n e e p o x i d a s e b i o s y n t h e s i z e s i n s e c t j u v e n i l e hormones (JH) from the analogous i n a c t i v e o l e f i n i c p r e c u r s o r , and the enzyme a c t i v i t y appears h i g h e r i n p r e c o c e n e - s e n s i t i v e s p e c i e s (32) · Subsequent d e t o x i f i c a t i o n o f JH o c c u r s p r i m a r i l y by EHs and e s t e r a s e s i n p e r i p h e r a l t i s s u e s , and p r e l i m i n a r y i n f o r m a t i o n does not i n d i c a t e major d i f f e r e n c e s f o r JH d e g r a d a t i o n r o u t e s between chewing and s u c k i n g h e r b i v o r e s , o r i n ­ sect c a r n i v o r e s (33,34). More s t u d y o f the r o l e o f d e t o x i f i c a t i o n i n r e g u l a t i n g the a c t i o n o f JH i n t a r g e t t i s s u e s i s r e q u i r e d . E s t e r a s e s f o r t r a n s - p y r e t h r o i d s are well-developed i n p i e r c ­ i n g - s u c k i n g h e r b i v o r e s s u c h as the milkweed bug (35) and greenpeach a p h i d ( 3 6 ) , and may e x p l a i n , i n p a r t , the g e n e r a l h i g h e r t o l e r a n c e o f these organisms over chewing h e r b i v o r e s f o r p y r e t h roids. C a r n i v o r o u s l a c e w i n g l a r v a e , moreover, a r e l e s s s u s c e p i b l e than h e r b i v o r e s to c i s - i s o m e r s of p y r e t h r o i d s such as d e l t a m e t h r i n and p e r m e t h r i n a p p a r e n t l y s i n c e they r e t a i n e s t e r a s e s t h a t d e t o x i f y the c i s - i s o m e r s f a s t e r than the l e s s i n s e c t i c i d a l trans-isomers (37). O t h e r enzyme-feeding g u i l d a s s o c i a t i o n s may be i m p o r t a n t to i n s e c t c o n t r o l . 3 - g l u c o s i d a s e s a r e more a c t i v e i n h e r b i v o r e s , p a r ­ ticularly the p i e r c i n g - s u c k i n g t y p e s , than c a r n i v o r e s , and are p r o b a b l y r e s p o n s i b l e f o r the s e l e c t i v e a c t i v a t i o n o f g l u c o s i d i c juvenogens i n some h e m i p t e r a n i n s e c t s ( 3 8 , 3 9 ) . In t h i s r e g a r d , i t i s i n t e r e s t i n g that g l u c o s i d e formation f o r precocene metabolites appears t o be r e t a r d e d i n s u c k i n g r e l a t i v e t o chewing h e r b i v o r e s ( 4 0 ) , and s u g g e s t s the involvement o f 3 - g l u c o s i d a s e s . In c o n t r a s t , g l u t a t h i o n e t r a n s f e r a s e s f o r a r y l h a l i d e s appear t o be h i g h e r i n chewing r e l a t i v e t o s u c k i n g h e r b i v o r e s (41)·

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

19.

MULLIN

Detoxification Enzymes in Arthropods

211

In summary, r e s u l t s o f i n v e s t i g a t i o n s on comparative d e t o x i f i ­ c a t i o n i n a r t h r o p o d s may h e l p e x p l a i n the f r e q u e n t i n c r e a s e s i n s u c k i n g p e s t s when c o n t r o l measures f o r the major chewing p e s t s a r e s u c c e s s f u l , and s h o u l d a i d the more e f f e c t i v e use of s e l e c t i v e c h e m i s t r y i n the management of crop p e s t s . Indeed, even t o x i c a n t s e n s i t i v i t y a t nerve t a r g e t s i t e s may be p a r t l y r e g u l a t e d by d e t o x ­ i f i c a t i o n such as i s i n d i c a t e d f o r d i e l d r i n (42) and p y r e t h r o i d s (43). C e r t a i n l y , i n h i b i t i o n of d e t o x i f i c a t i o n a c t i v i t i e s e s s e n t i a l t o an o r g a n i s m i s a v i a b l e s t r a t e g y f o r b i o r a t i o n a l control (44,45); i n d e e d the h i g h l y e f f e c t i v e a c t i o n of organophosphates and c a r b a m a t e s i n a n i m a l s i s b a s e d on i m p e d i m e n t o f a c e t y l c h o l i n e d e t o x i f i c a t i o n a t the nerve t a r g e t . Acknowledgments The t e c h n i c a l a i d of Ε . M. Reeves and R. J . S t e i g h n e r , and the s u p p o r t of the N a t i o n a l S c i e n c e F o u n d a t i o n ( g r a n t BSR-8306008) a r e much a p p r e c i a t e d . Thi A g r i c . Exp. S t n . j o u r n a

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Saito, T. Residue Rev. 1969, 25, 175-186 Weiden, M. H. J. Bull. Wld. Hlth. Org. 1971, 44, 203-213. Hollingworth, R. M. In "Insecticide Biochemistry and Physiology"; Wilkinson, C. F., Ed.; Plenum: New York, 1976; pp. 431-506. Croft, Β. Α.; Brown, A. W. A. Ann. Rev. Entomol. 1975, 20, 285-335. Kulkarni, A. P.; Hodgson, E. Ann. Rev. Pharmacol. Toxicol. 1984, 24, 19-42. Casida, J. E. In "Pesticide Chemistry: Human Welfare and the Environment, Mode of Action, Metabolism and Toxicology"; Matsunaka, S.; Hutson, D. H.; Murphy, S. D., Eds.; Pergamon: New York, 1983; Vol. III, pp. 239-246. Conney, A. H. Cancer Res. 1982, 42, 4875-4917. Manson, M. M. Br. J. Ind. Med. 1980, 37, 317-336. Brooks, G. T. Gen. Pharmac. 1977, 8, 221-226. Bowers, W. S. Ent. Exp. Appl. 1982, 31, 3-14. Brattsten, L. B. In "Plant Resistance to Insects"; Hedin, P. Α., Ed.; ACS SYMPOSIUM SERIES No. 208, American Chemical Society: Washington, D. C., 1983; pp. 173-195. Dowd, P. F.; Smith, C. M.; Sparks, T. C. Insect Biochem. 1983, 13, 453-468. Wilkinson, C. F. In "Xenobiotic Metabolism: In Vitro Meth­ ods"; Paulson, G. D.; Frear, D. S.; Marks, E. P., Eds.; ACS SYMPOSIUM SERIES No. 97, American Chemical Society: Washing­ ton, D.C., 1979; pp. 249-284. Croft, Β. Α.; Mullin, C. A. Environ. Entomol. 1984, 13, in press. Mullin, C. Α.; Matsumura, F.; Croft, B. A. Comp. Biochem. Physiol. 1984, 79C, 85-92.

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33.

34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

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McKey, D. In "Herbivores - Their Interaction with Secondary Plant Metabolites"; Rosenthal, G. Α.; Janzen, D. H., Eds.; Academic: New York, 1979; pp. 55-133. Waller, G. R.; Nowacki, Ε. K. "Alkaloid Biology and Metabo­ lism in Plants"; Plenum: New York, 1978; pp. 121-141. Giaquinta, R. T., this volume. Chamberlain, K.; Burrell, M. M.; Butcher, D. N.; White, J. C. Pestic. Sci. 1984, 15, 1-8. Duffey, S. S. Ann. Rev. Entomol. 1980, 25, 447-477. Mullin, C. Α.; Croft, B. A. Experientia 1984, 40, 176-178. Cross, A. D. Quart. Rev., Chem. Soc. Lond. 1960, 14, 317-335. Kolattukudy, P. E. Science 1980, 208, 990-1000. Manitto, P. "Biosynthesis of Natural Products"; Ellis Horwood Ltd.: Chichester, England, 1981; pp. 287-429. Bell, Ε. Α.; Charlwood, Β. V. "Secondary Plant Products"; Springer-Verlag: New York, 1980. Harwood, J. L. In "The Biochemistry of Plants"; Stumpf, P. K., Ed.; Academic: Tuntiwachwuttikul, P.; Reutrakul, V. Phytochemistry 1981, 20, 1164-1165. Yu, S. J. Pestic. Biochem. Physiol. 1983, 19, 330-336. Mullin, C. Α.; Croft, Β. Α.; Strickler, K.; Matsumura, F.; Miller, J. R. Science 1982, 217, 1270-1272. Mullin, C. Α.; Hammock, B. D. Arch. Biochem. Biophys. 1982, 216, 423-439. Bowers, W. S., this volume. Pratt, G. E. In "Natural Products for Innovative Pest Manage­ ment"; Whitehead, D. L.; Bowers, W. S., Eds.; Pergamon: New York, 1983; pp. 323-355. Hammock, B. D.; Quistad, G. B. In "Progress in Pesticide Biochemistry"; Hutson, D. H.; Roberts, T. R., Eds.; Wiley: New York, 1981; Vol. I, pp. 1-83. Ajami, A. M.; Riddiford, L. M. J. Insect Physiol. 1973, 19, 635-645. Jao, L. T.; Casida, J. E. Pestic. Biochem. Physiol. 1974, 4, 465-472. Devonshire, A. L.; Moores, G. D. Pestic. Biochem. Physiol. 1982, 18, 235-246. Casida, J. E.; Gammon, D. W.; Glickman, A. H.; Lawrence, L. J. Ann. Rev. Pharmacol. Toxicol. 1983, 23, 413-438. Robinson, D. Biochem. J. 1956, 63, 39-44. Slama, K.; Wimmer, Z.; Romanuk, M. Hoppe-Seyler's Ζ. Physiol. Chem. 1978, 359, 1407-1412. Bergot, B. J.; Judy, K. J.; Schooley, D. Α.; Tsai, L. W. Pestic. Biochem. Physiol. 1980, 13, 95-104. Cohen, A. J.; Smith, J. N.; Turbert, H. Biochem. J. 1964, 90, 457-464. Brooks, G. T. In "Insect Neurobiology and Pesticide Action"; Society of Chemical Industry: London, 1980; pp. 41-55. Soderlund, D. M.; Hessney, C. W.; Helmuth, D. W. Pestic. Biochem. Physiol. 1983, 20, 161-168. Hedin, P. A. J. Agric. Food Chem. 1982, 30, 201-215. Menn, J. J.; Henrick, C. A. Phil. Trans. R. Soc. Lond. 1981, 295B, 57-71.

RECEIVED November 8, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

20 δ-Endotoxin of Bacillus thuringiensis var. israelensis Broad-Spectrum Toxicity and Neural Response Elicited in Mice and Insects R. MICHAEL A. RANDALL

1

2

ROE , PETER Y. K. CHEUNG , ALFORD

2

BRUCE D. HAMMOCK ,

2

DAN BUSTER , and

3

1

Department of Entomology, North Carolina State University, Raleigh, NC 27695-7613 Department of Entomology, University of California, Davis, CA 95616 Department of Entomology, University of Maine, Orono, ME 04469

2

3

The alkaline-dissolved Bacillus thuringiensis israelensis (BTI) δ-endotoxin when introduced by injection was biologically active against a wide spectrum of host animals including insects from four orders and mice. The LD for dissolved BTI δ-endotoxin in mice was 1.31 PPM and in Trichoplusia ni (Lepidoptera: Noctuidae) 3.71 PPM. Neuromuscular effects like heart cessation, lost coordination, tremor, and paralysis were observed in test animals. Using the appearance of lactate dehydrogenase in insect hemolymph post-injection as a cytosolic marker, we found that dissolved BTI δ-endotoxin was cytotoxic. In vivo recordings of activity in the ventral nerve cord post-injection indicated that dissolved BTI δ-endotoxin at the T. ni LD elicited hyperexcitability and then nerve death as was also the case for the organophosphate, methamidophos. The cytotoxin phospholipase-A when injected at its LD elicited no neural response. BTI poisoning was also temperature dependent while BTI cytotoxicity was not. Proteins at 24, 27, 35, 49 and 68K daltons were resolved from the dissolved BTI δ-endotoxin. These were introduced in various combinations by injection and ingestion into mice and insects and compared to the alkaline-dissolved Bacillus thuringiensis kurstaki δ-endotoxin. 50

50

2

50

W i t h i n t h e sporangium o f t h e b a c t e r i u m B a c i l l u s t h u r i n g i e n s i s (BT) i s s y n t h e s i z e d a p a r a s p o r a l , p r o t e i n a c e o u s c r y s t a l ( 1 - 2 ) t h a t has found w i d e s p r e a d use as a b i o l o g i c a l c o n t r o l agent ( 3 ) . T h i s c r y s t a l i s commonly r e f e r r e d t o as t h e "δ-endotoxin" as s u g g e s t e d by Heimpel ( 4 ) . The taxonomy o f BT i s based on t h e s e r o l o g y o f t h e f l a g e l l a r Η a n t i g e n 0 5 ) , and 29 s u b s p e c i e s and 26 s e r o t y p e s have been i d e n t i f i e d (6). The δ-endotoxins from t h e

0097-6156/85/0276-0279$06.00/0 © 1985 American Chemical Society In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR

280

PEST CONTROL

m a j o r i t y o f the s e r o t y p e s a r e t o x i c when i n g e s t e d by more than 182 s p e c i e s o f i n s e c t s , p a r t i c u l a r l y i n the e c o n o m i c a l l y i m p o r t a n t o r d e r , L e p i d o p t e r a (3). The m a j o r i t y o f the r e s e a r c h has c e n t e r e d on B a c i l l u s t h u r i n g i e n s i s s u b s p e c i e s k u r s t a k i (BTK) because o f i t s l a r v i c i d a l a c t i v i t y a g a i n s t major a g r i c u l t u r a l p e s t s i n the o r d e r L e p i d o p t e r a . The s e r o t y p e H14, B a c i l l u s t h u r i n g i e n s i s s u b s p e c i e s i s r a e l e n s i s ( B T I ) , d i f f e r s from the o t h e r s e r o t y p e s , however, by b e i n g h i g h l y t o x i c to members o f the i n s e c t o r d e r D i p t e r a (7-8) and y e t has l i t t l e known t o x i c i t y t o Lepidoptera. The p r o s p e c t o f employing BTI t o c o n t r o l m o s q u i t o e s , b l a c k f l i e s , or o t h e r m e d i c a l l y important i n s e c t p e s t s has s t i m u l a t e d g r e a t i n t e r e s t i n e l u c i d a t i n g the m o l e c u l a r b a s i s f o r i t s mode o f a c t i o n (_3). A l l o f the BT δ-endotoxins are a l s o o f s p e c i a l i n t e r e s t because they appear to be h i g h l y s e l e c t i v e a g a i n s t i n s e c t s and seem t o pose no h e a l t h r i s k s to humans or livestock. The δ-endotoxin o f ingestio b l a r v a l Lepidopter is quickly activated b ( 9 - 1 1 ) ; gut e p i t h e l i a l c e l l c e l l s s e p a r a t e from the basement membrane and each o t h e r u l t i m a t e l y d i s r u p t i n g the gut-hemocoel b a r r i e r (12-15). Similar o b s e r v a t i o n s i n mosquito l a r v a e f e d BTI (1_6) l e d to the g e n e r a l a c c e p t a n c e t h a t the BT i n s e c t i c i d a l a c t i v i t y was d i r e c t e d a g a i n s t , i f not r e s t r i c t e d to the gut e p i t h e l i u m o f the h o s t (12-13). An i n i t i a l symptom o f BTK p o i s o n i n g i s gut p a r a l y s i s (17) and i t was h y p o t h e s i z e d t h a t an i n c r e a s e i n the hemolymph pH from the leakage o f a l k a l i n e gut c o n t e n t s (17) or the i n f l u x o f K i n t o the hemocoel caused t h i s p a r a l y s i s ( 1 8 - 1 9 ) . These s t u d i e s l e d to the d i s c o v e r y t h a t BTK d i g e s t s a p p l i e d to the v e n t r a l nerve c o r d o f the c o c k r o a c h , P e r i p l a n e t a americana ( O r t h o p t e r a : B l a t t i d a e ) caused e x c i t a t i o n and then nerve b l o c k a g e (20-21 ) which appeared to be p r e s y n a p t i c i n o r i g i n (20). Other s t u d i e s have shown t h a t the a l k a l i n e - d i s s o l v e d BTI δ-endotoxin i s c y t o t o x i c to a number o f d i f f e r e n t c e l l l i n e s from i n s e c t s and mammals (3,22-24) and has a h i g h a f f i n i t y f o r s p e c i f i c p h o s p h o l i p i d s i n the plasma membrane (25). Thus, the o b j e c t i v e o f t h i s s t u d y i s to a s s e s s the t o x i c i t y o f a l k a l i n e - d i s s o l v e d BTI i n t r o d u c e d i n t o i n s e c t s and mice by f e e d i n g and i n j e c t i o n and t o a s s e s s the r o l e o f c y t o t o x i c i t y and n e u r o t o x i c i t y i n m o r t a l i t y when d i s s o l v e d δ-endotoxin i s i n j e c t e d i n t o i n s e c t s . +

SDS-PAGE A n a l y s i s

o f BTK

and

BTI

δ-Endotoxin

BTK and BTI s t r a i n IFC-1 were p r o v i d e d by Biochem P r o d u c t s - US D i v i s i o n ( S a l s b u r y Labs., I n c . ) . BTI was a l s o i s o l a t e d from a commercial p r e p a r a t i o n p r o v i d e d by Sandoz Inc., c u l t u r e d on GYS medium ( 2 6 ) . BTK and BTI t o x i n was p r e p a r e d i n an analogous manner. Spores and c r y s t a l s were s e p a r a t e d from c e l l d e b r i s by r e p e a t e d washing w i t h water and c e n t r i f u g a t i o n . BTI c r y s t a l s were s u b s e q u e n t l y s e p a r a t e d from spores by R e n o g r a f i n d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (27_) and BTK c r y s t a l s by d i s c o n t i n u o u s sucrose gradient c e n t r i f u g a t i o n Ο ) . C r y s t a l s were then d i s s o l v e d by i n c u b a t i o n f o r 3 h i n 0.5% Na2C03 (pH 11.0) and d i a l y z e d i n t o 0.025 M sodium phosphate (pH 8.0) f o r s t o r a g e a t

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

20.

ROE ET AL.

Effects of δ-Endotoxin in Mice and Insects

-60°C. BTI (Sandoz) was further p u r i f i e d by DEAE-Cellulose (DE-52, Whatman, 30 ml) i n 0.025 M Tris-HCL (pH 8.00), eluted with a 10 h, 100 ml, 0.0-0.5 M NaCl linear gradient; by acid p r e c i p i t a t i o n where the DEAE elutant was dialyzed into 0.05 M sodium acetate (pH 4.5) and the percipitate removed by centrifugation; and by Sephadex G-75 super fine (Pharmacia) gel permeation chromatography (95 χ 1.3 cm i . d . column) i n 0.025 M sodium phosphate (pH 8.0). Alkaline-dissolved and p a r t i a l l y p u r i f i e d δ-endotoxin was analyzed by 12.5% SDS-PAGE (2j8), stained with Coomassie b r i l l i a n t blue (Figure 1). Incubation of BTK and BTI crystals i n 0.5% Na2C03 (pH 11.0) s o l u b i l i z e d a number of protein components (Figure 1). For BTK, there were a number of proteins at 64K daltons and higher (Track 1). The standard procedure of washing BTK crystals with 1 M NaCl before s o l u b i l i z a t i o n removes endogenous proteinases and results in an enriched 130K dalton protein as the predominant component. This procedure had l i t t l e effect on the immunoreactivity of the s o l u b i l i z e d c r y s t a l or i t (Sandoz, Track 2) there daltons. Of the lower molecular weight Sandoz BTI components, the 27K proteins were the prédominent component i n the Salsbury BTI (Track 8). Differences i n the protein p r o f i l e of the ό-endotoxin from different BT v a r i e t i e s have been reported previously (11,30-31). A l l a l k a l i n e - s o l u b i l i z e d 6-endotoxin of BTI (Sandoz) adsorbed to DE-52 and eluted i n one peak (Track 3) with an apparent concentration of the 68K component. The acid precipitate (Track 4) was enriched with the 35K component which was r e - s o l u b i l i z e d only at high pH. Because of i t s limited s o l u b i l i t y , the acid precipitate could not be bioassayed i n later studies. The soluble f r a c t i o n was enriched with the 24K and 27Κ· components (Track 5). Gel permeation chromatography enriched the 27 and 24K proteins (Tracks 6 and 7, r e s p e c t i v e l y ) . 6-Endotoxin Toxicity i n Mice and Insects BTI and BTK alkaline-dissolved and p a r t i a l l y p u r i f i e d δ-endotoxins were injected and/or fed to insects of 6 orders and to mice (Tables I and I I ) . The δ-endotoxin was injected i n 0.15 M NaCl, 0.05 M Na2HP04, and 0.02 M K H 2 P O 4 at pH 7.2 into the insect hemocoel or intraperitoneally into mice. In feeding experiments, the toxin was dissolved i n 5% sucrose and force-fed in 2 μΐ volumes, to Trichoplusia n i and Heliothis zea (Lepidoptera: Noctuidae). Aedes aegypti (Diptera: Culicidae) larvae fed for a standard incubation period i n water containing BT toxin preparations; adults were given r e c t a l injections (32). Mice were also given BT by gavage. Data collected were subjected to Probit analysis (33). The alkaline-dissolved 6-endotoxin of BTI (Sandoz, Track 2) was toxic by i n j e c t i o n to a l l animals tested except Tenebrio molitor (Coleoptera: Tenebrionidae) (Table I ) . Mice, Trichoplusia n i , and Periplaneta americana were the most sensitive, the L D 5 0 being 1.3, 3.7 and 4.4 PPM, respectively. The s u s c e p t i b i l i t y to BTI poisoning also varied s i g n i f i c a n t l y within a single insect family (the Noctuidae) with the L D 5 0

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F i g u r e 1. SDS-PAGE a n a l y s i s o f a l k a l i n e - d i s s o l v e d B a c i l l u s t h u r i n g i e n s i s s u b s p e c i e s k u r s t a k i (BTK) and i s r a e l e n s i s ( B T I ) δ-endotoxin a t 25 y g per t r a c k : ( 1 ) BTK δ-endotoxin from Biochem P r o d u c t s - US D i v i s i o n ( S a l s b u r y Labs., I n c . ) , (2) BTI δ-endotoxin from Sandoz I n c . , (3) BTI (Sandoz) δ-endotoxin p u r i f i e d by DEAE-anion exchange chromatography, ( 4 ) p e r c i p i t a t e formed a f t e r d i a l y s i s o f BTI (Sandoz) δ-endotoxin i n t o pH 4.5 sodium a c e t a t e b u f f e r , (5) s o l u b l e f r a c t i o n a f t e r d i a l y s i s o f BTI (Sandoz) δ-endotoxin i n t o pH 4.5 sodium a c e t a t e b u f f e r , (6) BTI (Sandoz) δ-endotoxin p u r i f i e d by Sephadex G-75 g e l f i l t r a t i o n chromatography a t Rf 1.35, (7) a t Rf 1.58, and (8) BTI s t r a i n IFC-1 δ-endotoxin from Biochem P r o d u c t s - US D i v i s i o n ( S a l s b u r y Labs., I n c . ) . S, m o l e c u l a r w e i g h t s as i n d i c a t e d X1000 f o r b o v i n e serum a l b u m i n (BSA), o v a l b u m i n (OA), t r y p s i n , and m y o g l o b i n . Reproduced w i t h p e r m i s s i o n from R e f . 29. C o p y r i g h t 1984, Academic P r e s s , I n c .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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ROE ET AL.

283

Effects of δ-Endotoxin in Mice and Insects

T a b l e I . I n j e c t e d t o x i c i t y o f a l k a l i n e - s o l u b i l i z e d BTI (Sandoz, T r a c k 2) and BTK ( S a l s b u r y , T r a c k 1) δ-endotoxin. Animal - A or L* Aedes a e g y p t i - A (Diptera: Culicidae) Musca domesticus - A ( D i p t e r a : Muscidae) Trichoplusia n i - L (Lepidoptera: Noctuidae) H e l i o t h i s zea - L (Lepidoptera: Noctuidae) Tenebrio m o l i t o r - L (Coleoptera: Tenebrionidae) Oncopeltus f a s c i a t u s - A (Hemiptera: L y g a e i d a e ) P e r i p l a n e t a american (Orthoptera: B l a t t i d a e Swiss-Webster Mice

*Adult +

24h BTI L D + 5 0

24h BTK L D + 5 0

11.6 + 2.2 (3.16) 10.9 + 2.2 (2.17) 3.71 + 0.32 (3.22) 73.6 + 3.0 (19.23) >100

>900

27.7 + 7.0 (1.96)

>300

1.31 + 0.23 (4.47)

>150 >130 >100 >100

>30

or Larva.

PPM o r mg/kg body weight + 1 S.D. w i t h analysis i n parenthesis.

slope of probit

r a n g i n g from 3.7 PPM f o r T. n i t o 73.6 PPM f o r H e l i o t h i s z e a . The b a s i s f o r t h i s d i f f e r e n c e i s unknown but these s p e c i e s d i f f e r e n c e s c o u l d be u s e f u l i n the e l u c i d a t i o n o f the mechanism for t o x i c i t y . BTI t o x i c i t y by i n j e c t i o n a l s o was n o t p e c u l i a r t o the Sandoz s t r a i n but was a l s o noted f o r the IFC-1 s t r a i n from S a l s b u r y ( T a b l e I I I ) . By c o n t r a s t the a l k a l i n e - d i s s o l v e d δ-endotoxin o f BTK ( T r a c k 1) showed no t o x i c i t y when i n j e c t e d i n t o the same s p e c i e s ( T a b l e I ) . Obvious fundamental d i f f e r e n c e s e x i s t between t h e d i s s o l v e d δ-endotoxin o f BTI and BTK. When the p u r i f i e d , p a r a s p o r a l c r y s t a l o f BTI (Sandoz) was f e d t o A. a e g y p t i l a r v a e , t h e L C 5 0 was 2.95 + 0.59 ng/ml. A l k a l i n e - s o l u b i l i z a t i o n d e c r e a s e d the t o x i c i t y s i g n i f i c a n t l y t o an L C 5 0 o f 2.29 + 0.06 yg/ml and r e c t a l i n j e c t i o n s i n a d u l t s produced a L D 5 0 o f 54.5 + 3.1 PPM ( T a b l e I I ) . These f i n d i n g s were c o n s i s t e n t w i t h p r e v i o u s work ( 3 4 ) . D i s s o l v e d BTI δ-endotoxin when f e d t o L e p i d o p t e r a and mice as e x p e c t e d ( 3 ) was not t o x i c ( T a b l e I I ) . The BTI IFC-1 s t r a i n was a l s o s i m i l a r t o the Sandoz s t r a i n i n t h a t b o t h were t o x i c when f e d t o A. a e g y p t i ( T a b l e I I I ) . BTK d i s s o l v e d δ-endotoxin when g i v e n o r a l l y was t o x i c o n l y t o t h e l e p i d o p t e r a n , T. n i ( T a b l e I I ) . The r e s u l t s from a l l o f t h e s e f e e d i n g experiments were c o n s i s t e n t w i t h p r e v i o u s r e p o r t s t h a t BTK when f e d t o l e p i d o p t e r a n s i s a c t i v e w h i l e BTI i s t o x i c t o o n l y c e r t a i n d i p t e r a n s ( 3 ) and i s s u p p o r t i v e e v i d e n c e t h a t the BTI and BTK p r e p a r a t i o n s used i n our s t u d i e s were s i m i l a r t o p r e p a r a t i o n s p r e v i o u s l y used by o t h e r investigators. F u r t h e r m o r e , c r o s s - c o n t a m i n a t i o n between BTI and

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284

BTK as determined by ELISA was less than 0.01%, the detectable l i m i t of the assay (35). The combined 24, 27, and 35K components of BTI (Sandoz) δ-endotoxin (Figure 1, Tracks 2 and 3) had an equivalent t o x i c i t y

Table I I . Oral t o x i c i t y of a l k a l i n e - s o l u b i l i z e d BTI (Sandoz, Track 2) and BTK (Salsbury, Track 1) δ-endotoxin. Animal A. aegypti Larva

24h BTI LD +

24h BTK

2.29 + 0.06* (12.41)

>40*

50

LD50+

Adult (ENEMA T. n i Larva H. zea Larva Swiss-Webster Mice

>5 >35

(2.85) >35

>30

>30

+

PPM or mg/kg body weight + 1 S.D. with slope of probit analysis i n parenthesis.

*yg/ml of water i n which larvae were incubated.

Table I I I . Toxicity of p a r t i a l l y p u r i f i e d a l k a l i n e s o l u b i l i z e d BTI δ-endotoxin fed and injected into A. aegypti and T. n i , respectively. A. aegypti Toxin Alkaline dissolved, Sandoz (Track 2) DEAE (Track 3) pH 4.5 soluble (Track 5, 27 & 24K) G-75, Rf 1.35 (Track 6, 27K) G-75, Rf 1.53 (Track 7, 24K) Alkaline dissolved, Salsbury (Track 8, 27K)

24h

LC50*

2.29 + 0.06 (12.4) 1.91 + 0.30 (2.92) 2.02 + 0.54 (2.06) 2.71 + 0.12 (5.72) >20 5.78 + 0.42 (4.76)

T. n i 24h

LD50+

3.71 + 0.32 (3.22) 1.96 + 0.64 (2.16) 1.95 + 0.14 (4.32) 3.54 + 0.50 (3.28) 2.97 + 0.18 (7.00) 6.09 + 0.46 (4.67)

*yg/ml +1 S.D. with slope of probit analysis i n parenthesis. Larvae were incubated i n water with BTI δ-endotoxin added. PPM or mg/kg body weight + 1 S.D. with slope of probit analysis i n parenthesis. Larvae were injected.

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before and after DEAE (Table III for both A. aegypti and T. n i ) , even though there appeared to be a concentration of the 68K com­ ponent i n this p u r i f i c a t i o n step. The 24 and 27K component i n d i v i d u a l l y (Figure 1, Tracks 7 and 6, respectively) also had an equivalent t o x i c i t y when injected into T. n i (Table III) but the 24K component (Figure 1, Track 7) was not toxic when fed to A. aegypti whereas the 27K component was toxic (Table I I I ) . This i n a c t i v i t y cannot be explained by the absence of the 35 and 68K components i n Track 7 (Figure 1) because these same components are also absent i n Track 5 and yet Track 5 retained oral t o x i c i t y to A. aegypti (Table I I I ) . In fact the absence of the 68 and 35K components (Figure 1, Tracks 5 and 7) likewise did not affect the T. n i a c t i v i t y (Table I I I ) . Thus i t appears that at least the 27K proteins are necessary for A. aegypti oral t o x i c i t y while both the 24 and 27K components can impart t o x i c i t y to T. n i when injected. The 27K component was also toxic i n both A. aegypti and T. n i regardless of the source (Track 6 and Track 8, Figure 1 and Table I I I ) . Neural Toxicity of BTI 6-Endotoxin The i n j e c t i o n of alkaline-dissolved BTI 6-endotoxin led to a number of immediate neuromuscular effects (Table IV) including,

Table IV. Symptoms e l i c i t e d after the i n j e c t i o n of a l k a l i n e dissolved BTI δ-endotoxin (Track 2) into mice and insects Trichoplusia ni (5 PPM)

Periplaneta americana (6 PPM)

Swiss-Webster Mice (1.5 PPM)

0-lh

Mouth palpation of i n j e c t i o n site Increased wandering Heart arrest Abdominal paralysis L i s t side to side when crawling Total paralysis & flaccidity

Loss of motor activity

Ruffled fur Lost alertness Not i n q u i s i t i v e Reduced responsiveness Slow i n righting themselves Breathing shallow Lost a c t i v i t y in hind legs

20-24h

Localized black­ ening of the body Total blackening of the body No response to head stimulation

In Survivors Failure to right themselves Tremor

In Survivors, Constipation Dead animals with a pinched waist

Time PostInjection

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i n the insects tested, l i s t i n g from side to side when crawling, heart arrest, paralysis, and tremors. These symptoms were observed for both the Sandoz and Salsbury BTI 6-endotoxin and were also observed for p a r t i a l l y p u r i f i e d BTI δ-endotoxin (Table III). The symptoms observed i n mice were somewhat similar to those observed i n botulism poisoning (a neurotoxin), which included a loss of alertness, shallow breathing, and i n some cases lost a c t i v i t y i n the hind legs. Dead mice had a pinched waist, a sign of diaphram arrest. The symptoms observed i n insects following the i n j e c t i o n of BTI 6-endotoxin were c l e a r l y d i f f e r e n t from those following the ingestion of BTK 6-endotoxin. When T. n i were fed BTK δ-endotoxin, there was regurgitation within 15 min, a t o t a l cessation of feeding u n t i l death, and no overt neuromuscular anomalies. The i n j e c t i o n of a l k a l i n e dissolved BTK 6-endotoxin produced no obvious adverse effects i n insects or mice. A number of other lines of evidence also suggested that there may be another mode-of-actio i n j e c t i o n other than i t (3,22-24). Using the appearance of cytosolic lactate dehydrogenase (LDH) i n insect hemolymph post-injection as a marker for c y t o t o x i c i t y (36-37), we found that dissolved BTI δ-endotoxin was a potent cytotoxin. When T. n i , however, were injected with dissolved BTI 6-endotoxin and then incubated at 28, 15, and 9°, there was an increase i n the L D 5 0 with a decrease i n temperature (Figure 2) but the LDH levels at 3.5 PPM BTI were unaffected by temperature. A pharmocological study of ventral nerve cord function i n T. n i also suggested that alkaline-dissolved BTI 6-endotoxin was a f f e c t i n g the insect nervous system as a nerve poison (Figures 3 and 4 ) . After 7-60 min post-injection of alkaline-dissolved BTI δ-endotoxin at the L D 5 0 concentration of 3.7 PPM, the ventral nerve cord of T. n i exhibited spontaneous-high frequency discharges. This was followed by a reduced baseline a c t i v i t y and s e n s i t i v i t y to sensory stimulation (S, Figure 4) at 24 h post-treatment. By 2-90 min post-injection of methamidophos, there also was spontaneous-high frequency discharge which lasted 20 min - 6 h and was followed by a reduced baseline a c t i v i t y and s e n s i t i v i t y to sensory stimulation (S, Figure 4) by 24 h. The response to methamidophos was s l i g h t l y more rapid and sustained than with BTI toxin. Studies also suggested that the primary s i t e of action for BTI might be the peripheral nervous system and that the mode-of-action of BTI and methamidophos on the insect nervous system were probably d i f f e r e n t . When the peripheral nervous system i s severed from the T. n i ventral nerve cord, the methamidophos application s t i l l e l i c i t s spontaneous discharge, but there i s no response to BTI toxin. When the cytotoxin, phospholipase-A2, i s injected into T. n i at i t s L D 5 0 of 35 PPM, there i s no spontaneous-high frequency discharge i n the ventral nerve cord and no reduction i n the stimulus-response (S, Figure 4) as was the case for BTI. A 25K dalton component was isolated at r e l a t i v e l y high purity as determined by SDS-PAGE (Figure 5, Track 3) from alkaline-dissolved BTI δ-endotoxin (Figure 5, Track 1). The

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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R1

R2

F i g u r e 3. Neurophysiological preparation of T r i c h o p l u s i a n i . Head, t h o r a x and gut a r e removed. Tungsten e l e c t r o d e s were p l a c e d i n t o the hemocoel a l o n g s i d e abdominal g a n g l i o n V I I I ( a t R l ) , the v e n t r a l nerve c o r d ( a t R2) and the abdominal w a l l ( g r o u n d , R£>. I n j e c t i o n s o f a l k a l i n e - d i s s o l v e d BTI δ-endotoxin, methamidophos and p h o s p h o l i p a s e - A 2 were i n t o the second p a i r o f abdominal p r o l e g s ( C h ) . Mechanical sensory s t i m u l a t i o n w i t h a g l a s s probe was a t the a n a l p r o l e g ( S ) . A c t i v i t y i n the v e n t r a l n e r v e c o r d was m o n i t o r e d through 24 h post-treatment (38-40) (see F i g u r e 4 ) .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST CONTROL

!

S

s

Sec

0 min

1

24 hr

7-60 min

10 PPM

s MMP

0 min

35 PPM

s P-A

j !

2

0 min

24 hr

2-90 min

!

;

s

60 min

j ;

S

24 hr

F i g u r e 4 . Time dependency o f nervous a c t i v i t y i n the v e n t r a l nerve c o r d o f T r i c h o p l u s i a n i i n j e c t e d w i t h 3 . 7 PPM a l k a l i n e d i s s o l v e d BTI δ-endotoxin ( S a n d o z ) , w i t h 1 0 PPM methamidophos (MMP) and w i t h 3 5 PPM p h o s p h o l i p a s e - A 2 ( P - A 2 ) . Mechanical s e n s o r y s t i m u l a t i o n i s g i v e n a t arrow j>. The c o n t r o l r e s p o n s e was the same as t h e r e c o r d i n g f o r P - A 2 . BTI and P-A2 were i n j e c t e d i n t o T. n i a t t h e i r r e s p e c t i v e L D 5 Q .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Effects of δ-Endotoxin in Mice and Insects

F i g u r e 5. SDS-Page a n a l y s i s o f a l k a l i n e - d i s s o l v e d Bacillus t h u r i n g i e n s i s i s r a e l e n s i s ( B T I ) δ-endotoxin from Sandoz I n c . a t 25 pg per t r a c k : (1) BTI δ-endotoxin as p r e p a r e d i n F i g u r e 1 ( T r a c k 2 ) , (2) s o l u b l e f r a c t i o n a f t e r d i a l y s i s o f BTI δ-endotoxin i n t o pH 4.5 sodium a c e t a t e b u f f e r , and (3) 25K component from BTI δ-endotoxin a f t e r pH 4.5 p e r c i p i t a t i o n and DEAE-anion exchange chromatography. S, m o l e c u l a r weight markers from t o p t o bottom b o v i n e serum a l b u m i n (68K d a l t o n s ) , ovalbumin (43K), and m y o g l o b i n (16K).

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s u p e r n a t a n t ( F i g u r e 5, T r a c k 2) a f t e r pH 4.5 p e r c i p i t a t i o n o f t h e crude e n d o t o x i n was f u r t h e r p u r i f i e d by D E A E - C e l l u l o s e (DE-52, Whatman, 30 m l ) i n 0.025 M T r i s - H C l (pH 8.00), e l u t e d w i t h a 48 h, 400 m l , 0.0-0.2 M NaCl l i n e a r g r a d i e n t ( F i g u r e 5, T r a c k 3 ) . The r e s u l t i n g 25K d a l t o n component ( F i g u r e 5, T r a c k 3) a t 0.85 pg/ml produced 50% c e l l l y s i s i n a 1% human r e d b l o o d c e l l s o l u t i o n (25°C., 15 min) and was a l s o h e m o l y t i c a g a i n s t sheep and r a b b i t r e d blood c e l l s . H e m o l y t i c a c t i v i t y i n c r e a s e d f o r the 25K component ( F i g u r e 5, T r a c k 3) a f t e r a 15 s e c i n c u b a t i o n a t 70°C and was i n a c t i v a t e d a t 90°C as was a l s o the case f o r the crude t o x i n ( F i g u r e 5, T r a c k 1 ) . At 25 yg/ml, however, t h e 25K p r o t e i n was n o t t o x i c o r a l l y t o A. a e g y p t i even a f t e r 15 s e c heat t r e a t m e n t s a t 45, 70 o r 90°C n o r was i t l e t h a l when i n j e c t e d i n t o T. n i l a r v a e a t 4.6 PPM ( t h e 48 h L D 5 0 > 4.6 PPM). Injections d i d d i s r u p t l a r v a l - p u p a l metamorphosis l a t e r i n development. The crude t o x i n ( F i g u r e 5, T r a c k 1 ) , however, was t o x i c t o A. a e g y p t i and T. n i a t 45 and 70° b u t n o t a f t e r the 90° t r e a t m e n t . So the 25K component i s c y t o t o x i to A. a e g y p t i o r i n j e c t e Conclusions I t appears from our s t u d i e s and r e p o r t s i n the l i t e r a t u r e t h a t the p a r a s p o r a l c r y s t a l o f BTI has g u t - t o x i c i t y when f e d t o t h e mosquito ( l j 6 ) , both i n v i t r o and i n v i v o c y t o t o x i c i t y (3,22-24) and i n v i v o n e u r o t o x i c i t y . Injected t o x i c i t y occurred i n a number o f i n s e c t s p e c i e s . The crude a l k a l i n e - d i s s o l v e d δ-endotoxin o f BTI when i n j e c t e d i n t o T. n i was s t r o n g l y c y t o t o x i c and a t i t s L D 5 0 a l s o n e u r o t o x i c . T h i s was u n l i k e the c y t o t o x i n , p h o s p h o l i p a s e - A 2 which demonstrated no n e u r o t o x i c i t y at i t s L D 5 0 . The t o x i c i t y o f BTI ό - e n d o t o x i n i n j e c t e d was a l s o temperature-dependent w h i l e i t s c y t o l y t i c a c t i v i t y was unchanged i n the same temperature r a n g e . A 25K component i s o l a t e d from BTI ( F i g u r e 5, T r a c k 3) was a l s o found t o be c y t o l y t i c b u t when i n j e c t e d had no t o x i c i t y . U n t i l each o f t h e components from the a l k a l i n e - d i s s o l v e d δ-endotoxin o f BTI ( F i g u r e 1, T r a c k 2) can be p u r i f i e d and s e p a r a t e l y t e s t e d f o r c y t o t o x i c i t y and n e u r o t o x i c i t y , the i n t e r r e l a t i o n s h i p s o f t h e s e m o d e s - o f - a c t i o n t o the many p o l y ­ p e p t i d e s found i n the δ-endotoxin o f BTI w i l l be i n q u e s t i o n . The v a r i a t i o n s o b t a i n e d w i t h d i f f e r e n t BTI δ-endotoxin p r e p a r a t i o n s and BTI s t r a i n s w i l l m a g n i f y t h e c o m p l e x i t y o f the problem. N e v e r t h e l e s s , a number o f l i n e s o f e v i d e n c e now e x i s t s t o suggest t h a t i n j e c t e d t o x i c i t y and n e u r o t o x i c i t y a r e not necessary a function of general c y t o l y t i c a c t i v i t y . The e v i d e n c e i s c l e a r t h a t t h e r e a r e a l s o c y t o l y t i c components t h a t demonstrate no gut t o x i c i t y . The f i n d i n g o f s p e c i e s d i f f e r e n c e w i t h i n a s i n g l e f a m i l y o f L e p i d o p t e r a t o the s u s c e p t i b i l i t y o f BTI δ-endotoxin p o i s o n i n g by i n j e c t i o n w i l l be an i m p o r t a n t t o o l f o r studying mode-of-action i n the f u t u r e . Reports on the n e r v e b l o c k i n g a c t i o n from d i g e s t s o f the δ-endotoxin o f BTK ( 2 0 - 2 1 ) , suggest t h a t a n e u r o t o x i c element may be common t o the a c t i o n o f o t h e r members o f the BT complex.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Acknowledgments Use o f t r a d e names i n t h i s p u b l i c a t i o n does n o t imply endorsement o f the p r o d u c t s named o r c r i t i c i s m o f s i m i l a r ones not mentioned. T h i s paper i s Number 9592 o f the J o u r n a l S e r i e s o f t h e N o r t h C a r o l i n a A g r i c u l t u r e Research S e r v i c e , R a l e i g h , N o r t h C a r o l i n a 27695. R. M. Roe was s u p p o r t e d i n p a r t by t h e Department o f H e a l t h and Human S e r v i c e s , N a t i o n a l S e r v i c e Award 1 F32 GM09223-02 from the N a t i o n a l I n s t i t u t e o f G e n e r a l S c i e n c e s ; and B. D. Hammock by NIEHS Research C a r e e r Development Award 5 K04 ES00107-05. P a r t i a l support f o r t h i s r e s e a r c h was p r o v i d e d by NIEHS Grant ES02710-04, the U n i v e r s i t y o f C a l i f o r n i a G e n e r a l Fund f o r M o s q u i t o R e s e a r c h , and the r e s p e c t i v e s t a t e a g r i c u l t u r a l e x p e r i ­ ment s t a t i o n s . The a s s i s t a n c e o f Dr. C h a r l e s L. Judson and Ms. Mary Ann Montague f o r t h e i r mosquito i n j e c t i o n s o f BTI and BTK i s most a p p r e c i a t e d . A l s o the a s s i s t a n c e o f K e n j i Ota, R a f a e l d e l V e c c h i o , J i m O t t e a , and f u l l y acknowledged.

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20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30. 31. 32. 33. 34. 35.

36. 37. 38. 39. 40.

FOR PEST CONTROL

Cooksey, K. E.; Donninger, C.; Norris, J. R.; Shankland, D. J. Invertebr. Pathol. 1969, 13, 461-2. Aronson, J. N.; Crowder, L. A. SIP 16th Annual Meeting Abstract. 1983. Murphy, D. W.; Sohi, S. S.; Fast, P. G. Science. 1976, 194, 954-6. Nishiitsutsuji-Uwo, J.; Endo, Y.; Himeno, M. J. Invertebr. Pathol. 1979, 34, 267-75. Johnson, D. E. J. Invertebr. Pathol. 1981, 38, 94-101. Thomas, W. E.; Ellar, D. J. FEBS Letters. 1983, 154, 362-8. Nickerson, K. W.; Bulla, L. A. Jr. Appl. Microbiol. 1974, 28, 124-8. Sharpe, E. S.; Nickerson, K. W.; Bulla, L. A. Jr.; Aronson, J. N. Appl. Microbiol. 1975, 30, 1052-3. Laemmli, U. K. Nature. 1970, 227, 680-5. Cheung, P. Y. K.; Roe, R. M.; Hammock, B. D.; Judson, C. L.; Montague, M. A. Pestic. Press. Yamamoto, T.; Iizuka, T; Aronson, J. N. SIP 16th Annual Meeting Abstract. 1983. Calabrese, D. M.; Nickerson, K. W. Can. J. Microbiol. 1980, 26, 1006-10. Spielman, Α.; Wong, J. Biol. Bull. 1974, 147, 433-42. Finney, D. J. "Probit Analysis"; Cambridge Univ. Press: Great Britian, 1971; pp. 1-333. Klowden, M. J.; Held, G. Α.; Bulla, L. A. Jr. Appl. Environ. Microbiol. 1983, 46, 312-5. Wie, S. I.; Andrews, R. E. Jr.; Hammock, B. D.; Faust, R. M.; Bulla, L. A. Jr. Appl. Environ. Microbiol. 1982, 43, 891-4. Bergmeyer, H. -U.; Bernt, E. Meth. Enzymatic Anal. 1974, 2, 574-9. Wing, K. D.; Sparks, T. C.; Lovell, V. M.; Levinson, S. O.; Hammock, B. D. Insect Biochem. 1981, 11, 473-85. Gammon, D. W. Pestic. Biochem. Physiol. 1977, 7, 1-7. Miller, T.; Kennedy, J. M. Pestic. Biochem. Physiol. 1973, 3, 370-83. Narahashi, T. Adv. Insect Physiol. 1971, 8, 1-93.

