Handbook of Natural Pesticides: Part A, Volume III [1 ed.] 9781138596955, 9780429487255, 9780429945410, 9780429945403, 9780429945427

Table of contents :

Part 1: Theory and Practice 1. Pests and Their Control 2. Integrated Crop Management Systems for Pest Control 3. Integrated Weed Management Systems Technology for Agroecosystem Management 4. Computers and Pest Management 5. Allelopathy - A Natural Protection, Allelochemicals 6. Chemical Messengers and Insect Behavior 7. Toxicological Evaluation and Registration Requirements for Biorational Pesticides 8. The Regulation of Pesticides 9. Naturally Occurring Pesticides and the Pesticide Crisis, 1945 to 1980 Part 2: Methods for Detection 10. Bioassays for Plant Hormones and Other Naturally Occurring Plant Growth Regulators 11. Insect Bioassays 12. Biological Assays with Insect Pathogens

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CRC Series in Naturally Occurring Pesticides Series Editor-in-Chief N. Bhushan Mandava

Handbook of Natural Pesticides: Methods Volume I: Theory, Practice, and Detection Volume II: Isolation and Identification Editor N. Bhushan Mandava

Handbook of Natural Pesticides Volume III: Insect Growth Regulators Volume IV: Pheromones Editors E. David Morgan N. Bhushan Mandava

Future Volumes

Handbook of Natural Pesticides Insect Attractants, Deterrents, and Defensive Secretions Editors E. David Morgan N. Bhushan Mandava

Plant Growth Regulators Editor N. Bhushan Mandava

Microbial Insecticides Editors Carl M. Ignoffo

CRC Handbook of Natural Pesticides Volume III

Insect Growth Regulators Part A Editors

E. David Morgan, D.Phil.

N. Bhushan Mandava, Ph.D.

Reader Department of Chemistry University of Keele Staffordshire, England

(ev e

Senior Associate Mandava Associates Washington, D.C.

CRC Press Taylor &. Francis Group Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

First published 1987 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2018 by CRC Press © 1987 by Taylor & Francis Group. CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Publisher’s Note The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies may be apparent. Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-1-138-59695-5 (hbk) ISBN 13: 978-0-429-48725-5 (ebk) Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

CRC Handbook Series in Naturally Occurring Pesticides

INTRODUCTION The United States has been blessed with high quality, dependable supplies of low cost food and fiber, but few people are aware of the never-ending battle that makes this possible. There are at present approximately 1,100,000 species of animals, many of them very simple forms, and 350,000 species of plants that currently inhabit the planet earth. In the U.S. there are an estimated 10,000 species of insects and related acarinids which at sometime or other cause significant agricultural damage. Of these, about 200 species are serious pests which require control or suppression every year. World-wide, the total number of insect pests is about ten times greater. The annual losses of crops, livestock, agricultural products, and forests caused by insect pests in the U.S. have been estimated to aggregate about 12% of the total crop production and to represent a value of about $4 billion (1984 dollars). On a world-wide basis, the insect pests annually damage or destroy about 15% of total potential crop production, with a value of more than $35 billion, enough food to feed more than the population of a country like India. Thus, both the losses caused by pests and the costs of their control are considerably high. Insect control is a complex problem for there are more than 200 insects that are or have been subsisting on our main crops, livestock, forests, and aquatic resources. Today, in the U.S., conventional insecticides are needed to control more than half of the insect problems affecting agriculture and public health. If the use of pesticides were to be completely banned, crop losses would soar and food prices would also increase dramatically. About 1 billion pounds of pesticides are used annually in the U.S. for pest control. The benefits of pesticides have been estimated at about $4/$l cost. In other words, chemical pest control in U.S. crop production costs an estimated $2.2 billion and yields a gross return of $8.7 billion annually. Another contributing factor for increased crop production is the effective control of weeds, nematodes, and plant diseases. Crop losses due to unwanted weed species are very high. Of the total losses caused by pests, weeds alone count for about 10% of the agricultural production losses valued at more than $12 billion annually. Farmers spend more than $6.2 billion each year to control weeds. Today, nearly all major crops grown in the U.S. are treated with herbicides. As in insect pest and weed control programs, several chemicals are used in the disease programs. Chemical compounds (e.g., fungicides, bactericides, nematicides, and viracides) that are toxic to pathogens are used for controlling plant diseases. Several million dollars are spent annually by American farmers to control the diseases of major crops such as cotton and soybeans. Another aspect for improved crop efficiency and production is the use of plant growth regulators. These chemicals that regulate the growth and development of plants are used by farmers in the U.S. on a modest scale. The annual sale of growth regulators is about $130 million. The plant growth regulator market is made up of two distinct entities — growth regulators and harvest aids. Growth regulators are used to increase crop yield or quality. Harvest aids are used at the end of the crop cycle. For instance, harvest aids defoliate cotton before picking or desiccate potatoes before digging. The use of modem pesticides has accounted for astonishing gains in agricultural production as the pesticides have reduced the hidden toll exacted by the aggregate attack of insect pests, weeds, and diseases, and also improved the health of humans and livestock as they control parasites and other microorganisms. However, the same chemicals have allegedly posed some serious problems to health and environmental safety, because of their high toxicity and severe persistence, and have become a grave public concern in the last 2 decades. Since the general public is very much concerned about their hazards, the U.S. Environmental