RECEIVED November 15, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

21 Bioassay of Anti Juvenile Hormone Compounds: An Alternative Approach 1

2

3

T H O M A S C. SPARKS , R. M I C H A E L ROE , A D R I A N BUEHLER , and B R U C E D. H A M M O C K 4

1

2

3

Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, L A 70803 Department of Entomology, North Carolina State University, Raleigh, N C 27695 Abteilung fuer Zoophysiologie, Universitaet Bern, Erlachstrasse 9a, CH-3012 Bern, Switzerland Department of Entomology, University of California, Davis, C A 95616

4

During the last larva cabbag looper, Trichoplusia ni, Hubner, the prepupal burst of juvenile hormone (JH) stimulates the appearance of juvenile hormone esterase (JHE) which, in turn, degrades the JH. Disruption of this prepupal burst of JH by anti-juvenile hormones (AJHs) such as fluoromevalonolactone, result in a variety of teratogenic effects including delayed tanning and/or pupation, malformed larvae and reduced JHE activity. Although general toxicants and esterase inhibitors may also cause malformed larvae, there is usually no delay in tanning and/or pupation. These observations provide the basis for a simple, rapid AJH bioassay using an economically important pest insect (T. ni). A simple 'key' was devised to help facilitate the use of this bioassay. At the h e a r t o f any s e a r c h f o r b i o a c t i v e m o l e c u l e s i s the need for e f f e c t i v e bioassays. S e v e r a l b i o a s s a y s have been d e v e l o p e d f o r the i d e n t i f i c a t i o n o f compounds w i t h a n t i - j u v e n i l e hormone (AJH) a c t i v i t y . The most common of these AJH b i o a s s a y s i n v o l v e s the treatment o f young l a r v a e or nymphs w i t h the p o t e n t i a l AJH by i n c o r p o r a t i o n i n t o the d i e t or c o n t a c t a p p l i c a t i o n , and then w a i t i n g f o r s e v e r a l days ( o r , i n some c a s e s , weeks) f o r p r e c o c i o u s development ( o r o t h e r AJH r e s p o n s e ) t o o c c u r (.1,2^)· A l t e r n a t i v e l y , AJH a c t i v i t y can be determined u s i n g in v i t r o a s s a y s such as c o r p o r a a l l a t a c u l t u r e s or e p i d e r m a l c e l l c u l t u r e s t o m o n i t o r f o r i n h i b i t i o n o f j u v e n i l e hormone (JH) b i o s y n t h e s i s (3-6) or b l o c k a g e o f the JH induced i n h i b i t i o n o f pupal commitment (J)» r e s p e c t i v e l y . A l t h o u g h the above assays have a l l proven u s e f u l , t h e r e a r e a number of d i s a d v a n t a g e s a s s o c i a t e d w i t h each o f them. F o r example, the c o n t a c t and f e e d i n g b i o a s s a y s can r e q u i r e a l a r g e q u a n t i t y of compound, compared to a t y p i c a l t o p i c a l b i o a s s a y , and i t u s u a l l y takes s e v e r a l days b e f o r e the AJH e f f e c t s a r e o b s e r v e d . The i n v i t r o assays a r e t e d i o u s , r e q u i r e s p e c i a l s k i l l s i n m i c r o s u r g e r y , dedicated s t e r i l e f a c i l i t i e s , are unsuitable f o r screening large numbers o f compounds, d e t e c t a l i m i t e d number of p o s s i b l e mechanisms,

0097-6156/85/0276-0293$06.00/0 © 1985 American Chemical Society In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

294

BIOREGULATORS FOR

PEST C O N T R O L

a n d in v i t r o a c t i v i t y may n o t t r a n s l a t e i n t o in v i v o a c t i v i t y due t o i n v i v o m e t a b o l i s m ( 8 ) . F i n a l l y , t h e i n s e c t s m o s t commonly u s e d f o r AJH b i o a s s a y s , the t o b a c c o hornworm, Manduca s e x t a ( L . ) , and the l a r g e m i l k w e e d bug, O n c o p e l t u s f a s c i a t u s ( D a l l a s ) , a r e o f l i m i t e d e c o n o m i c i m p o r t a n c e , a n d b o t h s p e c i e s seem t o be h y p e r s e n s i t i v e s u c h t h a t compounds a c t i v e o n t h e m ( e . g . ETB, p r e c o c e n e ) d i s p l a y l i t t l e a c t i v i t y on m a j o r p e s t i n s e c t s , e s p e c i a l l y among t h e L e p i d o p t e r a (2,9,10). The c a b b a g e l o o p e r , T r i c h o p l u s i a n i H u b n e r , i s a p e s t o f a w i d e v a r i e t y of a g r i c u l t u r a l c r o p s (11-13) and r e p r e s e n t s a m a j o r f a m i l y of i n s e c t p e s t s , the N o c t u i d a e , which a l s o i n c l u d e s the c o r n e a r w o r m , H e l i o t h i s z e a ( B o d d i e ) , t h e t o b a c c o budworm, H e l i o t h i s v i r e s c e n s ( F . ) , the soybean l o o p e r , P s e u d o p l u s i a i n c l u d e n s ( W a l k e r ) , t h e b e e t armyworm, S p o d o p t e r a e x i g u a ( H u b n e r ) , t h e f a l l armyworm, S p o d o p t e r a f r u g i p e r d a ( J . E. S m i t h ) a n d o t h e r s . There i s a l s o a g r o w i n g b o d y o f k n o w l e d g e c o n c e r n i n g t h e e n d o c r i n o l o g y o f T. n i ΟΛ,Ι^)· R e c e n t s t u d i e s on J H a n d J H e s t e r a s e ( J H E ) r e g u l a t i o n i n wandering l a s t stadiu s u g g e s t e d an a l t e r n a t i v A J H a c t i v i t y t h a t c i r c u m v e n t s some o f t h e d i s a d v a n t a g e s a s s o c i a t e d w i t h o t h e r AJH b i o a s s a y s . F r o m a more f u n d a m e n t a l p o i n t o f v i e w , t h i s r e p o r t i l l u s t r a t e s how k e y x e n o b i o t i c s c a n be u s e d t o d i s s e c t developmental p r o c e s s e s d u r i n g c r i t i c a l phases of i n s e c t d e v e l o p ­ ment · The

Bioassay

The b i o a s s a y p r e s e n t e d h e r e i n i s b a s e d on w h a t a p p e a r s t o be a c o n s i s t e n t set of t e r a t o g e n i c m o r p h o l o g i c a l e f f e c t s that are p r o d u c e d i n r e s p o n s e t o d i s r u p t i o n s of the JH m e d i a t e d r e g u l a t i o n o f d e v e l o p m e n t d u r i n g t h e l a t e l a s t s t a d i u m o f T. n i . To f a c i l i t a t e t h e u s e o f t h i s b i o a s s a y , a s i m p l e key has b e e n d e v i s e d and i s p r e s e n t e d below a l o n g w i t h hypotheses f o r the p h y s i o l o g i c a l b a s i s for each s t e p . S i n c e the r e s p o n s e s o b s e r v e d i n t h i s b i o a s s a y (and o t h e r s ) c a n be t h e r e s u l t o f a v a r i e t y o f e f f e c t s , n o t o n l y on t h e e n d o c r i n e s y s t e m , b u t a l s o o n t h e n e r v o u s and o t h e r s y s t e m s ( 1 9 ) , a s e l e c t e d g r o u p o f J H s , AJHs and p e s t i c i d e s (Table I) were u s e d i n d e v e l o p i n g t h i s b i o a s s a y i n an e f f o r t t o t e s t and e l i m i n a t e r e s p o n s e s due t o n o n - A J H c o m p o u n d s . L i k e w i s e , i t i s i m p o r t a n t i n any s u c c e s s f u l b i o a s s a y t o s i m p l y and r a p i d l y e l i m i n a t e i n a c t i v e and i n a p p r o p r i a t e compounds ( i . e . n o n - A J H s ) . Thus, the f i r s t steps i n our b i o a s s a y a t t e m p t t o e x c l u d e non-AJHs and i n a c t i v e compounds, l e a v i n g the l a t e r s t e p s f o r t h e c o n f i r m a t i o n o f A J H a c t i v i t y t o t h o s e few compounds t h a t a r e m o s t l i k e l y A J H s . The I n s e c t and A s s a y s . L a s t ( f i f t h ) s t a d i u m ( L 5 ) l a r v a e o f T. n i were used throughout. L a r v a e w e r e r e a r e d on a 1 4 L : 1 0 D p h o t o p e r i o d ( l i g h t s on a t 5 AM) a t 27°+2°C., a n d s t a g e d a s d e s c r i b e d p r e v i o u s l y (31). L a r v a e w e r e t r e a t e d w i t h t h e d e s i r e d compounds e i t h e r by i n j e c t i o n a l o n g the m i d - d o r s a l l i n e of abdominal segments 1 & 2 ( 1 μΐ i n d i s t i l l e d w a t e r ) , o r by t o p i c a l t r e a t m e n t ( 1 - 2 μΐ i n a c e t o n e o r e t h a n o l ) on t h e dorsum o f t h e l a s t t h o r a c i c segment. C o n t r o l s r e c e i v e d a c e t o n e o r e t h a n o l ( t o p i c a l l y , 1-2 μ ΐ ) , o r w a t e r ( i n j e c t e d , 1 μΐ). O b v i o u s l y , t h e m e t h o d o f t r e a t m e n t w i l l be a

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

21.

SPARKS ET AL.

Bioassay of Anti Juvenile Hormone Compounds

function of the s o l u b i l i t y of the test compound and/or i t s suspected a b i l i t y to penetrate the insect c u t i c l e . Injection i s easily accomplished using either a sharpened 10 μΐ Hamilton syringe or a finely-drawn glass c a p i l l a r y tube. The c a p i l l a r y tube has the advantage of p r a c t i c a l l y eliminating any bleeding after i n j e c t i o n . Treated larvae were examined for behavioral and developmental changes or abnormalities. A pooled (3-5 larvae) hemolymph sample was collected by c l i p ­ ping either anal or thoracic legs of the larvae. JHE a c t i v i t y i n the hemolymp^ was monitored as described previously (32,33) using JH III (H_^at C ^ , 11 Ci/mmol., New England Nuclear) as the substrate (5x10 M). A l l assays were run i n duplicate on at least two (usually three or more) occasions. The Key. The bioassay i s divided into 5 steps which form a key for the i d e n t i f i c a t i o n of AJH a c t i v i t y . At each step examples are provided of compounds that exhibit the possible responses. Since' these compounds wer necessarily follow the sam see i f the bioassay was being used to evaluate their AJH a c t i v i t y . I d e n t i f i c a t i o n of AJH A c t i v i t y 1. Are there larval-pupal intermediates or malformed larvae? Treatment : L5D3 (last stadium, day 3) larvae treated ca. 9 AM; 200 nmol./larva. Expected Result : A majority (>50%) of the treated larvae become tanned, malformed larvae without displaying any obvious toxic response (see explanation below). Pupation/tanning i n controls generally occurs on L5D4 ca. 3 PM (ca. 30 hr. posttreatment). Results are expressed as the percentage of larvae displaying the above effect r e l a t i v e to the controls (solvent only). A) YES - GO TO 2. Examples: FMev, EPPAT, DEF (Table I ) . B) NO - Increase dose and/or change the time of treatment or mode of application and try again; otherwise STOP. Examples: Epofenonane, hydroprene, ETB, precocene I I , ethoxy-precocene, DFP, pipernoyl butoxide, chlordimeform, TFT, carbaryl (Table I ) . Explanation: The appearance of tanned, malformed larvae following treatment with an exogenous compound can be the result of AJH a c t i v i t y or a response on the part of the insect to an external stress such as an i n s e c t i c i d e . Since a variety of molecular struc­ tures are l i k e l y to be tested for AJH a c t i v i t y , i t i s important to eliminate compounds that are general toxicants at the beginning of the bioassay. I f the compound i s causing the insect to be stressed or intoxicated, the insect larva ( i n this case a prepupa) may have trouble casting the old c u t i c l e , yet be capable of tanning. Thus, the insect becomes a tanned larva or what appears to be a half pupa/half larva (due, i n part, to incomplete ecdysis). For example, larvae treated with paraoxon (200 nmol./larva; data not included i n Table I due to high mortality) caused the formation of tanned larvae i n a l l larvae treated. However, a l l of these larvae had obviously been adversely affected (intoxicated) by the paraoxon

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

295

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985. Dose used for key (nmol.)

200 200

200

200

200

200

2. Juvenile hormone III

3. Epofenonane: l - ( 4 - e t h y l phenoxy)-6,7-epoxy-3-ethyl7 -methyl no na ne

4. Hydroprene: ethyl (2E,4E)3,7,11-trimethy1-2,4-dodecadienoate

5. FMev: tetrahydro-4-fluorome t hy 1 -4- hy dr oxy - 2 H- py r a n2-one

6. ETB: ethyl 4-(2-(tert-butylcarbonyloxy)butoxy)benzoate

7. Precocene I I : 6,7-dimethoxy2,2-dimethylchromene

f

200

1. Juvenile hormone I

112+9

105+23

365+32

45+21

365+139

96+19

7.8-

1.0+

0.1-

100

87+25

87+26

99+7

100+25

445+226 110+31

164+54

304+83

JHE A c t i v i t y (% Control) L5D3 L5D1

1.5+

1.5-

0.0

4.0+

Q

T^ f o r tannine (hrs.)

0

0

0

0

% Malformed Larvae

1, 2, 3, 10 (for review), 28

2, 7, 9 22, 24, 26 28

4, 9, 18, 22-26

9, 21

20

Previously tested for AJH a c t i v i t y

used to Develop and Test the AJH Bioassay, and Their Effect on T. n i .

Used i n the Development of the AJH Bioassay

Compound

Table I. Compounds

Η 73 Ο

η Ο

"Ό

m C O H

3

73

ι

73 m Ο c r

6

to

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1

Con t. Dose used for key (nmol.)

200

11. 200

200

200

12. Piperonyl butoxide: 3,4methylenedioxy-6-propylbenzyl η-butyl diethylglycol ether

13. Chiordimeform: N,N-dimethylN'-[2-methyl-4-chlorophenyl]formamidine

14. TFT: 1,1,1-trifluorotetradecan-2-one

phosphorotrithiolate

DEF: j>,S,J5-tri-n-butyl-

200

200

10. DFP: 0,0-diisopropyl phosphorofluoridate

EPPAT: 0-ethyl S-phenyl phosphoramidothiolate

8. Ethoxy-precocene: 7-ethoxy200 6-methoxy-2,2-dimethylchromene

Compound

Table 1.

2, 3, 9 20

10, (for review), 29

Previously tested for AJH a c t i v i t y

71

72

% Malformed Larvae f o r

122+28

50+19

111+29

34+13

98+7

17+20

87+14

89+24

98+15

94+27

81+22

105+15

11+8

91+10

JHE A c t i v i t y (% Control) L5D3 L5D1

Continued on next page

0.8-

4.4-

0.1-

1.8-

0.8-

1.5-

1.1-

(hrs.r

tannine

bo

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1

Con t.

—

5

18. L-643,049-00H03: 6(R)-[2[8(S)-(2,2-diethylbutyryloxy)2(S),6(R)-diemthyl-l,2,6,7,8, 8a(R)-hexahydronaphthyl-l(S)jethy1]-4(R)-hy droxy-3,4,5,6tetrahydro-2H-pyran-2-one

56

—

5, 6, 29

5

100

—

Previously tested for AJH a c t i v i t y

17. L-643,049-01K01: 7-[l(S),2(S), 100 6(R),7,8(S),8a(R)-hexahydro2,6-dimethyl-8-(2,2-diethylbutyryloxy)-naphthaleny1-1]3(R),5(R)-hydroxyheptanic acid sodium salt

16. Methyl compact i n : methyl 7[1,2,6,7,8,8a-hexahydro-2methyl-8-(2-methylbutyryloxy) naphthylenyl-l]-3,5-hydroxyheptanate

200

Dose used for key (nmol.)

Evaluated Using the AJH Bioassay

15. Carbaryl: 1-naphthyl Nmethylcarbamate

Compound

Table 1.

10

89

0

7

% Malformed Larvae

1.7-

5.7-

1.3-

0.8-

Q

T^ for tannine (hrs.)

57+9

111+21

103+17

89+5

JHE A c t i v i t y (% Control) L5D3 L5D1

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1

Con t.

—

Previously tested for AJH a c t i v i t y

83

% Malformed Larvae

3.8-

T ^ for tanning (hrs.)

68+41

124+52

JHE A c t i v i t y (% C o n t r o l ) L5D3 L5D1

from l a r v a e t r e a t e d on L5D3 or L5D1.

A l l o t h e r s were a p p l i e d t o p i c a l l y .

the c o n t r o l s . JHE a c t i v i t y measured i n v i t r o

standard devations. 5. Compound was i n j e c t e d .

4.

Values

a r e means +

their

1. Sources of the compounds were as f o l l o w s ; 1,2 C a l b i o c h e m - B e r h i n g ; 3, P. Masner, Dr. Maag L t d . ; 4-6, G. Q u i s t a d , D. Schooley and G. S t a a l , Zoecon C o r p o r a t i o n ; 7,8, Sigma; 9, P. Magee, Chevron Chemical Company; 10,11, A l d r i c h ; 11,12,13,15, Chem S e r v i c e ; 14, s y n t h e s i s z e d as d e s c r i b e d i n ( 3 0 ) ; 16-19, R. Dybas, Merck, Sharp & Dohme. 2. Tanned and/or malformed l a r v a e due t o i n c o m p l e t e e c d y s i s or o t h e r c a u s e s . 3. Time f o r 50% of the t r e a t e d l a r v a e t o t a n and/or pupate r e l a t i v e (+ = b e f o r e ; - = a f t e r ) t o

5

Dose used f o r key (nmol.)

19. L-643,737-00S03: 6 ( R ) - [ 2 58 [8(S)-(2-ethyl-2-methylbutyryloxy)-2(S),6(R)-diemthyl-l,2,6, 7,8,8a(R)-hexahydronaphthyl1(S)]ethyl]-4(R)-hydroxy-3,4, 5,6-tetrahydro-2H-pyran-2-one

Compound

Table I.

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treatment ( i . e . f l a c c i d , hemolymph a c c u m u l a t i o n i n the abdomen, f a i l u r e t o m i g r a t e t o the top o f the r e a r i n g c o n t a i n e r s , dehydra­ tion). L i k e w i s e , c h l o r d i m e f o r m m o d i f i e d the b e h a v i o r o f the l a r v a e i n the form o f abnormal s p i n n i n g and wandering b e h a v i o r . Thus, n e i t h e r o f t h e s e compounds ( a t the dose t e s t e d ) would be c o n s i d e r e d as a p o t e n t i a l AJH. I t s h o u l d be remembered, however, that f o r some i n s e c t s p e c i e s the e f f e c t i v e dose f o r a p a r t i c u l a r AJH may be near the t o x i c dose f o r t h a t compound ( s e e 2 2 ) . One d i s a d v a n t a g e o f any i n v i v o b i o a s s a y , i n c l u d i n g t h i s one, i s t h a t they w i l l not d e t e c t the AJH a c t i v i t y of t o x i c m o l e c u l e s w h i c h , i n an i n v i t r o system, would f u n c t i o n as AJHs. As w i t h t h e i n s e c t i c i d e s , FMev (a h i g h l y a c t i v e AJH f o r the L e p i d o p t e r a ; 22) a l s o causes the f o r m a t i o n o f tanned l a r v a e . While t h e s e tanned l a r v a e may a l s o be the r e s u l t o f g e n e r a l t e r a t o g e n i c or s t r e s s e f f e c t s , the removal o f the c o r p o r a a l l a t a ( s i t e o f JH b i o s y n t h e s i s and r e l e a s e ) a l s o causes s i m i l a r e f f e c t s ( 3 4 ) . A l t h o u g h the p r e s e n c e o f tanned l a r v a e seems t o c o n s i s t e n t l y c o r r e l a t e w i t h AJH a c t i v i t y , i t i s i m p e r a t i v not be used as the s o l activity. R a t h e r , i t i s a s i n g l e marker that i s o n l y o f s i g n i f i ­ cance when c o u p l e d w i t h Step 2 ( b e l o w ) . 2. I s t h e r e a d e l a y i n time o f p u p a t i o n ? Treatment : L5D3 l a r v a e (9 AM); 200 n m o l . / l a r v a . Expected R e s u l t : Treated larvae d i s p l a y a d i s t i n c t ( c a . 4 h r . ) d e l a y i n the time o f p u p a t i o n / t a n n i n g . Results are expressed as the advancement ' + ' o r d e l a y ( i n h r s ) i n the time o f t a n n i n g / p u p a t i o n r e l a t i v e t o the c o n t r o l s ( s o l v e n t o n l y ) . C o n t r o l s u s u a l l y p u p a t e / t a n about 30 h r . p o s t t r e a t m e n t (L5D4, c a . 3 PM). A ) YES - Compound may possess AJH a c t i v i t y - GO TO 3 f o r verification. Examples: FMev, c h l o r d i m e f o r m ( T a b l e I ) . B) NO - STOP. Examples: JH I , JH I I I , epofenonane, h y d r o p r e n e , ETB, precocene I I , e t h o x y - p r e c o c e n e , EPPAT, DFP, DEF, p i p e r o n y l b u t o x i d e , TFT, c a r b a r y l ( T a b l e I ) . E x p l a n a t i o n : I n many l e p i d o p t e r a n s , i n c l u d i n g T. n i , the p r e s e n c e o f JH i n the prepupa seems t o a c c e l e r a t e the time o f e c d y s i s t o the pupa (18,35-38). C o n v e r s e l y , a r e d u c t i o n i n the JH t i t e r due t o an AJH causes a d e l a y i n the time o f e c d y s i s t o the pupae and/or the t a n n i n g p r o c e s s (_18,37). T h i s p a r t i c u l a r e f f e c t can be p r e v e n t e d , i n p a r t , by the c o a p p l i c a t i o n o f JH I ( 1 8 ) . Thus, a d e l a y i n the time o f t a n n i n g / p u p a t i o n seems to be r e l a t e d t o the a b i l i t y o f a compound t o b l o c k JH b i o s y n t h e s i s / r e l e a s e o r a c t i o n , and hence a c t as an AJH. V e r i f i c a t i o n o f AJH A c t i v i t y 3. I s t h e r e reduced JHE a c t i v i t y i n L5D4 l a r v a e ? Treatment : L5D3 (9 AM) l a r v a e ; 200 n m o l . / l a r v a . E x p e c t e d R e s u l t : L a r v a e a r e a s s a y e d 24 h r . p o s t t r e a t m e n t (L5D4, 9 AM, l a r v a e a r e now p r e p u p a e ) f o r JHE a c t i v i t y . A compound w i t h AJH a c t i v i t y s h o u l d cause the l e v e l o f JHE a c t i v i t y t o be lower than normal. R e s u l t s a r e e x p r e s s e d as a p e r c e n t a g e of the JHE a c t i v i t y p r e s e n t i n l a r v a e t r e a t e d w i t h o n l y the solvent.

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A) YES - GO TO 4. Examples: FMev, EPPAT, chlordimeform, DEF (Table I ) . B) NO - STOP. Not l i k e l y to be an AJH for T. n i . Examples: JH I, JH III, epofenonane, hydroprene, ETB, precocene I I , ethoxy-precocene, DFP, piperonyl butoxide, TFT, carbaryl (Table I ) . Explanation: Hemolymph JHE a c t i v i t y in late last stadium larvae of T. n i i s d i r e c t l y regulated by the JH t i t e r (16-18). Thus, any compound that affects JH biosynthesis /release or action at JH receptor sites w i l l cause a change i n the prepupal peak of JHE a c t i v i t y . Compounds with JH a c t i v i t y stimulate JHE production, often to greater than normal levels (e.g. JH I, JH I I I , epofenonane, hydroprene and ETB; Table I ) . For an AJH, a reduction i n the JHE a c t i v i t y i s the expected outcome. However, a reduction i n JHE a c t i v i t y can also be the result of an inhibitor that interacts d i r e c t l y with the JHE (see below) 4. Normal JHE a c t i v i t y Treatment : L5D1 larvae (3 PM); 200 nmol./larva. Expected Result : Treated larvae are assayed for JHE a c t i v i t y 1-2 hr. posttreatment. An AJH should have no effect on JHE a c t i v i t y i n this short of a time period. Results are expressed as a percentage of the normal (control) JHE a c t i v i t y . A) YES - Compound i s an AJH. GO TO 5 for determination of mode of action. Example: FMev (Table I ) . B) NO - STOP. Compound is a general toxicant and/or JHE i n h i b i t o r , and i s not an AJH. Example: EPPAT (Table I ) . Explanation: Step 4 insures that the potential AJH i s not merely acting as a JHE i n h i b i t o r . Since there i s always the p o s s i b i l i t y of bioactivation, an in vivo test i s used. AJHs w i l l have no effect on JHE a c t i v i t y in the time-frame used in this step (1 hour) while this amount of time should be more than s u f f i c i e n t for JHE inhibitors to act (e.g. EPPAT; ^19,39,40). I f the compound under consideration makes i t through this part of the bioassay key, then i t has some AJH a c t i v i t y . AJH C l a s s i f i c a t i o n

(Optional)

5. Does the AJH block the juvenoid induced increase in JHE a c t i v i t y ? Treatment: L5D3 (9 AM) larvae; selected doses of the AJH (100 nmol./larva i s a good starting place) are coapplied with the juvenoid, epofenonane (100 nmol./larva). Expected Result: Larvae are assayed for JHE a c t i v i t y on L5D4 (9 AM). The JHE induction should not be affected i f the AJH acts by blocking JH biosynthesis. Results are expressed as a percentage of the JHE a c t i v i t y induced by epofenonane (100 nmol./larva) alone. A) YES - AJH i s probably a suboptimal JH or a JH receptor antagonist. Example: ETB (Figure 1). B) NO - AJH probably functions by d i r e c t l y blocking JH bio­ synthesis and/or release. Example: FMev.

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Explanation: This section i s included i n an effort to provide a means to gain some insights into the mode of action of the test compound. Epofenonane appears to be a JH agonist for T. iii that i s somewhat less active than JH I (16,26). Unlike JH I, epofenonane is not susceptible to ester hydrolysis, which eliminates the problem of the JHE a f f e c t i n g the level of i t s own induction. Like epo­ fenonane, the JH agonist-antagonist ETB was able to induce the appearance of the hemolymph JHE i n T. n i , but only at much higher dosages (Figure 1). However, at lower doses ETB caused a dose dependent reduction i n the JHE a c t i v i t y induced by epofenonane i n T. n i (Figure 1). Since other tests demonstrated that ETB was not a direct JHE i n h i b i t o r (26), i t appeared that ETB was acting as a JH receptor antagonist (26). Unlike ETB, FMev appeared to have no effect on the JHE induction by epofenonane (41). This observation i s consistent with other studies that show i t to func­ tion as an i n h i b i t o r of JH biosynthesis (22,23). Thus, this assay can potentially provide information on the mode of action of an AJH. Running similar test that the action being observe for JH receptors (probably the fat body), and not due to other interactions with the brain, corpora a l l a t a , etc. Using the Key to Test for AJH A c t i v i t y . Besides the compounds used to develop the key, methyl compactin and three of i t s analogues (Table I) were evaluated for AJH a c t i v i t y (through step 4). Of the 4 compounds tested, both L-643,049-01K01 and L-643,73700S03 (tested at 100 and 58 nmol./larva, respectively; injected) caused the formation of tanned malformed larvae (89 and 83%, respectively; Step 1) and a large delay (ca. 4-6 hr.) i n the time of tanning/ pupation (Step 2) (Table I ) . Thus, both L-643,049-01K01 and L-643,737-00S03 appeared to possess AJH a c t i v i t y and were tested further. Treatment (100 and 58 nmol./larva, respectively; injected) of L5D3 larvae with either of these compounds resulted i n a 30-40% decrease i n the prepupal JHE a c t i v i t y peak (Step 3), while L5D1 larvae treated (as above) with either compound possessed normal l e v e l s of JHE a c t i v i t y one hour posttreatment (Step 4) (Table I ) . These results confirm that both L-643,049-01K01 and L-643,737-00S03are AJHs for T. n i . Discussion The bioassay-key presented here provides a r e l a t i v e l y simple approach to i d e n t i f y i n g compounds with AJH a c t i v i t y . Obviously, the s e n s i t i v i t y of the assay i s , i n part, a function of the number and magnitude of the dosages used. In using the bioassay and key, i t i s best to start with as high of a dose as possible to rapidly eliminate the inactive compounds. In our assays 200 nmol./larva i s t y p i c a l l y used as the starting dose. In doing so, however, i t should be kept i n mind that JH agonists/antagonists, such as ETB (2^j),26), may only display AJH a c t i v i t y over a narrow dose range which may be far below our 200 nmol./larva starting dose. At this high dose only JH l i k e a c t i v i t y may be seen. For example, i t has been demonstrated that ETB can antagonize the JH action of the juvenoid epofenonane i n T. iii (26, Figure 1) and yet be

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nmol.

ETB/La

rva

F i g u r e 1: JHE a c t i v i t y i n L5D3 (9 AM) l a r v a e of T. n i t r e a t e d w i t h epofenonane (100 n m o l . / l a r v a ) and s e l e c t e d doses o f ETB (1-120 n m o l . / l a r v a ) compared t o l a r v a e t r e a t e d w i t h ETB (1-900 n m o l . / l a r v a ) a l o n e . JHE a c t i v i t y i s e x p r e s s e d as a p e r c e n t a g e of the JHE a c t i v i t y produced by epofenonane (100 n m o l . / l a r v a ) alone. Data i s adapted from ( 2 6 ) .

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d e v o i d of AJH a c t i v i t y at doses t e s t e d i n both the c l a s s i c a l AJH b i o a s s a y s a s w e l l as the one p r e s e n t e d here (41 and see a b o v e ) . L i k e w i s e , ETB d i s p l a y s J H - l i k e a c t i v i t y a t h i g h doses (_> 200 nmol.; 18,26, F i g u r e 1 ) . I n L5D3 l a r v a e o f T. n i , JH a c t i v i t y i n the whole animal i s m a n i f e s t e d by normal wandering and p r e p u p a l a c t i v i ­ t i e s l e a d i n g t o a p p a r e n t l y normal pupae from which, however, t h e r e i s no a d u l t emergence ( 1 8 ) . Thus, i f a p p a r e n t l y normal pupation o c c u r s , but no a d u l t e c l o s i o n i s f o r t h c o m i n g , then i t i s p o s s i b l e t h a t t h e compound, a t the dose b e i n g t e s t e d , i s a c t i n g as a JH. W i t h any new p r o c e d u r e , t h e r e i s always the c o n c e r n f o r i t s proper use. I n our hands, the r e s u l t s o f the f i r s t two s t e p s o f the b i o a s s a y , which e a s i l y c a n be combined i n t o a s i n g l e t e s t , have p r o v i d e d a v e r y c o n s i s t e n t t e s t o f AJH a c t i v i t y t h a t h a s , t o d a t e , always been c o n f i r m e d by the l a t e r s t e p s i n the b i o a s s a y . However, c a r e must be taken i n the e v a l u a t i o n o f compounds that display toxic effects. As a l r e a d y mentioned, p e s t i c i d e s l i k e paraoxon can produce tanne be very g r e a t l y d e l a y e d However, s i n c e the l a r v a e t h a t d i s p l a y these e f f e c t s were b e h a v i n g i n an a b n o r m a l / i n t o x i c a t e d manner ( i . e . f l a c c i d , l a c k o f a c t i v i t y when probed, hemolymph a c c u m u l a t i o n , f a i l u r e t o s p i n , e t c . ) , the r e s u l t s s h o u l d be d i s c o u n t e d . As a s a f e t y measure, however, i t i s important t o t e s t a l l s u s p e c t e d AJHs as d e s c r i b e d i n s t e p s 3 and 4. T h i s p r o c e s s w i l l i n s u r e t h a t compounds that a r e g e n e r a l t o x i c a n t s w i l l n o t be m i s t a k e n l y a s s i g n e d as AJHs. F i n a l l y , i t i s suggested t h a t a p p r o p r i a t e c o n t r o l s ( s o l v e n t o n l y ) be r u n a t a l l times s i n c e t h e r e can be some day t o day v a r i a t i o n i n the exact time o f e c d y s i s t o the pupa and/or t a n n i n g . I f at a l l p o s s i b l e , a s t a n d a r d such as FMev s h o u l d a l s o be t e s t e d o c c a s i o n a l l y t o i n s u r e t h a t the assay i s w o r k i n g p r o p e r l y . U n l i k e some o f the more c l a s s i c AJH b i o a s s a y s , the one p r e s e n t e d here a l l o w s f o r the r a p i d ( c a . 36 h r s ) e l i m i n a t i o n o f i n a c t i v e compounds, s a v i n g the more i n t e n s i v e l a b o r f o r o n l y the more p r o m i s i n g compounds. An a d d i t i o n a l advantage i s t h a t o n l y a r e l a t i v e l y s m a l l amount o f m a t e r i a l i s needed f o r the b i o a s s a y . S i n c e the t e s t organism, T. n i , i s a major economic pest i n s e c t w i t h a b r o a d host range, i t can be assumed t h a t , u n l i k e Μ · s e x t a or 0. f a c i a t u s , i t has a r a t h e r w e l l developed d e t o x i f i c a t i o n systems. Thus, compounds a c t i v e on T. n i a r e more l i k e l y t o be a c t i v e on o t h e r e c o n o m i c a l l y important i n s e c t p e s t s . The b i o a s s a y p r e s e n t e d here f o r T. n i i s based on a growing body o f knowledge t h a t has been accumulated d u r i n g the l a s t decade. W h i l e few o t h e r i n s e c t s have been so w e l l s t u d i e d i n terms o f e n d o c r i n e r e g u l a t i o n o f metamorphosis, i t may be p o s s i b l e t o adapt t h i s b i o a s s a y - k e y t o o t h e r l e s s s t u d i e d , but e c o n o m i c a l l y more important i n s e c t p e s t s , such as H. v i r e s c e n s , S. f r u g i p e r d a and Ζ · i n c l u d e n s . L i k e w i s e , t h i s b i o a s s a y s h o u l d be e a s i l y adapted t o M. s e x t a f o r which t h e r e i s a w e a l t h o f e n d o c r i n o l o g i c a l i n f o r m a t i o n (42,43). Compactin e x h i b i t s AJH a c t i v i t y i n s e v e r a l i n s e c t s (5,6,29) and seems t o f u n c t i o n as an i n h i b i t o r o f 3-hydroxy1-3-methy1gluta r y l - C o A r e d u c t a s e i n both t h e r a t (44,45) and M. s e x t a C5). A l ­ though the methyl e s t e r o f compactin was i n a c t i v e as an AJH i n our b i o a s s a y , two o f i t s a n a l o g s (L-643,049-01K01 and L-643,737-

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00S03) d i d d i s p l a y AJH a c t i v i t y . Thus, f o r T. n i , a n a l o g s o f compactin would appear t o be good c a n d i d a t e s f o r c o n t i n u e d e x p l o r a t i o n as p o t e n t i a l AJHs. Acknowledgment s We thank B. Bondy f o r a s s i s t a n c e i n r e a r i n g the i n s e c t s , and G. Q u i s t a d , D. S c h o o l e y , G. S t a a l (Zoecon C o r p . ) , P. Masner ( D r . R. Maag L t d . ) , P. Magee (Chevron Chemical C o . ) , and R. A. Dybas (Merck, Sharp and Dohme Research L a b o r a t o r i e s ) f o r p r o v i d i n g many o f the compounds used i n t h i s s t u d y . T h i s r e s e a r c h was s u p p o r t e d , i n p a r t , by Merck, Sharp and Dohme, the L o u i s i a n a A g r i c u l t u r a l E x p e r i ­ ment S t a t i o n , B a s i c Research Grant No. 32-003 from L o u i s i a n a S t a t e U n i v e r s i t y ( t o TCS), N o r t h C a r o l i n a S t a t e U n i v e r s i t y , Department o f H e a l t h and Human R e s o u r c e s , N a t i o n a l S e r v i c e Award ( t o RMR) F32 GM09223-02 from the N a t i o n a l I n s t i t u t e o f G e n e r a l M e d i c a l S c i e n c e , and Grant No. 2 Ro 1 ES02710-04 and Research C a r e e r Development Award 5 K04 ES00107-05 ( t Environmental Health Sciences

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15. Hammock, B. D. In "Comprehensive Insect Physiology, Biochemistry and Pharmacology"; Kerkut, G. Α.; Gilbert, L. I. Eds.; Pergamon Press: New York, 1984; Vol. 7, In Press. 16. Sparks, T. C.; Hammock, B. D. J. Insect Physiol. 1979, 25, 551-60. 17. Jones, G.; Hammock, B. D. J. Insect Physiol. 1983, 29, 471-5. 18. Sparks, T. C. J. Insect Physiol. 1984, 30, 225-34. 19. Sparks, T. C.; Hammock, B. D. Pestic. Biochem. Physiol. 1980, 14, 290-302. 20. Pratt, G. E.; Finney, J. R. In "Crop Protection Agents - Their Biological Evaluation"; McFarlane, N. R. Ed.; Academic Press: New York, 1976; pp. 113-32. 21. Edwards, J. P.; Bergot, B. J.; Staal, G. B. J. Insect Physiol. 1983, 29, 83-9. 22. Quistad, G. B.; Cerf, D. C.; Schooley, D. Α.; Staal, G. B. Nature. 1981, 289, 176-7. 23. Quistad, G. B.; Staiger, L. E.; Cerf, D. C. J. Agric. Food Chem. 1982, 30, 1151-4 24. Beckage, N. E.; Riddiford 633-7. 25. Farag, A. I.; Varjas, L. Ent. exp. & appl. 1983, 34, 65-70. 26. Sparks, T. C.; Wing, K. D.; Hammock, B. D. Life Sciences. 1979, 25, 445-50. 27. Kiguchi, K.; Mori, T.; Akai, H. J. Insect Physiol. 1984, 30, 499-506. 28. Hammock, B. D.; Kuwano, E.; Ketterman, Α.; Scheffrahn, R. H.; Thompson, S. N.; Sallume, D. J. Agric. Food Chem. 1978, 26, 166-70. 29. Hiruma, K.; Yagi, S.; Endo, A. Appl. Ent. Zool. 1983, 18, 111-5. 30. Hammock, B. D.; Wing, K. D.; McLaughlin, J.; Lovell, V. M.; Sparks, T. C. Pestic. Biochem. Physiol. 1982, 17, 76-88. 31. Sparks, T. C.; Willis, W. S.; Shorey, Η. H.; Hammock, B. D. J. Insect Physiol. 1979, 25, 125-32. 32. Hammock, B. D.; Sparks, T. C. Analyt. Biochem. 1977, 82, 573-9. 33. Hammock, B. D.; Roe, R. M. Methods in Enzymology, 1984, In Press. 34. Jones, G.; Hammock, B. D. J. Insect Physiol. 1985, 31, In Press. 35. Cymborowski, B.; Stolarz, G. J. Insect Physiol. 1979, 25, 939-42. 36. Hiruma, K. Gen. Comp. Endocr. 1980, 41, 392-9. 37. Sieber, R.; Benz, G. Physiol. Ent. 1980, 5, 283-90. 38. Safranek, L.; Cymborowski, B.; Williams, C. M. Biol. Bull. mar. biol. Lab. Woods Hole 1980, 158, 248-56. 39. Abdel-Aal, Υ. A. I.; Roe, R. M.; Hammock, B. D. Pestic. Biochem. Physiol. 1984, 21, 232-41. 40. Prestwich, G. D.; Eng, W. -S.; Roe, R. M.; Hammock, B. D. Arch. Biochem. Biophys. 1984, 228, 639-45. 41. Sparks, T. C.; Wing, K. D.; Hammock, B. D. Unpublished data. 42. Granger, Ν. Α.; Bollenbacher, W. E. In "Metamorphosis, A Problem in Developmental Biology"; Gilbert, L. I.; Frieden, E. Eds.; Plenum Press: New York, 1981; pp. 105-37. 43. Sparks, T. C.; Hammock, B. D.; Riddiford, L. M. Insect Biochem. 1983, 13, 529-41. 44. Endo, Α.; Kuroda, M.; Tanzawa, K. FEBS Letters 1976, 72, 323-6. 45. Endo, A. TIBS 1981, 6, 10-13. RECEIVED November 8,

1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

22 Applications of Immunoassay to Paraquat and Other Pesticides J . M. VAN EMON, J. N. S E I B E R , and B. D .

HAMMOCK

Department of Environmental Toxicology, University of California, Davis, CA 95616

The enzyme-linked immunosorbent assay (ELISA) is a rapid immunochemical procedure which can be used for trace analysis. W paraquat and othe the more classical methods. The immunoassay for paraquat shows the practicality of the method for fortified and actual residue samples, and is being compared with a gas chromatography procedure. Our work with the ELISA illustrates that the immunochemical technology can be used to solve problems encountered in pesticide residue analysis. I t h a s been s t a t e d t h a t p r o g r e s s i n p e s t i c i d e a n a l y s i s w i l l no l o n g e r be made i n s e a r c h o f t h e p r o v e r b i a l z e r o r e s i d u e l e v e l o f d e t e c t a b i l i t y , but r a t h e r w i l l l i e i n d e v i s i n g methods o f g r e a t e r s e l e c t i v i t y f o r t h e p o s i t i v e i d e n t i f i c a t i o n o f nanogram q u a n t i t i e s o f p e s t i c i d e r e s i d u e s (J_). We c o u l d add t o t h i s statement t h e need f o r a s s a y s o f g r e a t l y reduced c o s t and i n c r e a s e d speed. Many r e s i d u e p r o c e d u r e s a r e t o o e x p e n s i v e t o be used r o u t i n e l y i n r e g u l a t o r y p r o c e d u r e s o r , perhaps o f g r e a t e r importance, t o be employed e f f e c t i v e l y i n o p t i m i z i n g p e s t i c i d e usage and m o n i t o r i n g worker h e a l t h and s a f e t y . Immunochemical methods o f a n a l y s i s o f f e r many advantages, i n c l u d i n g s e n s i t i v i t y , s p e c i f i c i t y , and speed o f a n a l y s i s (2). Compounds which a r e most d i f f i c u l t t o a n a l y z e by c l a s s i c a l p r o c e d u r e s due t o h i g h p o l a r i t y o r low v o l a t i l i t y a r e f r e q u e n t l y amenable t o a n a l y s i s by immunochemistry. We a r e now i n v e s t i g a t i n g enzyme-linked immunosorbent a s s a y s (ELISAs) r a t h e r t h a n radioimmunoassays (RIAs) f o r p e s t i c i d e r e s i d u e work. ELISAs a r e q u i c k e r , cheaper, and s a f e r than RIAs a s r a d i o a c t i v i t y i s n o t used. However, a s a l l immunoassays f u n c t i o n on t h e p r i n c i p l e o f mass a c t i o n , t h e same immunochemical t o o l s c a n be used t o d e v i s e a number o f d i f f e r e n t a s s a y p r o c e d u r e s . G e n e r a l ELISA Methodology Immunoassays a r e p h y s i c a l r a t h e r than b i o l o g i c a l a s s a y s ; they p o s s e s s t h e s p e c i f i c i t y and s e n s i t i v i t y o f b i o a s s a y s w i t h t h e speed

0097-6156/ 85/ 0276-0307S06.00/ 0 © 1985 American Chemical Society

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and p r e c i s i o n o f p h y s i c a l a s s a y s . S p e c i f i c antibodies are raised i n an e x p e r i m e n t a l a n i m a l i n r e s p o n s e t o a l a r g e f o r e i g n m o l e c u l e ( a n t i g e n ) . Most p e s t i c i d e m o l e c u l e s a r e n o t l a r g e enough t o s t i m u l a t e the immune system, and must be c o n j u g a t e d t o a l a r g e m o l e c u l e such as a p r o t e i n . A n t i b o d i e s a g a i n s t t h e p e s t i c i d e a r e then obtained u s i n g t h i s p e s t i c i d e - p r o t e i n conjugate. A small m o l e c u l e which becomes immunogenic a f t e r attachment t o t h e l a r g e c a r r i e r m o l e c u l e i s termed a h a p t e n . The p e s t i c i d e may a l r e a d y have a f u n c t i o n a l i t y , such as -OH, -SH, -C00H, o r -NHg, u s e f u l f o r c o n j u g a t i o n t o the c a r r i e r , but f r e q u e n t l y a d e r i v a t i v e o f t h e p e s t i c i d e p o s s e s s i n g such f u n c t i o n a l i t y must f i r s t be s y n t h e s i z e d b e f o r e c o n j u g a t i o n can o c c u r . I n e i t h e r c a s e a n t i b o d i e s can be o b t a i n e d which a r e d i r e c t e d a g a i n s t t h e o r i g i n a l p e s t i c i d e . Serum a n t i b o d i e s a r e heterogeneous m o l e c u l e s o f v a r y i n g a n t i g e n s p e c i f i c i t y and a f f i n i t y . Within t h i s p o l y c l o n a l p o p u l a t i o n , there are probably a n t i b o d i e s present which r e c o g n i z e t h e c a r r i e r p r o t e i n , but t h e c o n t r i b u t i o n o f t h e s e a n t i b o d i e s t o a s s a y b i n d i n g can be e l i m i n a t e (£). Monoclonal antibod the c l o n e with the h i g h e s t r e c o g n i t i o n t o hapten. Although p o l y c l o n a l a n t i b o d i e s a r e adequate f o r most ELISAs, m o n o c l o n a l a n t i b o d i e s c o u l d be d e v e l o p e d a g a i n s t p e s t i c i d e h a p t e n s y i e l d i n g an a n a l y t i c a l r e a g e n t t h a t i s p h y s i c a l l y , c h e m i c a l l y , and i m m u n o l o g i c a l l y homogeneous. As m o n o c l o n a l a n t i b o d i e s would be i n a v i r t u a l l y u n l i m i t e d s u p p l y , ELISAs c o u l d be e a s i l y s t a n d a r d i z e d as s e v e r a l l a b o r a t o r i e s would be u s i n g t h e same a n t i b o d y c l o n e . A l t h o u g h a v e r y s m a l l amount o f a n t i b o d y i s needed p e r ELISA, having a l a r g e supply o f monoclonals could a l l e v i a t e f e a r s o f e v e n t u a l l y e x h a u s t i n g one's s u p p l y . Hammock and Mumma (£) d i s c u s s some o f t h e advantages o f hybridoma t e c h n o l o g y . Yet, i t i s i m p o r t a n t t o c o n s i d e r t h a t , i n some c a s e s , p o l y c l o n a l s w i l l be s u p e r i o r t o m o n o c l o n a l a n t i b o d i e s , u n l e s s one i s d e v e l o p i n g a s o p h i s t i c a t e d system f o r a v o i d i n g t h e s e p a r a t i o n s t e p i n p e s t i c i d e Immunoassay, i t w i l l be a r a r e s i t u a t i o n where p r o d u c t i o n o f m o n o c l o n a l a n t i b o d i e s f o r p e s t i c i d e r e s i d u e a n a l y s i s can be j u s t i f i e d on a p u r e l y s c i e n t i f i c b a s i s . However, as immunoassay o f p e s t i c i d e s moves i n t o t h e p r i v a t e s e c t o r , t h e r e w i l l be c o m p e l l i n g a d m i n i s t r a t i v e and l e g a l p r e s s u r e s t o employ m o n o c l o n a l a n t i b o d i e s . F o r t h e s e r e a s o n s t h e y may dominate t h e f i e l d i n a few y e a r s . The ELISA i s based on t h e f a c t t h a t a n t i g e n o r a n t i b o d y can be attached t o a solid-phase support while r e t a i n i n g immunological a c t i v i t y , and t h a t e i t h e r a n t i g e n o r a n t i b o d y can be l i n k e d t o an enzyme w i t h t h e complex r e t a i n i n g both immunological and enzymatic activity. A v a r i e t y o f enzymes, i n c l u d i n g a l k a l i n e phosphatase, h o r s e r a d i s h p e r o x i d a s e , and g l u c o s e o x i d a s e have been l i n k e d t o a n t i b o d i e s and a n t i g e n s . T h i s method has been u s e d s u c c e s s f u l l y f o r d e t e c t i o n o f e i t h e r a n t i g e n o r a n t i b o d y (3-4) and i t has been u s e d by us f o r t h e d e t e c t i o n o f n u c l e o t i d e s , i n s e c t i c i d e s , s u r f a c t a n t s and a v a r i e t y o f o t h e r compounds. To p e r f o r m a m i c r o p l a t e ELISA f o r the measurement o f a n t i g e n , p l a t e s s e n s i t i z e d with the s p e c i f i c antigen are incubated with a m i x t u r e o f r e f e r e n c e a n t i b o d y and the t e s t sample. I f a n t i g e n i s p r e s e n t i n t h e t e s t s o l u t i o n , i t combines w i t h t h e r e f e r e n c e a n t i b o d y which cannot then r e a c t w i t h the s e n s i t i z e d p l a t e . The amount o f a n t i b o d y a t t a c h e d t o t h e s o l i d phase i s t h e n i n d i c a t e d by f

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an enzyme l a b e l l e d anti-immunoglobulin conjugate and enzyme substrate. There i s a proportional r e l a t i o n s h i p between the amount of i n h i b i t i o n of substrate converted to products i n the t e s t sample and to the amount of antigen i n the test system. We routinely run samples i n p l a s t i c plates containing 50 wells with which a l l of the procedures can be executed very r a p i d l y . The end point of the assay i s the bright yellow color of p-nitrophenol, an end product from the a l k a l i n e phosphatase-mediated hydrolysis of p-nitrophenyl phosphate; t h i s product can be v i s u a l l y estimated f o r semi-quantitative answers or r a p i d l y and p r e c i s e l y measured i n an inexpensive colorimeter f o r quantitation. Immunoassays can be designed to analyze parent compounds and metabolites separately or as a group. Ercegovich (5.) obtained antibodies against parathlon which also detected i t s metabolite p-nitrophenol. Other workers were successful i n r a i s i n g antibodies to DDT and malathion metabolites (6-7). Although the s p e c i f i c i t y of immunoassays are usually very high there i s no guarantee against cros techniques require controls immunoassays are quickly and e a s i l y performed so that the necessary controls can be run to check f o r interferences. The s e n s i t i v i t y and s e l e c t i v i t y of immunoassays can also greatly reduce the cost of analysis by minimizing the amount of sample preparation. Examples of Pesticide Immunoassays We w i l l f i r s t review several of the immunoassays developed i n t h i s laboratory as they i l l u s t r a t e some of the advantages of immunoassay i n pesticide residue analysis. Then we w i l l move on to a more detailed discussion of our recent a n a l y t i c a l work with the herbicide paraquat. Diflubenzuron. The benzoylphenyl urea insect growth regulators, f o r example, pose a formidable residue analysis problem. The compounds are nonvolatile and thus must be derivatized f o r GC analysis by a rather arduous chemical procedure. The immunoassay developed i n t h i s laboratory i s much more s e n s i t i v e and reproducible at a f r a c t i o n of the cost and can be used to analyze the more d i f f i c u l t matrices such as milk. For instance, a s e n s i t i v i t y of 1 ppb i s routinely obtained when milk i s added d i r e c t l y to the assay (£.)· A s e r i e s of p a r t i t i o n steps can also be added to further clean diflubenzuron milk extracts y i e l d i n g a s e n s i t i v i t y i n the low ppt range (£.). However t h i s increase i n s e n s i t i v i t y may not be needed since methods i n current use provide a detection l i m i t of only 10-50 ppb. An impressive aspect of Immunoassays i s t h e i r s p e c i f i c i t y . One immunoassay f o r diflubenzuron can d i s t i n g u i s h i t from the very c l o s e l y r e l a t e d BAY SIR 8514 as well as a v a r i e t y of other c l o s e l y related materials (£)· High resolution HPLC columns can resolve these compounds but the analysis i s slow and expensive. The ELISA can d i s t i n g u i s h these materials when applied d i r e c t l y , or l e s s s p e c i f i c assays can be used as a highly selective detector when used as an adjunct to HPLC.