Protection Agency enforced strong regulations for use, application, and handling of the pesticides. Moreover, such toxic pesticides as DDT 2,4,5 -T and toxaphene were either completely banned or approved for limited use. They were, however, replaced with less dangerous chemicals for insect control. Newer approaches for pest control are continuously sought, and several of them look very promising. According to a recent study by the National Academy of Sciences, pesticides of several kinds will be widely used in the foreseeable future. However, newer selective and biode­ gradable compounds must replace older highly toxic persistent chemicals. The pest control methods that are being tested or used on different insects and weeds include: (1) use of natural predators, parasites, and pathogens, (2) breeding of resistant varieties of species, (3) genetic sterilization techniques, (4) use of mating and feeding attractants, (5) use of traps, (6) development of hormones to interfere with life cycles, (7) improvement of cultural practices, and (8) development of better biodegradable insecticides and growth regulators that will effectively combat the target species without doing damage to beneficial insects, wildlife, or man. Many leads are now available, such as the hormone mimics of the insect juvenile and molting hormones. Synthetic pyretheroids are now replacing the conventional insecticides. These insecticides, which are a synthesized version of the extract of the pyrethrum flower, are much more attractive biologically than the traditional insecticides. Thus, the application rates are much lower in some cases, one tenth the rates of more traditional insecticides such as organophosphorus pesticides. The pyrethroids are found to be very specific for killing insects and apparently exhibit no negative effects on plants, livestock, or humans. Another apparent benefit is that there is no resistance to these compounds accumulated in the insects. The use of these compounds is now widely accepted for use on cotton, field corn, soybean, and vegetable crops. For the long term, integrated pest management (IPM) will have tremendous impact on pest control for crop improvement and efficiency. Under this concept, all types of pest control — cultural, chemical, inbred, and biological — are integrated to control all types of pests and weeds. The chemical control includes all of the traditional pesticides. Cultural controls consist of cultivation, crop rotation, optimum planting dates, and sanitation. Inbred plant resistance involves the use of varieties and hybrids that are resistant to certain pests. Finally, the biological control involves encouraging natural predators, parasites, and microbials. Under this system, pest-detection scouts measure pest populations and determine the best time for applying pesticides. If properly practiced, IPM could reduce pesticide use up to 75% on some crops. The naturally occurring pesticides appear to have a prominent role for the development of future commercial pesticides not only for agricultural crop productivity but also for the safety of the environment and public health. They are produced by plants, insects, and several microorganisms, which utilize them for survival and maintenance of defense mech­ anisms, as well as for growth and development. They are easily biodegradable, often times species-specific and also sometimes less toxic (or nontoxic) on other non-target organisms or species, an important consideration for alternate approaches of pest control. Several of the compounds, especially those produced by crop plants and other organisms, are consumed by humans and livestock, and yet appear to have no detrimental effects. They appear to be safe and will not contaminate the environment. Hence, they will be readily accepted for use in pest control by the public and the regulatory agencies. These natural compounds occur in nature only in trace amounts and require very low dosage for pesticide use. It is hoped that the knowledge gained by studying these compounds is helpful for the development of new pest control methods such as their use for interference with hormonal life cycles and trapping insects with pheromones, and also for the development of safe and biodegradable chemicals (e.g., pyrethroid insecticides). Undoubtedly, the costs are very high as compared to the presently used pesticides. But hopefully, these costs would be compensated for by the benefits derived through these natural pesticides from the lower volume of pesticide use

and elimination of risks. Furthermore, the indirect or external costs resulting from pesticide poisoning, fatalities, livestock losses, and increased control expenses (due to the destruction of natural enemies and beneficial insects as well as the environmental contamination and pollution from chlorinated, organophosphorus, and carbamate pesticides) could be assessed against benefits vs. risks. The development and use of such naturally occurring chemicals could become an integral part of IPM strategies. As long as they remain endogenously, several of the natural products presented in this handbook series serve as hormones, growth regulators, and sensory compounds for growth, development, and reproduction of insects, plants, and microorganisms. Others are useful for defense or attack against other species or organisms. Once these chemicals or their analogs and derivatives are applied by external means to the same (where produced) or different species, they come under the label “ pesticides” because they contaminate the environment. Therefore, they are subject to regulatory requirements, in the same way the other pesticides are handled before they are used commercially. However, it is anticipated that the naturally occurring pesticides would easily meet the regulatory and environmental requirements for their safe and effective use in pest control programs. A vast body of literature has been accumulated on naturally occurring pesticides during the last 2 or 3 decades; we plan to assemble this information in this handbook series. However, we realize that it is a single handbook series. Therefore, we have limited our attempts to chemical and a few biological aspects concerned with biochemistry and physiology. Wher­ ever possible, we tried to focus our attention on the application of these compounds for pesticidal use. We hope that the first volume which deals with theory and practice will serve as an introductory volume and will be useful to everyone interested in learning about the current technology that is being adapted from compound identification to the field trials. The subsequent volumes deal with the chemical, biochemical, and physiological aspects of naturally occurring compounds, grouped under such titles as insect growth regulators, plant growth regulators, etc. In a handbook series of this type with diversified subjects dealing with plant, insect, and microbial compounds, it is very difficult to achieve either uniformity or complete coverage while putting the subject matter together. This goal was achieved to a large extent with the understanding and full cooperation of chapter contributors who deserve my sincere appreciation. The editors of the individual volumes relentlessly sought to meet the deadlines and, more importantly, to bring a balanced coverage of the subject matter, but, however, that seems to be an unattainable goal. Therefore, they bear full responsibility for any pitfalls and deficiencies. We invite comments and criticisms from readers and users as they will greatly help to update future editions. It is hoped that this handbook series will serve as a source book for chemists, biochemists, physiologists, and other biologists alike — those engaged in active research as well as those interested in different areas of natural products that affect the growth and development of plants, insects, and other organisms. The editors wish to acknowledge their sincere thanks to the members of the Advisory Board for their helpful suggestions and comments. Their appreciation is extended to the publishing staff, especially Pamela Woodcock, Amy Skallerup, and Sandy Pearlman for their ready cooperation and unlimited support from the initiation to the completion of this project. N. Bhushan Mandava Editor-in-Chief

FOREWORD Pests of crops and livestock annually account for multi-billion dollar losses in agricultural productivity and costs of control. Insects alone are responsible for more than 50% of these losses. For the past 40 years the principal weapons used against these troublesome insects have been chemical insecticides. The majority of such materials used during this period have been synthetic organic chemicals discovered, synthesized, developed, and marketed by commercial industry. In recent years, environmental concerns, regulatory restraints, and problems of pest resistance to insecticides have combined to reduce the number of materials available for use in agriculture. Replacement materials reaching the marketplace have been relatively few due to increased costs of development and the general lack of knowledge about new classes of chemicals having selective insecticidal activity. In response to these trends, it is gratifying to note that scientists in both the public and private sectors have given significant attention to the discovery and evaluation of natural products as fertile sources of new insecticidal agents. Not only are these materials directly useful as insect control agents, but they also serve as models for new classes of chemicals with novel modes of action to attack selective target sites in pest species. Such new control agents may also be less susceptible to the cross resistance difficulties encountered with most classes of currently used synthetic pesticide chemicals to which insects have developed immunity. Natural products originating in plants, animals, and microorganisms are providing a vast source of bioactive substances. The rapid development and application of powerful analytical instrumentation, such as mass spectrometry, nuclear magnetic resonance, high performance liquid chromatography, reverse phase liquid chromatography, immunoassay, and radioim ­ munoassay, have greatly facilitated the identification of miniscule amounts of active bio­ logical chemicals isolated from natural sources. These new science approaches and tools are addressed and reviewed extensively in these volumes. Some excellent examples of success in this research involve the discovery of insect growth regulators, especially the so-called juvenoids, which are responsible for control of insect metamorphosis, reproduction, and behavior. Pheromones which play essential roles in insect communication, feeding, and sexual behavior represent another important class of natural products holding great promise for new pest insect control technology. All of these are discussed in detail in Volumes 1, 2, and 3. It is hoped that the science described in these volumes will serve researchers in industry, government, and academia, and stimulate them to continue to seek even more useful natural materials that produce effective, safe, and environmentally acceptable materials for use against insect pests affecting agriculture and mankind. Orville G. Bentley Assistant Secretary Science and Education U.S. Department of Agriculture