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^ 0 Η 0 H

^ 0 Η 0 H -N-C-N-^^-0CF

Diflubenzuron

F

3

BAY SIR 8514

Surfactants have many i n d u s t r i a l applications and are found i n such diverse products as pesticide formulations and cosmetics. These nonionic compounds are d i f f i c u l t to extract, clean up, and analyze and, consequently, no s e n s i t i v e method existed f o r t h e i r analysis. An ELISA was developed i n t h i s laboratory which distinguishes between the nonionic surfactants T r i t o n X and T r i t o n Ν (10). These compounds, mixtures o f ethoxylates o f varying length of 4-(1,1,3f3-tetramethylbutyl)phenol and 4-nonylphenol, give numerous overlapping peaks upon chromatographic analysis and have almost i d e n t i c a l UV and IR spectra An ELISA has been developed f o r class s e l e c t i v e detectio detects the 4-(1,1,3 3-tetramethylbutyl)pheny distinguish among molecules with ethoxylated side chains of varying lengths. f

CH

3

HC 3

CH -Ç-CH -ÇH^^-(OCH CH )^OH 3

2

2

CH

3

2

C H -^^-(0CH CH )^0H 9

I 9

2

H C ~~ Triton X

2

3

Triton Ν ,

S - B i o a l l e t h r i n . The pyrethroid S - b i o a l l e t h r i n (1R,3R,4 S) and i t s i n a c t i v e isomer (1S,3S,4 R) can be r e a d i l y distinguished by another ELISA procedure, i l l u s t r a t i n g the assay's a b i l i t y to determine c h i r a l i t y at the residue l e v e l (Figure 1). Antibodies were raised against S - b i o a l l e t h r i n using the a l l e t h r i n hemisuccinate conjugated to various proteins (11-12). f

0 S-Bioallethrin

0 1S,3S,4'R Allethrin

Pyrethroids, as well as some carbamate and organophosphate i n s e c t i c i d e s , are marketed as isomer mixtures, each isomer having a d i f f e r e n t degree of a c t i v i t y . I t can be expected that environmental degradation and metabolism w i l l occur p r e f e r e n t i a l l y with some isomers. Thus the a b i l i t y to distinguish between o p t i c a l isomers a t the residue l e v e l may be of c r i t i c a l importance i n monitoring the safety of treated substances.

Paraquat During a recent year, over 950,000 pounds o f paraquat dichloride

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( I j I ' - d i m e t h y l - ^ ' - b i p y r i d i n i u m d i c h l o r i d e ) was used i n C a l i f o r n i a a g r i c u l t u r e (11). Much o f t h i s h e r b i c i d e was used on c o t t o n , p r i m a r i l y as a h a r v e s t a i d . The v a l u e o f t h i s c o t t o n p r o d u c t i o n i s e s t i m a t e d t o exceed one b i l l i o n d o l l a r s ( H ) . Although i t i s e x t e n s i v e l y used, l i t t l e i s known r e g a r d i n g t h e l o n g term c h r o n i c e f f e c t s o f paraquat exposure. P a r a q u a t has a number o f b i o l o g i c a l e f f e c t s , but i t s main b i o c h e m i c a l a c t i o n i s a p p a r e n t l y as a p o t e n t redox u n c o u p l e r . R e g a r d l e s s o f t h e r o u t e o f a d m i n i s t r a t i o n , symptoms o f p a r a q u a t p o i s o n i n g a r e c e n t e r e d i n the l u n g s l e a d i n g t o f i b r o s i s and pneumonia (15.). Submicrogram q u a n t i t i e s o f p a r a q u a t d e p o s i t e d i n t h e l u n g can cause f i b r o t i c l e s i o n s (16) which c o u l d l e a d t o asthma and emphysema symptoms i n c h r o n i c a l l y exposed i n d i v i d u a l s (11). u n f o r t u n a t e l y , the c o n v e n t i o n a l methods o f a n a l y s i s o f p a r a q u a t a r e t o o l a b o r i o u s and/or i n s e n s i t i v e t o h a n d l e t h e l a r g e sample l o a d g e n e r a t e d by r i g o r o u s s t u d i e s needed t o e v a l u a t e human exposure. Immunochemical p r o c e d u r e difficulty. F a c t o r ! an f o r p a r a q u a t , and N i e w o l a e t a l . , (H) r e p o r t e d an ELISA f o r e s t i m a t i n g p a r a q u a t i n serum. T h i s ELISA i s s i m i l a r t o o u r s but u s e s a d i f f e r e n t enzyme, i n c u b a t i o n t e m p e r a t u r e and time, and shows a h i g h degree o f c r o s s r e a c t i v i t y w i t h e t h y l p a r a q u a t . Methods s i m i l a r t o t h o s e p r e v i o u s l y r e p o r t e d were u s e d t o g e n e r a t e p a r a q u a t h a p t e n s f o r our s t u d y . N - M e t h y l - 4 - ( 4 - p y r i d y l ) p y r i d i n i u m bromide was r e a c t e d w i t h e t h y l 5 - b r o m o v a l e r a t e f o r m i n g a p a r a q u a t analogue c a p a b l e o f c o n j u g a t i n g w i t h a p r o t e i n . New Z e a l a n d w h i t e r a b b i t s were i n j e c t e d w i t h a p a r a q u a t - p r o t e i n c o n j u g a t e and a n t i b o d i e s c a p a b l e o f r e c o g n i z i n g p a r a q u a t were o b t a i n e d . The a n t i b o d i e s a r e q u i t e s e l e c t i v e showing no c r o s s r e a c t i v i t y t o compounds s t r u c t u r a l l y r e l a t e d t o paraquat (e.g. e t h y l paraquat, N j N ' - d i m e t h y l - i j j l p - b i p i p e r i d i n e ) o r compounds used i n c o n j u n c t i o n w i t h paraquat (e.g. d i q u a t ) . Using the s e l e c t i v e antibody f o r paraquat, environmental samples can be a n a l y z e d w i t h l i t t l e o r no c l e a n u p . Sample t h r o u g h p u t can be measured i n samples p e r hour r a t h e r t h a n days p e r sample. T h i s was a r e m a r k a b l e d i s c o v e r y as p a r a q u a t i s n o t o r i o u s f o r r e q u i r i n g e x t e n s i v e sample p r e p a r a t i o n . The l i m i t o f d e t e c t i o n (2 ng/ml) i n the ELISA p r o c e d u r e i s a l s o much l o w e r t h a n the c o l o r i m e t r i c p r o c e d u r e , which has a s e n s i t i v i t y o f 200 ng/ml ( 2 0 ) , and l o w e r t h a n t h e GC p r o c e d u r e ( F i g u r e 2) ( 2 1 ) . Perhaps the g r e a t e s t advantage i s t h e speed o f t h e ELISA e n a b l i n g l a r g e numbers o f samples t o be a n a l y z e d w i t h o u t r e q u i r i n g an e x t e n s i v e c l e a n u p procedure, thus r e d u c i n g a n a l y t i c a l time. G a s - l i q u i d chromatography f o l l o w i n g r e d u c t i o n o f p a r a q u a t t o t h e mono- and d i u n s a t u r a t e d d e r i v a t i v e s (£1) i s o f adequate s e n s i t i v i t y f o r most work when N - s e l e c t i v e d e t e c t o r s a r e employed. S e i b e r and Woodrow (22.) m o d i f i e d t h i s method f o r a s s a y i n g p a r a q u a t i n a i r samples. The method i s time consuming and l a b o r i n t e n s i v e , i n v o l v i n g a c i d e x t r a c t i o n and many c o n c e n t r a t i o n and evaporation steps. The maximum sample o u t p u t per a n a l y s t p e r day i s 6-8 w i t h no d u p l i c a t e s . The r e p o r t e d r e c o v e r y e f f i c i e n c y was 75$ (22.) a l t h o u g h an e f f i c i e n c y c l o s e r t o 50$ i s f r e q u e n t l y encountered i n p r a c t i c e . A m o d i f i e d a c i d e x t r a c t i o n combined w i t h a n a l y s i s by t h e ELISA p r o v i d e s r e c o v e r i e s o f 75$ ( F i g u r e 3 ) . This 9

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

VAN EMON ET AL.

Immunoassay Applications to Pesticides

PARAQUAT

PARAQUAT

CATION

CATION

ng/ml

ug/mI

F i g u r e 2. S t a n d a r d c u r v e s f o r p a r a q u a t u s i n g ELISA ( m i d d l e ) , and c o l o r i m e t r y ( b o t t o m ) .

(top),

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST CONTROL

Incubate with Antibody Glass Fiber F i l t e r

^ELISA

60$ E x t r a c t i o n

I

Efficiency

2 steps

E x t r a c t w i t h 6N HC1 Concentrate & C e n t r i f u g e

Paraquat i n 6N HC1

Evaporate 5 Steps Extract with sat'd

NH HC0 4

3

Centrifuge

P a r a q u a t i n s a t ' d NH^HCO,

Evaporate D i s s o l v e i n NaOH (NaBH^) Heat (60°C) f o r 12 min Cool, extract with ethyl acetate

Reduced P a r a q u a t

(monoene & d i e n e ) i n e t h y l a c e t a t e

Gas Chromatography

50$ E x t r a c t i o n E f f i c i e n c y 11 S t e p s DB-1 30 m c a p i l l a r y NP d e t e c t i o n (HP 5710A) Sum monoene and d i e n e peaks

F i g u r e 3. Comparison o f sample p r e p a r a t i o n s t e p s f o r a n a l y s i s of p a r a q u a t on a i r f i l t e r s u s i n g ELISA and gas chromatography.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

22.

VAN EMON ET AL.

Immunoassay Applications to Pesticides

315

i l l u s t r a t e s t h e s u c c e s s f u l a d a p t a t i o n o f an e x t r a c t i o n t e c h n i q u e i n t e n d e d f o r use w i t h GC d e t e r m i n a t i o n t o an immunochemical method o f a n a l y s i s . A l t e r n a t i v e l y , t h e a n t i b o d y i t s e l f can be used t o e x t r a c t p a r a q u a t from a g l a s s f i b e r f i l t e r , e l i m i n a t i n g the time-intensive e x t r a c t i o n procedure. Using the s p e c i f i c a n t i b o d i e s f o r e x t r a c t i o n w i t h subsequent d e t e c t i o n by ELISA, 50 samples can be a n a l y z e d I n t r i p l i c a t e i n one day w i t h a r o u t i n e e x t r a c t i o n e f f i c i e n c y o f 60$ ( F i g u r e 3). However, t h i s method u s e s a r a t h e r l a r g e amount o f a n t i b o d y making t h e m o d i f i e d a c i d e x t r a c t i o n p r o c e d u r e p r e f e r a b l e f o r a n a l y z i n g a l a r g e sample l o a d . T h i s r a p i d s a m p l e - p r o c e s s i n g c a p a b i l i t y makes i t f e a s i b l e t o measure exposure o f f i e l d w o r k e r s l i t e r a l l y on t h e same day t h e exposures occur. I n o r d e r t o t e s t t h i s c a p a b i l i t y a i r s a m p l i n g was c o n d u c t e d i n t h e San J o a q u i n v a l l e y d u r i n g a p a r a q u a t a p p l i c a t i o n on c o t t o n , and t h e samples a r e now b e i n g a n a l y z e d u s i n g both ELISA and GC methods t o compare t h e two t e c h n i q u e s i n terms o f speed, p r e c i s i o n (by c a l c u l a t i n g t h e p e r c e n t c o e f f i c i e n t o f v a r i a t i o n ) and a c c u r a c y (by comparing r e s u l t techniques). Preliminar w e l l w i t h GC l i t e r a t u r e v a l u e s i n t h e s e t h r e e p a r a m e t e r s and a d d i t i o n a l l y has the a n t i c i p a t e d g r e a t e r sample throughput capability. Conclusions I t i s p r o b a b l e t h a t i n c e r t a i n s i t u a t i o n s immunochemical methods w i l l provide d i s t i n c t advantages over c o n v e n t i o n a l a n a l y t i c a l methods. However, i t i s u n l i k e l y t h a t immunochemical methods w i l l c o m p l e t e l y r e p l a c e c u r r e n t , e s t a b l i s h e d a n a l y t i c a l methods o f p e s t i c i d e a n a l y s i s (JL). T h i s i s i n s p i t e o f the f a c t t h a t c h e m i c a l c l a s s e s c u r r e n t l y a s s a y e d by immunochemical t e c h n i q u e s i n c l i n i c a l a n a l y t i c a l l a b s c o n t a i n t h e same t y p e o f f u n c t i o n a l groups as many p e s t i c i d e s . ELISA c o u l d p o t e n t i a l l y be used a d v a n t a g e o u s l y i n many t y p e s o f exposure and m o n i t o r i n g s i t u a t i o n s , f o r p a r a q u a t and o t h e r p e s t i c i d e s amenable t o ELISA a n a l y s i s . An o b v i o u s use o f ELISA i s t h e d e t e c t i o n o f p e s t i c i d e r e s i d u e l e v e l s i n p l a n t and a n i m a l t i s s u e s , o r f o o d e x t r a c t s . B i o l o g i c a l specimens such as plasma and u r i n e c u r r e n t l y a n a l y z e d by RIA seem p a r t i c u l a r l y amenable t o a n a l y s i s by ELISA. P o r t a b l e f i e l d k i t s c o u l d be d e v e l o p e d t o d e t e r m i n e s a f e worker r e - e n t r y t i m e s i n t o t r e a t e d f i e l d s . E n v i r o n m e n t a l samples s u c h as s o i l , water, and a i r , can be a n a l y z e d by t h e ELISA. P e s t i c i d e c o n j u g a t e s have been p r o p o s e d f o r s k i n t e s t i n g o f i n d i v i d u a l s suspected o f s e n s i t i v i t y t o p e s t i c i d e s (£) ; the ELISA c o u l d be used t o d e t e c t s p e c i f i c a n t i b o d i e s i n t h e s e i n d i v i d u a l s and a i d i n exposure s t u d i e s . A n t i b o d i e s have been r a i s e d a g a i n s t r e p r e s e n t a t i v e compounds from t h e major c l a s s e s o f p e s t i c i d e s . A l t h o u g h t h e ELISA w i l l be u s e f u l f o r i n d i v i d u a l a n a l y s i s o f a wide v a r i e t y o f compounds, i f one needed t o a n a l y z e s e v e r a l d i f f e r e n t compounds s i m u l t a n e o u s l y i n one m a t r i x immunoassay may n o t be the method o f c h o i c e , due t o the l a r g e amount o f c o n t r o l s and s t a n d a r d s needed. However, i t c o u l d be s u c c e s s f u l l y used f o r t h e r a p i d s c r e e n i n g o f a l a r g e number o f samples f o r t h e p r e s e n c e o f s p e c i f i c t y p e s o f p e s t i c i d e s and f o r c o n f i r m a t o r y t e s t s (JL). The work r e p o r t e d h e r e w i t h p a r a q u a t ,

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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allethrin, diflubenzuron, Triton X and Triton Ν provides evidence of the ELISA's ability to distinguish closely related compounds. The ELISA promises to be a good supplement to current methods of residue analysis. Literature Cited 1. Zweig, G. In "Essays in Toxicology"; Blood F.R. Ed.; Academic Press: New York, 1970; Chap. 3. 2. Hammock, B. D.; Mumma R. O. In "Recent Advances in Pesticide Analytical Methodology"; Harvey J.; Zweig, G., Eds.; ACS SYMPOSIUM SERIES No. 136, American Chemical Society: Washington, D.C., 1980; Chap. 18. 3. Voller, Α.; Bidwell, D.E. ; Bartlett, A.BullWorldHealth Org. 1976, 53, 55-65. 4. Van Weeman, B.K.; Schuurs, A.H.W.M. FEBS Letters 1971, 15, 232-6. 5. Ercegovich, C.D. I Level"; Gould, R.F., Ed.; ADVANCES IN CHEMISTRY SERIES No. 104, American Chemical Society: Washington, D.C., 1971; Chap. 11. 6. Centeno, E.R.; Johnson, W.J.; Shehon, A.H. Int. Arch. AllergyAppl.Imm.1970, 37, 1-13. 7. Haas, G.J.; Guardia, E.J.Proc.Soc.Expt.Biol.Med. 1968, 129, 546-51. 8. Wie, S.I.; Hammock, B.D. J. Agric. Food Chem. 1984, Accepted. 9. Wie, S.I.; Hammock, B.D. J, Agric. Food Chem. 1982. 30, 949-57. 10. Wie, S.I.; Hammock, B.D. Anal. Biochem. 1982, 125, 168-176. 11. Wing, K.D.; Hammock, B.D.; Wuster, D.A. J. Agric. Food Chem. 1978, 26, 1328-33. 12. Wing, K.D.; Hammock, B.D. Experientia.1979,35, 1619-20. 13. "Pesticide Use Report by Commodity." California Department of Food and Agriculture, 1983. 14. "California Agriculture." California Department of Food and Agriculture, 1982. 15. Smith, P.; Heath, D. "Paraquat" CRC Crit Revs. Toxicol. 4, 411-45. 16. Zavala, D.C.; Rhodes, M.L. Chest 1978, 74, 418-20. 17. Maddy, K.T. "Human Health Problems with the Herbicide Paraquat in California 1965 through 1974." California Department of Food and Agriculture, 1975. 18. Fatori, D.; Hunter, W.M.ClinicChimicaActa1980,100, 81-90. 19. Niewola, Ζ.; Walsh, S.T.; Davies, G.E.Int.J.Immunopharmac. 1983, 5, 211-18. 20. Lott, P.F.; Lott, J.W. J. Chrom. Sci. 1978, 16, 390-5. 21. van Dijk, Α.; Ebberink, R.; deGroot, G.; Maes, R.A.A.; Douze, J.M.C.; van Heyst, A.N.P. J. Analy. Tox. 1977, 1, 151-54. 22. Seiber, J.N.; Woodrow, J.E. Arch. Environm. Contam. 1981, 10, 133-49. RECEIVED November 8, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

A the

Direct C o r r e l a t i o n Between B i o a s s a y and ELISA for Bacillus thuringiensis var. israelensis δ-endotoxin.

P e t e r Y. K. Cheung and Bruce D. Hammock

Department of Entomology University of California Davis, California 95616

A partially purified Bacillus thuringiensis var. israelensis (Bti) δ-endotoxin was used to immunize rabbits. The antisera obtained have an improved spe­ cificity towards the mosquito larvacidal activity of the toxin, as opposed to antiserum raised when the whole crystal was used as immunogen. Using a two step/indirect ELISA (enzyme linked immunosorbent assay) procedure developed in our laboratory, fourteen experimental formulations were tested, and the results were compared with bioassays. An average of 69.1 international units ± 20% c.v. was found to associate with each ug of toxin detected by the ELISA. Our data indicate that when toxin specific antisera are available, immunoassays can be used to predict the biological activity of Bti samples with reasonable accuracy.

RECEIVED December 17, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

E u p l e c t r u s plathypenae Parasitization of Trichoplusia ni. on Weight G a i n , E c d y s t e r o i d Titer and M o l t i n g .

Thomas A. Coudron and Thomas

J.

Kelly

Effect

1/

U. S. Department o f Agriculture Biological C o n t r o l of I n s e c t s Research L a b o r a t o r y , P. O. Box A Columbia, MO 65205

E u p l e c t r u s spp. (Hymenoptera: E u l o p h i d a e ) a r e g r e ­ g a r i o u s e c t o p a r a s i t e s of s e v e r a l l e p i d o p t e r a n s p e c i e s that are pests to agricultural crops. Parasitization of Trichoplusia ni l a r v a e by E u p l e c t r u s p l a t h y p e n a e results in an inhibition of the larval larval molting p r o c e s s in t h e h o s t . After parasitization the host t e m p o r a r i l y r e f r a i n s from e a t i n g and l a g s b e h i n d t h e s y n c h r o n i z e d n o n - p a r a s i t i z e d l a r v a e in weight g a i n . However t h e parasitized l a r v a e does resume e a t i n g and c o n t i n u e s t o i n c r e a s e its body w e i g h t p r i o r t o t h e development o f t h e p a r a s i t e l a r v a e . There is no s i g n o f new cuticle formation in parasitized larvae. A p r o l o n g e d a s s o c i a t i o n between t h e parasite and h o s t is not n e c e s s a r y t o elicit an effect on t h e h o s t . Larvae w h i c h a r e i m m e d i a t e l y s e p a r a t e d from the parasitic egg will fail t o molt i n t o the next instar. The inhibi­ tion of h o s t molt may be r e l a t e d t o t h e absence o f peak in t h e e c d y s t e r o i d titer i n t h e hemolymph which o c c u r s 20 h r s b e f o r e e c d y s i s in n o n - p a r a s i t i z e d T. ni. T h i s u n i q u e t y p e o f h o s t development c o n t r o l appears t o be distinct from the p a r a l y z i n g effect o f many Hymenoptera venoms and the a l g o g e n i c effects o f some Hemiptera salivary toxins.

RECEIVED February 11, 1985

* Current

address:

I n s e c t R e p r o d u c t i o n L a b o r a t o r y , BARC-E, B l d g . 3 0 6 , B e l t s v i l l e , MD 20705

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

23 Role of Natural Product Chemistry J. R.

PLIMMER

Agrochemicals and Residues Section, Joint

FAO/IAEA

Division, International A t o m i c Energy

Agency, Vienna, Austria

The rapidly expanding growth of knowledge of natural product structures now provide clearer understanding of biochemical mechanisms "biorational" approache control agents. Natural products may be of potential value in pest control in several ways. They may be a source of structures for screening. They may possess activity that is applicable to pest control directly or after structural modification of the original structure. Finally, the recognition of their function in nature may suggest new approaches to pest control. However, their practical application may be limited by economics. Resistant plants are important in managing insect pests and their resistance may arise from many factors. Some plants contain insecticidal principles that may be exploited. Compounds that modify insect behaviour are not directly lethal, but may be valuable in pest control. However, their efficacy may be difficult and costly to determine. During t h e l a t t e r h a l f o f t h e n i n e t e e n t h century and t h e e a r l y decades o f t h e t w e n t i e t h c e n t u r y , t h e groundwork o f s t r u c t u r a l o r g a n i c c h e m i s t r y was l a i d . I n 1828, t h e theory t h a t a " v i t a l f o r c e * was u t i l i z e d i n p l a n t s a n d a n i m a l s t o e l a b o r a t e n a t u r a l p r o d u c t s w a s d i s p r o v e d , when W o h l e r a n n o u n c e d t h a t u r e a c o u l d b e f o r m e d b y h e a t i n g ammonium c y a n a t e . The i n v e s t i g a t i o n o f t h e t h e o r e t i c a l basis o f organic chemistry received i t s stimulus not only from the contemporary s p i r i t o f p h i l o s o p h i c a l i n q u i r y , b u t a l s o from t h e p r o s p e c t s o f a p r a c t i c a l outcome. The i n d u s t r i a l r e v o l u t i o n had as i t s b a s i s t h e a v a i l a b i l i t y o f energy i n t h e form o f c o a l . C o a l g a s was u s e d f o r l i g h t i n g e a r l y i n t h e nineteenth century. C o a l t a r was r e c o g n i z e d a s a s o u r c e o f o r g a n i c c h e m i c a l s , a n d W.H. P e r k i n ' s d i s c o v e r y o f t h e f i r s t c o a l t a r d y e i n 1 8 5 6 o c c u r r e d when h e a t t e m p t e d p r e p a r a t i o n o f q u i n i n e by o x i d a t i o n o f a n i l i n e . Subsequently, s y n t h e t i c d y e s t u f f s r a p i d l y r e p l a c e d those from n a t u r a l sources. The a b i l i t y t o 1

0097-6156/ 85/0276-0323$06.00/0 © 1985 American Chemical Society

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produce a v a r i e t y o f s y n t h e t i c chemicals from c o a l t a r and subsequently from petroleum feedstocks has g r e a t l y i n f l u e n c e d t h e development o f t h e chemical i n d u s t r y . The p r e v a l e n c e o f s t r u c t u r a l t y p e s b a s e d o n a r o m a t i c o r h e t e r o a r o m a t i c n u c l e i among c h e m i c a l s a v a i l a b l e f r o m c o a l t a r o r petroleum contrasts with t h e s t r u c t u r a l types that represent t h e major groups o f n a t u r a l products. Chemicals from n a t u r a l sources are a r i c h source o f s t r u c t u r a l d i v e r s i t y and r e f l e c t t h e c o m p l e x i t y o f b i o l o g i c a l systems i n which t h e processes o f b i o s y n t h e s i s and t h e b i o l o g i c a l r o l e s o f m o l e c u l e s have undergone c o n t i n u a l change and m o d i f i c a t i o n . N a t u r a l product chemistry remains a c h a l l e n g i n g branch o f study. Progress i n both theory and p r a c t i c e o f o r g a n i c chemistry has h a s t e n e d t h e p r o c e s s o f e l u c i d a t i o n o f m o l e c u l a r s t r u c t u r e . The p a u c i t y o f s t a r t i n g m a t e r i a l a n d r e l a t i v e l y p r i m i t i v e t e c h n o l o g y h a d , i n some c a s e s , m e a n t t h a t a l m o s t a c e n t u r y was required t o establish details o f molecular structure. Molecules of even moderate c o m p l e x i t modern i n s t r u m e n t a l t e c h n i q u e s e p a r a t i o n , c h a r a c t e r i z a t i o n , and s t e r e o c h e m i s t r y . W i t h i n t h e l a s t t h r e e d e c a d e s , i t h a s become p o s s i b l e t o d e r i v e s t r u c t u r a l i n f o r m a t i o n f r o m a f e w nanograms o f m a t e r i a l and t h e e l u c i d a t i o n o f s t r u c t u r e a n d s y n t h e s i s o f b i o l o g i c a l p o l y m e r s h a s now become almost r o u t i n e . I n addition, a better understanding o f c o n f o r m a t i o n a l p r i n c i p l e s and t h e i n f l u e n c e o f s t e r e o c h e m i s t r y on r e a c t i v i t y o f m o l e c u l e s now c o n t r i b u t e s g r e a t l y t o r a p i d elucidation of structure. Biorational

Approaches

Such i n f o r m a t i o n has l e d t o e x p l o s i v e growth i n t h e u n d e r s t a n d i n g of biochemical processes. Knowledge o f metabolism, b i o s y n t h e t i c p r o c e s s e s , n e u r o c h e m i s t r y , r e g u l a t o r y m e c h a n i s m s , a n d many o t h e r aspects o f p l a n t , animal and i n s e c t b i o c h e m i s t r y has p r o v i d e d a b a s i s o n w h i c h t h e mode o f a c t i o n o f a p e s t i c i d e may o f t e n b e more c l e a r l y u n d e r s t o o d . The e x p l o i t a t i o n o f b i o l o g i c a l i n f o r m a t i o n c a n l e a d t o t h e s y n t h e s i s o f a new m o l e c u l e d e s i g n e d to act a t a p a r t i c u l a r s i t e o r block a key step i n a biochemical process. The p o t e n t i a l v a l u e o f t h i s a p p r o a c h h a s g e n e r a t e d increasing interest i nthefunctioning of insect neurosecretory systems. T h a t s e l e c t i v e c h e m i c a l c o n t r o l methods m i g h t emerge f r o m a n i n c r e a s e d u n d e r s t a n d i n g o f i n s e c t b i o c h e m i s t r y became apparent w i t h t h e d i s c o v e r y o f t h e i n s e c t j u v e n i l e hormones. I n v e s t i g a t i o n o f s e c r e t i o n s from the corpora a l l a t a l e d t o t h e i d e n t i f i c a t i o n o f i n s e c t j u v e n i l e hormones and p r o v i d e d a stimulus f o r t h e study o f the molecular b a s i s o f i n s e c t p h y s i o l o g y ( 1 , 2, 3 ) . J u v e n i l e hormone a n a l o g u e s a r e p r o v i n g very valuable f o r selective control o f insects. A potentially more a t t r a c t i v e method a p p e a r s t o be t h e u s e o f c h e m i c a l s t h a t w o u l d b l o c k j u v e n i l e hormone p r o d u c t i o n , b e c a u s e s u c h compounds would shorten t h e l i f e t i m e o f t h e l a r v a e , t h e l i f e stage o f t h e i n s e c t w h i c h i n f l i c t s m o s t damage o n t h e h o s t . Screening of

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

23.

PLIMMER

Role of Natural Product Chemistry

p l a n t e x t r a c t s l e d to the d i s c o v e r y t h a t precocenes (from the p l a n t A g e r a t u m h o u s t o n i a n i u m ) p o s s e s s e d t h i s p r o p e r t y (4), but t h e a c t i v i t y i s c o n s i d e r e d t o r e s u l t f r o m c y t o t o x i c i t y and d e g e n e r a t i o n o f t h e c o r p o r a a l l a t a (5, 6) and n o t f r o m s p e c i f i c interference with biosynthetic routes. I n v e s t i g a t i o n of the b i o s y n t h e t i c pathways i n v o l v e d i n the p r o d u c t i o n o f j u v e n i l e hormones (tetrahydro-4-fluoromethyl-4hydroxy-2H-pyran-2-one) i n the c o r p o r a a l l a t a r e v e a l e d the i n v o l v e m e n t o f m e v a l o n o l a c t o n e as an i n t e r m e d i a t e and l e d t o t h e s y n t h e s i s of p o t e n t i a l b l o c k i n g agents w i t h the subsequent d i s c o v e r y of the a c t i v i t y of fluoromeralonolactone as an i n h i b i t o r o f j u v e n i l e hormone b i o s y n t h e s i s (7). E x p l o i t a t i o n of t h e s e f i n d i n g s has c o n t i n u e d i n a s e a r c h f o r more p o t e n t compounds o f p r a c t i c a l v a l u e (8). Such d i s c o v e r i e s gave r i s e t o the term " b i o r a t i o n a l " to describe s y n t h e t i c i n s e c t i c i d e s t h a t a r e d e s i g n e d by c o n s i d e r a t i o n o f s p e c i f i c b i o c h e m i c a l targets. T h e s e d e v e l o p m e n t s i n d i c a t e p o t e n t i a l new modes o f s e l e c t i v e insecticidal action. Throug physiological processes and t h e i n c r e a s e d k n o w l e d g e o f t h e d i v e r s e b i o c h e m i c a l pathways s u g g e s t s new a p p r o a c h e s t o t h e d e s i g n o f p e s t i c i d e s . Natural

Products

i n Pest

Control

The u t i l i z a t i o n o f n a t u r a l p r o d u c t s i n p e s t c o n t r o l may be c o n s i d e r e d f r o m a number o f s t a n d p o i n t s . F i r s t , the v a r i e t y of s t r u c t u r a l t y p e s p r o v i d e s a r i c h s o u r c e o f compounds o r m o d e l s f o r c o n v e n t i o n a l s c r e e n i n g programmes. Second, c o n s i d e r a t i o n of t h e known b i o l o g i c a l a c t i v i t y o f a n a t u r a l p r o d u c t may l e a d t o i t s a p p l i c a t i o n i n p e s t management, e i t h e r d i r e c t l y o r a f t e r structural modification. T h i r d , the r e c o g n i t i o n and u n d e r s t a n d i n g o f t h e f u n c t i o n o f a c h e m i c a l i n n a t u r e may reveal p o t e n t i a l a p p r o a c h e s t o p e s t management. F o r c o n t r o l o f some p e s t p r o b l e m s , m a t e r i a l s f r o m n a t u r a l s o u r c e s have p r o v e d e x t r e m e l y s u c c e s s f u l . I n p a r t i c u l a r , many p l a n t , human, a n d a n i m a l d i s e a s e s a r e c o n t r o l l e d b y a n t i b i o t i c s p r o d u c e d by m i c r o o r g a n i s m s . I n s e c t i c i d e s from p l a n t sources have b e e n u s e d e f f e c t i v e l y f o r many y e a r s , b u t t h e s c a l e on w h i c h t h e y have been u s e d does not compare w i t h t h a t of the s y n t h e t i c o r g a n i c i n s e c t i c i d e s i n t r o d u c e d i n t h e y e a r s f o l l o w i n g W o r l d War II. A p p l i c a t i o n of n a t u r a l products to pest c o n t r o l i s subject to s i m i l a r considerations to those that a f f e c t synthetic compounds. E l u c i d a t i o n o f s t r u c t u r e may be a r e l a t i v e l y r o u t i n e m a t t e r , b u t s y n t h e s i s may p r e s e n t d i f f i c u l t i e s , p a r t i c u l a r l y where t h e r e are s e v e r a l s t e r e o c h e m i c a l possibilities. There are o f t e n g r e a t d i f f e r e n c e s i n b i o l o g i c a l a c t i v i t y among d i f f e r e n t s t e r e o i s o m e r s and t h e p r e s e n c e o f i n a c t i v e i s o m e r s i n a p r o d u c t may be u n d e s i r a b l e . H o w e v e r , s u c h t e c h n i c a l p r o b l e m s c a n be o v e r c o m e as i n t h e c a s e o f t h e s y n t h e t i c p y r e t h r o i d s w h e r e s p e c i f i c s t e r e o i s o m e r s o f h i g h p u r i t y a r e now p r o d u c e d on a commercial s c a l e . Screening f o r b i o l o g i c a l a c t i v i t y also presents a major

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problem. B i o l o g i c a l t e s t i n g f o r many t y p e s o f a c t i v i t y i s important. I n s c r e e n i n g f o r i n s e c t i c i d a l a c t i v i t y , t h e number o f compounds p a s s i n g t h r o u g h r e s e a r c h a n d d e v e l o p m e n t s t e p s f r o m s y n t h e s i s t o m a r k e t p e r c o m m e r c i a l p r o d u c t was 1 4 , 5 0 0 i n 1 9 7 9 c o m p a r e d w i t h 1,800 i n 1956 ( 9 ) . The number o f compounds t h a t must be s c r e e n e d w i l l r i s e c o n s i d e r a b l y as t h e end o f t h e c e n t u r y approaches. Thus, s c r e e n i n g must be r a p i d o r m o d i f i e d p r o c e d u r e s must be d e v i s e d . The p r o b l e m o f t e s t i n g f o r t y p e s o f b i o l o g i c a l a c t i v i t y other than d i r e c t t o x i c i t y t o t h e t a r g e t o f t e n presents additional complexity. I f t h e t y p e o f a c t i v i t y t o be examined i s n o t d i r e c t l y l e t h a l , i . e . control o f thepest o r suppression o f pest p o p u l a t i o n i s a c h i e v e d through e f f e c t s on b e h a v i o u r , development o r r e p r o d u c t i o n , t h e p r o b l e m o f b i o l o g i c a l t e s t i n g may become much m o r e d i f f i c u l t , a s may a l s o t h e p r o b l e m o f d e m o n s t r a t i n g efficacy. I t i s i nthese areas i n which t h e spectrum o f b i o l o g i c a l a c t i v i t y o f n a t u r a l p r o d u c t s i s most l i k e l y t o be concentrated. The i n t e r f a c m o d i f i e d by a v a r i e t y o these complex i n t e r r e l a t i o n s h i p s i s a d i f f i c u l t g o a l , b u t t h e e x p l o i t a t i o n o f " n a t u r a l products** f o r t h e b e n e f i t o f a g r i c u l t u r e w i l l depend on p r o g r e s s towards i t . Economic

Aspects

A major problem i n achieving t h i s goal i st h equestion o f resources. T h e U.S. l o s e s a c o n s i d e r a b l e p e r c e n t a g e o f i t s a g r i c u l t u r a l p r o d u c t i o n t o p e s t s and a l s o pays h e a v i l y t o c o n t r o l pests. Such l o s s e s have been e s t i m a t e d a t about $10 b i l l i o n annually (10). However, t h e b e n e f i t o f r e d u c i n g l o s s e s cannot be measured c o m p l e t e l y i n monetary terms. F a c t o r s such as improved environmental q u a l i t y , c o n s e r v a t i o n and u t i l i z a t i o n o f l a n d and water resources a r ed i f f i c u l t t o include i n the a r i t h m e t i c o f potential benefits. The supremacy o f p e s t i c i d e c h e m i c a l s as c o n t r o l agents has i n f l u e n c e d t h eprocess o f c o n t r o l l i n g pests by emphasizing t h e p r o f i t a b i l i t y o f a market as a major c r i t e r i o n f o r development. Such a c r i t e r i o n i s u s e f u l , b u t l i m i t e d i n i t s applicability. The e l i m i n a t i o n o f t h e screwworm f l y f r o m t h e s o u t h e r n U n i t e d S t a t e s p r o v i d e s an example o f a s u c c e s s f u l a l t e r n a t i v e approach t o c o n t r o l o f a major pest. Benefits to the farmer and consumer a r e a measure o f t h e degree o f success o f t h i s programme. The d e v e l o p m e n t o f i n d i v i d u a l n a t u r a l p r o d u c t s t r u c t u r e s a s p e s t i c i d e s w i l l b e s u b j e c t t o t h e same e c o n o m i c f a c t o r s t h a t affect synthetic pesticides. N a t u r a l p r o d u c t s do n o t d i f f e r f r o m c o m p o u n d s s y n t h e s i z e d i n t h e l a b o r a t o r y , b u t t h e y may, a s p r o d u c t s o f b i o l o g i c a l p r o c e s s e s , be more r e a d i l y d e g r a d e d t h a n many man-made s t r u c t u r e s . A l t h o u g h t h e p o t e n t i a l f o r f a c i l e d e g r a d a t i o n may h a v e f a v o u r a b l e i m p l i c a t i o n s f o r e n v i r o n m e n t a l safety, there i sl i t t l e j u s t i f i c a t i o n f o r t h e assumption a p r i o r i . t h a t b e c a u s e a compound i s a n a t u r a l p r o d u c t , i t p o s s e s s e s no u n d e s i r a b l e t o x i c o l o g i c a l p r o p e r t i e s . Toxicological t e s t s m u s t b e p e r f o r m e d f o r b o t h n a t u r a l a n d man-made c o m p o u n d s b e f o r e r e g i s t r a t i o n as p e s t i c i d e s .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

23.

PLIMMER

Role of Natural Product Chemistry

The c o s t o f d e v e l o p i n g a n d r e g i s t e r i n g a p e s t i c i d e f o r t h e U.S. m a r k e t i s a b o u t $20 m i l l i o n (11). T h e r e f o r e , t h e compounds d e v e l o p e d must have a l a r g e p o t e n t i a l market. F o r an i n s e c t i c i d e , t h i s i m p l i e s b r o a d s p e c t r u m a c t i v i t y and use on s e v e r a l major crops. W o r l d w i d e , o n l y a few m a j o r i n d u s t r i a l c o r p o r a t i o n s possess adequate resources to undertake such activity. T h u s , i t w i l l be d i f f i c u l t t o r e a l i s e t h e p o t e n t i a l o f natural products f o r pest c o n t r o l without recourse to a broader i n s t i t u t i o n a l framework. The b i o l o g i c a l a c t i v i t y a s s o c i a t e d w i t h n a t u r a l p r o d u c t s i s o f t e n s p e c i e s - s p e c i f i c . For example, s u p p r e s s i o n of i n s e c t p o p u l a t i o n by m a s s - t r a p p i n g o r by m a t i n g d i s r u p t i o n r e l i e s on technology t h a t i s s p e c i f i c f o r the t a r g e t i n s e c t . Such s p e c i f i c p e s t c o n t r o l m e t h o d s h a v e t h e m e r i t t h a t t h e y do n o t adversely a f f e c t b e n e f i c i a l i n s e c t s , but they are r a r e l y appealing to the major commercial investor. The s p e c i f i c i t y o f s u c h m e t h o d s a n d t h e i r low p o t e n t i a l f o r e n v i r o n m e n t a l p o l l u t i o n appears t o o f f e r g r e a t p r o m i s e and, t o s t i m u l a t an a p p r o p r i a t e r e s p o n s t o x i c i t y and o t h e r t e s t i n g p r o c e d u r e s h a v e b e e n p r o p o s e d by t h e U.S. E n v i r o n m e n t a l P r o t e c t i o n Agency f o r b i o c h e m i c a l s to f a c i l i t a t e r e g i s t r a t i o n (12). A l t h o u g h the r e g u l a t o r y a g e n c i e s have i n d i c a t e d t h e i r i n t e r e s t i n s t i m u l a t i n g new m e t h o d s o f p e s t c o n t r o l as a l t e r n a t i v e s t o the use o f c o n v e n t i o n a l p e s t i c i d e s , t h e p r o b l e m s o f e c o n o m i c and s c i e n t i f i c d e v e l o p m e n t o f s u c h t e c h n i q u e s remain. T h e r e i s o f t e n much d i s p a r i t y i n p r o g r e s s i n d i f f e r e n t f i e l d s of science. B r e a k t h r o u g h s o f t e n a r i s e when new techniques o r i d e a s a r e t r a n s f e r r e d f r o m one f i e l d o f e n d e a v o u r t o a n o t h e r . Such a p r o c e s s o f c r o s s f e r t i l i z a t i o n i s h i n d e r e d by r i g i d academic s e p a r a t i o n of b a s i c d i s c i p l i n e s . The information r e q u i r e d f o r s u c c e s s f u l p e s t c o n t r o l does not r e s i d e s o l e l y i n the hands o f the c h e m i s t , but the c o o p e r a t i o n o f s e v e r a l s p e c i a l i s t s i s e s s e n t i a l . T h i s a p p l i e s p a r t i c u l a r l y t o methods of insect c o n t r o l that involve m o d i f i c a t i o n of behaviour through t h e use o f a t t r a c t a n t s , r e p e l l e n t s o r r e s i s t a n t p l a n t s . Resistant

Plants

and

Ecological

Chemistry

N a t u r a l p r o d u c t c h e m i s t r y has e v o l v e d t h r o u g h t h e s t a g e s o f s t r u c t u r a l e l u c i d a t i o n to the examination of the f u n c t i o n of n a t u r a l compounds w i t h i n e c o l o g i c a l s y s t e m s . Ecological chemistry i s a growing f i e l d of science i n which p l a n t - i n s e c t , p l a n t - p l a n t , and o t h e r i n t e r o r g a n i s m a l r e l a t i o n s h i p s c a n be e x a m i n e d i n t e r m s o f t h e e f f e c t s o f c h e m i c a l s on b i o l o g i c a l f u n c t i o n s , t h u s p r o v i d i n g a more f u n d a m e n t a l a p p r e c i a t i o n o f p l a n t r e s i s t a n c e and t h e i n t e r r e l a t i o n s h i p b e t w e e n p e s t s a n d a g r i c u l t u r a l crops. There i s a chemical b a s i s f o r the interdependence of i n s e c t a n d p l a n t l i f e w i t h i n an e c o s y s t e m . Although the i n f l u e n c e of h e r b i v o r e s , d i s e a s e s , e t c . a n d t h e m o r p h o l o g y o f i n s e c t s and p l a n t s are a l s o important, the n u t r i t i o n a l s t a t u s of the p l a n t h o s t s s i g n i f i c a n t l y a f f e c t s t h e s u r v i v a l o f an i n s e c t c o m m u n i t y

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and i t s v i g o r . Despite s i m i l a r i t i e s i n the major c o n s t i t u e n t s o f p l a n t s , i n s e c t s a r e h i g h l y s e l e c t i v e i n t h e i r f e e d i n g and other b e h a v i o u r s , such as o v i p o s i t i o n , t h a t o c c u r i n a s s o c i a t i o n w i t h a s p e c i f i c host. I n t h i s respect, secondary plant products a r e e x t r e m e l y i m p o r t a n t a n d many t h o u s a n d s o f s e c o n d a r y p l a n t compounds have been e l a b o r a t e d d u r i n g t h e c o u r s e o f p l a n t evolution. Swain (13) d i s c u s s e d t h e o c c u r r e n c e and v a r i a t i o n o f s e c o n d a r y compounds i n t e r m s o f p l a n t a n d i n s e c t c o e v o l u t i o n . Some o f t h e c o n t r o v e r s y c o n c e r n i n g t h e r o l e o f i n s e c t s i n c o e v o l u t i o n has been summarized i n an a r t i c l e by Feeny ( 1 4 ) . I n terms o f a g r i c u l t u r a l p e s t c o n t r o l , he s u g g e s t s t h a t r e s e a r c h s h o u l d c o n c e n t r a t e o n t h e d i v e r s i t y o f compounds i n p l a n t s t h a t are t o x i c a t the metabolic l e v e l ( a n t i b i o s i s ) r a t h e r than a t t h e b e h a v i o u r a l l e v e l (nonpreference r e s i s t a n c e ) . Feeny a l s o discusses t h e concept o f "apparency", which i st h e s u s c e p t i b i l i t y o f p l a n t s o r p l a n t t i s s u e t o be d i s c o v e r e d b y i n s e c t s . A g r i c u l t u r a l p r a c t i c e s often favour t h e "apparency" o f plants when m o n o c u l t u r e s e x t e n d i n a t t r a c t i v e host f o r insects needed o v e r and above t h o s e r e q u i r e d i n a n a t u r a l community. Defences i n c l u d e crop p r o t e c t i o n chemicals and t h e c u l t i v a t i o n o f p l a n t s w i t h a high degree o f i n s e c t r e s i s t a n c e . The r e d u c t i o n o f "apparency", e.g. by c r o p r o t a t i o n , would reduce t h e need f o r d e f e n s i v e measures and might improve agronomic q u a l i t y by f a v o u r i n g p l a n t s i n which c o n s i d e r a b l e m e t a b o l i c r e s o u r c e s were n o t expended on s y n t h e s i s o f d e f e n s i v e compounds. Resistant crop plants arethe subject o f intensive programmes o f r e s e a r c h . S t u d y o f t h e r e l a t i o n s h i p s among c h e m i c a l c o m p o s i t i o n , v a r i e t y a n d i n s e c t r e s i s t a n c e h a s , i n many i n s t a n c e s , made i t p o s s i b l e t o c o r r e l a t e o b s e r v e d r e s i s t a n c e t o insect attack with plant chemistry. H o w e v e r , r e s i s t a n c e may b e conferred by f a c t o r s other than chemical composition, n o r i s p l a n t r e s i s t a n c e always compatible w i t h agronomic q u a l i t y . This p r o b l e m a n d t h e p r o b l e m o f d i s c o v e r i n g new s o u r c e s o f r e s i s t a n t germplasm are major l i m i t a t i o n s o f t h e technique. Although the h o s t - p l a n t r e s i s t a n c e method o f c o n t r o l u t i l i z e s mechanisms t h a t have e v o l v e d n a t u r a l l y , t h e s e l e c t i o n o f a g r o n o m i c a l l y valuable s p e c i e s f o r c u l t i v a t i o n b y man i n t e r f e r e s s u b s t a n t i a l l y w i t h t h e evolutionary process. The mechanisms o f r e s i s t a n c e have been summarized (15) as n o n p r e f e r e n c e r e s i s t a n c e , a n t i b i o t i c r e s i s t a n c e , and t o l e r a n c e . I n t h e case o f nonpreference r e s i s t a n c e , t h eplant i sl e s s s u i t a b l e f o r food, o v i p o s i t i o n o r s h e l t e r a n d r e s i s t a n c e may i n v o l v e c h e m i c a l o r p h y s i c a l f a c t o r s . The c h e m i c a l b a s i s o f r e s i s t a n c e i s g o v e r n e d b y t h e c o m p l e x o f compounds t h a t a f f e c t i n s e c t b e h a v i o u r , s u c h a s f e e d i n g deterrents, oviposition deterrents, attractants, etc. i n contrast t o a n t i b i o t i c r e s i s t a n c e where t h e presence o r absence o f compounds t h a t a f f e c t t h e d e v e l o p m e n t o r s u r v i v a l o f t h e i n s e c t is involved. Tolerance t o insects i sthea b i l i t y o f theplant t o s u r v i v e a t t a c k b e c a u s e p e s t damage i s r e p a i r a b l e o r d o e s n o t impair thevigour o f theplant. K n i p l i n g (16) d i s c u s s e s t h e e f f e c t o f t h e t y p e s o f r e s i s t a n c e on i n s e c t p o p u l a t i o n and c o n s i d e r s t h a t a n t i b i o t i c r e s i s t a n c e s h o u l d have t h e g r e a t e s t

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effect. High m o r t a l i t y should cause a d e c l i n e i n i n s e c t populations. T h i s w i l l , o f c o u r s e , d e p e n d on t h e r a t e o f i n c r e a s e o f i n s e c t p o p u l a t i o n s and t h e l e v e l o f a n t i b i o t i c r e s i s t a n c e , among o t h e r f a c t o r s . E x p e r i m e n t a l e v a l u a t i o n o f e f f e c t s on i n s e c t p o p u l a t i o n s p r e s e n t s a m a j o r s o u r c e o f d i f f i c u l t y and i t was e m p h a s i z e d i n t h e p r e c e d i n g d i s c u s s i o n t h a t a t o t a l e c o s y s t e m s t u d y w o u l d be r e q u i r e d t o determine the e f f e c t i v e n e s s of such c o n t r o l measures and t h a t s m a l l p l o t s a r e i n a d e q u a t e . Resistant plant varieties a r e a d v a n t a g e o u s b e c a u s e t h e y r e d u c e numbers o f i n s e c t s , b u t f r o m the p o i n t of view of the chemist seeking leads to s t r u c t u r e s of h i g h b i o l o g i c a l a c t i v i t y , t h e s t u d y may be d i s a p p o i n t i n g p a r t i c u l a r l y i f a number o f f a c t o r s a r e i n v o l v e d . In studies of r e s i s t a n c e , t e s t s f o r i n s e c t i c i d a l a c t i v i t y p e r s e may y i e l d o n l y limited information. Although pyrethroids, rotenoids, n i c o t i n e , r y a n o d i n e , and a number o f o t h e r p l a n t c o m p o n e n t s a r e i n s e c t i c i d a l , i t i s i m p o r t a n t i n a s c r e e n i n g programme t o r e c o g n i z e compounds t h a the precocenes ( 4 ) , ecdysone as w e l l as c o m p o u n d s t h a t a f f e c t i n s e c t b e h a v i o u r , s u c h as t h e t a n n i n s , a z a d i r a c h t i n , and a n u m b e r o f t e r p e n o i d s w h i c h a c t as insect antifeedants. Chemical f a c t o r s are a l s o i n v o l v e d i n the r e s i s t a n c e of p l a n t s t o d i s e a s e and i n t h e c o m p e t i t i v e a b i l i t y o f a p l a n t t o s u r v i v e w i t h i n a community of p l a n t s . P l a n t s t r e s s may also g e n e r a t e a c h e m i c a l r e s p o n s e g i v i n g r i s e t o c o m p o u n d s known as t h e p h y t o a l e x i n s , t h e n a t u r e o f w h i c h w i l l d e p e n d on t h e chemistry of the host p l a n t (18, 19). Such response t o i n j u r y i n f e c t i o n i s o f g r e a t i n t e r e s t b e c a u s e i t has stimulated i n v e s t i g a t i o n s of the nature of the b i o r e g u l a t o r y processes involved.

or

T h i s i s i l l u s t r a t e d i n the case o f g r a p e v i n e s where the e x p l o i t a t i o n of the defences of the p l a n t a g a i n s t f u n g a l a t t a c k b y v i n e downy m i l d e w ( P l a s m o p a r a v i t i c o l a ) and B o t r y t i s c i n e r e a has been s t u d i e d ( 2 0 ) . A group of p h y t o a l e x i n s , the v i n i f e r i n s , w h i c h may be f o r m e d b y o l i g o m e r i z a t i o n o f r e s v e r a t r o l ( a t r i h y d r o x y - t r a n s - s t i l b e n e ) were found o n l y i n i n f e c t e d or i n j u r e d v i n e l e a v e s and had m o d e r a t e a n t i f u n g a l a c t i v i t y i n v i t r o . Pryce (21) a l s o d i s c u s s e d t h e f o r m a t i o n o f a n t i f u n g a l compounds i n r i c e l e a v e s when t h e r i c e was t r e a t e d w i t h 2,2~dichloro-3,3-dimethylcyclopropane carboxylic acid. Responses t h a t a r e e l i c i t e d when t h e n a t u r a l d e f e n c e s o f t h e p l a n t a r e s t i m u l a t e d s u g g e s t new a p p r o a c h e s t o t h e c o n t r o l o f d i s e a s e s o r pests. Natural

P r o d u c t s as

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N a t u r a l p r o d u c t s h a v e p r o v i d e d l e a d s t o new p e s t i c i d e s . K n o w l e d g e o f t h e b i o l o g i c a l a c t i v i t y o f many p l a n t s h a s b e e n r e c o r d e d i n t h e a n c i e n t p h a r m a c o p o e i a s o f I n d i a and C h i n a a n d i s p r e s e r v e d i n the f o l k l o r e of most n a t i o n s . The practical a p p l i c a t i o n o f much o f t h i s k n o w l e d g e h a s p r o v e d d i f f i c u l t b e c a u s e i t h a s b e e n a c c u m u l a t e d t h r o u g h o u t t h e a g e s and i t i s

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b a s e d on a m i x t u r e o f e x p e r i e n c e , t r a d i t i o n , and m a g i c . Natural p r o d u c t s c o n t i n u e t o be s o l d as d r u g s o r as i n s e c t i c i d e s i n many p a r t s o f t h e w o r l d and t h e m a r k e t p l a c e i s e v i d e n c e o f t h e i r value. N i c o t i n e i s an e x a m p l e o f a n a t u r a l i n s e c t i c i d e t h a t h a s b e e n i n u s e f o r many y e a r s . P y r e t h r i n , a crude mixture of n a t u r a l p y r e t h r o i d s , was u s e d b y C a u c a s i a n t r i b e s as an i n s e c t i c i d e b e f o r e 1800. The e x t r a c t o f p y r e t h r u m f r o m c h r y s a n t h e m u m c i n a e r i a e f o l u m c o n t a i n s p y r e t h r i n s , c i n e r i n s and jasmolins. A l t h o u g h t h i s p r o d u c t was a v a l u a b l e i n s e c t i c i d e when few e f f e c t i v e i n s e c t i c i d e s were a v a i l a b l e , i t s use i n a g r i c u l t u r e has been l i m i t e d by i t s p h o t o c h e m i c a l i n s t a b i l i t y . Replacement o f s u c h n a t u r a l l y o c c u r r i n g i n s e c t i c i d e s by compounds s y n t h e s i z e d f r o m p e t r o c h e m i c a l s was f a v o u r e d b y w a r t i m e c o n d i t i o n s . S y n t h e t i c i n s e c t i c i d e d e v e l o p m e n t and o f f i c i a l a c t i o n t o a p p r o v e t h e i r use have r e c e i v e d a d d i t i o n a l s t i m u l i because s u p p l i e s o f i m p o r t e d i n s e c t i c i d e s have b e e n r e s t r i c t e d f r o m t i m e t o t i m e by p o l i t i c a l or economic f a c t o r p y r e t h r o i d s were c o n t i n u e a n a l o g u e s have been produced s i n c e the e l u c i d a t i o n of t h e i r structure. Some o f t h e e a r l i e s t o f t h e s e w e r e s y n t h e s i z e d as s o o n as t h e n a t u r e o f t h e a c t i v e m a t e r i a l s h a d b e e n e s t a b l i s h e d (23). I n t h e f o r e f r o n t o f r e s e a r c h on s y n t h e t i c p y r e t h r o i d s h a v e been E l l i o t t and h i s c o - w o r k e r s . As a r e s u l t o f t h e i r s u s t a i n e d e f f o r t , t h e p y r e t h r o i d s h a v e now a c h i e v e d i m p o r t a n t p o s i t i o n i n agriculture. The m a j o r s t r u c t u r a l f e a t u r e s n e c e s s a r y f o r t h e i r a c t i v i t y h a v e b e e n e l u c i d a t e d ( 2 4 ) , a n d t h e r e a r e now many r e l a t e d compounds c o m m e r c i a l l y a v a i l a b l e . The new p y r e t h r o i d i n s e c t i c i d e s h a v e r e q u i r e d many y e a r s f o r s u c c e s s f u l development, notwithstanding the s t r u c t u r a l i n f o r m a t i o n a v a i l a b l e i n 1924. The e n v i r o n m e n t a l p e r s i s t e n c e o f t h e new p y r e t h r o i d s i s g e n e r a l l y l o w e r t h a n t h a t o f t h e o r g a n o c h l o r i n e i n s e c t i c i d e s , and t h e s t r u c t u r e i s c a p a b l e o f extensive m o d i f i c a t i o n with r e t e n t i o n of a c t i v i t y . Fortunately, t h e y w e r e i n t r o d u c e d i n t o a g r i c u l t u r a l u s e a t a t i m e when h e a v y r e l i a n c e on o r g a n o c h l o r i n e i n s e c t i c i d e s was no l o n g e r f e a s i b l e and t h e r e was an u r g e n t n e e d f o r new insecticides. T h i s group i s perhaps the most o u t s t a n d i n g example o f n a t u r a l p r o d u c t s t h a t have p r o v i d e d l e a d s f o r m o d i f i c a t i o n . Other groups o f n a t u r a l l y o c c u r r i n g i n s e c t i c i d e s have been i n v e s t i g a t e d , b u t w i t h o u t t h e same s p e c t a c u l a r d e g r e e o f success. Q u a s s i a , r o t e n o n e s , s a b a d i l l a , r y a n i a , mamey, and s e v e r a l o t h e r p l a n t p r o d u c t s h a v e b e e n u s e d as i n s e c t i c i d e s . The c h e m i s t r y o f t h e m a j o r g r o u p s i s k n o w n , b u t many p l a n t p r i n c i p l e s r e m a i n t o be e x p l o r e d ( 2 5 ) . Screening of n a t u r a l products p r e s e n t s a p r o b l e m and r a i s e s t h e i s s u e o f s u i t a b l e b i o a s s a y i n t e r m s o f i n s e c t s p e c i e s , t h e p a r t o f t h e p l a n t t h a t s h o u l d be e x a m i n e d and t h e a p p r o p r i a t e t e s t s f o r a c t i v i t y . The n u m b e r o f d i s c o v e r i e s o f new i n s e c t i c i d e s b a s e d on n a t u r a l p r o d u c t s t r u c t u r e s as p r o t o t y p e s i s l i m i t e d , although i n s e c t i c i d a l a c t i v i t y i s widely d i s t r i b u t e d i n nature. For e x a m p l e , c a r b a m a t e s o c c u r i n n a t u r e and i t h a s l o n g b e e n known t h a t p h y o s t i g m i n e , an a l k a l o i d f r o m t h e c a l a b a r b e a n , i s t o x i c t o

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

23.