PREFACE Naturally occurring pesticides are those with which man began. Forty years ago science improved on these with the introduction of synthetics. Now the attention is again on natural pesticides. As we assess the results of those 40 years, we recognize that effective insect pest control has eluded us. The introduction of synthetics has led to new problems of contamination, resistance, toxicity, and pest resurgence. Despite the achievements of science, it has failed to protect food provision adequately. Food is the only indispensible product produced by man, yet we saw our inadequacies in that pursuit sharply revealed by the tragic famine in the Sahel of Africa in 1984-85. The present loss of rice to pests is estimated to be 46% of the potential crop. If this loss could be reduced to 20%, about another 177 million people could be adequately fed, without bringing any new land into cultivation. Similar, though less dramatic figures can be quoted for wheat, com, potatoes, soybeans, and many other staple crops. Losses due to pests, even in the U.S., with its intensive use of pesticides, continue at a level of 30% of total sales value. The incidence of malaria in India and Sri Lanka is rising again, despite near eradication by 1970, due to resistant strains of mosquito and plasmodium. We now have to reassess our knowledge to devise selective, safe pesticides. “ Natural” methods of biological control have in some notable cases been successful but these are exceptional. Pyrethrum is an effective natural insecticide, relatively nontoxic to mammals, nonpolluting, nonpersistent. From a systematic study of structure and activity, a new gen ­ eration of pesticides, with special applications has arisen. But since the days of natural pyrethrum and derris, a whole new world of substances has been discovered; substances which affect pest growth, behaviour, feeding, oviposition, aggregation, mating, and so on. Much might be learned from these substances, if only the scattered information can be brought together and organized in a way that will permit careful re-examination of these substances, often in a context or application quite foreign to the original discoveries or investigator’s concept of these chemicals. In a recent survey of plant species which have been recorded as possessing pest-control properties, 1005 species were listed as having insecticidal activity, 384 with antifeeding activity, and 279 with repellent activity. The definition of these activities is not exact, but these figures indicate that a great mass of material is waiting to be checked and evaluated. This Handbook Series is trying to achieve some order and illumination in the half-explored world of Naturally Occurring Pesticides. The present work is concerned with that great army of pests, the insects; Insect Growth Regulators brings together all those substances, which occur naturally, whether in plant or animal, which are known to affect insect growth and development. This beings with a thorough look at those hormones which occur naturally in insects and which regulate their development. Not many such compounds are known, the presence of many more has been inferred or suggested, but the known ones have been studied intensively, there is a great accumulation of literature on them, and many specialized books and reviews. This volume is different in that it attempts to assess our knowledge from the point of view of the reader wishing to know more about pest control and wishing to engender new ideas. As well as the naturally occurring hormones, substances related by structure from other sources are considered as well as materials from all origins which are known to have effects upon growth and development of insects. The subject is introduced by a brief consideration of the physiological and endocrinological aspects of insect hormones at our present state of knowledge, by two world experts in that subject. They provide a glossary of terms from their subject that may be unclear or unfamiliar to the chemist, entomologist, or agriculturalist.