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Role of Natural Product Chemistry

PLIMMER

mammals. H o w e v e r , many o t h e r l i n e s o f t h o u g h t m i g h t h a v e l e d t o t h e i n c l u s i o n o f c a r b a m a t e s i n an i n s e c t i c i d e s c r e e n i n g p r o g r a m m e , a s s u m i n g t h a t t h e y w e r e n o t t o be i n c l u d e d o n a r a n d o m basis. The p o w e r f u l a c t i o n o f some n a t u r a l p r o d u c t s on t h e c e n t r a l n e r v o u s s y s t e m may s u g g e s t t h e i r p o t e n t i a l v a l u e i n i n s e c t control. I f t h e i r m a m m a l i a n t o x i c i t y c a n be r e d u c e d b y s t r u c t u r a l m o d i f i c a t i o n t h e y may be u s e f u l i n s e c t i c i d e s . C a r t a p , a r i c e i n s e c t i c i d e , i s a bisthiocarbamate. I t s s t r u c t u r e was b a s e d on t h a t o f t h e n a t u r a l p r o d u c t , n e r e i s t o x i n , a n e u r o t o x i n i s o l a t e d f r o m s h e l l f i s h , a n d i t i s l i k e l y t h a t an i n v i v o m e t a b o l i c c o n v e r s i o n o f c a r t a p t o n e r e i s t o x i n o r r e l a t e d compound is responsible f o r i t s a c t i v i t y (11).

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As s c r e e n i n g t e c h n i q u e s become m o r e s o p h i s t i c a t e d , t h e r a n g e o f c o m p o u n d s t h a t a r e p o t e n t i a l l y v a l u a b l e as l e a d s should increase. A s , an e x a m p l e , t h e a n t i j u v e n i l e h o r m o n e a c t i o n o f t h e p r e c o c e n e s has been c i t e d . T h e i r spectrum of a c t i v i t y i s l i m i t e d , but t h e s t u d y o f t h e i r a c t i o n has r e v e a l e d p o t e n t i a l biochemical target sites. N a t u r a l l y o c c u r r i n g i n s e c t i c i d e s are subject to f l u c t u a t i o n s i n p r o d u c t i o n and a v a i l a b i l i t y and, i n d e v e l o p e d c o u n t r i e s , t h e y can r a r e l y compete e f f e c t i v e l y w i t h m a n u f a c t u r e d p r o d u c t s . A l l new s t r u c t u r e s m u s t f a c e t h e same s e a r c h i n g t o x i c o l o g i c a l e x a m i n a t i o n w h e t h e r t h e y a r e n a t u r a l o r s y n t h e t i c i n o r i g i n and t h e i r e c o n o m i c p o t e n t i a l m u s t , t h e r e f o r e , be e q u a l l y as attractive. A l t h o u g h s u c h f a c t o r s may a p p e a r d i s c o u r a g i n g , c h e m i c a l s t h a t p o s s e s s u n u s u a l modes o f a c t i o n s u c h as b e h a v i o u r - m o d i f y i n g c h e m i c a l s h a v e f o u n d an a c c e p t a b l e p l a c e i n p e s t management. Behavioural

Compounds

Many i n t e r a c t i o n s b e t w e e n i n s e c t s a n d i n s e c t s o r b e t w e e n p l a n t s a n d i n s e c t s a r e m e d i a t e d b y c h e m i c a l s a n d i n t h e s e c t i o n on p l a n t r e s i s t a n c e , t h e r o l e o f n o n p r e f e r e n c e r e s i s t a n c e was discussed. T h e r e has b e e n c o n s i d e r a b l e p r o g r e s s i n i s o l a t i n g and i d e n t i f y i n g compounds t h a t a f f e c t i n s e c t b e h a v i o u r . A recent r e v i e w c o n t a i n s m o r e t h a n 800 r e f e r e n c e s t o t h e l i t e r a t u r e a n d l i s t s o v e r 300 c o m p o u n d s as i n s e c t a t t r a c t a n t s , a t t r a c t a n t p h e r o m o n e s and r e l a t e d c o m p o u n d s t o g e t h e r w i t h t h e c o r r e s p o n d i n g insect species (26). O t h e r pheromones ( t r a i l pheromones, h a i r pencil secretions, e t c . ) , feeding deterrents, oviposition d e t e r r e n t s , an o t h e r t y p e s o f b e h a v i o u r a l c o m p o u n d s a r e n o t i n c l u d e d i n t h i s comprehensive r e v i e w , but the volume of i n f o r m a t i o n on b e h a v i o u r a l c o m p o u n d s i s c o n s t a n t l y b e i n g e x p a n d e d as new c o m p o u n d s a r e i d e n t i f i e d . T h e r e a r e many w a y s i n w h i c h b e h a v i o u r a l c o m p o u n d s may be

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u s e d i n p e s t management s y s t e m s . Potential application of p h e r o m o n e s a n d a t t r a c t a n t s a n d t h e s t a t u s o f r e s e a r c h was r e c e n t l y summarized (10). A n t i f e e d a n t s h a v e a l s o r e c e i v e d much a t t e n t i o n , p a r t i c u l a r l y e x t r a c t s o f t h e neem t r e e ( M e l i a azdirachta). V o l a t i l e a t t r a c t a n t s , a r r e s t a n t s , and s t i m u l a n t s from p l a n t s a l s o appear promising as topes f o r f u t u r e r e s a r c h . In p r a c t i c e , t h e predominant a p p l i c a t i o n o f i n s e c t pheromones and a t t r a c t a n t s i s f o r d e t e c t i o n and s u r v e y o f infestations. Pheromone t r a p s a r e an e x t r e m e l y v a l u a b l e c o m p o n e n t o f many p e s t m a n a g e m e n t s y s t e m s a n d p r o v i d e information on a n a r e a - w i d e b a s i s t h a t p e r m i t s t i m e l y a p p l i c a t i o n o f c o n t r o l measures. For suppression o r c o n t r o l o f i n s e c t populations, a t t r a c t a n t p h e r o m o n e s o r a t t r a c t a n t s may b e u s e d i n s e v e r a l w a y s : (a) i n combination with b a i t s , t o x i c a n t s , s t e r i l a n t s o r pathogens; (b) mass-trapping; o r (c) t o d i s r u p t mating. The a p p l i c a t i o n o f p h e r o m o n e s t o a r e a - w i d e s u p p r e s s i o n o f p o p u l a t i o n by permeatin communication has been pest insects. T h e r e h a s b e e n c o n s i d e r a b l e r e s e a r c h a n d some c o m m e r c i a l i n t e r e s t i n t h i s approach. There a r e , however, major p r a c t i c a l p r o b l e m s i n t h e way o f s u c c e s s f u l d e v e l o p m e n t . I thas a major a d v a n t a g e t h a t o n l y a f e w grammes o f p h e r o m o n e p e r h e c t a r e i s needed f o r a p p l i c a t i o n . S i n c e , pheromones a r e g e n e r a l l y degraded r a p i d l y , e n v i r o n m e n t a l burden i s minute compared w i t h t h a t t y p i c a l o f many i n s e c t i c i d e s . T h e p r o b l e m o f p h e r o m o n e f o r m u l a t i o n h a s b e e n t h e s u b j e c t o f much r e s e a r c h b e c a u s e t h e y a r e u s u a l l y l a b i l e a n d v o l a t i l e compounds ( 2 7 ) . One o f t h e m a j o r d i f f i c u l t i e s i n t h e u s e o f pheromones ( o r o t h e r n o n l e t h a l methods of c o n t r o l ) i st h ed i f f i c u l t y o f demonstrating e f f i c a c y (28). Extremely large p l o t sizes areusually necessary t o acquire s u f f i c i e n t i n f o r m a t i o n , p a r t i c u l a r l y i f i t becomes n e c e s s a r y t o measure t h e impact o f t h e t r e a t m e n t on m a t i n g and p o p u l a t i o n . R e s e a r c h o n t h e g y p s y moth o v e r a number o f y e a r s h a s demonstrated t h e d i f f i c u l t i e s and d e f i n e d t h e l i m i t s o f t h e technique f o r t h i s species (29). Although thetechnique suffers from the d i s a d v a n t a g e t h a t c o n s i d e r a b l e knowledge o f t h e p o p u l a t i o n dynamics and mating behaviour o f t h e t a r g e t species are e s s e n t i a l p r e r e q u i s i t e s f o r a p p l i c a t i o n , l i m i t e d successes are encouraging continued t r i a l s and development. The u s e o f b e h a v i o u r - m o d i f y i n g c o m p o u n d s o f f e r s g r e a t p r o m i s e f o r i n t e g r a t e d s y s t e m s o f p e s t management, b u t i t r e q u i r e s s u b s t a n t i a l knowledge o f t h e t a r g e t s p e c i e s . M o d i f i c a t i o n o f s t r u c t u r e o f b e h a v i o u r - m o d i f y i n g compounds f o r p o p u l a t i o n s u p p r e s s i o n i s a p o t e n t i a l a p p r o a c h a n d , i n some c a s e s , a n a l o g u e s a r e o f v a l u e i n m a t i n g d i s r u p t i o n . However, t h e r e s p o n s e o f an i n s e c t t o pheromones depends on v e r y p r e c i s e s t r u c t u r a l requirements and even s l i g h t m o d i f i c a t i o n o f s t r u c t u r e normally reduces t h eb i o l o g i c a l a c t i v i t y by s e v e r a l orders o f magnitude. I n v e s t i g a t i o n o f t h e p h y s i o l o g y o f pheromone p r o d u c t i o n h a s

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

23.

PLIMMER

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shown t h a t i n the f e m a l e c o r n earworm moth ( H e l i o t h i s zea) pheromone p r o d u c t i o n i s c o n t r o l l e d by a b r a i n hormone ( 3 0 ) . The s t i m u l a t i o n o f p h e r o m o n e p r o d u c t i o n b y t h i s h o r m o n e i s an important stage i n the r e p o r d u c t i v e process. Such key e v e n t s i n t h e r e p r o d u c t i v e p r o c e s s s u g g e s t new p o t e n t i a l f o r i n s e c t i c i d a l modes o f a c t i o n . Conclusion N a t u r a l products are a r i c h source of s t r u c t u r a l v a r i e t y . They may p r o v i d e s o u r c e m a t e r i a l s f o r s c r e e n i n g p r o g r a m m e s t o d e t e c t b i o l o g i c a l a c t i v i t y and n a t u r a l p r o d u c t s t h a t p o s s e s s k n o w n a c t i v i t y may s e r v e as t e m p l a t e s f o r s t r u c t u r a l m o d i f i c a t i o n . An o u t s t a n d i n g example of the o p t i m i z a t i o n of s t r u c t u r e t o improve p o t e n c y and s t a b i l i t y i s t h e s y n t h e s i s and s u c c e s s f u l c o m m e r c i a l i z a t i o n o f p y r e t h r o i d s and t h e i r a n a l o g u e s . B a c t e r i a p r e s e n t g r e a t p o t e n t i a l as s o u r c e s o f b i o l o g i c a l l y active chemicals. Development increase the f e a s i b i l i t in quantity. As a f u t u r e a p p r o a c h t o i n s e c t c o n t r o l , t h e prospect of t r a n s f e r r i n g the c a p a b i l i t y to produce s e l e c t i v e i n s e c t t o x i n s f r o m b a c t e r i a t o p l a n t s may l i e w i t h i n t h e p o t e n t i a l o f new g e n e t i c e n g i n e e r i n g techniques. S u c h d e v e l o p m e n t s i n b i o l o g y as t h e e l u c i d a t i o n o f b i o r e g u l a t o r y p r o c e s s e s and t h e d i s c o v e r y o f new r e c e p t o r s i t e s and r e c e p t o r s h a v e b e e n i n s t r u m e n t a l i n t h e d e v e l o p m e n t o f b i o r a t i o n a l p e s t i c i d e s capable of blocking e s s e n t i a l steps i n metabolic processes t h a t are p e c u l i a r to the organism or o r g a n i s m s t h a t a r e t o be c o n t r o l l e d . N a t u r a l p r o d u c t s are the key t o u n d e r s t a n d i n g e c o l o g i c a l systems. T h e r e has been r a p i d p r o g r e s s i n e l u c i d a t i o n o f t h e c h e m i s t r y o f p r i m a r y a n d s e c o n d a r y p l a n t c o m p o n e n t s and i n t h e growth of knowledge of i n s e c t b i o c h e m i s t r y . For i t s a p p l i c a t i o n to pest c o n t r o l , p a r a l l e l developments i n physiology and b e h a v i o u r a l s t u d i e s are e s s e n t i a l f o r r e v e a l i n g the u n d e r l y i n g c o m p l e x o f c h e m i c a l l y m e d i a t e d i n t e r a c t i o n s among o r g a n i s m s . B i o r a t i o n a l m e t h o d s o f c o n t r o l b a s e d on a l t e r e d i n s e c t - p l a n t r e l a t i o n s h i p s a r e b e i n g e f f e c t i v e l y u s e d , i f we e x t e n d t h i s t e r m to describe a p p l i c a t i o n of host plant resistance. Such c o n t r o l methods have been d e v e l o p e d e m p i r i c a l l y ; but the r a t i o n a l b a s i s f o r f u r t h e r d e v e l o p m e n t w i l l be t h e o u t c o m e o f new genetic studies. S u c c e s s i n d e c i p h e r i n g t h e s t r u c t u r e and f u n c t i o n o f n a t u r a l m o l e c u l e s has d r a m a t i c a l l y c h a n g e d t h e a p p r o a c h t o b i o l o g i c a l problems. The s t r u c t u r e a n d f u n c t i o n s o f b i o l o g i c a l m a c r o m o l e c u l e s , p a r t i c u l a r l y n u c l e i c a c i d s and p r o t e i n s a r e r a p i d l y b e c o m i n g known and t h e a b i l i t y t o e n g i n e e r o r a l t e r t h e i r subunits i n l i v i n g organisms provides p o t e n t i a l technology f o r a l t e r i n g b i o l o g i c a l outputs. Thus, the n u t r i t i v e value of a p l a n t p r o t e i n may be i m p r o v e d by c h a n g i n g t h e s e q u e n c e o f a m i n o acids. The i n s e r t i o n o f g e n e s i n t o p l a n t s a n d t h e i r s u b s e q u e n t expression i s a concept that promises a great future development. However, t h e q u e s t i o n o f r e s i s t a n c e t o p e s t s by

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334

p l a n t s i s c o m p l i c a t e d b y t h e c o n t r i b u t i o n o f many f a c t o r s , e a c h a f f e c t e d by d i f f e r e n t genes. The e v o l u t i o n o f h o s t - p l a n t p r e f e r e n c e s and p a r t i c u l a r l y , t h e a d a p t a t i o n o f i n s e c t behaviour, a r e i m p o r t a n t f a c t o r s w h i c h r e q u i r e more s t u d y and D e t h i e r ( 3 1 ) has a r g u e d t h a t t h i s i s i m p o r t a n t a t t h e g e n e t i c l e v e l . The behaviour o f i n s e c t s a t t h e f e e d i n g and r e p r o d u c t i v e stages i s c r i t i c a l l y important i ndetermining thepest status o f a species. A l t h o u g h c h e m i c a l s t h a t modify b e h a v i o u r c u r r e n t l y have a r o l e i n p e s t c o n t r o l , t h e i r a p p l i c a t i o n on an area-wide s c a l e i n a t t e m p t s t o s u p p r e s s i n s e c t p o p u l a t i o n s by u s i n g pheromones t o a l t e r m a t i n g b e h a v i o u r s , may m e e t w i t h l i t t l e s u c c e s s u n l e s s t h e b i o c h e m i c a l and b e h a v i o u r a l aspects o f t h e system are w e l l understood. One o f t h e m o s t i m p o r t a n t r o l e s o f n a t u r a l p r o d u c t s i n p e s t c o n t r o l may b e t h e i r r o l e i n s t i m u l a t i n g t h o u g h t . Pest control may b e c o n c e i v e d a s a n a c t i v i t y d i r e c t e d t o w a r d s c h a n g i n g t h e e x i s t i n g c o n d i t i o n s i n an e c o l o g i c a l system f o r t h e b e n e f i t o f man. A better understandin i n t e r a c t i o n s among o r g a n i s m p o s s i b l e t o d e v i s e w a y s i n w h i c h m i n i m a l c h a n g e s y i e l d maximum benefits. However, t h i s cannot be a c h i e v e d by t h e c h e m i s t a l o n e , e v e n t h o u g h new m o l e c u l a r b i o l o g y p r o v i d e s a n e x p l a n a t i o n o f b i o l o g i c a l phenomena i n c h e m i c a l t e r m s . Behaviour, population dynamics and o t h e r f a c t o r s a s s o c i a t e d w i t h whole organisms and communities a r e a l s o e s s e n t i a l components o f t h e c o o p e r a t i v e s t u d y r e q u i r e d t o a c h i e v e c o n t r o l o f p e s t s i n an e c o l o g i c a l l y sound and e f f e c t i v e manner.

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In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

24 Do Plants "Psychomanipulate" Insects? L. L. MURDOCK, G. BROOKHART, R. S. EDGECOMB, T. F. LONG, and L. SUDLOW Department of Entomology, Purdue University, West Lafayette, I N 47907

The hypothesis tha against herbivory by "psychomanipulation of insect herbivores is presented. In brief, the psychomanipulation hypothesis asserts that certain secondary plant chemicals imbibed in sublethal doses by insects feeding on plant parts interfere with information processing in the insectan central nervous system and thereby modify insect-herbivore behavior in ways that reduce or prevent further herbivory. Behavioral changes could include the evocation of unadaptive behaviors, inhibition of hunger, onset of narcosis, etc. Evidence will be presented that numerous plant substances do have "psychomanipulative" effects at sublethal levels and that the pyschomanipulation mechanism might operate in nature. One important d e f e n s i v e s t r a t e g y o f p l a n t s i s t h e s y n t h e s i s and accumulation o f chemicals i n t h e i r t i s s u e s that discourage o r stop f e e d i n g by h e r b i v o r e s . The mechanisms by which t h e s e c h e m i c a l s a f f e c t p o t e n t i a l h e r b i v o r e s a r e c u r r e n t l y b e i n g e l u c i d a t e d . Two g e n e r a l modes o f a c t i o n seem t o be w i d e l y a c c e p t e d : ( i ) d e t e r r e n c y and ( i i ) t o x i c i t y . Deterrents are gustatory stimulants that n e g a t i v e l y a f f e c t t h e i n s e c t ' s b i t i n g , f e e d i n g , and maintenance o f f e e d i n g (I) » D e t e r r e n c y i n v o l v e s an a c t i o n o f t h e p r o t e c t a n t c h e m i c a l on t h e s e n s o r y nervous system o f t h e unadapted h e r b i v o r e . The message sent t o t h e i n s e c t c e n t r a l nervous system i s a s e r i e s o f a c t i o n p o t e n t i a l s from a s p e c i f i c s e n s o r y neuron o r a c e r t a i n p a t t e r n e d output from a s e t o f s e n s o r y neurons (.2,30. I t i s i n t e r p r e t e d as "danger" o r "no good" and i t s e f f e c t i s t o cause f e e d i n g t o cease (2^3). T o x i c i t y , on t h e o t h e r hand, d i s r u p t s t h e f u n c t i o n i n g o f p h y s i o l o g i c a l and b i o c h e m i c a l systems w i t h i n t h e herbivore. T o x i c a c t i o n i n t h e i n s e c t may cause s i c k n e s s o r weakness. The i n s e c t may n o t grow, mature, o r reproduce n o r m a l l y and perhaps d i e p r e m a t u r e l y . Deterrents

and t o x i n s d i f f e r

i n t h e i r s i t e s and modes o f

0097-6156/85/0276-0337S06.00/0 © 1985 American Chemical Society

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action. D e t e r r e n t s a c t on t h e p e r i p h e r a l nervous system, c h i e f l y on the chemosensory a p p a r a t u s , r e s u l t i n g i n a b e h a v i o r a l change i n t h e insect. In c o n t r a s t , t o x i n s d i s r u p t c e l l u l a r , b i o c h e m i c a l o r p h y s i o l o g i c a l processes i n the i n s e c t . The h y p o t h e s i s p r e s e n t e d h e r e suggests a t h i r d g e n e r a l mode o f a c t i o n f o r secondary p l a n t compounds. I t b e g i n s w i t h the premises ( a ) t h a t t h e i n s e c t c e n t r a l nervous system (CNS) i s an e x c e l l e n t t a r g e t f o r p l a n t d e f e n s i v e c h e m i c a l s — a t l e a s t as s u i t a b l e as t h e chemosensory p o r t i o n o f t h e p e r i p h e r a l nervous system; (b) t h a t t h e importance o f many p l a n t c h e m i c a l s i s not t h e i r l e t h a l i t y but r a t h e r t h e changes they evoke i n t h e i n s e c t h e r b i v o r e ' s b e h a v i o r by i n t e r f e r i n g w i t h c e n t r a l nervous f u n c t i o n . We suggest t h e term " p s y c h o m a n i p u l a t i o n " t o d e s c r i b e t h e b e h a v i o r - m o d i f y i n g e f f e c t s o f such c h e m i c a l s a c t i n g i n the CNS. Our purpose i n t h i s paper i s t o d e f i n e t h e psychomanipulation h y p o t h e s i s , provide evidence i n support o f i t , and suggest how p o t e n t i a l p s y c h o m a n i p u l a n t s might be s t u d i e d . The p r i m a r y q u e s t i o n i s how p l a n t c h e m i c a l s m o d i f y i n s e c t b e h a v i o r through c e l l u l a r and m o l e c u l a q u e s t i o n might open approache c o n t r o l agents. The

Psychomanipulation

Scenario

Imagine a hungry i n s e c t a l i g h t i n g on t h e l e a f o f a non-host p l a n t t h a t accumulates a p s y c h o m a n i p u l a n t . Because the l e a f c o n t a i n s s u f f i c i e n t p o s i t i v e s t i m u l i and i n s u f f i c i e n t d e t e r r e n t s , f e e d i n g begins. Some o f t h e imbibed b e h a v i o r - m o d i f y i n g c h e m i c a l escapes t h e d e t o x i c a t i o n m a c h i n e r y i n t h e gut w a l l , e n t e r s c i r c u l a t i o n , and p e n e t r a t e s the b l o o d - b r a i n b a r r i e r i n t o t h e CNS. There i t r e a c t s w i t h p a r t i c u l a r n e u r o n a l types t o e x c i t e , i n h i b i t , modulate, o r o t h e r w i s e a f f e c t normal p a t t e r n s o f a c t i v i t y . This modified n e u r o n a l a c t i v i t y i s m a n i f e s t e d as a change i n t h e i n s e c t ' s b e h a v i o r such t h a t i t no l o n g e r consumes p l a n t m a t e r i a l , o r a t l e a s t consumes l e s s than i t would o t h e r w i s e . The I n s e c t CNS as T a r g e t . The i n s e c t c e n t r a l nervous system i s com­ posed o f a d o r s a l and r o s t r a l s u p r a e s o p h a g e a l g a n g l i o n o r b r a i n , which communicates v i a c i r c u m e s o p h a g e a l c o n n e c t i v e s t o t h e f i r s t o f a c h a i n o f v e n t r a l l y l o c a t e d g a n g l i a ( f u s e d t o d i f f e r e n t degrees i n different species). Sensory i n f o r m a t i o n from t h e environment e n t e r s g a n g l i a o f t h e CNS v i a the axons o f s e n s o r y neurons, whose c e l l b o d i e s a r e l o c a t e d i n t h e p e r i p h e r y o f the organism. The s e n s o r y axons p r o j e c t i n t o t h e n e u r o p i l and form synapses w i t h s p e c i f i c c l a s s e s o f neurons. I n f o r m a t i o n i s t r a n s m i t t e d between neurons i n most i n s t a n c e s by means o f c h e m i c a l t r a n s m i t t e r s u b s t a n c e s r e l e a s e d at p r e s y n a p t i c t e r m i n a l s . These t r a n s m i t t e r m o l e c u l e s d i f f u s e a c r o s s the narrow gap ( s y n a p t i c c l e f t ) between t h e p r e s y n a p t i c c e l l and the p o s t s y n a p t i c c e l l and i n t e r a c t w i t h s p e c i f i c r e c e p t o r s exposed on t h e s u r f a c e o f t h e l a t t e r . The t r a n s m i t t e r - r e c e p t o r i n t e r a c t i o n a l t e r s the l e v e l o f e x c i t a b i l i t y o f the p o s t s y n a p t i c cell. E x c i t a t i o n o r i n h i b i t i o n o f t h a t neuron c a n have b e h a v i o r a l consequences i f t h e change i n i t s e x c i t a b i l i t y a f f e c t s o t h e r neurons to which i t communicates. Motor neurons a r e the immediate d e t e r m i n a n t s o f movement and communicate d i r e c t l y w i t h muscle f i b e r s

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

24.

MURDOCK ET AL.

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v i a chemical t r a n s m i s s i o n . Motor neuron c e l l b o d i e s are l o c a t e d i n the p e r i p h e r y o f CNS g a n g l i a . The d e n d r i t e s o f these neurons a r e o f t e n e l a b o r a t e a r b o r i z a t i o n s l o c a t e d w i t h i n the c e n t r a l r e g i o n s o f the g a n g l i o n , c o l l e c t i n g i n f o r m a t i o n from o t h e r n e u r o n s . The n a t u r e and i n t e n s i t y o f t h a t i n f o r m a t i o n d e t e r m i n e s whether the motor neuron becomes a c t i v e and f i r e s a c t i o n p o t e n t i a l s . Interneurons r e s i d e c o m p l e t e l y w i t h i n the CNS. They c o o r d i n a t e the a c t i v i t i e s o f s e n s o r y and motor neurons and subserve a l l major i n t e g r a t i v e and h i g h e r f u n c t i o n s o f the nervous system. E v i d e n c e i s a c c u m u l a t i n g t h a t i n s e c t s a l s o make use o f m o d u l a t o r y neurons, neurons whose a c t i v i t i e s change the q u a l i t y o f i n f o r m a t i o n p a s s i n g t h r o u g h synapses, or m o d i f y the spontaneous a c t i v i t y o f r e c e p t i v e neurons or muscle c e l l s ( 4 , 5 ) . The b e s t - c h a r a c t e r i z e d m o d u l a t o r y neuron i s the Dumeti c e l l , which i n n e r v a t e s the e x t e n s o r t i b i a e muscle i n the m i g r a t o r y l o c u s t . T h i s c e l l , whose u n p a i r e d soma i s l o c a t e d i n the m i d l i n e o f the m e t a t h o r a c i c g a n g l i o n , r e l e a s e s octopamine from i t s terminals. Neurons o f t h i s type may be w i d e s p r e a d i n i n s e c t s and some o f them may be i n t e r n e u r o n The range o f n e u r o c h e m i c a i s remarkably s i m i l a r t o those i n mammals. In i n s e c t s , a c e t y l c h o l i n e seems t o be the n e u r o t r a n s m i t t e r r e l e a s e d by most types o f s e n s o r y neurons and L - g l u t a m i c a c i d a p p a r e n t l y s e r v e s as the t r a n s m i t t e r o f motor neurons i n n e r v a t i n g s k e l e t a l muscles ( 4 ) . P e r i p h e r a l i n h i b i t o r y neurons may r e l e a s e gamma-aminobutyric a c i d (GABA) at t h e i r t e r m i n a l s . I n a few i n s t a n c e s t h e r e i s e v i d e n c e t h a t a r o m a t i c b i o g e n i c amines s e r v e as neuromessengers i n the p e r i p h e r y , e.g., octopamine i s the m o d u l a t o r substance r e l e a s e d by Dumeti neurons r e f e r r e d t o above ( 4 ) , and the l i g h t organs o f the f i r e f l y a l s o seem t o be c o n t r o l l e d by neurons r e l e a s i n g octopamine (6). The s a l i v a r y g l a n d s o f v a r i o u s i n s e c t s , i n c l u d i n g the l o c u s t , S c h i s t o c e r c a g r e g a r i a ( 7 ) , the c o c k r o a c h , Nauphoeta c i n e r i a ( 8 ) , and the moth, Manduca s e x t a (9) may be i n n e r v a t e d by c a t e c h o l a m i n e r g i c nerve f i b e r s . A few muscles appear t o be i n n e r v a t e d by neurons t h a t r e l e a s e n e u r o a c t i v e p e p t i d e s , n o t a b l e examples b e i n g the c o c k r o a c h h i n d g u t , where p r o c t o l i n i s r e l e a s e d ( 1 0 ) , and the c o x a l d e p r e s s o r muscles i n n e r v a t e d by the Ds motoneuron i n P e r i p l a n e t a a m e r i c a n a (11). A l l o f these neuromessengers as w e l l as o t h e r s u n d o u b t e d l y a l s o s e r v e t o communicate i n f o r m a t i o n between neurons w i t h i n the i n s e c t c e n t r a l nervous system. P l a n t P r o t e c t i o n by B e h a v i o r M o d i f i c a t i o n . B e h a v i o r i s g e n e r a l l y equated w i t h the movement o f an o r g a n i s m or body p a r t and the c e s s a ­ t i o n o f such movements ( 1 2 ) . To be c l a s s i f i e d as b e h a v i o r , movement r e q u i r e s the p a r t i c i p a t i o n and d i r e c t i o n o f the CNS. The i n t i m a t e involvement o f the CNS i n the c o o r d i n a t i o n o f muscles and body p a r t s r e s u l t s i n b e h a v i o r s r a n g i n g from the fundamental ( e . g . , w a l k i n g and grooming) t o the complex ( e . g . , n a v i g a t i o n and l e a r n i n g ) . Even r e l a t i v e l y simple b e h a v i o r s such as jumping or g r a s p i n g f o o d , r e q u i r e the p a r t i c i p a t i o n o f neurons on many l e v e l s . S i n c e b e h a v i o r depends on the p r o p e r f u n c t i o n i n g o f e v e r y neuron i n v o l v e d i n i t s e x p r e s s i o n , d i s r u p t i o n o f any p a r t i c i p a t i n g neuron c o u l d have s e v e r e consequences. These consequences, however, may be important without b e i n g o b v i o u s . For example, g r e a t l y i n c r e a s e d locomotor a c t i v i t y might be d e t r i m e n t a l beause the i n s e c t

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i s spending f a r l e s s time f e e d i n g and c o n c u r r e n t l y expending food r e s e r v e s as u n n e c e s s a r y a c t i v i t y . O t h e r b e h a v i o r a l changes might cause an i n c r e a s e i n t h e r e s p o n s e time needed f o r escape, thus i n ­ c r e a s i n g t h e chances f o r t h a t i n s e c t t o end up as a n o t h e r ' s meal. By a f f e c t i n g neurons i n v o l v e d i n s p e c i f i c b e h a v i o r s , p l a n t psychom a n i p u l a n t s c o u l d n e g a t i v e l y a f f e c t i n s e c t b e h a v i o r and thus a i d i n the d e f e n s e o f the p l a n t . A c t i o n i n the CNS o f P l a n t - d e r i v e d Compounds S i n c e communication between neurons by means o f c h e m i c a l n e u r o messenger s u b s t a n c e s i s b a s i c t o t h e f u n c t i o n i n g o f the i n s e c t CNS, and s i n c e t h e CNS i s t h e w e l l s p r i n g o f a l l complex and p a t t e r n e d i n s e c t b e h a v i o r , i n t e r f e r e n c e w i t h c h e m i c a l communication w i t h i n t h e CNS s h o u l d prevent b e h a v i o r s from a p p e a r i n g when they n o r m a l l y would, o r evoke b e h a v i o r s when t h e y n o r m a l l y would n o t . Psychomanipulant c h e m i c a l s r e a c h i n g the i n s e c t CNS c o u l d d i s r u p t c h e m i c a l communication b antagonists, synaptic clearanc r e l e a s e r s , and neuromessenger s u p p l y s u p p r e s s a n t s . A wide v a r i e t y o f p l a n t s e c o n d a r y s u b s t a n c e s f i t t i n g these c a t e g o r i e s c o u l d i n t e r f e r e w i t h i n s e c t n e u r o c h e m i c a l messenger systems and e f f e c t b e h a v i o r a l changes. Our purpose h e r e i s t o mention a few examples. Receptor A g o n i s t s . By m i m i c k i n g t h e n a t u r a l c h e m i c a l messenger a t i t s r e c e p t o r s , p s y c h o m a n i p u l a n t s c o u l d cause s p u r i o u s e x c i t a t i o n o r i n h i b i t i o n i n the CNS. Two examples o f r e c e p t o r a g o n i s t s a r e n i c o t i n e found i n the l e a v e s o f t h e t o b a c c o p l a n t ,

NICOTINE N i c o t i a n a tabacum, and l o b e l i n e , p r e s e n t i n t h e d r i e d l e a v e s and tops o f the h e r b , L o b e l i a i n f l a t a ( 1 3 ) . Both n i c o t i n e and l o b e l i n e a c t as a g o n i s t s on a s p e c i f i c type o f a c e t y l c h o l i n e r e c e p t o r , t h e n i c o t i n i c cholinergic receptor. I n mammals, n i c o t i n i c c h o l i n e r g i c r e c e p t o r s mediate c h o l i n e r g i c n e u r o t r a n s m i s s i o n i n s k e l e t a l m u s c l e s , autonomic g a n g l i a , and t h e c e n t r a l nervous system. At these s i t e s n i c o t i n e ' s a c t i o n has two phases, e x c i t a t i o n and d e p r e s s i o n . Insect c e n t r a l nervous t i s s u e s a r e r i c h i n n i c o t i n i c c h o l i n e r g i c r e c e p t o r s ( c f . 1 4 ) , but t h e i r r o l e i n b e h a v i o r i s n o t w e l l u n d e r s t o o d . Among the known a g o n i s t s o f m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s i s o l a t e d from p l a n t s a r e p i l o c a r p i n e , which i s i s o l a t e d from l e a f l e t s o f South American shrubs o f t h e genus P i l o c a r p u s , and a r e c o l i n e , i s o l a t e d from t h e seeds o f A r e c a c a t e c h u , t h e b e t e l n u t . C h o l i n e r g i c receptors with pharmacological p r o p e r t i e s c l o s e l y r e s e m b l i n g those o f v e r t e b r a t e m u s c a r i n i c r e c e p t o r s a r e found i n t h e i n s e c t CNS ( 1 4 ) . I t i s i n t e r e s t i n g t h a t m u s c a r i n i c agents appear t o

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MURDOCKETAL.

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possess l e s s k i l l i n g p o w e r than n i c o t i n e a g o n i s t s ( 1 5 ) . Many o t h e r p l a n t - d e r i v e d c h o l i n e r g i c a g o n i s t s are known, i n c l u d i n g the a l k a l o i d s c y t i s i n e and m a t r i n e . There are n a t u r a l l y - o c c u r r i n g o p i o i d a g o n i s t s from p l a n t s , such as morphine, which owes i t s d r a m a t i c p s y c h o p h a r m a c o l o g i c a l effects i n mammals to an i n t e r a c t i o n w i t h r e c e p t o r s f o r e n k e p h a l i n s and endorphins. As e v i d e n c e i n c r e a s e s t h a t i n s e c t s and o t h e r a r t h r o p o d s u t i l i z e n e u r o a c t i v e p e p t i d e s as neuromessengers ( 1 6 ) , i t becomes more l i k e l y t h a t some o f the p l a n t o p i a t e s are d e f e n s i v e c h e m i c a l s having a "psychomanipulant type o f mechanism. 11

Receptor A n t a g o n i s t s . By b l o c k i n g neuromessenger r e c e p t o r s , r e c e p t o r a n t a g o n i s t s may evoke i n a p p r o p r i a t e b e h a v i o r s t h r o u g h i n h i b i t i o n or d i s i n h i b i t i o n o f a neuron or group o f n e u r o n s . GABA r e c e p t o r s i n a r t h r o p o d s and mammals are b l o c k e d by two c h e m i c a l s o f p l a n t o r i g i n , b i c u c c u l i n e , and p i c r o t o x i n ( 1 7 ) . Scopolamine, common i n many Solanaceae ( i n c l u d i n g the famous d e a d l y n i g h t s h a d e Atropa b e l l a d o n n a ) , i s an e x c e l l e n cholinergic receptors, a the more n o t o r i o u s p l a n t c h e m i c a l s i s c u r a r e (used f o r p o i s o n a r r o w s ) , which i s p r e p a r e d from a v a r i e t y o f s p e c i e s o f S t r y c h n o s (18). The a c t i v e compound common to a l l p r e p a r a t i o n s o f c u r a r e i s D-tubocurarine. Another antagonist ( n i c o t i n i c ) of p l a n t o r i g i n i s 3 - e r y t h r o i d i n e , from the seeds and o t h e r p l a n t p a r t s o f s e v e r a l E r y t h r i n a species (13). One o f the more s p e c i f i c a n t a g o n i s t s o f 01-2 a d r e n e r g i c r e c e p t o r s i s yohimbine, the p r i n c i p a l a l k a l o i d i n e x t r a c t s o f the b a r k o f P a u s i n y s t a l i a yohimbe ( 1 9 ) . Yohimbine a l s o b l o c k s one type o f o c t o p a m i n e r g i c r e c e p t o r i n l o c u s t s ( 2 0 ) . Synaptic Clearance Antagonists. By p r e v e n t i n g the removal o f n a t u r a l l y - r e l e a s e d t r a n s m i t t e r from the r e g i o n o f i t s r e c e p t o r s , the e f f e c t o f the neuromesssenger on the r e c e i v i n g c e l l w i l l be p r o l o n g e d and i n t e n s i f i e d . There are t h r e e p r i n c i p a l r o u t e s by which neuromessengers are removed from the s y n a p t i c c l e f t : ( i ) enzymatic d e s t r u c t i o n o f the t r a n s m i t t e r ( e . g . , a c e t y l c h o l i n e (ACh) which i s h y d r o l y z e d i n the s y n a p t i c c l e f t by a c e t y l c h o l i n e s t e r a s e ) ; ( i i ) uptake i n t o p r e - and p o s t - s y n a p t i c c e l l s by membrane-associated pumps t h a t have s u b s t a n t i a l s p e c i f i c t y f o r m o l e c u l e s they w i l l c a r r y ; ( i i i ) d i f f u s i o n away from the c l e f t . The c l a s s i c a l s y n a p t i c c l e a r a n c e a n t a g o n i s t i s the c h o l i n e s t e r a s e i n h i b i t o r p h y s o s t i g m i n e , from the c a l a b a r bean, Physostigma

PHYSOSTIGMINE venenosum, a West A f r i c a n p e r e n n i a l ( 1 3 ) . Physostigmine r e v e r s i b l y i n h i b i t s a c e t y l c h o l i n e s t e r a s e and t h e r e b y p e r m i t s the b u i l d u p o f

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a b n o r m a l l y - h i g h l e v e l s o f ACh i n the s y n a p t i c c l e f t . This r e s u l t s i n p r o l o n g e d and i n t e n s i f i e d s t i m u l a t i o n o f t h e p o s t s y n a p t i c c e l l , that can lead to d y s f u n c t i o n o f the p h y s i o l o g i c a l process c o n t r o l l e d by t h e c h o l i n e r g i c i n p u t . L e s s e r known, b u t p o s s i b l y s i g n i f i c a n t as a p s y c h o m a n i p u l a n t , i s g u v a c i n e ( 2 1 ) , from b e t e l n u t s , which b l o c k s uptake o f GABA i n mammalian CNS p r e p a r a t i o n s . C o c a i n e i s a p o w e r f u l CNS s t i m u l a n t as w e l l as l o c a l a n e s t h e t i c (13). I t i s i s o l a t e d from t h e l e a v e s o f E r y t h r o l o n c o c a , a t r e e i n d i g e n o u s t o P e r u and B o l i v i a . Cocaine e f f e c t i v e l y blocks the uptake o f c a t e c h o l a m i n e s i n t o p r e s y n a p t i c neurons and thus promotes the a c t i v i t y o f synapses ( b o t h c e n t r a l and p e r i p h e r a l ) i n v o l v i n g t h e s e amines ( 2 2 ) . Neuromessenger R e l e a s e r s . Normal CNS f u n c t i o n i n g c o u l d be d i s r u p t e d by e v o k i n g premature o r c o n t i n u e d r e l e a s e o f neuromessengers from p r e s y n a p t i c s t o r e s . A l t h o u g h D(+)-amphetamine, a well-known p s y c h o ­ s t i m u l a n t and a p p e t i t e d e p r e s s a n t , does n o t o c c u r i n p l a n t s , a c l o s e l y - r e l a t e d substance the l e a v e s o f t h e khat s h r u b A f r i c a and i n t h e Arab p e n i n s u l a . C a t h i n o n e evokes t h e r e l e a s e o f n o r e p i n e p h r i n e from c e n t r a l and p e r i p h e r a l p r e s y n a p t i c s t o r e s and has c a r d i o v a s c u l a r and a p p e t i t e d e p r e s s i n g e f f e c t s s i m i l a r t o Damphetamine ( 2 3 ) .

CATHINONE Neuromessenger S u p p l y S u p p r e s s a n t s . By r e d u c i n g t h e a v a i l a b i l i t y o f neuromessengers from p r e s y n a t i c s t o r e s , neuromessenger s u p p l y sup­ p r e s s a n t s c o u l d a l t e r t h e p h y s i o l o g y and b i o c h e m i s t r y o f the p r e ­ s y n a p t i c neuron such t h a t t h e s e s t o r e s a r e d e p l e t e d . The r e d u c t i o n o f neuromessenger s u p p l y c o u l d be caused by the drug r e s e r p i n e , i s o l a t e d from t h e I n d i a n p l a n t , R a u w o l f i a s e r p e n t i n a ( 1 3 ) . The

RESERPINE

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e f f e c t o f r e s e r p i n e i s the d e p l e t i o n o f stores o f aromatic b i o g e n i c amine t r a n s m i t t e r s , i n c l u d i n g dopamine, n o r e p i n e p h r i n e , 5-hydroxyt r y p t a m i n e , and octopamine. In i n s e c t s , the e f f e c t s o f r e s e r p i n e are r e l a t i v e l y slow i n onset and c a n p e r s i s t f o r many days ( 2 4 ) . R e s e r p i n e i s a c t i v e i n b o t h i n s e c t s and mammals. In mammals, r e s e r p i n e can reduce b l o o d p r e s s u r e and produce a s t a t e o f s e d a t i o n i n which the i n d i v i d u a l i s i n d i f f e r e n t t o e n v i r o n m e n t a l s t i m u l i (25). Others. There a r e o t h e r p l a n t s u b s t a n c e s which c o u l d a c t as p s y c h o m a n i p u l a n t s v i a mechanisms t h a t do n o t f i t n e a t l y i n t o t h e above categories. Some c o u l d e n t e r and d i s r u p t t h e normal p r o c e s s e s o p e r a t i n g i n a neuron. These would i n c l u d e s u b s t a n c e s l i k e c a f f e i n e and theobromine, b o t h o f which i n h i b i t i n t r a n e u r o n a l p h o s p h o d i ­ e s t e r a s e , thereby a f f e c t i n g the e x c i t a b i l i t y o f neurons. P r e s y n a p t i c s u p p l y o f t r a n s m i t t e r c o u l d be a f f e c t e d by t h e d i e t a r y content o f t r a n s m i t t e r precursors L-DOPA t h e p r e c u r s o r o f dopamine, a prominent i n s e c species of plants. L-5-Hydroxytryptophan p r e c u r s o r o f the i n d o l e a m i n e t r a n s m i t t e r 5 - h y d r o x y t r y p t a m i n e , a l s o o c c u r s a t h i g h l e v e l s i n some p l a n t m a t e r i a l s . Although the con­ sequences o f a l t e r i n g l e v e l s o f t r a n s m i t t e r s v i a d i e t a r e n o t c l e a r , such a l t e r a t i o n s have been shown t o o c c u r w i t h s e v e r a l t r a n s m i t t e r s i n mammals (26) and c o u l d a l t e r the b e h a v i o r o f an i n s e c t h e r b i v o r e . Candidate Psychomanipulants o f Plant

Origin

Physostigmine. There i s e v i d e n c e t h a t c h o l i n e r g i c drugs a t s u b l e t h a l l e v e l s can indeed modify i n s e c t b e h a v i o r . A d u l t male L o c u s t a m i g r a t o r i a t h a t have passed t h e i m a g i n a i molt a t l e a s t t h r e e weeks e a r l i e r f l y w i t h a wing-beat f r e q u e n c y o f 23 Hz. The e l e g a n t work o f D.M. W i l s o n (27) e s t a b l i s h e d t h a t the r e g u l a r wing b e a t i n g in l o c u s t s i s the r e s u l t o f a c e n t r a l p a t t e r n generator l o c a t e d i n the t h o r a c i c g a n g l i a . While t h e p a t t e r n g e n e r a t o r i s c a p a b l e o f p r o d u c i n g the motor program f o r f l i g h t i n t h e absence o f s e n s o r y feedback from t h e wings and t h o r a x , such feedback can, n e v e r t h e l e s s , i n f l u e n c e the f r e q u e n c y w i t h which t h e wings a r e f l a p p e d . Wind d e t e c t o r s on t h e head can a l s o m o d i f y t h e output o f t h e c e n t r a l pattern generator (28). When a d u l t JL. m i g r a t o r i a were i n j e c t e d w i t h 5 yg/g body weight o f p h y s o s t i g m i n e s a l i c y l a t e , they e x h i b i t e d a p e r i o d o f h y p e r a c t i v i t y l a s t i n g s e v e r a l m i n u t e s , f o l l o w e d by a 2-3 hour p e r i o d of hyperresponsiveness f o l l o w i n g handling or mechanical s t i m u l a t i o n (29). T h e r e a f t e r t h e y appeared o u t w a r d l y normal w i t h l i t t l e o r no m o r t a l i t y a t 24 h o u r s . T h i s s u b l e t h a l dose o f p h y s o s t i g m i n e had a d r a m a t i c e f f e c t on w i n g b e a t i n g f r e q u e n c y ( F i g . 1 ) . D u r i n g t h e e a r l y t e s t i n t e r v a l s many i n d i v i d u a l s o n l y e x h i b i t e d b r i e f p e r i o d s o f f l i g h t w i t h v i s i b l y reduced f r e q u e n c y . At 4 hours p o s t i n j e c t i o n , the f r e q u e n c y o f those i n d i v i d u a l s t h a t e x h i b i t e d s u s t a i n e d f l i g h t s f e l l t o about 16 Hz, r e t u r n i n g s l o w l y t h e r e a f t e r toward t h e c o n t r o l value. The a v a i l a b l e d a t a cannot demonstrate t h a t s l o w i n g t h e wing-beat f r e q u e n c y f o l l o w i n g i n j e c t i o n o f p h y s o s t i g m i n e i s s o l e l y or even p a r t l y due t o e l e v a t e d l e v e l s o f a c e t y l c h o l i n e a t

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J_ 4

-L 8

12

After injection,

16

20

24

48

hr

F i g u r e 1. Wing-beat f r e q u e n c y o f L o c u s t a a d u l t s b e f o r e (arrow on a b s c i s s a ) and a f t e r i n j e c t i o n o f 5 y l / g H^O ( A — A ) o r 5 yg/g p h y s o s t i g m i n e ( · — · ) . Reproduced w i t h p e r m i s s i o n from Ref. 29. C o p y r i g h t 1973, Pergamon P r e s s .

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sensory-central synapses i n the CNS. I t i s possible that some or a l l of the effect of physostigmine on the central pattern generator is a result of acetylcholine accumulation at other types of central cholinergic synapses, synapses that participate i n the pattern generation mechanism. However, this example does demonstrate that a naturally-occurring anticholinesterase agent acting at sublethal levels could s i g n i f i c a n t l y affect a c r u c i a l behavior. A locust able to f l y only a short distance or to f l y only with reduced speed i s much more susceptible to prédation. Reserpine. As mentioned e a r l i e r , the Rauwolfia alkaloid reserpine is noted for i t s a b i l i t y to deplete aromatic biogenic amines i n nervous tissue of mammals and insects. One of the more remarkable effects of reserpine i n humans i s to render individuals indifferent to environmental s t i m u l i . Reserpine appears to have a similar effect on insects, although information i s s t i l l r e l a t i v e l y scanty. In the cockroach, Periplaneta americana, reserpine at 50 ug/g causes strong and long-lastin dopamine, octopamine, an authors have noted that reserpine has t r a n q u i l i z i n g effects on insects, e.g., the ant Formica rufa (30) and P^. americana (24,31). In our own experiments with adult Phormia regina, feeding reserpine mixed with powdered milk and sucrose or injecting i t at doses of 100 yg/g body weight and higher seemed to induce a state of lethargy and inactivity. To obtain clearer understanding of the behavioral effects of reserpine and their possible r e l a t i o n s h i p to levels of aromatic biogenic amines i n the insect central nervous system, we injected unfed, 2-day old adult ]?. regina with 2 yg/fly of reserpine. Because of the i n s o l u b i l i t y of reserpine, we f i r s t dissolved i t i n a small volume of dimethylacetamide (DMAD) and mixed the DMAD with corn o i l i n a 1:10 r a t i o . Two groups of control f l i e s were used, one untreated and the other injected with 300 n l of the DMAD/corn o i l vehicle. The f l i e s were held for one day with water available ad l i b . , but no food. To determine the effectiveness of the drug, two behaviors were observed: ( i ) meal size; ( i i ) proboscis-extension responses to t a r s a l stimulation. Meal size was determined by weighing the f l i e s (plus the applicator sticks attached to them as handles) before feeding and after allowing them to feed for 30 minutes on 1 M sucrose. The net weight gain was taken as the meal s i z e . Proboscis-extension responsiveness was estimated as the mean acceptance threshold (M.A.T., the concentration of sucrose to which the average f l y i n the population w i l l respond) and was determined following the up-and-down procedure described by Thompson (32). Brains were dissected from control and reserpinized f l i e s and assayed for octopamine and dopamine by high performance l i q u i d chromatography (HPLC). The column used was a Brownlee RP-18 5 y p a r t i c l e size cartridge (250 mm χ 2.1 mm i . d . ) . The amines were eluted i s o c r a t i c a l l y with 0.1 M KH^PO^ containing 2% methanol and 0.3 mM heptanesulfonic acid, sodium s a l t ; mobile phase pH was 3.05 (33). The flow rate was 0.6 ml/min. Octopamine and dopamine were detected and quantitated using an RC-3 electrochemical detector (Bioanalytical Systems, W. Lafayette, Indiana) equipped with a glassy carbon electrode operated at +0.95 V r e l a t i v e to a Ag/AgCl

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reference electrode. Under these conditions the retention time of octopamine was 8 min and dopamine was 16 min. Vehicle-injected f l i e s exhibited a mildly elevated M.A.T. and a moderate meal size; concomitantly, there was a decline in brain octopamine content but no change i n brain dopamine levels (Table I ) . Reserpine treated f l i e s exhibited a 10-fold r i s e i n threshold r e l a t i v e to the vehicle-injected controls and a large increase in meal size. These f l i e s were hyperphagic i n that they consumed t h e i r own body weight in 1 M sucrose, more than double that of the un­ treated controls and 60 percent higher than the solvent controls. Brain levels of octopamine i n the group injected with reserpine were s i g n i f i c a n t l y depleted r e l a t i v e to both untreated and solvent controls. Clearly, i n j e c t i o n of adult blowflies with reserpine caused them to become less responsive to food s t i m u l i , while at the same time inducing them to eat more when they were offered 1 M sucrose, a highly stimulating food. Similar observations have been made with blowflies fed on reserpin ascribe the behavioral e f f e c t actions i n the CNS, i t seems quite l i k e l y that the CNS plays a major r o l e , p a r t i c u l a r l y in l i g h t of the demonstrated capacity for reserpine to deplete CNS aromatic biogenic amines. Both the meal size increase and the decreased responsiveness to t a r s a l stimulation with sucrose caused by reserpine can be explained as a requirement for increased i n t e n s i t y of sensory information to TABLE I.

EFFECTS OF RESERPINE ON BLOWFLY FEEDING BEHAVIOR AND BIOGENIC AMINES UNTREATED CONTROL

SOLVENT CONTROL

a

M.A.T. (mM)

5.2 *

a

MEAL SIZE (mg)

18

+ 6.3

OCTOPAMINE (pmol/brain)

7.6

a

+ 1.8

DOPAMINE (6) (pmol/brain)

2.8

a

+ 0.85

24

b

(10)

25

(6)

4.8

(6)

3.6

b

RESERPINE (2 yg/fly)

b

+ 6.4

BRAIN

250

(10)

C

40° +_ 5.8

(10)

+ 1.9

(6)

2.4° + 0.44

+ 1.7

(6)

0.94° +

(6)

0.24

*Values within a row followed by d i f f e r e n t l e t t e r s are s i g n i f i c a n t l y different (p < 0.05).

i n i t i a t e and terminate feeding. The higher M.A.T. r e f l e c t s that higher concentrations of sucrose are needed to evoke proboscisextension responses i n P. regina adults. Higher concentrations of

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s u c r o s e evoke h i g h e r - f r e q u e n c y r e s p o n s e s i n the t a r s a l sugar receptors (35). In o t h e r words, more i n t e n s e s e n s o r y i n f o r m a t i o n must r e a c h the a m i n e - d e p l e t e d CNS b e f o r e the b e h a v i o r ensues. Sensory i n f o r m a t i o n i s a l s o c r u c i a l t o t e r m i n a t e a meal. As the f l y i n i t i a t e s consumption, the c r o p b e g i n s f i l l i n g . As i t expands, the c r o p s t i m u l a t e s s t r e t c h r e c e p t o r s i n a nerve net a s s o c i a t e d w i t h the c r o p and abdominal w a l l ( 3 6 ) . I f the i n h i b i t o r y s e n s o r y i n f o r m a t i o n i s not f o r t h c o m i n g , h y p e r p h a g i a ensues (37-39). A c c o r d i n g t o our hypothe s i s , i n the r e s e r p i n e — a f f e c t e d CNS more i n t e n s e s e n s o r y s t i m u l i i s r e q u i r e d i n o r d e r t o b r i n g the meal t o an end. The n e c e s s a r y e x t r a s t i m u l i would be f o r t h c o m i n g as the c r o p c o n t i n u e s t o expand due t o c o n t i n u e d f e e d i n g . T h i s would impose a d d i t i o n a l s t r e t c h on the abdominal s t r e t c h r e c e p t o r s , which would respond by f i r i n g at h i g h e r and h i g h e r f r e q u e n c i e s . At some p o i n t the f i r i n g r a t e o f these r e c e p t o r s becomes s u f f i c i e n t l y h i g h t o t r i g g e r the t e r m i n a t i o n o f f e e d i n g i n the CNS. These o b s e r v a t i o n s combined w i t h those i n the l i t e r a t u r e support the concept t h a e x e r t a profound and l o n g - l a s t i n e f f e c t which would d e c r e a s e the i n s e c t ' s m o b i l i t y and f i t n e s s t o respond t o e n v i r o n m e n t a l s t i m u l i , and t o p r e d a t o r s . That e f f e c t , p r o b a b l y due l a r g e l y t o a c e n t r a l a c t i o n o f the s u b s t a n c e , c o u l d r e s u l t i n p l a n t p r o t e c t i o n by " p s y c h o m a n i p u l a t i o n . 11

L-canavanine and L - c a n a l i n e . Secondary p l a n t s u b s t a n c e s may evoke p a r t i c u l a r types o f s t e r e o t y p e d i n s e c t b e h a v i o r v i a a c t i o n s w i t h i n the CNS. An e x c e l l e n t example i s the work o f Kammer, Dahlman, and R o s e n t h a l ( 4 0 ) , who o b s e r v e d t h a t i n j e c t i o n o f a d u l t Manduca s e x t a w i t h L-canavanine and L - c a n a l i n e l e d , w i t h i n m i n u t e s , t o s u s t a i n e d f l i g h t s l a s t i n g many h o u r s . The s i t e o f a c t i o n o f L-canavanine and L - c a n a l i n e was b e l i e v e d t o be the CNS. T h i s produced c o n t i n u o u s motor o u t p u t , which became l e s s c o o r d i n a t e d w i t h t i m e . L-canavanine and L - c a n a l i n e are two o f some 260 n o n - p r o t e i n amino a c i d s a c ­ cumulated by v a r i o u s p l a n t s ( 4 1 ) . I f an unadapted i n s e c t a c q u i r e d

CANALINE s u f f i c i e n t L - c a n a l i n e o r L - c a n a v a n i n e t o e x h i b i t the above b e h a v i o r a l symptoms, the e n s u i n g u n c o n t r o l l e d l o c o m o t i o n would not o n l y l i k e l y c a r r y the i n s e c t away from i t s h o s t p l a n t , i t would a l s o p r o b a b l y a t t r a c t the a t t e n t i o n o f a p r e d a t o r . The m o l e c u l a r mode o f a c t i o n o f L-canavanine and L - c a n a l i n e i n a d u l t M. s e x t a remains t o be d e t e r m i n e d . Kammer e t a l . , however, n o t e d t h a t L - c a n a l i n e , which was more potent i n e v o k i n g f l i g h t b e h a v i o r than L - c a n a v a n i n e , s t r u c t u r a l l y resembles the i n h i b i t o r y t r a n s m i t t e r GABA ( 4 0 ) . N o t a b l y , i n j e c t i o n o f the formamidine i n s e c t o s t a t , N - d e m e t h y l c h l o r d i m e f o r m (DCDM), i n t o a d u l t M. s e x t a and H e l i o t h i s zea a l s o caused p r o l o n g e d , u n c o o r d i n a t e d f l i g h t s ( 4 2 ) .

American Chemical Society Library 1155 16th St., N.W. Washington, D.C. 20036 In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Nectar of the Yellow Kowhai. The yellow kowhai (Sophora microphylla A i t . ) occurs i n the North and South Islands of New Zealand. I t s yellow blossoms, which appear from July to October (late winter through early spring), secrete abundant nectar and are very attractive to honey bees. Bees only c o l l e c t nectar from the flowers, ignoring the pollen. New Zealand a p i a r i s t s , who maintained hives i n the v i c i n i t y of kowhai groves, observed that hives f a i l e d to build up normally i n the spring. These f a i l u r e s could not be attributed to the presence of Nosema or to pollen deficiency. In some cases f i e l d bee m o r t a l i t i e s were observed i n the v i c i n i t y of the hives. In seeking the cause of the poor hive development, Clinch, Palmer-Jones, and Forster (43) observed that nectar from S_. microphylla had a narcotic effect on the bees. When fed 10 y l of S_. microphylla nectar, about 70 percent of the 70 bees tested became narcotized within 30 min. When the treated bees were held i n an incubator at 30 deg. C., they a l l recovered within 4 hours with low 24-hour mortality (3-13%). The 24 hour mortality was much higher i f the treated bees were hel Clinch and coworkers (43 contain as major alkaloids methylcytisine, matrine, and smaller amounts of cytisine and suggested that the observed narcosis and mortality could be due to alkaloids reaching the nectar.

CYTISINE

Our own observations with pure c y t i s i n e support t h i s . Worker bees taken d i r e c t l y from a colony were held approximately 5 hours without food, then offered c y t i s i n e i n 40% aqueous sucrose. They were fed by hand using a micropipette and allowed to consume volumes of 10 y l . Cytisine at 10 yg/ 1 acted quickly: the bees fed avidly on the solution, but within one to two minutes reared back, made vigorous cleaning movements of their proboscises, f e l l over, made wild leg movements, and exhibited poorly coordinated f l i g h t . Within a few minutes they lay motionless on the bottom of the cage. Similar, but less dramatic, symptoms were observed with bees offered 1 yg c y s t i s i n e / y l . Bees fed 0.1 yg c y t i s i n e / y l were affected by c y t i s i n e , but immobilization of a l l 10 treated bees was not attained u n t i l 5-6 hours after treatment. The following day, 5 of the 10 bees treated with 1.0 yg had recovered and were maintained alive for 3 days by feeding them aqueous sucrose. While further observations are needed, i t i s evident that honey bees are susceptible to oral doses of c y t i s i n e as low as 1-10 yg. It i s interesting to note that narcotic or poisonous nectar i s not an isolated phenomenon. Honey bees are also poisoned by the nectar of the karaka tree (Cornyocarpus laevigata J.R. et G. Forst.), which induces weakness and i n a b i l i t y to f l y , as well as mortality (44). These observations raise the question: why do certain plants produce narcotic or toxic nectar? In the case of S^. microphylla the

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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e x p l a n a t i o n may be r e l a t i v e l y s i m p l e . As noted by C l i n c h e t a l . ( 4 0 ) , t h e honey bee i s n o t an e f f e c t i v e p o l l i n a t o r o f JS. m i c r o p h y l l a even though i t a v i d l y c o l l e c t s n e c t a r . Rather, C l i n c h e t a l . ( 4 0 ) r e p o r t e d t h a t b i r d s consume l a r g e q u a n t i t i e s o f kowhai n e c t a r , and t h a t they may be t h e most important p o l l i n a t o r s . Thus, p s y c h o ­ m a n i p u l a n t s i n n e c t a r c o u l d f u n c t i o n as a d e f e n s e o f t h e p l a n t a g a i n s t t h e honey bee and o t h e r n o n - p o l l i n a t i n g n e c t a r t h i e v e s . T h i s h y p o t h e s i s i s c o n s i s t e n t w i t h t h e o b s e r v a t i o n s t h a t b i r d s seem unharmed by kowhai n e c t a r and t h a t p r e p a r a t i o n s o f kowhai n e c t a r s are n o t t o x i c when f e d t o w h i t e mice ( 4 0 ) . Summary The p s y c h o m a n i p u l a t i o n h y p o t h e s i s p r e d i c t s t h a t p l a n t s o b t a i n a measure o f p r o t e c t i o n from i n s e c t h e r b i v o r e s by a c c u m u l a t i n g s e c o n d a r y s u b s t a n c e s which a c t i n t h e CNS t o change t h e h e r b i v o r e ' s b e h a v i o r i n ways t h a t reduce o r prevent f u r t h e r h e r b i v o r y Secondary p l a n t s u b s t a n c e s modify i n s e c t behavior, i n t e g r a t i v e processes, suppressing appetite, blocking learning or memory, o r d i s t o r t i n g v i s i o n , w i t h o u t k i l l i n g t h e h e r b i v o r e . E v i d e n c e t h a t s u b s t a n c e s c a n a c t as p s y c h o m a n i p u l a n t s has been p r e s e n t e d h e r e , but f u r t h e r r e s e a r c h i n t h e a r e a i s n e c e s s a r y . To d e f i n i t i v e l y test this hypothesis i t i s e s s e n t i a l to obtain q u a n t i t a t i v e estimates o f the i n d i v i d u a l psychomanipulative s u b s t a n c e s o c c u r r i n g i n p l a n t s , t o demonstrate t h a t they n e c e s s a r i l y and s u f f i c i e n t l y account f o r t h e change i n b e h a v i o r , and f i n a l l y t o show t h a t p s y c h o m a n i p u l a t i o n i s t h e r e s u l t o f an a c t i o n o f t h e s e s u b s t a n c e s w i t h i n t h e i n s e c t CNS. The f i r s t two c r i t e r i a c a n be met u s i n g c u r r e n t l y a v a i l a b l e methods. L o c a t i n g t h e s i t e o f a c t i o n w i t h i n t h e CNS i s more d i f f i c u l t and w i l l r e q u i r e i n n o v a t i v e a p p l i c a t i o n o f combined b e h a v i o r a l , n e u r o p h y s i o l o g i c a l and neuropharmacological techniques. I n s e c t s may be c o n t i n u a l l y p r o b i n g beyond t h e i r h o s t r a n g e s , t h e r e b y e n c o u n t e r i n g new c h e m i c a l s w i t h t h e p o t e n t i a l t o d i s r u p t t h e i r CNS i n f o r m a t i o n p r o c e s s i n g . Some o f t h e s e c h e m i c a l s may a l s o have the p o t e n t i a l t o be i n s e c t i c i d a l . When i n s e c t i c i d a l compounds are i n v o l v e d , we would suggest t h a t a phase o f p s y c h o m a n i p u l a t i o n w i l l always precede l e t h a l i n t o x i c a t i o n , as t h e f o l l o w i n g illustrates: Imagine a w i l d N i c o t i a n a p l a n t , w i t h l e a v e s c o n t a i n i n g s u b s t a n t i a l l e v e l s o f n i c o t i n e , a c l a s s i c n a t u r a l i n s e c t i c i d e . The n i c o t i n e may be s i g n i f i c a n t t o t h e p l a n t n o t so much because i t has the c a p a c i t y t o k i l l unadapted i n s e c t s , b u t because i t c a n m o d i f y t h e i r b e h a v i o r a f t e r t h e y have e a t e n s m a l l amounts o f t h e p l a n t ' s tissue. That b e h a v i o r m o d i f i c a t i o n , e.g., i n c r e a s e d e x c i t a b i l i t y and locomotory a c t i v i t y , c o u l d i n c r e a s e t h e l i k e l i h o o d t h a t t h e insect departs the plant. O n l y when t h a t i n s e c t were r e f r a c t o r y and p e r s i s t e d i n f e e d i n g on t h e p l a n t would i t r e c e i v e a k i l l i n g dose. The c h e m i c a l - e c o l o g i c a l l i t e r a t u r e abounds w i t h d e s c r i p t i o n s o f e f f e c t s o f secondary p l a n t s u b s t a n c e s on i n s e c t s , v a g u e l y lumped under t h e h e a d i n g " t o x i c " . I f we p e r s i s t i n b e i n g s a t i s f i e d w i t h " t o x i c i t y " as a d e s c r i p t i o n cum e x p l a n a t i o n o f t h e e f f e c t o f p l a n t s u b s t a n c e s on i n s e c t s , we s h a l l remain v e r y much i n t h e d a r k as t o the a c t i o n o f t h e s e important compounds. The o n l y way we c a n

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possibly discover subtle neurophysiological and neurochemical effects of secondary compounds i s to a c t i v e l y search for them.

Literature Cited 1. Hsiao, T. In "Insect and Mite Nutrition"; Rodriguez, J. G., Ed.; North-Holland: Amsterdam, 1972; p. 225-40. 2. Dethier, V. G. Entomol. Exp. & Appl. 1982, 31, 49-56. 3. Schoonhoven, L. M. Proc. Kon. Nedar Akad. (Amsterdam) Ser. C 1977, 80(4), 341-50. 4. Florey, E. Fed. Proc. 1967, 26, 1164-78. 5. Evans, P. D. Adv. Insect Physiol. 1980, 15, 317-473. 6. Carlson, A. D. Adv. Insect Physiol. 1969, 6, 51-97. 7. Klemm, N. Comp. Biochem. Physiol. 1972, 43A, 207-11. 8. Bland, K. P.; House, C. R.; Ginsbourg, B. L.; Lazlo, I. Nature: New biology 1973, 244, 26-7. 9. Robertson, H. A. J. Exp. Biol. 1975, 63, 413-19. 10. Brown, B. E.; Starratt 1879-81. 11. O'Shea, M.; Bishop, C. A. J. Neurosci. 1982, 2, 1242-51. 12. Hoyle, G. Adv. Insect Physiol. 1970, 7, 349-444. 13. Taylor, C. In: "The Pharmacological Basis of Therapeutics, Sixth Ed."; Gilman, A. G.; Goodman, L. S.; Gilman, Α., Eds.; MacMillan: New York, 1980; Chap. 6,10. 14. Sattelle, D. B. Adv. Insect Physiol. 1980, 15, 215-315. 15. Dudai, Y.; Nahum-Zvi, S.; Haim-Granot, N. Comp. Biochem. Physiol. 1980, 65C, 135-8. 16. Greenberg, M. J.; Price, D. A. Ann. Rev. Physiol. 1983, 45, 271-88. 17. Windholz, M.; Budavari, S.; Blunetti, R. F.; Otterbein, E. S., Eds. "The Merck Index, Tenth Ed."; Merck & Co.: Rahway, NJ, 1983; p. 1209. 18. Cooper, J. R.; Bloom, F. E.; Roth R. H. "The Biochemical Basis of Neuropharmacology, Fourth Ed."; Oxford University: New York, 1982. 19. Goldberg, M. R.; Robertson, D. Pharmacol. Rev. 1983, 35, 143-80. 20. Evans, P. D. J. Physiol. 1981, 99-122. 21. Johnston, G. A. R. Ann. Rev. Pharmacol. Toxicol. 1978, 18, 269-89. 22. Nielsen, J. Α.; Chapin, D. S.; Moore, Κ. E. Life Sciences 1983, 33, 1899-907. 23. Kalix, P. Psychopharmacology 1981, 74, 269-70. 24. Weiner, N. In "The Pharmacological Basis of Therapeutics, Sixth Ed."; Gilman, A. G.; Goodman, A. G.; Gilman, Α., Eds; MacMillan: New York, 1980; Chap. 9. 25. Sloley, D.; Owen, M. Insect Biochem. 1988, 12, 469-76. 26. Fernstrom, J. D.; Wurtman, R. J. Adv. Biochem Psychopharmacol. 1974, 11, 133-42. 27. Wilson, D. M. J. Exp. Biol. 1961, 38, 471-90. 28. Weis-Fogh, T. Phil. Trans. R. Soc. 1956, B239, 553-84. 29. Kutsch, W.; Murdock, L. L. J. Insect Physiol. 1973, 19, 1519-25.

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30. Kostowski, W.; Beck, J.; Mesaroy, J. J. Pharm. Pharmacol. 1965, 17, 253-55. 31. Frontali, N. J. Insect Physiol. 1968, 14, 881-6. 32. Thompson, A. J. Can. J. Zool. 1977, 53, 451-5. 33. Hopkins, T. L. Personal communication. 34. Green, J.; Murdock, L. L., unpublished data. 35. Shiraishi, Α.; Tanabe; Y. J. Comp. Physiol. 1974, 92, 161-79. 36. Gelperin, A. Z. Vergl. Physiol. 1971, 72, 17-31. 37. Gelperin, A. Annu. Rev. Entomol. 1971, 16, 365-78. 38. Dethier, V. G.; Gelperin, A. J. Exp. Biol. 1967, 47, 191-200. 39. Nuñez, J. A. Naturwissenschatter 1964, 17, 419. 40. Kammer, Α.; Dahlman, D. L.; Rosenthal, G. A. J. Exp. Biol. 1978, 75, 123-9. 41. Rosenthal, G. Α.; Bell, A. E. In: "Herbivores: Their Interaction with Secondary Plant Metabolites"; Rosenthal, G. Α.; Janzen, D. H., Eds.; Academic: New York, 1979; chap. 9. 42. Lund, Α.; Hollingworth R M.; Murdock L L In "Advances in Pesticide Science" 1979; Part 3, p. 465 43. Clinch, P. G.; Palmer-Jones, T.; Forster, I. W. N.Z.J. Agric. Res. 1972, 15, 194-201. 44. Palmer-Jones, T.; Line, L. J. S. N.Z.J. Agric. Res. 1963, 5, 433-6. RECEIVED February 5, 1985

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

25 Protein Hydrolysate Volatiles as Insect Attractants KENT

E.

MATSUMOTO,

RON

G.

BUTTERY,

ROBERT

A.

FLATH,

T.

RICHARD

MON,

and

ROY TERANISHI Biocommunication Chemistry Research Unit, Western Regional Research Center, Agricultural Research Service, U . S . Department of Agriculture, Albany, CA 94710

Hydrolysed protei attract various insects insect attractant use both in nature and by man is introduced, with particular reference to the Tephritid family of fruit flies. The work of the Biocommunication Chemistry Research Unit on the identification of the active attractant compounds in the hydrolysed corn protein, Nu-Lure Insect Bait (NLIB) is discussed. Different isolates have been obtained by running simultaneous steam distillation-extractions (SDE) under vacuum and atomospheric pressure and under basic and acidic conditions. Chemical fractionation of these isolates has also been accomplished. Chemical identification by gas chromatography/mass spectrometry (gc/ms) is discussed. M e t c a l f a n d M e t c a l f (1) h a v e q u o t e d R a c h e l C a r s o n (2) a s d e s c r i b i n g a t t r a c t a n t s a s "new, i m a g i n a t i v e , a n d c r e a t i v e a p p r o a c h e s t o t h e problem o f sharing o u r earth with other creatures". Many o f t h e s e a t t r a c t a n t s a r e n a t u r a l p r o d u c t s . We w o u l d l i k e t o d i s c u s s : 1) t h e u s e o f a t t r a c t a n t s i n p e s t c o n t r o l , 2) t h e e c o n o m i c i m p o r t a n c e o f the Tephritid family of fruit f l i e s , which i s t h e focus o fo u r research, and t h e use o f a t t r a c t a n t s i n i t s control, 3) t h e approach t h e Biocommunication Chemistry Research Unit i s taking toward finding new a t t r a c t a n t s , o u r progress t o date, ando u r p l a n s , a n d 4 ) some p r o b l e m s we h a v e e n c o u n t e r e d i n o u r r e s e a r c h . We w i l l t o u c h o n l y i n p a s s i n g u p o n t h e v e r y l a r g e a n d i m p o r t a n t f i e l d s o f pheromones a n d k a i r o m o n e s a s t h e y a r e c o v e r e d i n more d e t a i l i n t h e c h a p t e r s b y T u m l i n s o n (3) a n d K l u n ( 4 ) . Uses o f A t t r a c t a n t s A t t r a c t a n t s w e r e d e f i n e d b y D e t h i e r (5) a s " c h e m i c a l s t h a t c a u s e insects t o make oriented movements toward the source". He differentiated them from arrestants which "cause insects to aggregate". F o r t h e p u r p o s e s o f t h i s p a p e r , we w i l l n o t b e t h i s

This chapter not subject to U.S. copyright. Published 1985 American Chemical Society

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s p e c i f i c i n our terminology, because, i n many cases, the b i o l o g i c a l observations necessary to make this d i f f e r e n t i a t i o n have not been made. Insect control professionals have used attractants as tools to monitor populations as part of integrated pest management (IPM) programs and as means of s e l e c t i v e l y reducing populations by l u r i n g individuals to traps, poisons, or even chemosterilants (6). Some work i s currently being done on the a t t r a c t i o n of natural enemies as well (7). (Please note that examples shown and references c i t e d are meant to be i l l u s t r a t i v e rather than exhaustive). But for what do insects use attractants? I t i s postulated that they are used for finding shelter, mates, oviposition s i t e s , and food (1, 8). I t i s here that the b i o l o g i s t becomes indispensible to the chemist. Only from h i s observations can chemists know where to look f o r naturally occuring attractants. I t i s also important to note that not a l l attractants are chemical. V i s u a l (9-11) and auditory (12) clues also play very important roles i n the behavior of some insects. Even with chemicals attractants as v o l a t i l are active only over very short distances or induce the appropriate behavior only upon contact (13). What are some examples of these attractants? (Figure 1) For mate finding, the insect-produced pheromones are the primary examples. However, environmental factors may also play an important role i n the effectiveness of attractants. In the southern pine beetle, Dendroctonus f r o n t a l i s , alpha-pinene released from an attacked tree i s necessary along with the endogenously produced f r o n t a l i n i n order to attract males for mating (14). For species whose larvae are s p e c i a l i s t feeders, finding suitable plants for oviposition i s of great importance. Corn earworm moths, H e l i o t h i s armigera. will oviposit on twine impregnated with an extract of com s i l k (1!5). The r i c e stemborer, Chilo p l e j a d e l l u s . female w i l l be attracted to and oviposit near a component of r i c e plants i d e n t i f i e d as £-methylacetophenone (16). Some of these o v i p o s i t i o n attractants are contact materials and, thus, are probably of no use i n p r a c t i c a l applications. This i s the case f o r many of the b u t t e r f l i e s of the Nymphalid family. The Indian butterfly, Papilio demoleus, seems to require some non-volatile component i n c i t r u s leaves to induce o v i p o s i t i o n , although i t seems to be attracted, at least p a r t i a l l y , to the odor of the leaves (17). Food finding i s another area i n which attractants play a major role i n insect behavior. This i s true for some larvae as well as adults. Saxena (18) has shown that the larvae of P. demo 1 eus are attracted to the leaves of c i t r u s and cotton plants by their odor. However, i t seems that most chemicals which p o s i t i v e l y affect insect feeding are gustatory stimulants such as s i n i g r i n f o r the cabbage b u t t e r f l y , P i e r i s brassicae (19-21). This insect feeds on plants of the family Crueiferae whose members are unique i n having high concentrations of this and other mustard o i l glycosides. S i n i g r i n , or, more l i k e l y , a v o l a t i l e r e l a t i v e , w i l l also attract the adults of the diamondback moth, P l u t e l l a maculipennis (22), and the cabbage root f l y , Delia brassicae (23). Leek v o l a t i l e s w i l l attract the Ichneumid Diadromus pulchellus, a p a r a s i t o i d of the pupae of the leek moth Acrolepiopsis a s s e c t e l l a (24).

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However, t h e u s e o f n a t u r a l a t t r a c t a n t s i s b e s t known when the a d u l t s are host feeders: b r a n and m o l a s s e s f o r g r a s s h o p p e r s , M e l a n o p l u s spp. and Camnula spp. ( 2 5 ) ; s y r u p s f o r a n t s , I r i d o m y r m e x humilis and Tapinoma s e s s i l e ( 2 6 ) ; and peanut b u t t e r f o r the imported f i r e ant, Solenopsis saevissima r i c h t e r i (27). F r o m some n a t u r a l a t t r a c t a n t s of t h i s type d i s c r e e t chemical a t t r a c t a n t s have been i s o l a t e d (Figure 2): sotolone f o r a n t s , house f l i e s , and cockroaches ( 2 8 ) ; g e r a n i o l and e u g e n o l f o r the Japanese b e e t l e , P o p i l l i a j a p o n i c a (29); methyl eugenol f o r the o r i e n t a l f r u i t f l y , Dacus d o r s a l i s ( 3 0 ) ; c a r b o n d i o x i d e and l a c t i c a c i d f o r m o s q u i t o e s , Aedes a e g y p t i ( 3 1 - 3 2 ) ; and 3 - h e x e n - l - o l and 2 - h e x e n - l - o l f o r t h e s i l k w o r m , Bombyx m o r i ( 3 3 ) . T h e s e l a s t two c o m p o u n d s b e l o n g t o t h e " g r e e n o d o u r c o m p l e x " ( 3 4 ) , and e l e c t r o p h y s i o l o g y work has shown the e x i s t e n c e of "green" r e c e p t o r s i n t h i s s p e c i e s (35-36) and other species (37-38). T h e r e h a v e a l s o b e e n many s y n t h e t i c a t t r a c t a n t s ( F i g u r e 3 ) t h a t have been c l a s s i f i e d as f o o d l u r e s ( 3 9 ) : p_-acetoxyphenethyl methyl ketone (cue-lure t e r t i a r y - b u t y l 2-methyl-4-chlorocyclohexanecarboxylat (41) f o r the M e d i t e r r a n e a n f r u i t f l y , C e r a t i t i s c a p i t a t a ; p h e n e t h y l propionate (42), which, along with eugenol, i s used for the Japanese b e e t l e ; p r o p y l l,4-benzodioxan-2-carboxylate (amlure) (43) for the European c h a f e r , Amphimallon m a j a l i s ; e t h y l 3-isobutyl2 , 2 - d i m e t h y l c y c l o p r o p a n e c a r b o x y l a t e (44) f o r the coconut r h i n o c e r o s beetle, Orcytes r h i n o c e r o s ; a n d h e p t y l b u t y r a t e (45,) f o r y e l l o w jackets of the genus Vespula. Wasp traps containing pentyl v a l e r a t e are even a v a i l a b l e i n r e t a i l garden shops. Although these have been c a l l e d food lures, c u e - l u r e , t r i m e d l u r e , and methyl eugenol a t t r a c t mainly males. They have a l s o been r e f e r r e d t o as parapheromones ( 4 6 ) . A synthetic attractant f o r a predator has a l s o been found: c y c l o h e x y l p h e n y l a c e t a t e (47) w i l l a t t r a c t the checkered flower b e e t l e , Trichodes ornatus. Tephritid Attractants Our s e a r c h f o r a t t r a c t a n t s i s f o c u s e d on t h e T e p h r i t i d f a m i l y o f f r u i t f l i e s which i n c l u d e s s p e c i e s t h a t are of economic importance i n E u r o p e , A s i a , A u s t r a l i a , and t h e A m e r i c a s . I t i s estimated that the o l i v e f l y , Dacus o l e a e , causes ten percent fruit drop in European o l i v e s . Of t h e i n f e s t e d f r u i t r e m a i n i n g o n t h e t r e e s , 25 percent of the f l e s h i s destroyed (48). A conservative estimate of the annual cost of the recent Medfly i n f e s t a t i o n i n C a l i f o r n i a , not i n c l u d i n g c a p i t a l o u t l a y s , i s $59 m i l l i o n f o r c h e m i c a l controls, $38 m i l l i o n f o r q u a r a n t i n e a n d f u m i g a t i o n , a n d $ 2 6 0 m i l l i o n i n c r o p losses (49). I t i s e s t i m a t e d t h a t 70% of the s u s c e p t i b l e f r u i t i n E g y p t i s i n f e s t e d b y t h e M e d f l y ( 5 0 ) a n d a $50 m i l l i o n control program has been s t a r t e d t h e r e . B e c a u s e o f t h e i r v e r y l a r g e p o t e n t i a l f o r c r o p damage a n d f o r economic l o s s e s t o t h e e x p o r t m a r k e t , t h e A n i m a l and P l a n t H e a l t h I n s p e c t i o n S e r v i c e (APHIS), as w e l l as t h e A g r i c u l t u r a l Research Service (ARS), i s very concerned w i t h the c o n t r o l of Tephritid species. APHIS has a very active program of traps to spot i n f e s t a t i o n s of the Mediterranean fruit f l y , the o r i e n t a l fruit

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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356

ATTRACTANTS

SPECIES

USE

MATE FINDING

DFNDROfTONUS FRONTALIS

OVIPOSITION

HELIOTHIS ARMIGFRA

V-METHYLACETOPHENONE (FROM RICE STALKS)

OVIPOSITION

CHILQ

CITRUS LEAF

OVIPOSITION (NON-VOLATILE)

PAPILIO niMÛLEJdi

a-PlNENE (FROM TREES) CORN SILK EXTRACT >COCH,

PLFJADFI

ι ιις

CITRUS LEAF

Figure 1.

Attractants f o r mate f i n d i n g , o v i p o s i t i o n and food.

•OH

ANTS HOUSEFLIES COCKROACHES

S0T0L0NE

GERANIOL CH o, 3

EUGENOL CH O 3

CH30

JAPANESE BEETLE

X

\\

/ΓΛ-

METHYLEUGENOL C0

ORIENTAL FRUIT FLY

2

CH3CHCOOH OH

LACTIC ACID

MOSQUITOES

CH CH CH=CHCH CH OH 3

2

2

2

3-HEXEN-l-OL SILKWORM CH CH CHC H = C H CH OH 3

2

2

2

2-HEXEN-1-0L Figure 2.

Compounds i s o l a t e d from food sources.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

MATSUMOTO ET AL.

Insect Attractants

CH C O O ^ ~ ~ ^ CH CH COCH 3

2

2

3

CUE-LURE

D A C U S

C U C U R B I T A E

Cl

>^^COO(CH ) TRIMEDLURE 3

Ο

3

G E R A T I Τ I S

C A P I T A T A

CH CH OCOC 2

2

EOPILLA JAPQNICA

COOC H -n 3

AMLURE

7

AMPHIMALLQN MAJALIS

X^^COOCzHj QRCYTES RHINOCEROS

CH (CH ) CH OCOC H -n 3

2

2

3

7

V E S P A

-OCOCH ^ 2

S P P .

β TRICHQDES QRNATUS

Figure 3.

Synthetic Attractants.

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BO IREGULATORS FOR PEST CONTROL

f l y , the melon f l y , the Mexican f r u i t f l y (Anastrepha ludens). and the Caribbean fruit f l y (A. suspensa). The California State Department of Food and Agriculture i s involved in a trapping program to halt the spread of the apple maggot (Rhagoletis pommenella). The ARS h a s p e r s o n n e l i n H o n o l u l u a n d H i l o ( H a w a i i ) , Weslaco (Texas), B e l t s v i l l e (Maryland), and M i a m i and G a i n e s v i l l e ( F l o r i d a ) , as w e l l as o u r f a c i l i t y i n A l b a n y ( C a l i f o r n i a ) w o r k i n g on t h e s e p r o b l e m s . A t t r a c t a n t s are a prominent p a r t of the work a t this laboratory. H i s t o r i c a l l y , s h o r t l y a f t e r the t u r n o f t h e c e n t u r y , an e i g h t y e a r o l d g i r l n o t i c e d t h a t f l i e s were a t t r a c t e d t o kerosene t h a t h e r m o t h e r h a d d a u b e d o n a h i t c h i n g p o s t t o k e e p a n t s away f r o m t h e jam she was cooling at the top of the post. On further investigation, the girl's father found that these flies were M e d f l i e s w h i c h w e r e i n d e e d a t t r a c t e d t o t h e k e r o s e n e and n o t t o t h e jam ( 5 1 ) . L a t e r , H o w l e t t (52) h e a r d a n e i g h b o r c o m p l a i n i n g t h a t he was b e i n g b o t h e r e d b y f l i e s a t t r a c t e d t o t h e o i l o f c i t r o n e l l a h e was u s i n g a s a m o s q u i t as m a l e Dacus z o n a t u s an e u g e n o l as t h e a c t i v e component o f t h e c i t r o n e l l a o i l ( 3 0 ) . Now, t r i m e d l u r e i s used f o r surveying f o r the Medfly, cue-lure f o r the m e l o n f l y , m e t h y l e u g e n o l f o r t h e O r i e n t a l f r u i t f l y , and protein hydrolysates for the Mexfly and Caribfly. In fact, protein h y d r o l y s a t e s c a n b e u s e d f o r many s p e c i e s o f f r u i t f l i e s . Protein hydrolysates a l s o d i f f e r s i g n i f i c a n t l y from the s y n t h e t i c l u r e s i n that they w i l l a t t r a c t female f l i e s , e s p e c i a l l y g r a v i d ones, as w e l l as m a l e s . The sex pheromones o f t h e M e d f l y and the olive fruit f l y have been i d e n t i f i e d (53-55), synthesized (53-56.) and tested in the field (57-59). Methyl eugenol, mixed with an i n s e c t i c i d e and applied to cardboard pieces (60) o r s p o t s p r a y e d (61.), h a s been used i n male-annihilation eradication programs. Protein hydrolysates m i x e d w i t h an i n s e c t i c i d e h a v e b e e n u s e d i n many M e d f l y (62-65.) a n d Mexfly (64, 66.) eradication projects, i n c l u d i n g r e c e n t ones i n C a l i f o r n i a (61). Experimental Approach. Results,

and

Discussion

The Biocommunication Chemistry Research Unit, WRRC, USDA, has d e v e l o p e d a p r o g r a m t o f i n d o t h e r a t t r a c t a n t s f o r members o f the Tephitid family allowing for the development of new lures, i m p r o v e m e n t o f c u r r e n t IPM p r o g r a m s , i m p a r t i n g s p e c i e s selectivity t o p r o g r a m s , and possible replacement of c u r r e n t l y used b a i t s . These are i n a d d i t i o n to f i n d i n g a t t r a c t a n t s f o r p e s t s currently uncontrolled i n this way. We are examining those commonly u s e d b a i t s , t h e protein hydrolysates. Initial studies have used the corn gluten h y d r o l y s a t e c o m m o n l y k n o w n a s P I B - 7 o r , now, as N u - L u r e I n s e c t B a i t (NLIB). T h i s m a t e r i a l was used i n the recent successful Medfly e r a d i c a t i o n program in California. Since the fruit flies are p r o b a b l y a t t r a c t e d t o the v o l a t i l e emanations from the b a i t , we h a v e u s e d e q u i p m e n t a n d t e c h n i q u e s p r e v i o u s l y d e v e l o p e d b y members of our group for f l a v o r research. For example, a modified Likens-Nickerson simultaneous steam distillation-extraction head was d e v e l o p e d b y F l a t h a n d F o r r e y (67.). A l s o , t h e r e i s a 90 l i t e r

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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p i l o t plant system (68) that can be used f o r i s o l a t i n g enough v o l a t i l e material f o r chemical and b i o l o g i c a l assays. This i s very important because without sufficient quantities of material, replicated b i o l o g i c a l testing and proper chemical fractionation, y i e l d i n g s u f f i c i e n t material for bioassays, would be impossible. Conventional vapor trapping techniques y i e l d only 10~ to 10" grams of material. Using a laboratory scale, 12 liter, simultaneous steam d i s t i l l a t i o n - e x t r a c t i o n system 10""* to 10~* grams can be collected. The 90 l i t e r p i l o t plant system w i l l y i e l d 10~ to 1 gram. Recent work has shown that the figures f o r these l a s t two methods can be increased by an order of magnitude. Thus, using the equipment and techniques already developed by our group, i t i s possible to produce material i n s u f f i c i e n t quantity to conduct both the b i o l o g i c a l and chemical investigations. Our f i r s t separation method involved running the simultaneous steam d i s t i l l a t i o n extraction under 100 mm vacuum i n order to minimize heat e f f e c t s This was followed by extraction under atmospheric pressure i atmospheric extraction wa solvent each day (68-69). Approximately 10 times as much material was collected each day at atmospheric pressure as was collected under vacuum. Since Schultz, et. a l . (70) showed that many nonwater-soluble alcohols, esters, aldehydes, and ketones can be recovered by this system i n less than 3 hours, the c o l l e c t i o n of a large amount of material after 10 days i s indicative of a very complex and probably dynamic system. Gas chromatograms f o r these extracts (68) and some compound i d e n t i f i c a t i o n s (69) have been reported. (Other reports on the i d e n t i f i c a t i o n of v o l a t i l e s from protein hydrolysates are given i n references 71-75). Prelminary results have shown that the vacuum extracts are more a t t r a c t i v e f o r the Medfly than the atmospheric ones. Next, i n agreement with the work of Bateman and Morton (76) on the Queensland f r u i t f l y , we found that increasing the pH of the hydrolysate from i t s normal 4.2 to 8.5 to 9 increases the a t t r a c tancy of the b a i t f o r our test species. Gas chromatography (Figure 4) and gc/mass spectrometry (Finnigan/Incos model 4500X GC/MS with 0V-101 fused s i l i c a c a p i l l a r y column) on the isolated v o l a t i l e s of pH 4.2 and pH 9 hydrolysate show considerable differences. The major v o l a t i l e components of the pH 4.2 b a i t are 2-methylpropanal, 3-methylbutanal, 2-methylbutanal, f u r f u r a l , 3-(methylthio)propanal, acetylfuran, phenylacetaldehyde, and acetophenone. The major v o l a t i l e components of the pH 9 bait are 2-methylpropanal, 3-methylbutanal, 2-methylbutanal, methylpyrazine, dimethylpyrazine, ethylpyrazine, 2,3-dimethylpyrazine, ethylmethylpyrazine, trimethylpyrazine, dimethylethylpyrazine, and d i e t h y l pyrazine. The v o l a t i l e s of the pH 4.2 bait are dominated by aldehydes and ketones while those of the pH 9 bait are dominated by pyrazines. Figure 5 shows the structures of some of these pyrazines. I t i s interesting to note that some of these pyrazines have also been found i n the r e c t a l gland secretion of the male melon f l y (77). We have separated the v o l a t i l e s from the pH 9 bait into basic and neutral fractions by extraction with acid, followed by neturalization of the acid extract (Figure 6). 6

2

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3

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pH 4.2

pH 9

J IL l *.u Ο Min.

20

10

SO

F i g u r e 4. Gas chromatograms o f v o l a t i l e s i s o l a t e d from NLIB; t o p c h r o m a t o g r a m , p H 4 . 2 ; b o t t o m c h r o m a t o g r a m , p H 9; 3 0 m χ 0.25 mm f u s e d s i l i c a DB-1 c o l u m n , t e m p e r a t u r e p r o g r a m m e d : 50°C f o r 0.1 m i n . , t h e n 4 C / m i n . t o 2 2 0 C , h e l d a t 220°C f o r 2 0 m i n . e

e

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Insect Attractants

Or XT XX

σ METHYLPYRAZINE

ETHYLPYRAZINE

2,3-DlMETHYLPYRAZINE 3-DIMETHY

2-ETHYL-6-HETHYLPYRAZINE ETHYL-6-METH

TRIMETHYLPYRAZINE

Ï-ETHYL-2.5-DIMETHYLPYRAZINE

CC XIX

F i g u r e 5.

S t r u c t u r e s o f some p y r a z i n e s

i d e n t i f i e d i n NLIB.

ASD-N

iiuJLii ASD-B

ijJlLi 10

20

30

F i g u r e 6. Gas chromatograms o f v o l a t i l e s i s o l a t e d from pH 9 NLIB; t o p chromatogram, n e u t r a l f r a c t i o n ; bottom chromatogram, basic fraction; 60 m χ 0.32 mm fused silica DB-1 column, temperature programmed: 5 0 C f o r 0.1 min., t h e n 4°C/min. t o 2 3 0 C , h e l d a t 2 3 0 C f o r 10 min. e

e

e

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BIOREGULATORS FOR PEST CONTROL

The major components of the neutral f r a c t i o n are 2-methylpropanal, 2-butanone, 3-methylbutanal, 2-methylbutanal, dimethyl d i s u l f i d e , and 3-methylpentanal. The major components of the basic f r a c t i o n are 3-methylbutanal, methylpyrazine, dimethylpyrazine, ethylpyrazine, 2-ethy1-6-methy1pyrazine, trimethylpyrazine, and 3-ethy1-2,5-dimethylpyrazine. (The aldehyde was probably carried over during the extractions because of i t s s o l u b i l i t y i n both water and organic solvents.) Preliminary results show that this f r a c t i o n has some attraction for the male o r i e n t a l f r u i t f l y . We are now further bioassaying these fractions and w i l l further separate components i n order to isolate the active materials. Our very able entomologist-cooperators and t h e i r insect test species are l i s t e d i n Table I. We are also setting up f a c i l i t i e s to study the neurophysiology and ethology of these insects. This w i l l greatly expand the types of assays i n our research program.

Table I Entomologist-Cooperators

Roy T. Cunningham ARS, H i l o , HI

Mediterranean F r u i t F l y Oriental F r u i t F l y Melon F l y

William G. Hart ARS, Weslaco, TX

Mexican F r u i t F l y

Peter Landolt Dennis Howard ARS, Miami, FL

Caribbean F r u i t F l y

Shmuel Gothilf Volcani Institute Bet-Dagan, Israel

Mediterranean F r u i t F l y

What are some of the problems that researchers on attractants might face? One i s the p o s s i b i l i t y that mixtures are necessary f o r activity. Their components may even need to be i n s p e c i f i c ratios. This i s something that has become very apparent i n pheromone work (78). In our investigations of feeding attractants, t h i s may also be true (79). As most of us know, people often f i n d the odor of a complex natural o i l more a t t r a c t i v e than that of i t s most c h a r a c t e r i s t i c single component. However, current chemical a n a l y t i c a l methodology i s designed to separate materials into single components, and we w i l l have to f i n d ways of determining how to combine materials most e f f i c i e n t l y to obtain the best a c t i v i t y . A further complication when working with feeding attractants i s that the combination may have to include non-volatile arrestants such as carbohydrates or peptides with the v o l a t i l e materials i n order to be useful.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Another problem with bioassays i s that many possible attractants can act as repellents at higher concentrations. Protocols must be designed to take this into acount, even though the concentration ranges over which the attractancy and/or repellancy occur may vary substantially from compound to compound. Putting a compound into a test at too high a concentration can swamp out even the standards, much i n the same way that a pheromone i s used i n a confusion technique. Also insects may be attracted to a compound only u n t i l i t reaches a certain concentration and then stop coming. I f this i s the case, insects may be attracted to a region around a trap but never enter the trap. How these problems are handled w i l l depend on whether the bioassay i s of the olfactometer type or the f i e l d type. Another problem i s the lack of knowledge about insect behavior. Although an insect i s attracted to a host plant, i t may be f o r reasons other than food. For instance, although an attractant i s isolated from a food source such as a f r u i t i t may actually be a signal f o congregation of males f o t h i s i s so, then the material may be e f f e c t i v e i n attracting insects to an area but may be i n e f f e c t i v e i n inducing them to enter into, or land on, a trap. Conclusions We have found that: 1) v o l a t i l e s from protein hydrolysates w i l l attract f r u i t f l i e s of various species, both male and female, 2) that v o l a t i l e s from protein hydrolysates which had been brought to pH 9 are more a t t r a c t i v e than v o l a t i l e s from protein hydrolysate as i t comes at pH A.2, 3) that isolates from protein hydrolysates are a t t r a c t i v e to various species of f r u i t f l i e s , and 4 ) that the basic i s o l a t e i s a t t r a c t i v e to male o r i e n t a l f r u i t f l i e s . We are continuing to pursue vigorously the i s o l a t i o n , i d e n t i f i c a t i o n , and b i o l o g i c a l testing of the active components of this mixture. I would also l i k e to reemphasize the usefulness of attractants research i n finding new ways of c o n t r o l l i n g insect pests; i t s e f f i c a c y has been shown. We must now refine the tools we have, as i s the case with the protein hydrolysates, and expand the range of pests than can be s e l e c t i v e l y controlled by t h i s technique. Acknowledgements I would again l i k e to acknowlege our entomologist--cooperators: Roy Cunningham, Shmuel G o t h i l f , B i l l Hart, Dennis Howard, and Peter Landolt; I would also l i k e to thank W. G. Schultz f o r h e l p f u l discussions and C. Caulins f o r the leading references on the history of Tephritid attractants. Reference to a company and/or product by name i s only f o r purposes of information and does not imply approval or recommendation of the product by the Department of Agriculture to the exclusion of others which may also be suitable.

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Steiner, L. F.; Hart, W. G.; Harris, E. J.; Cunningham, R. T.; Ohinata, K.; Kamakahi, D. C. J. Econ. Entomol. 1970, 63, 131-5. Dowel1, R. Department of Entomology Seminar, University of California, Berkeley; 30 January 1984. Steiner, L. F. J. Econ. Entomol. 1952, 45, 838-48. Stephenson, B. C.; McClung, Β. B. Bull. Entomol. Soc. Amer. 1966, 12, 374. Hart, W. G.; Ingle, S.; Reed, D.; Flitters, N. J. Econ. Entomol. 1967, 60, 1264-5. Steiner, L. F. Proc. 1st Int. Citrus Symp. 1968, 2, 881-7. Lopez-D., F.; Chambers, D. L.; Sanchez-R., M.; Kamasaki, H. J. Econ. Entomol. 1969, 62, 1255-7. Flath, R. Α.; Forrey, R. R. J. Agric. Food Chem. 1977, 25, 103-9. Teranishi, R.; Buttery, R. G.; Mon, T. R. In "Isolation and Characterization of Biologically Active Natural Products"; Acree, T., Ed.; Walte Buttery, R. G.; Ling Agric. Food Chem. 1983, 31, 689-92. Schultz, T. H.; Flath, R. Α.; Mon, T. R.; Eggling, S. B.; Teranishi, R. J. Agric. Food Chem. 1977, 25, 446-9. Manley, C. H.; Fagerson, I. S. J. Food Sci. 1970, 18, 340-2. Manley, C. H.; Fagerson, I. S. J. Food Sci. 1970, 35, 286-91. Markh, A. T.; Vinnikova, L. G. Appl. Biochem. Microbiol. (Eng. Trans.) 1973, 9, 774-8. Withycombe, D. Α.; Mookherjee, Β. D.; Hruza, A. In "Analaysis of Food and Beverages"; Charalambous, G., Ed.; Academic Press: New York; 1978; p. 81-94 Morton, T. C.; Bateman, M. A. Aust. J. Agric. Res. 1981, 32, 905-16. Bateman, Μ. Α.; Morton, T. C. Aust. J. Agric. Res. 1981, 32, 883-903. Baker, R.; Herbert, R. H.; Lomer, R. A. Experientia 1982, 38, 232-3. Roelofs, W. In "Chemical Ecology: Odour Communication in Animals"; Ritter, F. J., Ed.; Elsevier: New York; 1979, p. 159-68. Yamamoto, I.; Yamamoto, R. In "Control of Insect Behavior by Natural Products"; Wood, D. L., Silverstein, R. Μ., Nakajima, Μ., Eds; Academic Press: New York; 1970; p. 331-45.

RECEIVED

November 8, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

26 Beetles: Pheromonal Chemists par Excellence J. H .

TUMLINSON

Insect Attractants, Behavior, and Basic Biology Research Laboratory, Agricultural Research Service, U . S . Department of Agriculture, Gainesville, FL 32604

Pheromones of insec are characterized by considerable structural diversity. Unlike the lepidopterous sex pheromones, which are nearly all fatty acid derivatives, coleopterous sex pheromone structures range in complexity from the relatively simple 3,5-tetradecadienoic acid of the black carpet beetle to the tricyclic terpenoid, lineatin, of the striped ambrosia beetle. While the sex pheromones of many beetles consist of mixtures of compounds that act synergistically to elicit a behavioral response, other Coleoptera species appear to use only a single compound for chemical communication between the sexes. In the latter case the compound usually has at least one chiral center and chirality plays a major role in determining pheromone specificity. Some interesting relationships within groups of coleopterous species, based on subtle differences in structures or mixtures, have been unraveled in the last few years. These and the variety of structures comprising coleopterous pheromones provide a challenging opportunity for the natural products chemist. T h e r e a r e more s p e c i e s o f C o l e o p t e r a t h a n o f a n y o t h e r o r d e r o f insects. I t h a s b e e n e s t i m a t e d t h a t o v e r 350,000 s p e c i e s o f b e e t l e s have been d e s c r i b e d , and t h i s o r d e r r e p r e s e n t s about h a l f o f t h e t o t a l n u m b e r o f s p e c i e s (I). Thus i t i s somewhat s u r p r i s i n g t o d i s c o v e r f r o m r e c e n t s u r v e y s o f t h e l i t e r a t u r e (2,3) t h a t p h e r o m o n e s h a v e b e e n i d e n t i f i e d f o r o n l y a b o u t 50 t o 75 species o f Coleoptera. I n c o n t r a s t , s e x pheromones o r s e x a t t r a c t a n t s (most o f t h e l a t t e r d i s c o v e r e d b y s c r e e n i n g p r o c e d u r e s ) a r e a v a i l a b l e f o r o v e r 500 s p e c i e s o f L e p i d o p t e r a . F u r t h e r m o r e , o v e r h a l f o f t h e known c o l e o p t e r o u s pheromones h a v e been i s o l a t e d and i d e n t i f i e d from s p e c i e s i n t h e f a m i l y Scolytidae.

This chapter not subject to U.S. copyright. Published 1985 American Chemical Society

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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PEST CONTROL

As p r e v i o u s l y n o t e d (4,5), c o l e o p t e r o u s p h e r o m o n e s a r e c h a r a c ­ t e r i z e d by g r e a t d i v e r s i t y and c o m p l e x i t y i n b o t h t h e c h e m i c a l s t r u c t u r e s and t h e c o m p o s i t i o n o f b l e n d s . Some s p e c i e s , p a r t i c ­ u l a r l y i n the f a m i l y Dermestidae, u t i l i z e very simple f a t t y a c i d ­ l i k e m o l e c u l e s s i m i l a r t o the l e p i d o p t e r o u s pheromones. In con­ t r a s t , Trypodendron lineatum ( O l i v i e r ) uses a t r i c y c l i c a c e t a l (6). T h e r e a r e a v a r i e t y o f o t h e r c o l e o p t e r o u s pheromone s t r u c ­ tures of intermediate complexity. A d d i t i o n a l l y , t h e phenomenon o f c h i r a l i t y i n t r o d u c e s e v e n more c o m p l e x i t y and d i v e r s i t y i n t o t h e s e p h e r o m o n e s t r u c t u r e s . S i l v e r s t e i n (5) l i s t s nine possible cate­ g o r i e s o f r e s p o n s e by i n s e c t s t o e n a n t i o m e r s . S o f a r we know o f e x a m p l e s f o r f i v e o f t h e s e c a t e g o r i e s . T h u s a n i n s e c t may p r o d u c e a s i n g l e e n a n t i o m e r a n d t h e o t h e r e n a n t i o m e r may b e a c t i v e , inactive, or i n h i b i t o r y . A l t e r n a t i v e l y , t h e i n s e c t may p r o d u c e b o t h e n a n t i o m e r s and r e s p o n d o p t i m a l l y t o t h e n a t u r a l r a t i o o f t h e two e n a n t i o m e r s o r more s t r o n g l y t o one t h a n t h e o t h e r For a more d e t a i l e d d i s c u s s i o Silverstein (7). I t w o u l d s e e m t h a t many o f t h e c o l e o p t e r o u s s p e c i e s c o u l d a c h i e v e a h i g h d e g r e e o f s p e c i f i c i t y i n t h e i r pheromone s i g n a l s w i t h s i n g l e c o m p o n e n t s , g i v e n t h e many d i v e r s e s t r u c t u r e s a v a i l ­ a b l e and t h e added d e g r e e o f s t r u c t u r a l s p e c i f i c i t y p r o v i d e d by d i f f e r e n c e s i n enantiomeric or s t e r e o i s o m e r i c forms o f a mole­ cule. I n f a c t , many c l o s e l y r e l a t e d s p e c i e s u s e d i f f e r e n t e n a n t i ­ omers o r s t e r e o i s o m e r s t o a c h i e v e s p e c i f i c i t y i n t h e i r c h e m i c a l signal. However, most o f t h e c o l e o p t e r o u s pheromones i d e n t i f i e d t h u s f a r a r e m u l t i c o m p o n e n t and i n t h e few c a s e s where pheromones have been i d e n t i f i e d f o r s e v e r a l c l o s e l y r e l a t e d s p e c i e s i n a g e n u s , we u s u a l l y f i n d t h a t t h e d i f f e r e n t s p e c i e s a r e u s i n g b l e n d s o f a few compounds and a c h i e v i n g s p e c i f i c i t y by v a r y i n g r a t i o s , number o r t y p e s o f c o m p o n e n t s , and t h e c h i r a l i t y o f i n d i v i d u a l components. D e s p i t e the g e n e r a l t r e n d toward multicomponent pheromones, s e v e r a l s i n g l e component c o l e o p t e r o u s pheromones have been i s o ­ l a t e d and i d e n t i f i e d . I n many o f t h e s e i n s t a n c e s i t a p p e a r s t h a t t h e pheromone i d e n t i f i c a t i o n i s i n c o m p l e t e , a l t h o u g h a s i n g l e com­ p o u n d may be a t t r a c t i v e a n d may i n f a c t be a v e r y e f f e c t i v e l u r e for f i e l d trapping tests. No d o u b t f u r t h e r w o r k w i l l t u r n up m o r e c o m p o n e n t s a n d m o r e e f f e c t i v e p h e r o m o n e s i n many i n s t a n c e s . How­ e v e r , t h e r e a r e c a s e s i n w h i c h v e r y c a r e f u l and t h o r o u g h investi­ g a t i o n s h a v e y i e l d e d a s i n g l e compound w i t h a p p a r e n t l y t h e t o t a l a c t i v i t y o f the o r i g i n a l e x t r a c t or o f the l i v e i n s e c t s . There i s s t i l l t h e p o s s i b i l i t y i n many o f t h e s e i n s t a n c e s t h a t t h e b i o a s s a y d i d not measure a l l b e h a v i o r a s s o c i a t e d w i t h pheromone communica­ tion. T h u s , m o r e t h o r o u g h s t u d i e s o f t h e b e h a v i o r may indicate t h e p r e s e n c e o f more pheromone components. A d d i t i o n a l l y , where a s p e c i e s i s i s o l a t e d f r o m i t s n a t i v e h a b i t a t and o t h e r r e l a t e d s p e c i e s , the r o l e o f o t h e r components i n p r o v i d i n g s p e c i f i c i t y t o t h e s i g n a l may n o t be e v i d e n t . T h e r e f o r e , a s a g e n e r a l r u l e we s h o u l d p r o b a b l y a l w a y s e x p e c t t h a t t h e c o m p l e t e pheromone w i l l c o n s i s t o f two o r more compounds. H o w e v e r , i t i s p o s s i b l e t h a t a f e w s p e c i e s may u t i l i z e s i n g l e c o m ­ ponent pheromones, p a r t i c u l a r l y i f the s t r u c t u r e i s s u f f i c i e n t l y u n i q u e and i f c h i r a l i t y p r o v i d e s s u f f i c i e n t d i v e r s i t y t o a l l o w

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

26.

TUMLINSON

Beetles

s p e c i f i c i t y of the s i g n a l i n the context o f the a s s o c i a t e d environment. C o n s i d e r a t i o n o f t h e e n v i r o n m e n t i n w h i c h a s p e c i e s l i v e s and reproduces i s very important, although o f t e n neglected i n studying the chemical communications system of t h a t s p e c i e s . I n many c a s e s i t i s v e r y d i f f i c u l t t o m o n i t o r t h e c h e m i c a l i s o l a t i o n and p u r i f i c a t i o n w i t h f i e l d b i o a s s a y s and t h u s l a b o r a t o r y b i o a s s a y s are o f t e n used. Obviously a laboratory bioassay i s quite a r t i f i c i a l i n t h a t many e n v i r o n m e n t a l f a c t o r s t h a t a f f e c t t h e i n s e c t ' s behavior are e l i m i n a t e d . In f a c t , bioassays conducted i n t h e f i e l d may n o t m e a s u r e a l l t h e b e h a v i o r a l c o m p o n e n t s e i t h e r . I n e i t h e r c a s e a pheromone i d e n t i f i c a t i o n s h o u l d be f o l l o w e d by a n i n v e s t i g a t i o n of the response o f other species t o s y n t h e t i c compounds, b l e n d s , i s o m e r s , e n a n t i o m e r s , e t c . , and c o m p a r i s o n o f the r e s u l t s w i t h the n a t u r a l i n t e r s p e c i f i c responses or behavior. A d d i t i o n a l l y , o d o r s from h o s t p l a n t s o r pheromones o f o t h e r s p e c i e s may a f f e c t t h e r e s p o n s pheromones. F o r a v a r i e t y o f r e a s o n s , some o f w h i c h h a v e a l r e a d y b e e n d i s c u s s e d , we c a n o f t e n l e a r n m o r e a b o u t a c o m m u n i c a t i o n s y s t e m b y s t u d y i n g t h e p h e r o m o n e s a n d r e l a t e d b e h a v i o r o f s e v e r a l members o f a genus o r f a m i l y t h a n by an i n - d e p t h s t u d y o f a s i n g l e s p e c i e s . The i n t e r s p e c i f i c i n t e r a c t i o n s a r e o f t e n q u i t e s u b t l e b u t v e r y important. For example, the importance o f enantiomers or s t e r e o i s o m e r s may o n l y b e c o m e e v i d e n t a f t e r i n v e s t i g a t i n g t h e r e s p o n s e s o f s e v e r a l c l o s e l y r e l a t e d s p e c i e s t o a pheromone o r pheromone b l e n d . T h e r e have been s e v e r a l good r e v i e w s — p a r t i c u l a r l y of the c h e m i s t r y and b e h a v i o r a s s o c i a t e d w i t h b a r k b e e t l e pheromones — i n t h e l a s t few y e a r s ( 6 , 9-12). Many a s p e c t s o f my d i s c u s s i o n h e r e a r e d i s c u s s e d i n more d e t a i l i n one o r more o f t h e s e reviews. In the remainder o f t h i s chapter, I i n t e n d t o i l l u s t r a t e w i t h s e l e c t e d examples the p o i n t s t h a t I d i s c u s s e d above. Scolytidae B a r k b e e t l e s a r e o f g r e a t e c o n o m i c i m p o r t a n c e , w h i c h i s one o f t h e r e a s o n s more r e s e a r c h has b e e n done on t h e pheromones o f t h e S c o l y t i d a e than on those o f any o t h e r f a m i l y o f C o l e o p t e r a . Their p h e r o m o n e s y s t e m s a l s o s e e m t o be t y p i c a l o f t h e C o l e o p t e r a i n t h a t w h i l e t h e r e i s c o n s i d e r a b l e d i v e r s i t y i n pheromone s t r u c t u r e w i t h i n t h i s f a m i l y , t h e r e a l s o seems t o be a p a t t e r n o f s t r u c t u r e s , p a r t i c u l a r l y w i t h i n a genus. The f i r s t p h e r o m o n e i d e n t i f i e d f r o m a c o l e o p t e r o u s s p e c i e s was f r o m I p s paraconfusus L a n i e r ( t h e n I . c o n f u s u s ) by S i l v e r s t e i n e t a l . ( 1 3 ) . Three compounds — i p s e n o l ( I ) , i p s d i e n o l ( I I ) , and c i s - v e r b e n o l ( I I I ) — were i d e n t i f i e d . These compounds a r e s y n e r g i s t i c , t h e combination of a l l three being required f o r a c t i v i t y with l i t t l e o r no a t t r a c t i o n b e i n g e l i c i t e d by any one o f them a l o n e .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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370

HO

OH H

I

II

III

E a c h o f t h e s e compounds i s c h i r a l a n d t h u s c a n e x i s t i n e i t h e r o f two enantiomeric forms. Thus, t h e c h e m i c a l s i g n a l c a n be v a r i e d b y (1) u s i n g d i f f e r e n t r a t i o s o f t h e s e t h r e e c o m p o u n d s , ( 2 ) u s i n g d i f f e r e n t b l e n d s e m p l o y i n g o n e , t w o , o r t h r e e o f t h e s e com­ p o u n d s , a n d (3) i m p o s i n g t h e e l e m e n t o f c h i r a l i t y o n e a c h o f t h e o t h e r two methods. The I . p a r a c o n f u s u s pheromone was i d e n t i f i e d as ( S ) ( - ) i p s e n o l , p r e d o m i n a n t l c i s - v e r b e n o l (13). S u b s e q u e n t l identified as (R)(-) ipsdieno (14), (S)(+) enantiomer c o m p l e t e l y i n t e r r u p t e d I . p i n i response t o t h e (R)(-). F u r t h e r m o r e , w h i l e I . p i n i i n t h e w e s t e r n U. S. p r o d u c e s a n d r e s p o n d s t o ( R ) ( - ) i p s d i e n o l , I . p i n i i n New Y o r k p r o d u c e s a 65:35 r a t i o o f ( + ) : ( - ) i p s d i e n o l a n d r e s p o n d s m o r e s t r o n g l y t o t h e racemic s y n t h e t i c i p s d i e n o l i n t h e f i e l d (15). A s u r v e y o f t h e l i t e r a t u r e o n I p s pheromones s u g g e s t s t h a t most i f n o t a l l I p s s p e c i e s u t i l i z e o n e o r more o f t h e s e t h r e e components i n v a r i o u s combinations and o f d i f f e r e n t c h i r a l i t i e s t o achieve t h e necessary s p e c i f i c i t y i ntheir chemical signal. The a g g r e g a t i o n p h e r o m o n e o f D e n d r o c t o n u s b r e v i c o m i s L e C o n t e c o n s i s t s o f a s y n e r g i s t i c mixture o f exo-brevicomin (IV) produced by f e m a l e s , myrcene ( V ) f r o m t h e h o s t t r e e , a n d f r o n t a l i n ( V I ) p r o d u c e d b y m a l e s w h i c h r e s p o n d t o c o m p o u n d s I V a n d V. T h e m i x ­ t u r e o f a l l t h r e e compounds t h e n a t t r a c t s m a l e s a n d f e m a l e s ( 1 0 ) . Subsequently, verbenone ( V I I ) and t r a n s - v e r b e n o l ( V I I I ) a r e p r o ­ duced and d e t e r f u r t h e r a t t a c k (11). A d d i t i o n a l l y verbenone d e t e r s t h e r e s p o n s e o f I . p a r a c o n f u s u s w h i c h u t i l i z e s t h e same h o s t t r e e s p e c i e s i n t h e same a r e a (16,17). T h u s t h e t w o s p e c i e s may b e f o u n d c o l o n i z i n g d i f f e r e n t p a r t s o f t h e same t r e e . Again, as i n t h e I p s pheromones, c h i r a l i t y p l a y s a r o l e . The n a t u r a l l y o c c u r r i n g enantiomers o f exo-brevicomin and f r o n t a l i n i n combina­ t i o n w i t h myrcene e l i c i t a g r e a t e r response from both male and f e m a l e D. b r e v i c o m i s t h a n d o t h e o t h e r e n a n t i o m e r s . T h e r e a r e many o t h e r s c o l y t i d s p e c i e s t h a t u t i l i z e t h e s e a n d s i m i l a r compounds t o e f f e c t i n t r a s p e c i f i c c o m m u n i c a t i o n . Francke e t a l . (18) h a v e d i s c o v e r e d s e v e r a l s p i r o k e t a l s a s a c t i v e c o m p o ­ nents o f s c o l y t i d pheromones. A d d i t i o n a l l y , several parasites and p r e d a t o r s u t i l i z e t h epheromones produced by t h e i r s c o l y t i d p r e y as kairomones. F o r example, t h e p r e d a t o r y b e e t l e , Temnochila c h l o r o d i a , responds s p e c i f i c a l l y t o exo-brevicomin produced by f e m a l e D. b r e v i c o m i s ( 1 9 ) . T h i s same p h e n o m e n o n h a s b e e n demonstrated r e c e n t l y i nL e p i d o p t e r a ; t h eegg p a r a s i t e Trichogramma s p . u s e s t h e H e l i o t h i s z e a ( B o d d i e ) s e x pheromone t o l o c a t e t h e H. z e a e g g s ( 2 0 ) .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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T h u s , t o q u o t e f r o m B i r c h (10), "Not o n l y s h o u l d t h e c h e m i c a l c o m m u n i c a t i o n s y s t e m w i t h i n a s p e c i e s be i n t i m a t e l y k n o w n i n o r d e r t o m a n i p u l a t e b e h a v i o r e f f e c t i v e l y , but the e f f e c t o f manipulating one s p e c i e s o n t h e c o m p l e x o f o t h e r s p e c i e s i n t h a t e n v i r o n m e n t s h o u l d a l s o be u n d e r s t o o d . "

IV

V

VI

OH

VII

VIII

Curculionidae R e l a t i v e l y few pheromones h a v e been i d e n t i f i e d f r o m s p e c i e s i n this family. The f i r s t c u r c u l i o n i d p h e r o m o n e i d e n t i f i e d , t h a t o f t h e b o l l w e e v i l , Anthonomus g r a n d i s Boheman, i s t e r p e n o i d i n character l i k e those o f the S c o l y t i d a e . Two t e r p e n e a l c o h o l s ( I X , X) and two a l d e h y d e s ( X I , X I I ) w e r e i s o l a t e d and i d e n t i f i e d f r o m m a l e w e e v i l s a n d f r o m f r a s s (21). A l l f o u r o f t h e s e compounds a r e r e q u i r e d t o e l i c i t optimum a t t r a c t i o n .

IX

X

XI

XII

P r o d u c e d by t h e m a l e , t h i s pheromone a c t s as an aggregating pheromone i n the f i e l d i n t h e s p r i n g , but i n m i d s e a s o n a p p e a r s t o a c t more l i k e a t r u e s e x pheromone i n t h a t i t p r i m a r i l y a t t r a c t s females. A l s o , as i n the b a r k b e e t l e s , components o f the e s s e n ­ t i a l o i l s o f the host p l a n t appear t o enhance the a t t r a c t i v e n e s s o f t h i s pheromone (22).

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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G r a n d i s o l (IX) and t h ecorresponding aldehyde, g r a n d i s a l , a l s o have been i s o l a t e d a n d i d e n t i f i e d a s components o f t h e pheromone of two other c u r c u l i o n i d s , P i s s o d e s s t r o b i (Peck), t h e white p i n e w e e v i l , a n d P. a p p r o x i m a t i f H o p k i n s , t h e n o r t h e r n p i n e w e e v i l (23). The pecan w e e v i l , C u r c u l i o caryae (Horn), a l s o h a s been r e p o r t e d t o r e s p o n d t o t h e s y n t h e t i c b o l l w e e v i l pheromone b l e n d a n d t o c o m p o u n d X a l o n e ( 2 4 ) , a n d H e d i n e t a l . (25) i s o l a t e d com­ pound X f r o m a n a c t i v e e x t r a c t o f female pecan w e e v i l s . A d d i t i o n ­ a l l y , t h e New G u i n e a s u g a r c a n e w e e v i l , R h a b d o s c e l u s o b s c u r u s ( B o i s d u v a l ) , was r e p o r t e d t o respond t o t h e s y n t h e t i c b o l l w e e v i l b l e n d ( 2 6 ) . T h u s t h e s e c o m p o u n d s a n d s i m i l a r o n e s may s e r v e a s pheromone components i n s e v e r a l c u r c u l i o n i d s p e c i e s f r o m d i f f e r e n t genera. I n t e r e s t i n g l y , females o f a cerambycid s p e c i e s Dectes t e x a n u s t e x a n u s LeConte have been r e p o r t e d t o be a t t r a c t e d b y t h e s e c o m p o u n d s ( 2 7 ) . When we c o n s i d e r t h a t t h e t r i c y c l i c a c e t a l l i n e a t i n , a s c o l y t i d p h e r o m o n e , h a s t h e same c a r b o n s k e l e t o n a s g r a n d i s o l , i t s e e m s p o s s i b l e t h a t t h i s t y p e o f s t r u c t u r e may b e f a i r l y w i d e s p r e a d among Very r e c e n t l y (R*,S i d e n t i f i e d a s t h e m a j o r component o f t h e a g g r e g a t i o n pheromone o f t h e r i c e w e e v i l , S i t o p h i l u s o r y z a e L . , a n d t h e m a i z e w e e v i l , S. z e a m a l s M o t s c h . ( 2 8 ) . T h i s compound i s o b v i o u s l y v e r y d i f f e r e n t i n s t r u c t u r e from t h ep r e v i o u s l y i d e n t i f i e d c u r c u l i o n i d phero­ mones. I n f a c t , i t i s v e r y s i m i l a r t o t h e pheromone o f two o t h e r stored-products p e s t s , t h edrugstore b e e t l e and t h e c i g a r e t t e beetle, which a r e i nthe f a m i l y Anobiidae (seel a t e r ) . Undoubt­ e d l y , we w i l l f i n d m o r e d i v e r s i t y i n s t r u c t u r e a s m o r e c u r c u l i o n i d pheromones a r e i d e n t i f i e d . S i n c e s o f e w c u r c u l i o n i d p h e r o m o n e s h a v e b e e n i d e n t i f i e d , we d o n o t y e t know w h a t r o l e c h i r a l i t y w i l l p l a y o r t h e i m p o r t a n c e o f blends i nmediating behavior w i t h i n t h i s family. Obviously, since t h e b o l l w e e v i l r e s p o n s e t o i n d i v i d u a l components o r p a i r s o f com­ ponents i salmost n i l , t h eblend i simportant. However, t h e b o l l weevil i s e s s e n t i a l l y an introduced pest, having migrated i n t o t h e U. S. i n a b o u t t h e s e c o n d d e c a d e o f t h i s c e n t u r y f r o m C e n t r a l America. Thus i n t e r a c t i o n s r e l a t e d t o c h e m i c a l c o m m u n i c a t i o n s between t h e b o l l w e e v i l and o t h e r c l o s e l y r e l a t e d s p e c i e s have n o t b e e n s t u d i e d . M u c h m o r e r e s e a r c h i s n e e d e d i n t h i s a r e a b e f o r e we w i l l b e g i n t o u n d e r s t a n d how t h i s i n s e c t a n d r e l a t e d s p e c i e s i n t e r a c t i nt h e ecosystem. Scarabaeidae The e x i s t e n c e o f c h i r a l i t y i n p h e r o m o n e m o l e c u l e s h a s b e e n r e c o g n i z e d s i n c e 1966, b u t a s S i l v e r s t e i n (7) e x p l a i n s , m o s t o f u s ignored i t because t h e i n s e c t s responded t o t h e s y n t h e s i z e d r a c e m i c compounds. Thus t h e i n s e c t s ' response t o t h e c h i r a l pheromones i d e n t i f i e d i n e a r l i e r work appeared t o f a l l i n t o t h e f i r s t category d e s c r i b e d by S i l v e r s t e i n (5), i . e . , t h e i n s e c t produced and responded t o a s i n g l e enantiomer and t h e other e n a n t i o m e r was i n a c t i v e . Furthermore, t h ep a u c i t y o f n a t u r a l pheromone o b t a i n a b l e f r o m t h e i n s e c t s makes i t d i f f i c u l t , a n d i n most c a s e s i m p o s s i b l e , t o d e t e r m i n e t h e s t e r e o c h e m i s t r y o f t h e natural material.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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I t was i n e v i t a b l e t h a t e v e n t u a l l y we w o u l d b e f o r c e d t o c o n s i d e r e f f e c t s o f c h i r a l i t y on pheromone a c t i v i t y . The importance o f geometrical isomerism i n determining the a c t i v i t y o f l e p i d o p t e r o u s p h e r o m o n e s was w e l l r e c o g n i z e d . H o w e v e r , i t was s t i l l s o m e w h a t s u r p r i s i n g w h e n we d i s c o v e r e d t h a t t h e s y n t h e s i z e d r a c e m i c J a p a n e s e b e e t l e , P o p i l l i a j a p o n l e a Newman, p h e r o m o n e was i n a c t i v e e v e n t h o u g h we h a d p u r i f i e d b o t h t h e n a t u r a l ( a c t i v e ) pheromone and t h e s y n t h e s i z e d pheromone t o g r e a t e r t h a n 99%, and t h e two w e r e i n d i s t i n g u i s h a b l e by e v e r y c h r o m a t o g r a p h i c and s p e c t r o s c o p i c a n a l y t i c a l method a v a i l a b l e ( 2 9 ) . I t was o n l y w h e n t h e ( R ) a n d ( S ) e n a n t i o m e r s o f X I I I w e r e s y n t h e s i z e d ( 3 0 ) t h a t we d i s c o v e r e d t h a t t h e p u r e ( R ) ( - ) e n a n t i o m e r o f X I I I was very a t t r a c t i v e t o m a l e s a n d t h e ( S ) ( + ) was a n i n h i b i t o r t h a t , e v e n when p r e s e n t i n o n l y a few p e r c e n t , s i g n i f i c a n t l y r e d u c e d t h e a c t i v i t y of the ( R ) ( - ) . T h e r e a r e two o t h e r f a c t o r s w o r t h n o t i n g i n r e g a r d t o t h e J a p a n e s e b e e t l e pheromone A s i n g l e compound a p p a r e n t l y p o s s e s s e s a l l the a c t i v i t y of th w h e n we n o t e t h a t t h e m a t e r i a a b o u t 1 5 % o f t h e ( E ) i s o m e r a n d 3% o f t h e a n a l o g w i t h a s a t u r a t e d side chain. However, t h e s e o t h e r two compounds had no e f f e c t o n t h e a c t i v i t y o f t h e ( R ) ( Z ) - i s o m e r when added i n t h e a p p r o p r i a t e r a t i o s whether the racemic o r the pure e n a n t i o m e r i c forms were used. S i n c e t h e J a p a n e s e b e e t l e i s an i m p o r t e d p e s t , i t r e m a i n s t o be s e e n w h e t h e r t h e s e o t h e r i s o m e r s a n d / o r e n a n t i o m e r s h a v e a m o r e s i g n i f i c a n t r o l e i n i n t e r a c t i o n s among c l o s e l y r e l a t e d s p e c i e s i n i t s n a t i v e h a b i t a t . O n l y one o t h e r s e x a t t r a c t a n t has b e e n i d e n t i f i e d for a scarab species. Phenol a t t r a c t s males of the grass grub b e e t l e , C o s t e l y t r a z e a l a n d i c a (White) (31.). T h u s , we d o n o t k n o w w h a t t y p e s o f c o m p o u n d s a r e l i k e l y t o be m o s t p r e v a l e n t among t h e p h e r o m o n e s o f t h e S c a r a b a e i d a e . However, i t i s worth n o t i n g t h a t a t t r a c t a n t s d e r i v e d from p l a n t s appear t o s y n e r g i z e t h e r e s p o n s e o f b o t h m a l e and f e m a l e J a p a n e s e b e e t l e s t o t h e p h e r o m o n e ( 3 2 , 3 3 ) , a n d t h u s i t i s s i m i l a r t o many o t h e r coleopterous species i n t h i s respect.

Chrysomelidae I n t h i s v e r y l a r g e c o l e o p t e r o u s f a m i l y the sex pheromone o f o n l y two s p e c i e s h a v e b e e n i d e n t i f i e d . Diabrotica virgifera virgifera L e C o n t e , t h e w e s t e r n c o r n r o o t w o r m (WCR), p r o d u c e s a n d r e s p o n d s t o

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(R,R)-8-methyl-2-decanol propanoate ( 3 4 ) . Sonnet e t a l . ( 3 5 ) s y n t h e s i z e d t h e f o u r s t e r e o i s o m e r s o f t h i s pheromone i n h i g h s t e r e o i s o m e r i c p u r i t y (Table I ) . Comparison o f t h ec a p t u r e s o f WCR m a l e s i n t h e f i e l d w i t h t r a p s b a i t e d w i t h t h e i n d i v i d u a l s t e r o i s o m e r s and w i t h t h e racemic mixture demonstrated that t h e males respond s t r o n g l y t o t h e (R,R)-isomer and t o a l e s s e r e x t e n t to t h e (2S,8R)-isomer. The o t h e r two i s o m e r s a r e i n a c t i v e ; i . e . , t h e y n e i t h e r a t t r a c t n o r i n h i b i t t h e r e s p o n s e o f WCR m a l e s , n o r d o they s y n e r g i z e t h e a c t i v i t y o f t h e (R,R)-isomer ( 3 6 ) . The ( R , R ) - i s o m e r a p p e a r s t o b e a s a t t r a c t i v e a s a n e q u a l amount o f t h e n a t u r a l pheromone i n v o l a t i l e s c o l l e c t e d f r o m f e m a l e s . Thus i t i s p o s s i b l e t h a t t h e WCR i s u t i l i z i n g a s i n g l e c o m p o n e n t p h e r o m o n e .

Table I . P u r i t y o f t h e s y n t h e s i z e d s t e r e o i s o m e r s o f 8-methyl-2d e c a n o l propanoate t h a t were t e s t e d i n t h e f i e l d f o r attractiveness t o several species o f Diabrotica. Gross p u r i t Isomer

(%)

2S,8S

99.3

2R,8S 2S,8R

2S,8S

2R,8S

2S,8R

98.8

0.4

0.8

99.1

0.9

99.3

98.3

1.9

-

97.4

0.7

2R,8R

95.3

-

1.9

0.3

97.8

2RS,8RS

99+

-

2R,8R

0.8

Evidence from s t u d i e s o f t h e response o f s e v e r a l c l o s e l y r e l a t e d D i a b r o t i c a s p e c i e s t o t r a p s i n t h e f i e l d b a i t e d w i t h WCR female v o l a t i l e s , t h e r a c e m i c s y n t h e t i c pheromone, and t h e f o u r s t e r e o i s o m e r s s u g g e s t s t h a t a number o f c l o s e l y r e l a t e d s p e c i e s may b e r e l y i n g o n t h e s t e r e o c h e m i s t r y o f t h i s m o l e c u l e t o e f f e c t s p e c i f i c communication. T h e M e x i c a n c o r n r o o t w o r m ( M C R ) , D. v . z e a K r y s a n & S m i t h r e s p o n d e d t o t h e same s t e r e o i s o m e r s a s t h e WCR. T h i s i s n o t t o o s u r p r i s i n g s i n c e t h e MCR a l s o r e s p o n d s t o v o l a t i l e s c o l l e c t e d f r o m WCR f e m a l e s , a n d t h e s e t w o t a x a a r e s u b s p e c i e s t h a t have been r e p o r t e d t o i n t e r b r e e d i n t h o s e a r e a s where t h e i r g e o g r a p h i c a l ranges abut ( 3 7 ) . I n o t h e r f i e l d t e s t s we f o u n d t h a t t h e n o r t h e r n c o r n r o o t w o r m ( N C R ) , D. b a r b e r i S m i t h & Lawrence, responded t o t h e racemic m a t e r i a l a t l o w c o n c e n t r a t i o n s ( o p t i m u m 0.1 p g o n c o t t o n w i c k s ) , b u t a s t h e d o s e w a s i n c r e a s e d t o 1 u g , t h e NCR r e s p o n s e d i m i n i s h e d r a p i d l y a n d a t 10 u g i t was a l m o s t n i l . C o n v e r s e l y , i n t h e same t e s t t h e WCR r e s p o n s e c o n t i n u e d t o i n c r e a s e t o 100 y g , t h e h i g h e s t d o s e t e s t e d . This b e h a v i o r w a s e x p l a i n e d w h e n c a p t u r e s o f NCR m a l e s i n t r a p s b a i t e d w i t h t h e s t e r e o i s o m e r s was i n v e s t i g a t e d . NCR m a l e s w e r e c a p t u r e d o n l y i n t r a p s b a i t e d w i t h t h e ( 2 R , 8 R ) - i s o m e r a t t h e 1 \ig d o s e .

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F u r t h e r m o r e , NCR m a l e r e s p o n s e t o t h e ( 2 R , 8 R ) - i s o m e r was r e d u c e d t o t h e l e v e l o f t h e b l a n k when an e q u a l amount o f t h e ( 2 S , 8 R ) — i s o m e r was a d d e d t o t h e ( 2 R , 8 R ) . The ( 2 S , 8 S ) - i s o m e r a l s o s i g n i f i ­ c a n t l y r e d u c e d r e s p o n s e t o t h e ( 2 R , 8 R ) , b u t n o t a s much a s (2S,8R), w h i l e (2R,8S) n e i t h e r enhanced nor d i m i n i s h e d r e s p o n s e t o (2R,8R) ( 3 8 ) . Two o t h e r s p e c i e s i n t h i s g r o u p , D. p o r r a c e a H a r o l d , w h i c h i s s y m p a t r i c w i t h t h e MCR b u t n o t w i t h WCR, a n d D. l o n g i c o r n i s ( S a y ) , s y m p a t r i c w i t h WCR a n d NCR, r e s p o n d t o t h e r a c e m i c s y n t h e t i c p h e r ­ omone, b u t o n l y t o t h e ( 2 S , 8 R ) - i s o m e r ( 3 6 , 3 8 ) . These f i e l d t r a p p i n g s t u d i e s w i t h the s t e r e o i s o m e r s have p r o ­ v i d e d s t r o n g c i r c u m s t a n t i a l e v i d e n c e t h a t t h e WCR f e m a l e p r o d u c e s o n l y t h e ( 2 R , 8 R ) - i s o m e r , a l t h o u g h WCR m a l e s r e s p o n d t o b o t h (2R,8R) and ( 2 S , 8 R ) . These r e s u l t s a r e summarized i n Table I I . S i n c e t h e NCR m a l e s , w h i c h a r e s t r o n g l y i n h i b i t e d b y ( 2 S , 8 R ) , r e s p o n d t o v o l a t i l e s f r o m WCR f e m a l e s t h i s s u g g e s t s t h a t t h e s e v o l a t i l e s do n o t c o n t a i n t h e i n h i b i t o r y i s o m e r Furthermore D p o r r a c e a responds t o (2S,8R i s n o t i n h i b i t e d by t h p o r r a c e a d o e s n o t r e s p o n d t o WCR f e m a l e v o l a t i l e s a l s o i n d i c a t e s t h a t t h e s e v o l a t i l e s do n o t c o n t a i n (2S,8R). A t t h i s t i m e we h a v e d i s c e r n e d n o o b v i o u s r e a s o n why WCR m a l e s r e s p o n d t o a n i s o m e r t h e i r f e m a l e s do n o t p r o d u c e . T h i s i s e v e n m o r e c u r i o u s w h e n we c o n s i d e r t h a t t h e ( 2 S , 8 R ) - i s o m e r may be t h e p h e r o m o n e o f a s y m p a t r i c s p e c i e s , D. l o n g i c o r n i s .

Table I I .

Species

A t t r a c t i v e n e s s o f v o l a t i l e s c o l l e c t e d from v i r g i n Diabrotica v i r g i f e r a v i r g i f e r a females, synthesized r a c e m i c , ( 2 R , 8 R ) - , and ( 2 S , 8 R ) - 8 - m e t h y l - 2 - d e c a n o l p r o ­ panoate t o males o f 4 D i a b r o t i c a s p e c i e s i n f i e l d t e s t s . D. v i r g i f e r a volatiles

0

8-Methvl-2-decvl Racemic 2R,8R

WCR D. v . virgifera

++b

4-4-

4-4-

MCR

4-4-

+4-

+4-

4-c

4-4-

D.

D.

v.

zea

porracea

NCR D. b a r b e r i

propanoate 2S,8R

Ob

—b

a

WCR = w e s t e r n c o r n r o o t w o r m , MCR = M e x i c a n c o r n r o o t w o r m , NCR = n o r t h e r n c o r n rootworm. 4- i n d i c a t e s a t t r a c t i o n ; 4-4- i n d i c a t e s s t r o n g a t t r a c t i o n ; — i n d i c a t e s s t r o n g i n h i b i t i o n ; 0 i n d i c a t e s no a c t i v i t y . T h e r a c e m i c m a t e r i a l i s a t t r a c t i v e t o NCR m a l e s a t l o w c o n ­ c e n t r a t i o n s ( c a . 0.1 p g ) b u t i n h i b i t o r y a t h i g h e r c o n c e n t r a t i o n s ( c a . 10 u g ) . b

c

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D i a b r o t i c a c r i s t a t a ( H a r r i s ) , a nonpest s p e c i e s taxonoraically c l a s s i f i e d i n the v i r g i f e r a s p e c i e s group, h a s a p p a r e n t l y developed a s l i g h t l y d i f f e r e n t mechanism f o r s p e c i e s i s o l a t i o n . The m a l e D. c r i s t a t a r e s p o n d t o 8 - m e t h y l - 2 - d e c a n o l acetate. H o w e v e r , w h e n t h e f o u r i s o m e r s o f t h e a c e t a t e w e r e t e s t e d , D. c r i s t a t a males responded o n l y t o t h e (2S,8R)-isomer (39). Thus both s t r u c t u r a l d i v e r s i t y and stereoisomerism are being used by t h i s group o f i n s e c t s t o achieve s p e c i f i c i t y i n t h e i r chemical signals. No e v i d e n c e o f m u l t i c o m p o n e n t p h e r o m o n e s i n t h i s g r o u p has y e t been d i s c o v e r e d . T h e p h e r o m o n e o f t h e s o u t h e r n c o r n r o o t w o r m ( S C R ) , D. u n d e c l m p u n c t a t a h o w a r d i i B a r b e r , was i d e n t i f i e d a s (R)-10-methyl-2-tridecanone (40). Since i t has o n l y one asymmetric carbon, i t c a n e x i s t i n o n l y two e n a n t i o m e r i c forms. S y n t h e s i s o f b o t h t h e ( R ) - a n d ( S ) - e n a n t i o m e r s b y S o n n e t (41.) high enantiomeric purity again provided material for f i e l d tests. SCR m a l e s r e s p o n d o n l y t o t h e ( R ) - e n a n t i o m e r i n t h e f i e l d . Although extensive f i e l p r e l i m i n a r y evidence suggest may r e s p o n d t o t h i s p h e r o m o n e . S i n c e fewer isomers a r e a v a i l a b l e , t h e number o f s p e c i f i c s i g n a l s t h a t c a n be f o r m u l a t e d i s r e d u c e d correspondingly. Thus i t might be n e c e s s a r y f o r t h i s group t o employ more components, a l t h o u g h n o e v i d e n c e i s a v a i l a b l e t o indicate that this occurs. O b v i o u s l y , much m o r e r e s e a r c h o n p h e r o m o n e c o m m u n i c a t i o n i n t h i s genus and i n o t h e r c h r y s o m e l i d s p e c i e s i s needed. However, i t seems p o s s i b l e t h a t t h e s e D i a b r o t i c a s p e c i e s a r e a c h i e v i n g s p e c i f i c i t y i n t h e i r s i g n a l s through t h euse o f stereoisomers as s i n g l e component pheromones. i

Other

n

Families

The o n l y o t h e r f a m i l y f o r w h i c h p h e r o m o n e s h a v e b e e n i d e n t i f i e d for s e v e r a l species i s Dermestidae. A p p r o x i m a t e l y 11 d e r m e s t i d pheromones have been i d e n t i f i e d t h u s f a r , and t h e y a r e o f two general types. Trogoderma s p e c i e s u t i l i z e 14-methyl-8-hexadecenal and t h e a n a l o g o u s a l c o h o l a n d c a r b o x y l i e a c i d m e t h y l e s t e r s . Apparently a l l o f t h es p e c i e s s t u d i e d thus f a r respond t o t h e ( R ) - e n a n t i o m e r o f t h e s e compounds ( 4 2 ) . O t h e r d e r m e s t i d p h e r o ­ mones i d e n t i f i e d t h u s f a r a r e f a t t y a c i d s o r m e t h y l e s t e r s o f f a t t y a c i d s , an example b e i n g t h e b l a c k c a r p e t b e e t l e , Attagenus megatoma ( F a b r i c i u s ) , pheromone ( E , Z ) - 3 , 5 - t e t r a d e c a d i e n o i c a c i d (43). O n l y two A n o b i i d a e pheromones have been i d e n t i f i e d thus f a r . The d r u g s t o r e b e e t l e , S t e g o b i u m p a n i c e u m ( L . ) , p h e r o m o n e w a s i d e n t i f i e d as 2,3-dihydro-2,3,5-trimethyl-6-(l-methyl-2-oxobutyl)-4H-pyran-4-one (XIV) (44) and t h ec i g a r e t t e b e e t l e , L a s i o d e r m a s e r r i c o r n e F., a s 4 , 6 - d i m e t h y l - 7 - h y d r o x y - n o n a n - 3 - o n e (XV) ( 4 5 ) . The s i m i l a r i t y o f t h e s e s t r u c t u r e s i s w o r t h n o t i n g .

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XIV

XV

Examples o f s t r u c t u r e s i d e n t i f i e d from s p e c i e s i n o t h e r f a m i l i e s i l l u s t r a t e even f u r t h e r the d i v e r s i t y o f c o l e o p t e r o u s pheromone c h e m i s t r y . The o n l y B r u c h i d a e sex a t t r a c t a n t pheromone known s o f a r i s methyl ( E ) - 2 , 4 , 5 - t e t r a d e c a t r i e n o a t e from A c a n t h o s c e 1 i d e s o b t e c t u s ( S a y ) . The a l l e n i c n a t u r e o f the o l e f i n i c bonds i n t h i s m o l e c u l e i m p a r t s c h i r a l i t y t o t h e structure. The pheromone o f t h e T e n e b r i o n i d a e s p e c i e s T r i b o l e u m castaneum H e r b s t has been r e p o r t e d as 4 , 8 - d i m e t h y l d e c a n a l w h i c h a l s o has two c h i r a l c e n t e r Rhyzopertha d o m i n i c a ( P a b r i c i u s ) f a m i l y , u s e s a m i x t u r e o f e s t e r s as i t s pheromone, ( S ) - l - m e t h y l b u t y l (E)-2-methyl-2-pentenoate and ( S ) - l - m e t h y l b u t y l (B)-2,4-dimethylpentenoate (49). V e r y r e c e n t l y seven m a c r o l i d e l a c t o n e s have been i s o l a t e d and i d e n t i f i e d from v o l a t i l e s c o l l e c t e d from males o f f i v e s p e c i e s o f g r a i n b e e t l e s i n the f a m i l y C u c u j i d a e . The number o f l a c t o n e s produced by each o f t h e s e s p e c i e s v a r i e s from two t o f i v e , a l t h o u g h f o u r o f the s p e c i e s use o n l y two components as t h e i r pheromone and the f i f t h u s e s o n l y t h r e e . Thus t h e r u s t y g r a i n b e e t l e , C r y p t o l e s t e s f e r r u g i n e u s (Stephens), uses a s y n e r g i s t i c blend of (E,E)-4,8-dimethyl-4,8-decadien-10~olide and ( 3 Z , H S ) - 3 - d o d e c e n - l l - o l i d e ( 5 0 ) ; the f l a t g r a i n b e e t l e , C. p u s i l l u s (Schônerr), u s e s ( Z ) - 3 - d o d e c e n o l i d e and ( Z ) - 5 - t e t r a d e c e n - 1 3 - o l i d e ( 5 1 ) ; and the f l o u r m i l l b e e t l e , C. t u r c i c u s ( G r o u v e l l e ) , u s e s ( Z , Z ) - 5 , 8 - t e t r a d e c a d i e n - 1 3 - o l i d e and ( Z ) - 5 - t e t r a d e c e n - 1 3 - o l i d e ( 5 2 ) . O r y z a e p h i l u s mercator ( F a u v e l ) u s e s a b l e n d o f ( 3 Z , l l R ) - 3 - d o d e c e n - l l - o l i d e and ( Z , Z ) - 3 , 6 - d o d e c a d i e n - l l - o l i d e , w h i l e O. s u r i n a m e n s i s ( L ) u s e s a t h r e e component b l e n d o f ( Z , Z ) - 3 , 6 - d o d e c a d i e n - l l - o l i d e , ( Z , Z ) - 3 , 6 - d o d e c a d i e n o l i d e , and ( Z , Z ) - 5 , 8 - t e t r a d e c a d i e n - 1 3 - o l i d e (53) . The c h i r a l i t y o f o n l y one o f t h e s e male produced pheromones has been d e t e r m i n e d , i . e . ( Z ) - 3 - d o d e c e n - l l - o l i d e . However, i t appears t h a t h e r e a g a i n b o t h multicomponent b l e n d s and c h i r a l i t y a r e b e i n g used t o a c h i e v e s p e c i f i c i t y i n the pheromone s i g n a l s (54) . In a t l e a s t some c a s e s where s i n g l e component pheromones have been i d e n t i f i e d the pheromone i d e n t i f i c a t i o n p r o b a b l y c a n be labeled incomplete. As more r e s e a r c h i s c o n d u c t e d on the b e h a v i o r o f t h e s e s p e c i e s and t h e i r i n t e r a c t i o n s w i t h o t h e r s p e c i e s , p a r t i c u l a r l y t h o s e i n the same f a m i l y and genus, more compounds w i l l u n d o u b t e d l y be i d e n t i f i e d t h a t a r e a c t i v e i n m e d i a t i n g t h e b e h a v i o r o f t h e s e s p e c i e s . A t t h i s time I c a n o n l y r e i t e r a t e t h a t c o n s i d e r a b l y more r e s e a r c h i s needed on c o l e o p t e r o u s pheromones b e f o r e we c a n b e g i n t o u n d e r s t a n d t h e s e complex i n t e r a c t i v e systems. For n a t u r a l p r o d u c t s c h e m i s t s t h i s s h o u l d be a f r u i t f u l a r e a f o r i n v e s t i g a t i o n f o r some t i m e .

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24. Polles, S. G.; Payne, J. Α.; Jones, R. L. Pecan South 1977, 4, 26-8. 25. Hedin, P. A.; Payne, J. A.; Carpenter, T. L.; Neel, W. Environ. Entomol. 1979, 8, 521-3. 26. Chang, V. C. S.; Curtis, G. A. Environ. Entomol. 1972, 1, 476. 27. Patrick, D. R. J. Ga. Entomol. Soc. 1974, 9, 17. 28. Schmuff, N. R.; Phillips, J. K.; Burkholder, W. E.; Fales, H. M.; Chen, C. V.; Roller, P. P.; Ma, M. Tetrahedron Lett. 1984, 25, 1533-4. 29. Tumlinson, J. H.; Klein, M. G.; Doolittle, R. E.; Ladd, T. L.; Proveaux, A. T. Science 1977, 197, 789-92. 30. Doolittle, R. E.; Tumlinson, J. H.; Proveaux, A. T.; Heath, R. R. J. Chem. Ecol. 1980, 6, 473-85. 31. Henzell, R. f.; Lowe, M. D. Science 1970, 168, 1005. 32. Klein, M. G.; Tumlinson, J. H.; Ladd, T. L., Jr.; Doolittle, R. E. J. Chem. Ecol. 1981, 7, 1-7. 33. Ladd, T. L.; Klein 1981, 74, 665-7. 34. Guss, P. L.; Tumlinson, J. H.; Sonnet, P. E.; Proveaux, A. T. J. Chem. Ecol. 1982, 8, 545-56. 35. Sonnet, P. E.; Carney, R. L.; Henrick, C. A. J. Chem. Ecol. 1985, (in press). 36. Guss, P. L.; Sonnet, P. E.; Carney, R. L.; Branson, T. F.; Tumlinson, J. H. J. Chem. Ecol. 1984, 10, 1123-1131. 37. Krysan, J. L.; Smith, R. F.; Branson, T. F.; Guss, P. L. Ann. Entomol. Soc. Am. 1980, 73, 123-30. 38. Guss, P. L.; Sonnet, P. E.; Carney, R. L.; Tumlinson, J. H.; Wilkin, P. J., J. Chem. Ecol. 1985, 11, 000-00. 39. Guss, P. L.; Carney, R. L.; Sonnet, P. E.; Tumlinson, J. H. Environ. Entomol. 1983, 12, 1296-7. 40. Guss, P. L.; Tumlinson, J. H.; Sonnet, P. E.; McLaughlin, J. R. J. Chem. Ecol. 1983, 9, 1363-75. 41. Sonnet, Philip E. J. Org. Chem. 1982, 47, 3793-6. 42. Silverstein, R. M.; Cassidy, R. F.; Burkholder, W. E.; Shapas, T. J.; Levinson, H. Z.; Levinson, A. R.; Mori, K. J. Chem. Ecol. 1980, 6, 911-17. 43. Silverstein, R. M.; Rodin, J. O.; Burkholder, W. E.; Gorman, J. E. Science 1967, 157, 85. 44. Kuwahara, Y.; Fukami, H.; Howard, R.; Ishi, S.; Matsumura, F.; Burkholder, W. E. Tetrahedron 1978, 34, 1769-74. 45. Chuman, T.; Kohno, M.; Kato, K.; Noguchi, M. Tetrahedron Lett. 1979, 2361. 46. Suzuki, T. Agric. Biol. Chem. 1980, 44, 2519-20. 47. Suzuki, T. Agric. Biol. Chem. 1981, 45, 1357-63. 48. Suzuki, T. Agric. Biol. Chem. 1981, 45, 2641-3. 49. Williams, H. J.; Silverstein, R. M.; Burkholder, W. E.; Khorramshahi, A. J. Chem. Ecol. 1981, 7, 759-80. 50. Wong, J. W.; Verigin, V.; Oehlschlager, A. C.; Borden, J. H.; Pierce, H. D., Jr.; Pierce, Α. M.; Chong, L. J. Chem. Ecol. 1983, 9, 451-74. 51. Millar, J. G.; Pierce, H. D., Jr.; Pierce, Α. M.; Oehlschlager, A. C.; Borden, J. H.; Barak, Α. V., J. Chem. Ecol. 1985, 11, 000-00.

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52. Millar, J. G.; Pierce, H. D., Jr.; Pierce, A. M.; Oehlschlager, A.C.;borden, J. H., J. Chem. Ecol. 1985, 11, 000-00. 53. Pierce, A. M.; Pierce, H. D., Jr.; Millar, J. G.; Borden, J. H.; Oehlschlager, A. C. Proc. Third Intern. Working Conf. on Stored Product Entomology, 1983 (in press). 54. Pierce, H. D., Jr.; Pierce, A. M.; Millar, J. G.; Wong, J. W.; Verigin, V. G.; Oehlschlager, A.C.;Borden, J. H. Proc. Third Intern. Working Conf. on Stored Product Entomology, 1983 (in press). RECEIVED

November 15, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

27 Sexual Messages of Moths: Chemical Themes Are Known and New Research Challenges Arise JEROME A. KLUN Organic Chemical Synthesis Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Female moths produce chemical signals to attract and sexually stimulat decades has define these messages. The females employ fairly simple aliphatic compounds in their communications; the major variables are carbon-chain length, olefinic site and geometry, functionality, and optical form, and various combinations of compounds involving these variables constitute the chemical message. Attainment of a fundamental understanding of these chemical themes has brought this field to a threshold of new research challenges. There is now a need to understand the intermediary metabolic processes that regulate composition of the molecular message, the endogenous factors that regulate timely onset of biosynthesis and release of the chemical message, the nature of the olfactory chemoreceptor, and the molecular basis of perception and transduction of chemical stimuli into electrophysiological events in the sensory neurons. E n t o m o l o g i s t s have been f a s c i n a t e d w i t h m o l e c u l a r messages o r " s e x s c e n t s " o f moths f o r a l o n g t i m e . Near t h e t u r n o f t h e 2 0 t h c e n t u r y , r e s e a r c h e r s p r o v i d e d v i v i d d e s c r i p t i o n s o f t h e amorous v i s i t a t i o n s o f male moths t o s i t e s c o n t a i n i n g c a p t i v e v i r g i n female moths ( 1 , 2 ) . However, t h e famous F r e n c h e n t o m o l o g i s t , Jean-Henri F a b r e , had d i f f i c u l t y p r o v i n g t h a t t h e b e h a v i o r e x h i b i t e d by t h e male moths was t r i g g e r e d by a n a i r b o r n e s c e n t emanating from t h e v i r g i n f e m a l e s , and he u n d e r r a t e d t h e i m p o r t a n c e o f o l f a c t o r y perception. I n 1900, w h i l e s t u d y i n g t h e peacock moth ( S a t u r n i a p y r i ) , F a b r e formed t h e f o l l o w i n g h y p o t h e s i s ( 1 ) : " P h y s i c a l s c i e n c e i s t o - d a y p r e p a r i n g t o g i v e us w i r e l e s s t e l e g r a p h y , by means o f t h e H e r t z i a n waves. Can t h e G r e a t Peacocks have a n t i c i p a t e d o u r e f f o r t s i n t h i s d i r e c t i o n ? I n order t o s e t the surrounding a i r i n motion and t o i n f o r m p r e t e n d e r s m i l e s away, c a n t h e n e w l y - h a t c h e d b r i d e have a t h e r d i s p o s a l e l e c t r i c o r

This chapter not subject to U.S. copyright. Published 1985 American Chemical Society

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magnetic waves, w h i c h one s o r t o f s c r e e n would a r r e s t and a n o t h e r l e t through? I n a word, does s h e , i n h e r own manner, employ a k i n d o f w i r e l e s s t e l e g r a p h y ? 1 see nothing impossible i n t h i s : I n s e c t s a r e accustomed to i n v e n t things q u i t e as wonderful." Fabre t e s t e d t h i s h y p o t h e s i s and s u b s e q u e n t l y r e j e c t e d the i d e a t h a t e l e c t r o m a g n e t i c r a d i a t i o n was i n v o l v e d i n the s e x u a l communi­ c a t i o n systems o f moths; however he remained s k e p t i c a l o f the i n v o l v e m e n t o f the sense o f s m e l l * S i x t y y e a r s l a t e r the case f o r s m e l l was f i n a l l y s e t t l e d when German c h e m i s t s , l e d by A. Butenandt ( 3 - 5 ) , were the f i r s t t o i s o l a t e , i d e n t i f y , and s y n t h e s i z e a n i n s e c t s e x pheromone, t h a t o f t h e female s i l k w o r m (Bombyx m o r i ) . They f o u n d t h a t the compound r e s p o n s i b l e f o r e l i c i t i n g s e x u a l b e h a v i o r from the male moth was (E,Z)-10,12-hexadecadien-l-ol* T h i s i d e n t i f i c a t i o n was no t r i v i a l accomplishment inasmuch as i t r e q u i r e d e x t r a c t i o n o f 500,000 s i l k w o r m f e m a l e s (most moths produce o n l y nanogram amounts o i n the days b e f o r e the other m i c r o - a n a l y t i c a l tools* Seven y e a r s l a t e r , i n 1966, the i d e n t i t y o f a s e x pheromone from a second s p e c i e s o f moth, the cabbage l o o p e r ( T r i c h o p l u s i a n i ) , was announced by R. S. B e r g e r (6)· By 1970, pheromonal components had been i d e n t i f i e d from t h r e e a d d i t i o n a l s p e c i e s o f moths; ( Z ) - 9 - t e t r a d e c e n - l - o l a c e t a t e (7) from the f a l l armyworm ( S p o d o p t e r a f u g i p e r d a ) and ( Z ) - l l - t e t r a d e c e n - l - o l a c e t a t e ( 8 , 9) from the redbanded l e a f r o l l e r ( A g y r o t a e n i a v e l u t i n a n a ) and the E u r o p e a n c o r n b o r e r ( O s t r i n i a nubilalis)· D i s c o v e r y o f the same compound i n the l e a f r o l l e r and c o m b o r e r was p e r p l e x i n g a t the time because i t was thought t h a t the sex pheromone o f a s p e c i e s s h o u l d be a s i n g l e compound w i t h a s t r u c t u r e u n i q u e t o e a c h s p e c i e s * T h i s naïveté was s h o r t - l i v e d and a n e x p l o s i v e growth p e r i o d i n the f i e l d o f moth sex pheromone c h e m i s t r y ensued* V i r t u a l l y a l l o f the e a r l i e s t pheromone i d e n t i f i c a t i o n s were c h e m i c a l l y i n c o m p l e t e and sometimes i n a c c u r a t e owing t o i n a d e q u a ­ c i e s o f e a r l y a n a l y t i c a l t e c h n i q u e s and i n s t r u m e n t a t i o n and methods o f b e h a v i o r a l assay* However, o v e r a p e r i o d o f 25 y e a r s much o f the c h e m i s t r y o f pheromones and the i n s e c t b e h a v i o r t h e y evoke was d e f i n e d w i t h r e a s o n a b l e completeness and a c l e a r p i c t u r e o f the c h e m i c a l themes used by the moths i n t h e i r s e x u a l communications has emerged t h r o u g h the c o l l e c t i v e f i n d i n g s o f s c i e n t i s t s throughout the w o r l d * We now know t h a t moth sex pheromone chemistry Involves r e l a t i v e l y simple s t r a i g h t carbon-chain compounds ( F i g u r e 1 ) ; c h a i n l e n g t h , f u n c t i o n a l i t y , s i t e s and geometry o f d o u b l e bonds, s p e c i f i c p r o p o r t i o n s o f compounds, and p e r m u t a t i o n s o f t h e s e v a r i a b l e s c o n s t i t u t e the e s s e n c e o f moths' s e x u a l messages* O p t i c a l form i s s e e m i n g l y r e l e g a t e d t o a minor r o l e i n moth s e x pheromone c h e m i s t r y * O n l y a h a n d f u l o f the s e v e r a l hundred i d e n t i f i e d moth pheromones (10, 11) i n v o l v e asymmetry o f the e p o x i d e m o i e t y (12, 1 3 ) , m e t h y l b r a n c h i n g (14, 15), o r an e s t e r o f a secondary a l c o h o l (16)* The c h e m i c a l p i c t u r e i s d e c e p t i v e l y s i m p l e u n t i l one a p p r e ­ c i a t e s the f a c t t h a t e a c h s p e c i e s may use v e r y s p e c i f i c permuta­ t i o n s and/or p r o p o r t i o n s o f compounds i n the a r r a y o f F i g u r e 1. It t h e n becomes a p p a r e n t t h a t moths have e v o l v e d a n e l e g a n t and

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27.

KLUN

Sexual Messages of Moths

F i g u r e 1 . S t r u c t u r e s o f r e p r e s e n t a t i v e compounds female moths a s s e x pheromones*

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s e c r e t e d by

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s p e c i f i c s i g n a l i n g system t h a t i s based upon m u l t i - d i m e n s i o n a l sets of simple chemical v a r i a b l e s . Development o f s y n t h e t i c r e p l i c a s o f the s e t s o f compounds i d e n t i f i e d as sex pheromone components has c o n s t i t u t e d a s i g n i f i ­ c a n t c h a l l e n g e to s y n t h e t i c o r g a n i c c h e m i s t s , because the r e q u i r e ­ ments f o r pheromonal a c t i v i t y i n c l u d e s t r i n g e n t c h e m i c a l and i s o m e r i c p u r i t y t h a t was n o t e a s i l y a t t a i n e d by t r a d i t i o n a l s y n t h e t i c o r g a n i c c h e m i c a l methods. These c h a l l e n g e s were met a d m i r a b l y by i n g e n i o u s and i m a g i n a t i v e o r g a n i c c h e m i s t s (17, 1 8 ) . Our r e c e n t s t u d y o f the female sex pheromones o f two c l o s e l y r e l a t e d moths, the p i c k l e w o r m ( D i a p h a n i a n i t i d a l i s ) (19) and melonworm (D. h y a l i n a t a ) ( 2 0 ) , p r o v i d e s an example o f how t r a c e components and m i x t u r e s o f compounds w i t h d i f f e r e n t f u n c t i o n a l i t y make up a moth*s pheromonal s i g n a l . F i g u r e 2 shows gas chromatograms o b t a i n e d by d i r e c t a n a l y s i s o f heptane e x t r a c t s o f sex pheromone g l a n d s ( l o c a t e d a t the t i p o f the f e m a l e abdomen) o f the two s p e c i e s . The chromatograms a r e a r r a n g e d as o p p o s i n g images to a l l o w comparison o f th species. Ten compounds s e v e n were found i n the p i c k l e w o r m . The major c h r o m a t o g r a p h i c peaks r e p r e s e n t c a . 50 ng compound. C o n s i s t e n t w i t h the c h e m i c a l theme d e p i c t e d i n F i g u r e 1, a l l o f the compounds were C^g s t r a i g h t - c h a i n compounds w i t h a l d e h y d e , a l c o h o l , o r a c e t a t e f u n c ­ t i o n a l i t y and w i t h g e o m e t r i c a l forms o f mono-enes and c o n j u g a t e d dienes. Figure 2 i n d i c a t e s that both s p e c i e s shared s i m i l a r phero­ monal components, but y e t the complement o f compounds from e a c h was u n i q u e . T h i s q u a l i t y makes f o r the s p e c i e s s p e c i f i c i t y o f p h e r o ­ monal s i g n a l s among moths. I n b e h a v i o r a l a s s a y s u s i n g male moths i n a wind t u n n e l , the s p e c i f i c i t y o f male r e s p o n s e c o u l d be e a s i l y demonstrated. As an example, when a p r o p o r t i o n a l amount o f melonworm pheromone component 6 [(E,£)-10,12-hexadecadlenal] was added t o a s y n t h e t i c m i x t u r e o f p i c k l e w o r m compounds the m i x t u r e was r e n d e r e d t o t a l l y u n a t t r a c t i v e to p i c k l e w o r m m a l e s . T r a c e components i n a s e t o f compounds produced by a female o f t e n a r e e s s e n t i a l f o r the e x p r e s s i o n o f b i o l o g i c a l a c t i v i t y o f a sex pheromone and the p i c k l e w o r m pheromone e x e m p l i f i e s t h i s q u a l i t y . F i g u r e 2 shows t h a t component 4 c o n s t i t u t e d o n l y 0.1% o f the t o t a l m i x t u r e o f compounds produced by the p i c k l e w o r m f e m a l e . Bioassays showed, however, t h a t i f t h i s compound was d e l e t e d from the s e t o f compounds produced by the f e m a l e , males were b e h a v i o r a l l y u n r e s p o n s i v e to the m i x t u r e d e s p i t e the f a c t t h a t a l l o t h e r components o f the pheromone complement were p r e s e n t . Similarly, when component 2 [ ( Z ) - l l - h e x a d e c e n a l ] , a l s o a t r a c e component ( c a . , 3%), was d e l e t e d from the s e t o f pickleworm compounds, the tendency o f males to f l y upwind to the s t i m u l u s was r e d u c e d by 70% r e l a t i v e to the r e s p o n s e e l i c i t e d by the complete s e t o f compounds. The development o f our u n d e r s t a n d i n g o f the c h e m i c a l themes o f moth sex pheromones has been d i r e c t l y l i n k e d to s p e c t a c u l a r advancements i n the t e c h n o l o g i e s o f a n a l y t i c a l c h e m i s t r y s u c h as open t u b u l a r c a p i l l a r y chromatography (OTCC), combined OTCC-mass s p e c t r o s c o p y , and m i c r o c h e m i c a l c h a r a c t e r i z a t i o n t e c h n i q u e s . S u r e l y , s u b s t a n c e s l i k e component 4 o f the p i c k l e w o r m pheromone would n e v e r have been d i s c o v e r e d w i t h o u t s u c h a n a l y t i c a l capability. Combined w i t h advancements i n a n a l y t i c a l c h e m i s t r y , i n c r e a s i n g l y o b j e c t i v e and e f f e c t i v e b e h a v i o r a l and

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F i g u r e 2. Chromatograms o b t a i n e d by d i r e c t a n a l y s i s o f heptane e x t r a c t s o f the sex pheromone g l a n d s o f pickleworm and melonworm females on a 60 m χ 0.25 mm (ID) DB-1 column ( J & W S c i e n t i f i c , I n c . , Rancho Cordova, CA 95670). 1. (E)-ll-hexadecenal; 2. ( Z ) - l l - h e x a d e c e n a l ; 3. hexadecanal; 4. ( E , Z ) - 1 0 , 1 2 - h e x a d e c a d i e n a l ; 5. (Z,Z)-10,12-hexadecadienal, 6. ( E , E ) - 1 0 , 1 2 - h e x a d e c a d i e n a l ; 7. (E)-ll-hexadecen-l-ol; 8. ( Z ) - l l - h e x a d e c e n - l - o l ; 9. h e x a d e c a n - l - o l ; 10. T E , E ) - 1 0 , 1 2 - h e x a d e c a d i e n - l - o l ; 11. (E)-ll-hexadecen-l-ol a c e t a t e ; 12. (E,E)-10,12-hexadecadien-l-ol acetate. Unnumbered c h r o m a t o g r a p h i c peaks a r e e i t h e r s o l v e n t contaminants o r components t h a t were n o t o b s e r v e d c o n s i s t e n t l y i n r e p l i c a t e d analyses. The g e o m e t r i c a l p r o p o r t i o n o f 11-hexadecenal i n pickleworm e x t r a c t was 96:4 ( E : Z ) .

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e l e c t r o p h y s i o l o g i c a l b i o a s s a y s (21-24) have been d e v e l o p e d , and c o n t i n u e t o be r e f i n e d , t h a t a l l o w f o r b e t t e r u n d e r s t a n d i n g o f the b i o l o g i c a l r e l e v a n c e o f the v a r i o u s c h e m i c a l s t h a t i n f l u e n c e the behavior of i n s e c t s * The moth sex pheromones a r e among the most b i o l o g i c a l l y p o t e n t c h e m i c a l s known and we have d e v e l o p e d an i n - d e p t h u n d e r s t a n d i n g o f the c h e m i c a l themes i n v o l v e d . The e n t h u s i a s m o f r e s e a r c h e r s i n the f i e l d o f i n s e c t sex pheromone c h e m i s t r y and b e h a v i o r i s n o t o n l y d i r e c t e d t o the e x p a n s i o n o f fundamental u n d e r s t a n d i n g o f t h i s phenomenal b i o l o g i c a l system b u t a l s o to how t h i s knowledge may e v e n t u a l l y be a p p l i e d p r a c t i c a l l y to r e l i e v e some o f the many p e s t i n s e c t problems t h a t c o n f r o n t u s . So f a r , our knowledge o f i n s e c t sex pheromone systems has had l i m i t e d i m p a c t a g r i c u l t u r a l l y ; however, f u n d a m e n t a l l y i m p o r t a n t r e s e a r c h c h a l l e n g e s l i e ahead i n t h i s a r e a o f b i o l o g y t h a t , i f met s u c c e s s f u l l y , may y i e l d unimagined and e f f e c t i v e a p p r o a c h e s to p e s t - i n s e c t m a n i p u l a t i o n . There a r e many p r o m i s i n g avenues o f r e s e a r c h . L i t t l e i s known o f pheromones a r e p r o d u c e d i n d i c a t e t h a t mono-unsaturated a c e t a t e moth sex pheromones may a r i s e v i a a b i o s y n t h e t i c pathway i n v o l v i n g Δ-11-desaturases t h a t o p e r a t e on l o n g c h a i n s a t u r a t e d f a t t y a c y l m o i e t i e s o f g l y c e r o l i p i d s to g e n e r a t e an o l e f i n i c s i t e and enyzmes t h a t s h o r t e n o r l e n g t h e n f a t t y a c y l c h a i n l e n g t h by 2 c a r b o n s . A t e n t a t i v e o u t l i n e f o r the b i o s y n t h e t i c o r i g i n o f a major component o f the sex pheromone o f the cabbage l o o p e r , ( Z ) - 7 - d o d e c e n - l - o l a c e t a t e , i s shown i n F i g u r e 3. Much remains to be done, however, to expand our u n d e r s t a n d i n g o f the I n t e r m e d i a r y m e t a b o l i c pathways t h a t r e g u l a t e the q u a l i t a t i v e and q u a n t i t a t i v e c o m p o s i t i o n o f the p r e c i s e s e t s o f compounds t h a t we know make up most pheromonal messages. As example, the f a c t o r s t h a t r e g u l a t e p r o d u c t i o n o f the s p e c i f i c m i x t u r e s o f g e o m e t r i c a l i s o m e r s , u s e d so f r e q u e n t l y by the moths, a r e u n d e f i n e d as i s the b i o s y n t h e t i c o r i g i n o f pheromones h a v i n g s k i p p e d and c o n j u g a t e d d i e n e f u n c t i o n a l i t y . R e l a t e d to the i s s u e o f the b i o s y n t h e t i c o r i g i n o f the p h e r o ­ mones, Ashok R a i n a and I r e c e n t l y d i s c o v e r e d t h a t f e m a l e sex p h e r o ­ mone p r o d u c t i o n i n a t l e a s t one s p e c i e s o f moth i s r e g u l a t e d by a neurohormonal p e p t i d e t h a t i s produced i n the f e m a l e b r a i n ( 2 7 ) . We o b s e r v e d t h a t when a d u l t f e m a l e s o f the c o r n earworm moth ( H e l i o t h i s zea) were l i g a t e d between the head and t h o r a x so t h a t normal hemolymph c i r c u l a t i o n between the b r a i n and the r e s t o f the body was i n t e r r u p t e d , they d i d n o t produce sex pheromone. However, the l i g a t e d f e m a l e s c o u l d be s t i m u l a t e d t o produce pheromone, i n amounts n o t d i f f e r e n t from t h a t produced by n o r m a l f e m a l e s , by an i n j e c t i o n o f a s a l i n e e x t r a c t o f the female b r a i n . Our s t u d i e s w i t h t h i s n o c t u r n a l moth i n d i c a t e t h a t the neurohormone i s s t o r e d i n the b r a i n and r e l e a s e d i n t o the hemolymph i n the s c o t o p h a s e c a u s i n g t i m e l y p r o d u c t i o n o f pheromone by the f e m a l e . A d d i n g t o the puzzlement o f t h i s system, we f o u n d t h a t p h e r o ­ mone p r o d u c t i o n c o u l d a l s o be i n d u c e d i n the g l a n d s o f l i g a t e d c o r n earworm females by i n j e c t i o n o f b r a i n e x t r a c t s o f the male c o r n earworm and e x t r a c t s o f f e m a l e s from f o u r d i f f e r e n t f a m i l i e s o f moths ( P y r a l i d a e , L y m a n t r i i d a e , Geometridae, and P s y c h i d a e ) ( 2 7 ) . The sex pheromones o f f e m a l e s r e p r e s e n t i n g t h e s e f a m i l i e s have l i t t l e o r no c h e m i c a l s i m i l a r i t y to the c o m earworm sex pheromone

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and the f u n c t i o n o f the neurohormone i n the male c o r n earworm i s unknown. Thus, the hormone may be w i d e l y d i s t r i b u t e d i n the o r d e r but t h e r e i s no o b v i o u s c o n n e c t i o n between i t s o c c u r r e n c e and the p h y s i o l o g i c a l f u n c t i o n s i t may have i n t h e s e a s s o r t e d i n s e c t s . E l u c i d a t i o n o f the c h e m i s t r y and p h y s i o l o g i c a l modes o f a c t i o n and f u n c t i o n a l s i g n i f i c a n c e o f t h i s n e u r o e n d o c r i n e s u b s t a n c e may n o t o n l y expand our knowledge o f endogenous f a c t o r s t h a t r e g u l a t e pheromone b i o s y n t h e s i s i n moths b u t a l s o s e r v e as a s t e p p i n g s t o n e to the u n d e r s t a n d i n g o f the mechanisms by w h i c h the b r a i n e x e r t s i t s i n f l u e n c e upon the v i t a l l i f e p r o c e s s e s o f the i n s e c t s . A n o t h e r p r o m i s i n g avenue f o r r e s e a r c h t h a t i s u n q u e s t i o n a b l y a t the f r o n t i e r o f s c i e n c e c o n c e r n s development o f an u n d e r s t a n d i n g of processes involved i n o l f a c t o r y perception. We know from our r e s e a r c h on the sex pheromones t h a t male moths p o s s e s s a phenomenal a b i l i t y to p e r c e i v e and r e s p o n d b e h a v i o r a l l y t o i n c r e d i b l y minute q u a n t i t i e s o f pheromonal compounds i n the a i r . The c h e m o r e c e p t i v e s e n s i t i v i t y o f t h i s d e t e c t o r system i s c e r t a i n l y u n r i v a l e d by any organic-chemical detecto We know t h a t the i n s e c t s l o c a t e d on the a n t e n n a e , and c o n s i d e r a b l e work has been done t o de­ s c r i b e the g e n e r a l m o r p h o l o g i c a l and f i n e s t r u c t u r e f e a t u r e s o f moth antennae ( 2 8 ) . A t y p i c a l moth antenna i s o f t e n f i l a m e n t o u s and m u l t i s e g m e n t e d . The v e n t r a l s u r f a c e o f e a c h segment i s h e a v i l y c l o t h e d w i t h h a i r - l i k e p r o j e c t i o n s c a l l e d s e n s i l l a ( F i g u r e 4) t h a t have p r o p r i o c e p t i v e , m e c h a n o r e c e p t i v e , o r c h e m o r e c e p t i v e s e n s o r y f u n c t i o n s , a c c o r d i n g t o m o r p h o l o g i c a l and e l e c t r o p h y s i o l o g i c a l studies. S e n s i l l a r e s p o n s i b l e f o r pheromone d e t e c t i o n a r e u s u a l l y the most numerous on the antennae o f male moths ( 2 9 ) . Microscopic s t u d i e s o f the c h e m o r e c e p t i v e s e n s i l l a i n d i c a t e t h a t they a r e porous e x t e n s i o n s o f the i n s e c t ' s c u t i c l e and e a c h s e n s i l i u m o f t e n houses one t o f i v e s e n s o r y n e u r o n c e l l s . E a c h s e n s o r y c e l l has d e n d r i t e s t h a t p r o j e c t from the c e l l body t o p o s i t i o n s n e a r the p o r e s o f the s e n s i l l u m and e a c h c e l l sends an axon, w i t h o u t s y n a p s e , t o a c e n ­ t r a l j u n c t i o n i n the b r a i n ( 3 0 ) . E l e c t r o p h y s i o l o g i c a l s t u d i e s (30, 31) show t h a t t h e s e s e n s o r y c e l l s send e l e c t r i c a l i m p u l s e s t o the b r a i n when the pheromone m o l e c u l e s a r e moved o v e r the a n t e n n a . It i s by t h e s e s e n s o r y - c e l l a c t i o n p o t e n t i a l i m p u l s e s t h a t the b r a i n r e c e i v e s i n f o r m a t i o n a b o u t i t s environment and t h e n , o s t e n s i b l y , orchestrates appropriate p h y s i o l o g i c a l - b e h a v i o r a l responses b e n e f i c i a l to the organism's s u r v i v a l . D e s p i t e t h i s s e e m i n g l y s t r a i g h t f o r w a r d d e s c r i p t i o n o f pheromone p e r c e p t i o n , l i t t l e i s a c t u a l l y known o f the m e c h a n i s t i c d e t a i l s o f the p r o c e s s . The e x a c t l o c a t i o n and b i o c h e m i c a l n a t u r e o f the r e ­ c e p t o r s i t e i s unknown. Because most pheromones a r e s p e c i f i c m i x t u r e s o f compounds, i t i s p r o b a b l e t h a t the o r g a n i z a t i o n o f the r e c e p t o r system may i n v o l v e an a r r a y o f s e v e r a l r e c e p t o r s , e a c h d e s i g n e d t o r e c e i v e c e r t a i n components o f the m i x t u r e , t h a t o p e r a t e i n concert (32). Bestmann and Vostrowsky (33) p r o p o s e d a h y p o t h e t i c a l model o f a pheromone r e c e p t o r b a s e d upon s t u d y o f the e l e c t r o p h y s i o l o g i c a l a c t i v i t y o f s e x pheromone a n a l o g s and the i n f l u e n c e o f p h e r o m o n e - l i k e compounds on temperature-dependent phase t r a n s i t i o n s o f s y n t h e t i c d i p a l m i t o y l l e c i t h i n m i c e l l e s . The model d e p i c t s the r e c e p t o r as b e i n g composed o f 4 o r more p r o t e i n s u b u n i t s s u r r o u n d e d by a c r y s t a l l i n e l i p i d m a t r i x and l o c a t e d on the d e n d r i t e s o f the s e n s o r y c e l l s . A c c o r d i n g to t h e i r

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C oate 16

Δ-11-desaturase 2H

Z-11-C oate 16

-

β-oxidation 2C

Z-9-C oate 14

2C

r

—

Z-7-C oate 12

1. reductase "2. acetylase

Z-7-C oac 12

F i g u r e 3. P r o b a b l e b i o s y n t h e t i component o f t h e cabbage l o o p e r moiety o f t r i a c y l g l y c e r o l s .

(Trichoplusla ni) involving acyl

F i g u r e 4. S c a n n i n g e l e c t r o n p h o t o m i c r o g r a p h o f a segment o f the male antenna o f t h e t o b a c c o budworm moth ( H e l i o t h i s virescens). The h a i r - l i k e p r o j e c t i o n s on the a n t e n n a a r e sensory s e n s i l i a r e s p o n s i b l e f o r d e t e c t i o n o f female s e x pheromone. P h o t o m i c r o g r a p h by M i c h a e l B l a c k b u r n , Entomology Dept., U o f MD, C o l l e g e P a r k .

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s p e c u l a t i o n s , pheromone m o l e c u l e s were thought t o impinge on the s e n s i l l a , d i f f u s e t h r o u g h t h e p o r e s o f t h e s e n s i l l a , and then i n t e r a c t w i t h the dendrite-based r e c e p t o r , causing the opening o f i o n c h a n n e l s v i a a c o n f o r m a t i o n a l change i n t h e r e c e p t o r p r o t e i n - l i p i d matrix. T h e i r model d i d n o t i n c l u d e any c o n s i d e r a t i o n o f how t h e r e c e p t o r m i g h t be c l e a r e d o f t h e pheromone and the i o n g a t e r e t u r n e d t o i t s o r i g i n a l c l o s e d c o n f o r m a t i o n . On the o t h e r hand, V o g t a n d R i d d i f o r d (33) have f o c u s e d t h e i r a t t e n t i o n upon p r o t e i n s o f t h e s e n s i l l a and s p e c i f i c a l l y on a p r o t e i n t h a t b i n d s pheromone and a n e s t e r a s e . They propose a m o l e c u l a r model i n w h i c h pheromone r e c e p t i o n may i n v o l v e a dynamic k i n e t i c e q u i l i b r i u m i n w h i c h t h e pheromone ( a n e s t e r ) c a n f o l l o w 3 p o s s i b l e pathways i n t h e r e c e p t i o n p r o c e s s : s e n s i l i u m pore t o b i n d i n g p r o t e i n to r e c e p t o r to the e s t e r a s e ; pore to b i n d i n g p r o t e i n t o e s t e r a s e ; and pore to e s t e r a s e . They s u g g e s t t h a t t h i s s h i f t a b l e - e q u i l i b r i a model system c a n e x p l a i n t h e b r o a d s t i m u l u s - r e s p o n s e range e x h i b i t e d by some s p e c i e s o f moths. The i d e a h e r e i s t h a t a s pheromon p r o p o r t i o n a l l y l a r g e r amount s e n s i l l u m a r e s h u n t e d d i r e c t l y t o t h e e s t e r a s e and t h e r e b y s a t u r a t i o n o f the r e c e p t o r i s prevented. A combination o f these h y p o t h e t i c a l models m i g h t b e g i n t o b e t t e r a p p r o x i m a t e t h e biochemical events i n v o l v e d i n the chemoreceptive process. There i s a n o b v i o u s need t o expand o u r knowledge o f t h e pheromone r e c e p t o r system. We c e r t a i n l y know much about t h e c h e m i c a l s t i m u l i t h a t the r e c e p t o r system i s d e s i g n e d t o r e c e i v e . I t i s time t o b e g i n t h e s e a r c h f o r s u b s t a n c e s t h a t might b i n d , a n t a g o n i z e , o r d e b i l i t a t e the r e c e p t o r . D i s c o v e r y o f s u c h s u b s t a n c e s would prove u s e f u l i n l o c a l i z a t i o n o f t h e r e c e p t o r s i t e and i n e l u c i d a t i o n o f t h e b i o l o g i c a l chemistry o f o l f a c t o r y perception. I t i s very possible that the chemistry o f substances t h a t w i l l antagonize the r e c e p t o r w i l l b e a r l i t t l e resemblance t o t h e c h e m i s t r y o f t h e n a t u r a l product i t i s designed to r e c e i v e . P r e c e d e n t s f o r t h i s i d e a may be found i n the neurosciences. As examples, o p i a t e s a r e n o t p e p t i d e s , but they b l o c k e n d o r p h i n r e c e p t o r s ( 3 4 ) ; and a v e r m e c t i n s , m a c r o c y c l i c l a c t o n e s d e r i v e d from a n a c t i n o m y c e t e ( S t r e p t o m y c e s a v e r m i t i l l i s ) , do n o t resemble t h e n e u r o t r a n s m i t t e r , γ-aminobutyric a c i d , b u t t h e y n e v e r t h e l e s s b l o c k r e c e p t o r s f o r t h a t compound ( 3 5 ) . I n t u i t i v e l y , one i s i n c l i n e d t o t h i n k t h a t many o f t h e t e c h n i q u e s a n d t e c h n o l o g i e s t h a t have been d e v e l o p e d and a p p l i e d i n t h e n e u r o s c i e n c e s ( 3 6 ) may have u t i l i t y i n p r o b i n g the n a t u r e o f t h e pheromonal o l f a c t o r y r e c e p t o r s y s t e m . A l t h o u g h the moth pheromones have l i t t l e s t r u c t u r a l s i m i l a r i t y t o t h e m o l e c u l a r messengers o f t h e n e u r o s c i e n c e s , a c e t y l c h o l i n e , e t c . , the machinery by w h i c h they a r e p e r c e i v e d may i n v o l v e v a r i a t i o n s on t h e n e r v o u s system r e c e p t o r theme r a t h e r than a n e n t i r e l y n o v e l mechanism. The momentum o f r e s e a r c h on the i d e n t i t y a n d f u n c t i o n a l s i g n i f i c a n c e o f t h e m o l e c u l a r messengers i n i n s e c t s (neurotransmit­ t e r s , pheromones, and neurohormones) and t h e i r r e c e p t o r s w i l l u n d o u b t e d l y b u i l d a n d , a s i t does, s o t o o w i l l t h e p r o b a b i l i t y t h a t n o v e l , e f f i c a c i o u s , and e n v i r o n m e n t a l l y sound methods o f p e s t - i n s e c t c o n t r o l w i l l emerge. F o r those o f us i n v o l v e d w i t h s t u d i e s o f i n s e c t s e x pheromones, t h e avenues f o r r e s e a r c h a r e c l e a r . We a r e

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no l o n g e r c o n t e n t t o a s k , "What I s t h e c h e m i s t r y o f t h e s e m o l e c u l a r messengers?" We must now a l s o a s k , "How a r e t h e y produced a n d how are they p e r c e i v e d ? " Acknowledgments I thank M. I n s c o e , J . N e a l , J . O l i v e r , A. R a i n a , R. Ridgway, M. Schwarz, a n d D. Warthen, f o r t h e i r c o n s t r u c t i v e comments a s I prepared t h i s paper.

Literature Cited 1. Fabre, J.-H. In "Souvenirs Entomologiques"; Librairie Ch. Delagrave; Paris, France, 1900; pp. 339-60. Translated by A. Teixeiro de Mattos, in "The Life of the Caterpillar"; Dodd, Mead, and Co.; New York, 1916; pp. 246-78. 2. Lintner, J. A. West N.Y. Hort. Soc. Proc. 1882, 27, 52-66. 3. Butenandt, Α.; Beckman Naturforsch. 1959, 4. Butenandt, Α.; Hecker, Ε.; Hopp, M.; Koch, W. Justus Liebigs Ann. Chem. 1962, 658, 39-64. 5. Truscheit, E.; Eiter, K. Ibid. 1962, 658, 65-90. 6. Berger, R. S. Ann. Entomol. Soc. Amer. 1966, 59, 767-71. 7. Sekul, Α. Α.; Sparks, A. N. J. Econ. Entomol. 1967, 60, 1270-2. 8. Roelofs, W. L.; Arn, H. Nature 1968, 219, 513. 9. Klun, J. Α.; Brindley, T. A. J. Econ. Entomol. 1970, 63, 779-80. 10. Klassen, W.; Ridgway, R. L.; Inscoe, M. In "Insect Suppression with Controlled Release Pheromone Systems"; Kydonieus, A. F.; Beroza; Μ., Eds. CRC Press, Inc. Boca Raton, Florida, 1982; Vol. 1, p. 13. 11. Baker, R.; Bradshaw, J. W. S. Aliphatic Rel. Nat. Prod. Chem. 1983, 3, 66-106. 12. Iwaki, S.; Marumo, S.; Saito, T.; Yamada, M.; Katagiri, K. J. Amer. Chem. Soc. 1974, 96, 7842-4. 13. Hill, A. S.; Kovaleν, B. G.; Nikolaeva, L. Ν.; Roelofs, W. L. J. Chem. Ecol. 1982, 8, 383-96. 14. Sugie, H.; Tamaki, Y.; Sato, R.; Kumakura, N. Appl. Ent. Zool. 1984, 19, 323-30. 15. Tamaki. Y.; Noguchi, H.; Horiike, M.; Hirano, C. Appl. Ent. Zool. 1984, 19, 245-51. 16. Leonhardt, Β. Α.; Neal, J. W.; Klun, J. Α.; Schwarz, M.; Plimmer, J. R. Science 1983, 219, 314-6. 17. Brand, J. M.; Young, J. C.; Silverstein, R. M. Prog. Chem. Org. Nat. Prod. 1979, 37, 1-190. 18. Henricks, C. A. Tetrahedron 1977, 33, 1845-89. 19. Klun, J. Α.; Leonhardt, Β. Α.; Schwarz, M.; Day, Α.; Raina, A. K. (In preparation.) 20. Raina, A. K.; Klun, J. Α.; Schwarz, M.; Day, Α.; Leonhardt, B. A. (In preparation.) 21. Silverstein, R. M. Pure and Appl. Chem. 1982, 54, 2479-88. 22. Plimmer, J. R.; Inscoe, M. N.; McGovern, T. P. Ann. Rev. Pharmacol. Toxicol. 1982, 22, 297-320.

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23. Cardé, R. T. In "Chemical Ecology of Insects"; Bell, W. J.; Cardé, R. T., Eds. Sinauer Associates, Inc. Sunderland, Massachusetts, 1984; Chap. 5. 24. Cardé, R. T.; Baker, T. C. Ibid; Chap. 13. 25. Bjostad, L. B.; Roelofs, W. L. Science 1983, 220, 1387-9. 26. Bjostad, L. B.; Roelofs, W. L. J. Chem. Ecol. 1984, 10, 681-91. 27. Raina, A. K.; Klun, J. A. Science 1984, 225, 531-3. 28. Zacharuk, R. Y. Ann. Rev. Entomol. 1980, 25, 27-47. 29. Cornford, M. E.; Rowley, W. Α.; Klun, J. A. Ann. Entomol. Soc. Amer. 1973, 66, 1079-88. 30. Mustaparta, H. In "Chemical Ecology of Insects"; Bell, W. J.; Cardé, R. T., Eds. Sinauer Associates, Inc. Sunderland, Massachusetts, 1984, Chap. 2. 31. O'Connell, R. J.; Grant, A. J.; Mayer, M. S.; Mankin, R. W. Science 1983, 220, 1408-10. 32. Boeckh, J. Proc. 15th Internat. Congress of Entomol., 1976, pp. 308-22. 33. Bestmann, H. J.; Vostrowsky Regulation"; Breipohl, W., Ed.; IRL Press Limited: Falconberg Court, London, 1982; Section 7, pp. 253-65. 34. Vogt, R. G.; Riddiford, L. M. XVII International Congress of Entomol., Abstract Volume, 1984, p. 465. 35. Krassner, M. B. Chem. Eng. News 1983, 61 (35), 22-33. 36. Campbell, W.C.;Fisher, M. H.; Stapley, E. O.; Albers-Schönberg, G.; Jacob, T. A. Science 1983, 221, 823-8. 37. In "Neurosciences"; Abelson, P. H.; Butz, E., Eds. Science 1984, 225, 1253-70. RECEIVED November 8, 1984

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28 Alkaloidal Ant Venoms: Chemistry and Biological Activities M U R R A Y S.

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Department of Entomology, University of Georgia, Athens, GA 30602

By producin chemical compounds which are employed as defensive agents, ants have prospered in the insect world. The alkaloidal ant venoms are biologically active natural products that produce a range of responses in insect ecosystems. Some possess bacterial and fungicidal properties; others are i n s e c t i c i d a l , or repellents, or dermal necrotoxins, and have diverse pharmacological a c t i v i t i e s . This report discusses the nitrogen heterocycles produced by ants in order of structural complexity (pyrrolidines, pyrrolines, pyrroles, piperidines, piperideines, pyridines, pyrrolizidines, indolizidines, pyrazines, and indoles), and allocates the corresponding biological properties. Solomon's e x h o r t a t i o n , "Get thee to the a n t s " , i s p r o b a b l y e v e n m o r e a p p r o p r i a t e t o d a y t h a n i t was a t t h e t i m e t h a t he u t t e r e d i t . A n t s a r e an e m i n e n t l y s u c c e s s f u l g r o u p o f i n s e c t s , and undoubtedly c o n s i t u t e t h e m a j o r g r o u p o f p r e d a t o r y a n i m a l s on e a r t h , a development o f t e n a t t r i b u t e d to the en masse a t t a c k s so c h a r a c t e r i s t i c o f t h e s e social arthropods. But t h e i r f o r m i d a b i l i t y as a d v e r s a r i e s is also correlated with their chemical arsenals, w h i c h , when u n l e a s h e d at their omnipresent a n t a g o n i s t s , may o f t e n p r o d u c e s e v e r e b i o c h e m i c a l

0097-6156/ 85/0276-0393$06.00/0 © 1985 American Chemical Society

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lesions. In p a r t i c u l a r , the venoms o f a n t s a r e f o r t i f i e d w i t h a p o t p o u r r i o f compounds t h a t o f t e n c o n s t i t u t e n o v e l d e t e r r e n t s , and the e v o l u t i o n o f t h e s e p o i s o n g l a n d p r o d u c t s must be r e g a r d e d as a m a j o r f a c t o r i n the g r e a t s u c c e s s o f t h e s e s o c i a l Lilliputians. P e r h a p s Solomon f o r e s a w t h a t man, l o c k e d i n h i s unending b a t t l e with pest i n s e c t s t h a t have f r e q u e n t l y d e v e l o p e d r e s i s t a n c e t o man-made i n s e c t i c i d e s , would e v e n t u a l l y need the p o w e r f u l t o x i c a n t s p r e s e n t i n t h e venoms o f a n t s f o r h i s own use. I n d e e d , the s u c c e s s f u l u t i l i z a t i o n o f t h e s e natural insecticides for countless millenia d e m o n s t r a t e s t h a t t h e y have s t o o d the t e s t o f t i m e as b i o c i d e s par e x c e l l e n c e . In the p r e s e n t r e v i e w , an e f f o r t has been made t o c h a r a c t e r i z e t h e m a j o r compounds t h a t have b e e n r e c e n t l y i d e n t i f i e d i n the venoms o f a n t s s p e c i f i c a l l y the a l k a l o i d s extraordinarily divers t h e s e venom p r o d u c t s a r e d e s c r i b e d . Hopefully, this b r i e f e x p o s i t i o n w i l l l e a v e l i t t l e doubt t h a t a n t s ' b i o s y n t h e t i c v i r t u o s i t y has p r o v i d e d them w i t h c h e m i c a l weaponry n o t o n l y f o r p r é d a t i o n , b u t f o r use i n b l u n t i n g t h e a t t a c k s o f t h e h o s t i l e o r g a n i s m s w i t h which t h e y s h a r e t h e i r w o r l d . Alkalodial

Chemistry

o f Ant

Venoms

F o r more t h a n 300 y e a r s (_1) a n t venoms were synonymized with f o r m i c a c i d , a well-known cytotoxin. However, t h i s p o i s o n - g l a n d p r o d u c t i s now known t o be l i m i t e d to s p e c i e s i n t h e most s p e c i a l i z e d o f the ant s u b f a m i l i e s — the F o r m i c i n a e — t h e members o f w h i c h do n o t p o s s e s s f u n c t i o n a l stings. In c o n t r a s t , the venoms o f s t i n g i n g a n t s have g e n e r a l l y been d e m o n s t r a t e d t o be a l k a l i n e , o f t e n c o n s i s t i n g o f complex m i x t u r e s o f enzymes, t o x i c p r o t e i n s , and b i o g e n i c amines (2, 3), i n common w i t h p o i s o n g l a n d p r o d u c t s o f b e e s and wasps. However, t h e venoms o f some a n t s a r e d o m i n a t e d by a d i v e r s i t y o f a l k a l o i d s , compounds whose known d i s t r i b u t i o n i s l i m i t e d to s p e c i e s i n a few g e n e r a . These n o v e l n i t r o g e n h e t e r o c y c l e s a r e now known t o p o s s e s s a wide r a n g e o f b i o l o g i c a l a c t i v i t i e s , endowing t h e i r p r o d u c e r s w i t h t r u l y f o r m i d a b l e defensive c a p a b i l i t i e s . A l k a l o i d s have o n l y been i d e n t i f i e d i n the venoms o f ant s p e c i e s i n the M y r m i c i n a e , the l a r g e s t of the f o r m i c i d s u b f a m i l i e s . T h i s d i v e r s e s u b f a m i l y i n c l u d e s f i r e a n t s and h a r v e s t e r a n t s , t h e s p e c i e s o f which p r o d u c e h i g h l y a l g o g e n i c venoms. In a d d i t i o n , t h i s ant t a x o n i n c l u d e s t h i e f a n t s and

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P h a r a o h ' s a n t , s p e c i e s w h i c h , w h i l e t h e y do n o t s t i n g , a r e known t o u t i l i z e t h e i r venoms i n o f f e n s i v e c o n t e x t s ^4). A l t h o u g h t h e venoms o f some m y r m i c i n e s p e c i e s may c o n t a i n up t o 95% a l k a l o i d s , m i n o r amounts o f enzymes, c h a r a c t e r i s t i c p r o d u c t s o f a n i m a l venoms, a r e a l s o p r e s e n t and a p p e a r t o c o n s t i t u t e the a l l e r g e n s a s s o c i a t e d with these venoms {5). I n d e e d , even t h e f o r m i c a c i d - r i c h venoms o f f o r m i c i n e a n t s a r e f o r t i f i e d w i t h nitrogen-containing constituents (i.e., peptides), a probable b i o c h e m i c a l legacy o f the nitrogenous m e t a b o l i s m t h a t has been e m p h a s i z e d i n t h e hymenopterous p o i s o n g l a n d . In p a r t i c u l a r , t h e venom c h e m i s t r y o f m y r m i c i n e s p e c i e s i n t h e g e n e r a S o l e n o p s i s and Monomorium a p p e a r s t o have d i g r e s s e d from t h a t o f s p e c i e s i n other myrmicine genera b e i n g c h a r a c t e r i z e d by an emphasis on t h e s y n t h e s i h e t e r o c y c l e s (β) constituents. These a l k a l o i d s , which a r e r e s t r i c t e d i n t h e i r animal d i s t r i b u t i o n to ant s p e c i e s i n s e v e r a l g e n e r a , a r e r e p r e s e n t e d by b o t h mono- and b i c y c l i c compounds. They may p o s s e s s some chemotaxonomic v a l u e f o r t a x a i n t h e s e m y r m i c i n e g e n e r a {]_) . In t h e p r e s e n t r e p o r t , t h e s e n i t r o g e n heterocycles are organized according to t h e i r s t r u c t u r e s , t h e s a t u r a t e d m o n o c y c l i c compounds b e i n g p r e s e n t e d f i r s t , f o l l o w e d by the b i c y c l i c a l k a l o i d s , and f i n a l l y t h e m o n o c y c l i c compounds c o n t a i n i n g more t h a n one n i t r o g e n atom, i . e . , p y r a z i n e s and h e t e r o c y c l i c amines, the i n d o l e s . Pyrrolidines. More t h a n a d o z e n 2 , 5 ^ d i a l k y l p y r r o l i d i n e s have been i d e n t i f i e d i n t h e venoms o f S o l e n o p s i s and Monomor ium s p e c i e s {!_) . A l l o f t h e s e compounds a r e o f t h e t r a n s c o n f i g u r a t i o n . Whereas S o l e n o p s i s s p e c i e s may p r o d u c e o n l y one compound i n t h e i r p o i s o n g l a n d s , t h e venoms o f Monomorium s p e c i e s g e n e r a l l y c o n s i s t of mixtures of these a l k a l o i d s . The d i a l k y l p y r r o l i d i n e s (I) c o n t a i n u n b r a n c h e d s i d e c h a i n s i n w h i c h one a l k y l g r o u p i s even-numbered whereas t h e o t h e r g r o u p i s odd-numbered. These compounds have been i d e n t i f i e d i n t h e venoms o f a v a r i e t y o f S o l e n o p s i s s p e c i e s (S, 2r 10), a l l members o f t h e subgenus D i p l o r h o p t r u m . A v a r i e t y o f Monomorium s p e c i e s i n t h e subgenus Monomor ium p r o d u c e t h e s e a l k a l o i d s (I) (j5, ]A, 12) , as does one s p e c i e s i n t h e subgenus Xeromyrmex (6) · Monomorium s p e c i e s a l s o p r o d u c e dialkylidenepyrrolidines containing a terminally

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u n s a t u r a t e d d o u b l e bond i n one s i d e c h a i n ( I I ) (£, 11, 12^) or t e r m i n a l l y u n s a t u r a t e d d o u b l e bonds i n b o t h s i d e c h a i n s ( I I I ) (12). These a r e i n a d m i x t u r e with p y r r o l i d i n e s i n which both s i d e c h a i n s a r e s a t u r a t e d (I) (6, 11, _12) . S e v e r a l N - m e t h y l p y r r o l i d i n e s have been i d e n t i f i e d i n t h e venoms o f Monomorium (Monomorium) species. M. l a t i n o d e , an O l d World s p e c i e s , i s d i s t i n c t i v e i n p r o d u c i n g an a l k a l o i d a l - r i c h venom c o n t a i n i n g two N - m e t h y l p y r r o l i d i n e s w i t h s a t u r a t e d s i d e c h a i n s (IV) (61). On t h e o t h e r hand, t h e p o i s o n gland s e c r e t i o n s o f s e v e r a l North American s p e c i e s c o n t a i n N - m e t h y l a t e d compounds i n w h i c h one (V) or b o t h s i d e c h a i n s (VI) a r e t e r m i n a l l y u n s a t u r a t e d {β); t h e y a r e accompanied by t h e c o r r e s p o n d i n g norpyrrolidines. Pyrrolines. Both p o s s i b l e isomers of the 2 , 5 - d i a l k y l 1- p y r r o l i n e s have been i d e n t i f i e d i n t h e venoms o f S o l e n o p s i s and Monomorium s p e c i e s . These compounds g e n e r a l l y accompany t h e c o r r e s p o n d i n g d i a l k y l p y r r o l i d i n e s as venom c o n s t i t u e n t s . The venom o f S. p u n c t a t i c e p s , an A f r i c a n s p e c i e s , c o n t a i n s b o t h S - and S -pyrrolines ( V I I ) (J3) as do the venoms o f t h r e e N o r t h A m e r i c a n Monomorium s p e c i e s {12) . The l a t t e r d i a l k y l p y r r o l i n e s ( V I I I ) a r e d i s t i n g u i s h e d by t h e p r e s e n c e o f two t e r m i n a l l y unsaturated side chains. The p y r r o l i n e s i n t h e venom o f M. l a t i n o d e a r e d i s t i n c t i v e i n c o n s t i t u t i n g the o n l y 5 - p y r r o l i n e s (IX) i n a n t venoms t h a t a r e not accompanied by t h e 1 - p y r r o l i n e s {6). 1

5

Pyrrole. A t r a c e c o n s t i t u e n t i n t h e venoms o f t h e m y r m i c i n e s A t t a t e x a n a , A. c e p h a l o t e s , A. s e x d e n s , and Acromyrmex o c t o s p i n o s u s i s m e t h y l 4-methylpyrrole2- c a r b o x y l a t e (X) (1J3, Ij4, 1!5) . T h i s s i m p l e p y r r o l e e s t e r i s a t r a i l pheromone f o r some o f t h e s e s p e c i e s (13, 14) . Piperidines. 2 - A l k y l - 6 - m e t h y l p i p e r i d i n e s have o n l y been d e t e c t e d i n t h e venoms o f S o l e n o p s i s w o r k e r s and t h e i r queens (16, 17, 1 8 ) . These compounds, w h i c h a r e sometimes r e f e r r e d t o as s o l e n o p s i n s , a r e c o n s i s t e n t p o i s o n g l a n d p r o d u c t s o f S o l e n o p s i s s p e c i e s i n t h e subgenus S o l e n o p s i s , t h e f i r e a n t s (JL7^ 1_8) . In a d d i t i o n , some s p e c i e s i n t h e subgenus D i p l o r h o p t r u m (thief

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

28.

BLUM

397

Alkaloidal Ant Venoms

C H

C H

3
r^^ H

m= 1

η= 6

m= 3

η = 4, 6

m= 5

η=4

Η η=4,6, 8

H.C=HC(CHJ 2

Ν j

2 7

(CH ) CH = C H ^4 2

2

Η 11

CH (CH )^V^(CH ) CH 2

3

2

CH

3

3

3

η = 4, 6

C H ( C H ) ^ ^ ^ ( C H ) C H = CH 3

2

2

CH

4

2

3

31

H C=HC(CH )^\^ 2

2

CH

3

3ΖΓ

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST CONTROL

398

CH CH^%j/^(CH ) CH 3

2

n

3

CH3CH^Sj^(CH ) CH 2

n

3

η = 4, 6 2ΊΓ

H C= 2

H C = H C ( C H ) ^ N ^ "(CH ) CH = C H 2

2

2

4

2

T7TTT

C H

3) , t h a t o f S. xenovenenum i s d i s t i n c t i v e i n c o n t a i n i n g a p y r r o l i z i d i n e as t h e sole alkaloid. -^H and C NMR s p e c t r o s c o p y show t h a t t h i s compoun i s t h e (5Z^8E) isome p y r r o l i z i d i n e (XVII) ( 2 3 ) . 1 3

Indolizidines. I n d o l i z i d i n e s have been d e t e c t e d as p o i s o n g l a n d p r o d u c t s o f b o t h Monomorium and S o l e n o p s i s s p e c i e s . Monomorine-1, 3 - b u t y 1 - 5 - m e t h y l o c t a h y d r ο i n d o l i z i d i n e ( X V I I I ) , i s a major c o n s t i t u e n t i n t h e venom o f M. p h a r a o n i s and i s a c c o m p a n i e d by i t s c o n g e n e r , 3 - ( 3 - h e x e n - l - y l ) - 5 - m e t h y l i n d o l i z i d i n e (XIX) (11). Monomorine-1, w h i c h i s p r o d u c e d on t h e p o i s o n g l a n d as t h e a l l c i s - i s o m e r , i s accompanied by p y r r o l i d i n e s , c h a r a c t e r i s t i c Monomorium a l k a l o i d s (12). On t h e o t h e r h a n d , 3 - e t h y l - 5 m e t h y l i n d o l i z i d i n e (XX), a major c o n s t i t u e n t i n the venom o f £>. c o n j u r a t a , i s a c o n c o m i t a n t o f 2 , 6 - d i a l k y l p i p e r i d i n e s ( 2 4 ) . Queens o f an u n i d e n t i f i e d Costa Rican S o l e n o p s i s (Diplorhoptrum) s p e c i e s produce a s i n g l e poison gland product, 3 - h e x y l - 5 - m e t h y l i n d o l i z i d i n e (XXI) (^4). Pyrazines. A l k y l p y r a z i n e s , w h i c h a r e commonly p r o d u c e d i n t h e m a n d i b u l a r g l a n d s o f a n t s and wasps (7^) , appear t o have a l i m i t e d d i s t r i b u t i o n i n t h e venoms o f a n t s . 2,5-Dimethyl-3-ethylpyrazine (XXII) has been i d e n t i f i e d as a t r a c e c o n s t i t u e n t i n t h e venom o f A t t a sexdens ( 1 5 ) , a myrmicine s p e c i e s t h a t a l s o p r o d u c e s a p y r r o l e (X) as a p o i s o n g l a n d product.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BLUM

Alkaloidal Ant Venoms

CH ^N--(CH ) CH 3

2

N

3

H η =8, 10, 12,14 XI

CH/Ç^(CH ) CH 2

CH

N

3

3

21

C H ^ N ^ ( C H ) C = C (CH ) CH 2

n

2

7

3

H η =3, 5 , 7

CH ^T^(CH ) 3

2

α

CH

1 0

3

*N^"(CH ) CH = CH 2

3

2

23?

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST CONTROL

402

Indoles. S k a t o l e , 3 - m e t h y l i n d o l e ( X X I I I ) , i s a major c o n s t i t u e n t i n the h y p e r t r o p h i é e p o i s o n g l a n d o f s o l d i e r s o f the m y r m i c i n e P h e i d o l e f a l l a x (25) and o f o t h e r P h e i d o l e s p e c i e s as w e l l ( 2 4 ) . Biological

Activities

of

Alkaloids

A wide v a r i e t y o f a c t i v i t i e s have been d e m o n s t r a t e d f o r the a l k a l o i d s i d e n t i f i e d i n myrmecine ant venoms, i n d i c a t i n g t h a t t h e s e s m a l l nitrogen h e t e r o c y c l e s have been a d a p t e d to s u b s e r v e m u l t i p l e functions. B o t h the p i p e r i d i n e s and p y r r o l i d i n e s possess d i v e r s e p h a r m a c o l o g i c a l a c t i v i t i e s (reviewed i n ]_) , and i t seems l i k e l y t h a t t h e i r r o l e s i n r e g u l a t i n g both i n t r a i n t e r a c t i o n s are ver Toxicology. S t u d i e s on the modes o f a c t i o n o f d i a l k y l p i p e r i d i n e s have shown t h a t t h e s e compounds c a u s e d i v e r s e biochemical lesions. These a l k a l o i d s w h i c h a r e p o w e r f u l h e m o l y s i n s (^6) and d e r m a l n e c r o t o x i n s (27, 28) , a l s o r e l e a s e h i s t a m i n e from mast c e l l s (29) , t h u s f u n c t i o n i n g as e f f e c t i v e a l g o g e n s . In a d d i t i o n , t h e s e f i r e ant p r o d u c t s i n h i b i t N a and K A T P a s e s (30) and a t low c o n c e n t r a t i o n s uncouple oxidative phosphorylation l e a d i n g to reduced mitochondrial respiration (31). 2,6-Disubstituted p i p e r i d i n e s a l s o i n t e r f e r e w T t h the c o u p l i n g between t h e i o n c h a n n e l and the r e c o g n i t i o n s i t e o f the vertebrate n i c o t i n i c a c e t y l c h o l i n e receptor ( 3 2 ) . Recent i n v e s t i g a t i o n s demonstrate t h a t these compounds i n t e r a c t w i t h the c l o s e d a c e t y l c h o l i n e r e c e p t o r / i o n c h a n n e l complex a t a s i t e w h i c h i s s e p a r a t e from the b i n d i n g s i t e o f p r e v i o u s l y known b l o c k i n g a g e n t s (33_) . These r e s u l t s e m p h a s i z e the b r o a d s p e c t r u m a c t i v i t i e s of 2 - a l k y l - 6 - m e t h y l p i p e r i d i n e s which o f f e r n o v e l p r o b e s f o r i d e n t i f y i n g and e x p o s i n g t a r g e t s i t e s f o r new c l a s s e s o f i n s e c t n e u r o t o x i n s +

(33) .

Insecticidal

Activities.

F i r e ants r a p i d l y immobilize s t i n g i n g , demonstrating that

i n s e c t p r e y by the venom i s s t r o n g l y

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

+

28.

BLUM

403

Alkaloidal Ant Venoms

H XXIII

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

404

BIOREGULATORS FOR PEST CONTROL insecticidal. T r o p i c a l a p p l i c a t i o n s o f n e a t venom to f l i e s and b e e t l e s r e s u l t s i n h i g h m o r t a l i t y o f the t a r g e t i n s e c t s , which e x h i b i t p a r a l y t i c symptoms s h o r t l y after treatment (34). On t h e o t h e r h a n d , s y n t h e t i c c i s and c i s - t r a n s m i x t u r e s o f 2-undecyl-6-methylpiperidine or 2 - t r i d e c y l 6-methylpiperidine were n o n t o x i c to a d u l t s o f t h e American cockroach, P e r i p l a n e t a americana (33). W o r k e r s o f Monomorium minimum r a p i d l y kill termite workers ( R e t i c u l i t e r m e s species) with their p o i s o n g l a n d s e c r e t i o n s (_35) · The venom o f t h i s species i s primarily f o r t i f i e d with 2,4-dialkylp y r r o l i d i n e s (1^) w h i c h must f u n c t i o n as c o n t a c t i n s e c t i c i d e s s i n c e t h e venom i s a p p l i e d t o p i c a l l y r a t h e r than being subdermally a d m i n i s t e r e d . The same r e s u l t s h a v e b e e n r e p o r t e d f o r E u r o p e a n s p e c i e s o f M o n o m o r i u m a t t a c k i n g t e r m i t e s (36) Repellent

(Deterrent

The d i s u b s t i t u t e d p y r r o l i d i n e s and p i p e r i d i n e s h a v e been d e m o n s t r a t e d t o be e x c e l l e n t r e p e l l e n t s f o r ants under f i e l d c o n d i t i o n s . W o r k e r s o f Monomorium pharaonis e f f e c t i v e l y repel other species of ants w i t h venom d r o p l e t s w h i c h a c c u m u l a t e on t h e t i p o f the s t i n g (37), a s t r a t e g y t h a t i s used w i t h equal s u c c e s s by w o r k e r s o f N o r t h A m e r i c a n Monomorium s p e c i e s (38) a n d t h e E u r o p e a n t h i e f a n t , S o l e n o p s i s fugax (37). The venom o f t h e l a t t e r species contains a single alkaloid, trans-2-butyl5 - h e p t y l p y r r o l i d i n e ( I , m=3, n=6) (JJ3) , a n d t h e s y n t h e t i c compound i s a s a c t i v e a s t h e n e a t venom i n r e p e l l i n g other ant species after being applied to food or ant l a r v a e . Some o f t h e d i a l k y l p i p e r i d i n e s a n d d i a l k y l i n d o l i z i d i n e s are outstanding ant r e p e l l e n t s . A l a r g e s e r i e s o f t h e s e compounds were r e c e n t l y e v a l u a t e d as f e e d i n g r e p e l l e n t s f o r f o u r s p e c i e s o f a n t s t h a t a r e c o n s i d e r e d t o be m a j o r p e s t s p e c i e s because of their aggressive h a b i t s . Hungry workers of the f i r e ant S o l e n o p s i s i n v i c t a , t h e A r g e n t i n e ant, Iridomyrmex h u m i l i s , Tapinoma melanocephalium, and P h a r a o h ' s a n t , Monomorium p h a r a o n i s , w e r e r e p e l l e d by t r a c e amounts o f s e l e c t e d p i p e r i d i n e s and i n d o l i z i d i n e s added t o d r o p l e t s o f s u c r o s e solution (35). These r e s u l t s c l e a r l y document the a b i l i t i e s o f t h e a n t - d e r i v e d a l k a l o i d s , a_t n a t u r a l c o n c e n t r a t i o n s , to r e p e l f e e d i n g by ant workers a t very a c c e p t a b l e food sources, notwithstanding their prolonged food d e p r i v a t i o n .

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BLUM

Alkaloidal Ant Venoms

Antimicrobial

Activities.

More than 25 y e a r s ago i t was d e m o n s t r a t e d t h a t f i r e a n t venom (S. i n v i c t a = s a e v i s s i m a ) possessed b a c t e r i c i d a l and f u n g i c i d a l a c t i v i t i e s {34). S u b s e q u e n t l y , the s y n t h e t i c d i a l k y l p i p e r i d i n e s were shown to p o s s e s s p r o n o u n c e d a b i l i t i e s to i n h i b i t the g r o w t h o f a v a r i e t y o f b a c t e r i a a t low c o n c e n t r a t i o n s {38). D e t a i l e d s t u d i e s on the f u n g i c i d a l a c t i v i t i e s of these a l k a l o i d s v i s - a - v i s c o m m e r c i a l f u n g i c i d e s i n d i c a t e d t h a t t h e y were eminently capable of m a n i f e s t i n g pronounced f u n g i s t a t i c and/or f u n g i c i d a l a c t i v i t y a g a i n s t a wide v a r i e t y of f u n g i (40^) . B o t h c i s and t r a n s - i s o m e r s o f a l l the a l k a l o i d s i d e n t i f i e d i n the venom of the f i r e ant S. i n v i c t a (16) were e v a l u a t e d a g a i n s t f u n g i t h a t c o n s t i t u t e d human and p l a n t p a t h o g e n s were i s o l a t e d fro In g e n e r a l , the s t e r e o i s o m e r s were o f e q u a l a c t i v i t y o v e r a wide r a n g e o f c o n c e n t r a t i o n s and r i v a l e d c o m m e r c i a l f u n g i c i d e s ( e . g . , J ^ - u n d e c e n o i c a c i d ) as f u n g a l growth i n h i b i t o r s . There i s a tendency f o r t h e a l k a l o i d s w i t h 6 - a l k y l i d i n e s i d e c h a i n s to e x h i b i t g r e a t e r f u n g i t o x i c i t y than t h e i r s a t u r a t e d counterparts (40). Conclusions A l t h o u g h r e l a t i v e l y few m y r m i c i n e s p e c i e s have been s u b j e c t e d t o a n a l y t i c a l s c r u t i n y , t h e members o f t h i s subfamily c l e a r l y demonstrate great v i r t u o s i t y i n the b i o s y n t h e s i s o f a l k a l o i d s {]_) . S p e c i e s o f Monomorium a r e p a r t i c u l a r l y abundant i n the O l d W o r l d (4JL) , and a n a l y s e s o f a few members o f t h i s genus {6, _11) i n d i c a t e t h a t g r e a t a l k a l o i d a l d i v e r s i t y may c h a r a c t e r i z e t h e i r p o i s o n g l a n d secretions. The same i s t r u e f o r the genus S o l e n o p s i s {&, 16). Furthermore, a l k a l o i d a l s u r p r i s e s w i l l p r o b a b l y be f o r t h c o m i n g when t h e venoms o f s p e c i e s i n a d d i t i o n a l m y r m i c i n e g e n e r a a r e examined. I n d e e d , a new p y r r o l i z i d i n e has been c h a r a c t e r i z e d i n a s p e c i e s i n the genus C h e l a n e r ( 2 4 ) , an O l d World t a x o n c l o s e l y r e l a t e d to Monomorium (41) . In s h o r t , t h e r e a r e s t r o n g g r o u n d s for concluding that myrmicine s p e c i e s , with t h e i r g r e a t e c o l o g i c a l d i v e r s i t y , w i l l c o n t i n u e to be an e x c e l l e n t source of i n t e r e s t i n g n i t r o g e n h e t e r o c y c l e s f o r some t i m e to come. In a d d i t i o n , i n t e r m s o f a l k a l o i d s , a n a l y s e s o f ant s p e c i e s i n n o n m y r m i c i n e s u b f a m i l i e s c o u l d a l s o p r o v e to be v e r y fruitful.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

405

BIOREGULATORS FOR PEST CONTROL

A v a r i e t y of a l k a l o i d a l c o n s t i t u e n t s , not produced in poison (venom) g l a n d s , h a v e b e e n i d e n t i f i e d as n a t u r a l p r o d u c t s o f ant s p e c i e s in four s u b f a m i l i e s , i n c l u d i n g the Myrmicinae (see r e v i e w i n 1) . T h i s d e m o n s t r a t e s t h a t the biogenesis of nitrogen heterocycles occurs widely in the F o r m i c i d a e and stresses that species in a l l s u b f a m i l i e s s h o u l d be r e g a r d e d as p o s s i b l e a l k a l o i d producers. S i g n i f i c a n t l y , novel nitrogen-containing c o m p o u n d s ( e . g . , a l i p h a t i c a m i n e s and a m i d e s ) h a v e r e c e n t l y b e e n i d e n t i f i e d as venom p r o d u c t s o f two species i n the s u b f a m i l y P o n e r i n a (42) , e s t a b l i s h i n g t h a t the p o n e r i n e p o i s o n gland can synthesize non-proteinaceous nitrogenous constituents. Thus, the p o i s o n g l a n d s of nonmyrmicine ants can be r e g a r d e d as p o s s i b l e s o u r c e s o f n i t r o g e n o u s compounds. Ants have prospere h a v e had to overcom i n c l u d e p a t h o g e n s as w e l l as i n v e r t e b r a t e and vertebrate predators. F o r many a n t s p e c i e s , i t appears t h a t t h e i r a b i l i t y to s u r v i v e i n such a m a n i f e s t l y h o s t i l e w o r l d i s i n no s m a l l way due to the a l k a l o i d a l a r s e n a l s s t o r e d i n t h e i r poison gland reservoirs. These compounds have been d e m o n s t r a t e d to possess a wide spectrum of t o x i c o l o g i c a l properties (J7) which include pronounced neurotoxic, a n t i m i c r o b i a l , and insecticidal activities. Living in moist s u b t e r r a n e a n environments, ants have been able to prevent omnipresent pathogenic b a c t e r i a and f u n g i f r o m o v e r w h e l m i n g t h e m , and a l k a l o i d s have played a major a n t i b i o t i c r o l e in t h i s context (39, 40) . The d e m o n s t r a t e d i n s e c t i c i d a l p o t e n c i e s of a l k a l o i d a l v e n o m s (34^, 35) f u r t h e r documents the b i o c i d a l p r o p e r t i e s o f t h e s e c o m p o u n d s and augurs w e l l f o r t h e i r f u t u r e c o n s i d e r a t i o n as v i a b l e insecticides. Indeed, mankind can i l l a f f o r d to ignore t h i s wondrous c r o p of n a t u r a l f u n g i c i d e s and i n s e c t i c i d e s that are a v a i l a b l e for h a r v e s t i n g . It i s time to g i v e Solomon h i s due.

Literature Cited 1. Wray, J. Phil. Trans. Roy. Soc. London 1670, 5, 2063. 2. Cavill, G. W. K.; Robertson, P. L.; Whitfield, F. B. Science 1964, 146, 79. 3. Schmidt, J. O.; Blum, M. S. Science 1978, 200, 1064. 4. Holldobler, K. Biol. Zbl. 1928, 48, 129.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

28. BLUM 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Alkaloidal Ant Venoms Baer, H.; Liu, T. Y.; Anderson, M. C.; Blum, M. S.; Schmid, W. H., James, F. J. Toxicon 1979, 17, 397. Jones, T. H.; Blum, M. S.; Fales, H. M. Tetrahedron 1982, 38, 1949. Jones, T. H.; Blum, M. S. In "Alkaloids: Chemical and Biological Perspectives"; Pelletier, S. W.; John Wiley and Sons, Inc., 1983; Vol. I, pp. 33-84. Pedder, D. J.; Fales, H. M.; Jaouni, T.; Blum, M. S.; MacConnell, J.; Crewe, R. M. Tetrahedron 1976, 32, 2275. Jones, T. H.; Blum, M. S.; Fales, H. M. Tetrahedron Letters 1979, 1031. Blum, M. S.; Jones, T. H.; Holldobler, B.; Fales, H. M.; Jaouni, T. Naturwissenschaften 1980, 67, 144 Ritter, F. J.; Verkuil, E. Pheromones and Defensive Secretions in Social Insects, 1975, pp. 99-103. Jones, T. H.; Blum, M. S.; Howard, R. W.; McDaniel, C. A.; Fales, H. M.; DuBois, M. B.; Torres, J. J . Chem. Ecol. 1982, 8. Tumlinson, J. H.; Silverstein, R. M.; Moser, J. C.; Brownlee, R. G.; Ruth, J. M. Nature 1971, 234, 348. Riley, R. G.; Silverstein, R. M.; Carroll, B; Carroll, R. J . Insect Physiol. 1974, 20, 651. Cross, J. H.; Byler, R. C.; David, U.; Silverstein, R. M.; Robinson, S. W.; Baker, P. M.; de Olveira, J. S.; Jutsum, A. R.; Cherett, J. M. J . Chem. Ecol. 1979, 5, 187. MacConnell, J. G.; Blum, M. S.; Fales, H. M. Tetrahedron 1971, 26, 1129. Brand, J. M.; Blum, M. S.; Fales, H. M.; MacConnell, J. G. Toxicon 1972, 10, 259. MacConnell, J. G.; Blum, M. S.; Buren, W. F.; Williams, R. N.; Fales, H. M. Toxicon 1976, 14, 79. Brand, J. M.; Blum, M. S.; Ross, H. H. Insect Biochem. 1973, 3, 45. MacConnell, J. G.; Williams, R. N.; Brand, J. M.; Blum, M. S. Ann. Ent. Soc. Am. 1974, 67, 134. Blum, M. S.; Fales, H. M.; Leadbetter, G.; Bierl, B. A. unpublished results, 1984. Wheeler, J. W.; Olubajo, O.; Storm, C. B.; Duffield, R. M. Science 1981, 211, 1051. Jones, T. H.; Blum, M. S.; Fales, H. M.; Thompson, C. R. J . Org. Chem. 1980, 45, 4778.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

407

BIOREGULATORS FOR PEST CONTROL 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

Jones, T. H.; Blum, M. S. unpublished results, 1984. Law, J. H.; Wilson, E. O.; McCloskey, J. A. Science 1965, 149, 544. Adrouny, G. A.; Derbes, V. J . ; Jung, R. C. Science 1959, 130, 449. Caro, M. R.; Derbes, V. J . ; Jung, R. Arch. Derm. 1957, 75, 475. Bufkin, D. C.; Russell, F. E. Toxicon 1972, 10, 526. Read, G. W.; Lind, Ν· K.; Oda, C. S. Toxicon 1978, 16, 361. Koch, R. B.; Desiah, D.; Foster, D.; Ahmed, K. Biochem. Pharm. 1977, 26, 983. Cheng, E. Y.; Cutkomp, L. K.; Koch, R. B. Biochem. Pharm. 1977, 26, 1179. Yeh, J. Z.; Narahashi, T.; Almon, R. R. J. Pharm. Exp David, J. A. Battersby, M.; Sattelle, D. B. J . Insect Physiol. 1984, 30, 191. Blum, M. S.; Walker, J. R.; Callahan, P. S.; Novak, A. F. Science 1958, 128, 306. Tomalski, M.; Blum, M. S.; Jones, T. H. Unpublished results, 1984. Clement, J. L. Unpublished results, 1984. Holldobler, B. Oecologica 1973, 11, 371. Urbani, C. B.; Kannowski, P. B. Environ. Ent. 1974, 3, 755. Jouvanez, D. P.; Blum, M. S.; MacConnell, J. G. Antimicrob. Ag. Chemother. 1972, 2, 291. Cole, L. K. Ph.D. Thesis, University of Georgia, 1973. Emery, C. Genera Insectorum 1922, 174, 166. Fales, H. M.; Blum, M. S.; Bian, Z.; Jones, T. H.; Don, A. W. J . Chem. Ecol. 1984, 10, 651.

RECEIVED December 14, 1984

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

29 Use of Natural Products and Their Analogues For Combating Pests of Agricultural and Public Health Importance in Africa D. L .

WHITEHEAD

International Centre of Insect Physiology and Ecology (I.C.I.P.E.), P.O. Box 30772, Nairohi, Kenya

Ectoparasites such as ticks mites and haematophagous flies together wit "managed" by us tones) or hormone analogues and by immunization of the hosts with antigens derived from the vectors or parasites themselves. Recently this combined approach has been used most effectively against helminth infestations as well as against ticks and flies. Use of components of host odors and pheromones to trap, sterilize or k i l l tsetse flies is described as well as inexpensive methods of crop and livestock protection, such as oils and plant extracts, used traditionally in less developed countries. T h e r e a r e u r g e n t economic reasons f o r t r y i n g t o c o n t r o l prédation i n A f r i c a by b i t i n g , s u c k i n g f l i e s and a c a r i n e s ( t i c k s and m i t e s ) . Among them a r e i d e n t i f i e d t h e v e c t o r s o f mammalian p a r a s i t i c and v i r u s d i s e a s e s such as t r y p a n o s o m i a s i s ( s l e e p i n g s i c k n e s s ) , f i l a r i a s i s s u c h as o n c h o c e r c i a s i s ( r i v e r b l i n d n e s s ) , l e i s h m a n i a s i s , m a l a r i a , Dengue f e v e r and E a s t Coast f e v e r ( T h e i l e r i o s i s ) ( E C F ) . One d i s e a s e a l o n e , caused by trypanosomes t r a n s m i t t e d by t h e t s e t s e f l y ( G l o s s i n a s p p . ) , i s e s t i m a t e d (1) t o l i m i t r a n c h i n g and human s e t t l e m e n t i n over 4 m i l l i o n square m i l e s o f A f r i c a . E f f e c t i v e t r y p a n o c i d a l drugs have been a v a i l a b l e f o r 35 y e a r s , b u t because o f t h e i r expense, t o x i c i t y and now r e s i s t a n c e t o them, t h e o n l y s u r e way t o e l i m i n a t e the d i s e a s e c o m p l e t e l y i s c o n s i d e r e d t o be by v e c t o r e r a d i c a t i o n (_2). A more r e a l i s t i c approach adopted i n West A f r i c a (3) i s aimed a t i n t r o d u c i n g t r y p a n o t o l e r a n t b r e e d s o f l i v e s t o c k i n a r e a s where t h e c h a l l e n g e from i n f e c t e d f l i e s i s n o t unduly high. Control of reproduction R e s e a r c h c a r r i e d o u t i n t h i s l a b o r a t o r y and a t t h e T s e t s e R e s e a r c h L a b o r a t o r y n e a r B r i s t o l i n England has f o c u s e d on attempts t o

0097-6156/85/0276-0409$07.00/0 © 1985 American Chemical Society

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST CONTROL

410

d i s r u p t r e p r o d u c t i o n o r s t e r i l i z e t s e t s e . T h e i r s p e c i a l i z e d mode o f a a d e n o t r o p h i c l a r v i p a r o u s r e p r o d u c t i o n , whereby a s i n g l e l a r v a ( s t a g e I I I ) i s d e p o s i t e d e v e r y 8 - 1 0 d a y s , would seem t o o f f e r an avenue whereby p o p u l a t i o n s i z e c o u l d be e f f e c t i v e l y and s e l e c t i v e l y c o n t r o l l e d ( 4 ) . The q u a n t i t y and f r e q u e n c y o f b l o o d meals a v a i l a b l e t o pregnant females does, o f c o u r s e , c o n s t i t u t e a l i m i t on the s i z e and t h e r e f o r e s u c c e s s o f t h e next g e n e r a t i o n . I n 1971 i t was r e p o r t e d (5) t h a t some s u b s t a n c e ( s ) p r e s e n t i n the d i e t o f the h o s t r e s u l t e d i n reduced f e c u n d i t y when G l o s s i n a m o r s i t a n s m o r s i t a n s were f e d on t h e s e a n i m a l s . Subsequently Turner and M a r a s h i r e p o r t e d s i m i l a r problems w i t h (î. m. m o r s i t a n s f e d on r a b b i t s w h i c h had e a t e n contaminated f o o d 0 6 ) . The t o x i n ( s ) were not i d e n t i f i e d i n e i t h e r case, but t h e e f f e c t s o f c o c c i d i o s t a t s added t o the d i e t were n o t r e a l i z e d u n t i l J o r d a n and Trewern r e p o r t e d t h a t s u l p h a q u i n o x a l i n e (75 ppm) w i t h pyrimethamine (7.5 ppm) t o g e t h e r markedly lowered f e c u n d i t y o f G. a u s t e n i and G* m. m o r s i t a n s when f e d t o t h e r a b b i t h o s t s ( 7 ) . U s i n g hormones as I n s e c These o b s e r v a t i o n s prompted an i n v e s t i g a t i o n i n t o the use o f i n s e c t growth r e g u l a t o r s (IGR) and i n s e c t hormones - a p p l i e d t o p i c a l l y o r added t o t h e d i e t o f t s e t s e f e d i n v i t r o ( 8 ) ( i . e . t h r o u g h a s t e r i l e s i l i c o n e membrane) - t o d i s r u p t r e p r o d u c t i o n . Further impetus was g i v e n t o these s t u d i e s i n B r i s t o l when D e l i n g e r r e p o r t e d from ICIPE t h a t 5-10 ug o f 20-hydroxyecdysone (20-OHE) o r j u v e n i l e hormone (JH) analogue, when i n j e c t e d o r a p p l i e d t o p i c a l l y ( r e s p e c t i v e l y ) t o pregnant (5. m. m o r s i t a n s , caused o v e r h a l f t o a b o r t ( 9 ) . By comparison, f e e d i n g o f 20-OHE t o pregnant (î. m. mor­ s i t a n s was s u r p r i s i n g l y more e f f e c t i v e ( F i g u r e 1) than i n j e c t i n g t h e hormone ( 1 0 ) . T h i s c o u l d mean t h a t a m e t a b o l i t e o f t h e s t e r o l i s t h e a c t i v e a b o r t i f a c i e n t , e s p e c i a l l y as e n t r y from t h e g u t i n t o the haemolymph o f l a b e l from 3H-20-OHE (NEN, Boston) was v e r y slow (10). Ecdysone i t s e l f and t h e p h y t o e c d y s o n e s , i n o k o s t e r o n e and m a k i s t e r o n e A, were much l e s s e f f e c t i v e as a b o r t i f a c i e n t s t h a n 20-OHE ( T a b l e I ) . C y a s t e r o n e and p o n a s t e r o n e A were i n a c t i v e ; t h e i m p l i c a t i o n b e i n g t h a t t h e h y d r o x y l group on C25 i s r e q u i r e d f o r a c t i v i t y (10). O b s e r v a t i o n o f t h e d i s t a l t u b u l e s o f the u t e r i n e g l a n d r e s p o n ­ s i b l e f o r n o u r i s h m e n t o f t h e l a r v a i n u t e r o showed ( F i g u r e 2) t h a t a d d i n g e c d y s t e r o i d s t o t h e d i e t o f female t s e t s e i n h i b i t e d g l a n d f u n c t i o n i n some way. As a r e s u l t , l e s s " m i l k " from t h e g l a n d was a v a i l a b l e t o n o u r i s h t h e l a r v a , hence s m a l l e r pupae were p r o d u c e d , o r t h e l a r v a was a b o r t e d . The d i a m e t e r o f d i s t a l t u b u l e s was a l s o s m a l l e r i n f e m a l e s t o which JH had been a p p l i e d ( T a b l e I I ) b u t , c o n t r a r y t o D e n l i n g e r ' s o b s e r v a t i o n ( 9 ) , i n t h i s experiment (where HPLC pure JH I I I was used i n s t e a d o f h i s crude m i x t u r e ) , no a b o r ­ tions resulted. Attempts t o u s e 20-OHE as a s y s t e m i c a b o r t i f a c i e n t - i t was i n j e c t e d i n t o r a b b i t s used as h o s t - was n o t s u c c e s s f u l ( 1 0 ) . The s t e r o l (mixed w i t h ^H-OHE) had r a p i d l y been removed from c i r ­ culation. T h e r e f o r e , analogues o f t h e s t e r o l which w i l l n o t r a p i d l y be m e t a b o l i z e d ( i n t o s u l p h a t e s o r g l u c u r o n i d e s ) i n t h e mam­ m a l i a n l i v e r might be l o n g e r a c t i n g . The 2, 2 2 - d i t o s y l d e r i v a t i v e

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

WHITEHEAD

Combating Pests with Natural Products

• = ABORTIONS/STILLBIRTHS ο = STERILE / INFERTILE

0.2

0.4

0.6 0.8

1

2

4

β

8

10

20

LOG DOSE pg IN 50 mg MEAL Figure 1 Dose response c u r v e f o r p e r c e n t a g e o b s e r v e d a b o r t i o n s o r s t i l l b i r t h s and f o r p e r c e n t a g e s t e r i l e females ( i n c l u d i n g u n r e c o r d e d egg a b o r t i o n s ) r e s u l t i n g from f e e d i n g 20-OHE i_n v i t r o t o p r e g n a n t G. m. m o r s i t a n s ( E D 5 0 - 2,5 ug f o r a b o r t i o n s a l o n e (Y = 37.07 + 17.34X) o r = 1.0 pg f o r a l l a b n o r m a l i t i e s ) "Reproduced w i t h p e r m i s s i o n from R e f . 10. C o p y r i g h t 1981* ' I n s e c t S c i . A p p l i c . Pergamon P r e s s , O x f o r d . " 1

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

15 30

Ponasterone

* A b o r t i o n s and f l i e s

15 15

Cyasterone

A

15 15 15

15 30 15

Makisterone

Inokosterone

30 30 30

Ecdysone

A

15 30 15 30

No. o f f l i e s fed

-

0.2 1.1 3.4 3.4

5.0 15.0

1.9 2.5

1.8 3.7 8.7

7 5

20 21

33 51 100

7 15 100

37 55 100

30 64 70 100

1st

cycle

0 0

23 30

18 14 35

-

_

-

_

14 14

11 8 12

-

_ _

-

_

25

100 42 42 90

20 40

_

o f abnormal l a r v a e * cycle 3rd cycle

50 56

-

Percentage 2nd

that d i d not d e p o s i t l a r v a e

5 10 35

0.8 - 1.1 1.5 - 2.0 3.6 - 3.8

0.1 0.8 1.5 2.8

E s t i m a t e d dose ( ug/50 mg meal)

The e f f e c t s o f p o t e n t i a l l a r v i c i d e s f e d i n v i t r o t o pregnant G. m. m o r s i t a n s . "Adapted from T a b l e 1, r e p r o d u c e d w i t h p e r m i s s i o n i n p a r t from réf. 10 C o p y r i g h t 1981 I n s e c t S c i e n c e & i t s A p p l i c a t i o n , Pergamon P r e s s , O x f o r d . "

20-HydroxyEcdysone

Substance

Table I .

WHITEHEAD

Combating Pests with Natural Products

Figure 2 V a r i a t i o n i n diameter of d i s t a l tubules of u t e r i n e gland d u r i n g second pregnancy c y c l e o f (3. m. m o r s i t a n s f e d i n v i t r o on day 1 w i t h 20-OHE (4 yg/50 mg b l o o d ) i n d e f i b r i n a t e d p i g b l o o d com­ p a r e d w i t h c o n t r o l ( a n a l y s i s of v a r i a n c e g i v e s Pl-octen-3-ol>MEK>pentanol. Auto-sterilization A t t e m p t s t o s t e r i l i z e t s e t s e caught i n the w i l d have been made using aerosol sprays w i t h i male (ï. m. m o r s i t a n s migh a z a r i d i n e ) was a p p l i e d t o decoys, a t t a c h e d to c l o t h or n e t t i n g s c r e e n s , t r e a t e d w i t h the sex s t i m u l a n t " M o r s i l u r e " (29) (15, 19, 23-trimethyl heptatriacontane). S u c c e s s f u l r e s u l t s i n the l a b o r a ­ t o r y had encouraged t h e s e f i e l d t r i a l s ( 3 0 ) . The s e x - s t i m u l a n t f o r male G. p a l l i d i p e s , the (13R, 23S) - 13, 23 - d i m e t h y l p e n t a t r i a c o n t r a n e ( F i g u r e 5) has been s y n t h e s i z e d (31) and t e s t e d ( F i g u r e 6) ( 3 2 ) . I t i s p r o b l e m a t i c a l whether t h i s s h o r t - r a n g e " P a l l i d i l u r e " pheromone can be used t o i m m o b i l i s e male f l i e s f o r l o n g enough t o a l l o w s u f f i c i e n t c h e m o s t e r i l a n t to be a b s o r b e d t h r o u g h the t a r s a l and abdominal c u t i c l e . The s e a r c h i s c o n t i n u i n g f o r a c h e m o s t e r i l a n t s a f e enough f o r humans t o use ( 3 3 ) . More r e a c t i v e a n a l o g u e s might t o t a l l y d i s r u p t mating b e h a v i o u r . D e r i v a t i v e s of n a t u r a l p r o d u c t s ( t h e b e n z y l p h e n o l s and b e n z y l - 1 , 3 - b e n z o d i o x o l e s ) ( F i g u r e 5) e f f e c t i v e l y s t e r i l i z e screwworm f l i e s ( 3 4 ) , i n s e c t i c i d e - r e s i s t a n t female house f l i e s (35) and female G. m. m o r s i t a n s ( 3 6 ) . In the l a t t e r c a s e , 2 u g / f l y of b e n z y l - 1 , 3 - b e n z o d i o x o l e was e f f e c t i v e . T h i s group of n a t u r a l p r o ­ d u c t s was found t o be nonmutagenic i n s t a n d a r d Ames b a c t e r i a l t e s t s and the compounds were r e l a t i v e l y n o n - t o x i c t o mammals (36)· T h e r e f o r e , i n c o r p o r a t i o n of t h e s e compounds i n s a l t b l o c k s might be e n v i s a g e d , or t r a p p e d t s e t s e might be exposed t o them b e f o r e r e l e a s e , as p a r t of an i n t e g r a t e d c o n t r o l programme. Sterilizing i n s e c t s r e d u c e s p o p u l a t i o n s f a r more q u i c k l y t h a n t r y i n g to k i l l them. I m m u n o l o g i c a l methods The s y s t e m i c r o u t e remains the most d e s i r a b l e method f o r c o n t r o l l i n g haematophagous p e s t s or e c t o p a r a s i t e s , e s p e c i a l l y i f t h e y happen t o be v e c t o r s of v i r u s , p r o t o z o a n or metazoan i n f l i c t e d diseases. I t has been u n d e r s t o o d f o r q u i t e some time t h a t the b i t e o f the t i c k t r a n s f e r s s u b s t a n c e s , l i k e an a n t i c o a g u l a n t f o r i n s t a n c e , which somehow e v e n t u a l l y l e a d s t o a c q u i r e d r e s i s t a n c e t o i n f e s t a t i o n by t h a t t i c k ( 3 7 ) . T h e r e f o r e , use of p r o t e i n s from the e c t o p a r a s i t e , as a n t i g e n t o immunize l i v e s t o c k l i k e l y t o be exposed

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

29.

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Table V I I .

423

Combating Pests with Natural Products

The e f f e c t o f v a r i o u s Ketones and l - 0 c t e n - 3 - o l on the s i z e of 6h c a t c h e s of G. p a l l i d i p e s i n B i c o n i c a l T r a p s s e t under Nkuruman Escarpment (25)

Index o f Increase

Dose r a t e (mg/h)

Detransformed mean c a t c h e s

Control Low dose Medium dose H i g h dose

0 139 463 2391

10.2 18.1 18.2 27.9

xl.8 xl.8 x2.7

Methylethylketone (MEK) Control Low dose Medium dose H i g h dose

190 1546

10.3 9.8

xl.7 xl.7

Me t h y 1 v i n y I k e t one (MVK) Control Low dose Medium dose H i g h dose

0 64 145 703

30.0 25.2 29.6 37.7

x0.8 xl.O xl.2

0 2216 1694 936

44.9 81.9 69.4 48.0

xl.8 xl.5 xl.l

0 2216 50

26.3 62.2 30.0

x2.4 xl.l

2216/50

53.7

xl.O

0 85 50 85/50

47.0 73.9 88.0 125.9

xl.6 xl.9 x2.7

Treatment

Acetone

H i g h dose o f 3 k e t o n e s Control Acetone MEK MVK Acetone+l-0cten-3-ol Control Acetone l-0cten-3-ol Acetone+1Octen-3-ol 1

MEK+l-0cten-3-ol Control MEK Octenol MEKfOctenol

*P ifi) » * there followed a d e s c r i p t i o n o f t h e s t r u c t u r e and s t e r e o c h e m i s t r y o f o p h i o b o l i n A (11 ,12) ( F i g u r e 4 ) , w h i c h was f o u n d t o be i d e n t i c a l to c o c h l i o b o l i n . Reports o f the i s o l a t i o n s of other ophiobolins followe o p h i o l o b i n C (ϋ_>11. ( c e p h a l o n i c a c i d ) ( J ^ ) f r o m Ç e p f r a l P v S j x Q r i u m caeruleng; and o p h i o l o b i n F ( \±) from CocklioKoJ^us k e t e r A s . t r PPvhu s . O p h i o b o l i n Ε r e m a i n s an enigma and one c a n o n l y s p e c u l a t e t h a t t h e s u b s t a n c e was i d e n t i c a l t o a p r e v i o u s p r o d u c t , was u n s t a b l e u p o n p u r i f i c a t i o n , o r was n o t a v a i l a b l e i n s u f f i c i e n t q u a n t i t i e s f o r p h y s i c a l , c h e m i c a l and b i o l o g i c a l studies. W h a t e v e r , t h e l i t e r a t u r e i s blank f o r o p h i o b o l i n E. A l l of these f u n g a l l y d e r i v e d o p h i o b o l i n s have one s t r u c t u r a l f e a t u r e i n common: t h e j u n c t i o n between r i n g s A and Β i s c i s .

oxygae

(ÇQçhXvivQvhQlus

a n