The chapters which then follow deal with the known insect hormones. The treatment of these necessarily varies. There are over 70 ecdysteroids known but only four juvenile hormones. The knowledge of peptide hormones is still very scanty, and other substances, chiefly from plants, which affect insect growth and development are difficult to categorize under just one heading. The same substance may turn up in more than one place, but the treatment and emphasis of the different authors are such that there is no serious overlap. We are fortunate in being able to include a chapter on the substances from the neem and chinaberry trees which affect insect feeding and development. The subject has received growing attention in the periodical literature, but this is the first comprehensive introduction to the subject to appear. The substance azadirachtin, obtained from neem seeds would appear to hold great promise, in the form of a crude extract, as a cheap pesticide in the Third World. It is already challenging the ingenuity of chemists to discover and mimic the rela ­ tionship between its structure and activity. We wish to thank all the contributors for their great efforts in bringing together all the information contained here. Our thanks are due also to Iris Jones and Margaret Fumival for their help throughout the editorial stage. E.D.M . N.B.M .

THE EDITORS E. David Morgan, D.Phil., is a Chartered Chemist, a Fellow of the Royal Society of Chemistry, and a Fellow of the Royal Entomological Society of London. He received his scientific training in Canada and England, and has worked for the National Research Council of Canada, Ottawa, The National Institute for Medical Research, London, the Shell Group of Companies and is now Reader in Chemistry at the University of Keele, Staffordshire, England. He is co-author of a textbook on aliphatic chemistry with the Nobel prizewinner, Sir Robert Robinson, and with him is a co-inventor of a number of patents. Dr. Morgan has contributed to over 100 papers, most of them on aspects of insect chemistry and has written a number of reviews on insect hormones and pheromones. N. Bhushan Mandava, holds B.S., M.S., and Ph.D. degrees in chemistry and has published over 120 papers including two patents, several monographs and reviews, and books in the areas of pesticides and plant growth regulators and other natural products. As editorial advisor, he has edited two special issues on countercurrent chromatography for the Journal o f Liquid Chromatography. He is now a consultant in pesticides and drugs. Formerly, he was associated with the U.S. Department of Agriculture and the Environmental Protection Agency as Senior Chemist. He has been active in several professional organizations, was President of the Chemical Society of Washington, and serves as Councilor of the American Chemical Society.

ADVISORY BOARD E. David Morgan N. Bhushan Mandava Editors Members Peter Karlson Institut fur Physiologische Chemie I University of Marburg Marburg, West Germany Laurence I. Gilbert Department of Zoology University of North Carolina Chapel Hill, North Carolina Heinz Schmutterer Institute of Phytopathology and Applied Zoology Justus Liebig University Giessen, West Germany Jan Koolman Institut fur Physiologische Chemie I University of Marburg Marburg, West Germany

CONTRIBUTORS Alexej B. Borkovec, Ph.D. Chief Insect Reproduction Laboratory U.S. Department of Agriculture Agricultural Research Center Beltsville, Maryland Jules A. Hoffman, Ph.D. Directeur de Recherche Laboratoire Biologie Generale University Louis Pasteur Strasbourg, France Caleb W. Holyoke, Ph.D. Senior Research Chemist Agricultural Products Department E.I. DuPont DeNemours & Company Wilmington, Delaware W. Mordue, D.Sc. Professor Department of Zoology University of Aberdeen Aberdeen, Scotland

Geoff Richards, Ph.D. Directeur de Recherche Laboratoire de Genetique Moleculaire des Eukaryotes Institut de Chimie Biologique Strasbourg, France

H. Schmutterer, D.Phil.Nat. Professor Institute of Phytopathology and Applied Zoology Justus Liebig University Giessen, Federal Republic of Germany

Nobel Wakabayashi, Ph.D. Research Chemist U.S. Department of Agriculture Agricultural Research Service Beltsville, Maryland

P. J. Morgan, Ph.D. Department of Zoology University of Aberdeen Aberdeen, Scotland

Rolland M. Waters, Ph.D. Research Chemist Science and Educational Administration Agricultural Research Service Beltsville, Maryland

John C. Reese, Ph.D. Associate Professor Department of Entomology Kansas State University Manhattan, Kansas

Ian D. Wilson, Ph.D. Safety of Medicines ICI Pharmaceuticals Division Macclesfield, England

TABLE OF CONTENTS Part A Introduction to the Insect Neuroendocrine System.................................................................. 1 Geoffrey Richards and Jules A. Hoffmann The Ecdysteroids........................................................................................................................... 15 Ian D. Wilson Juvenile Hormones and Related Com pounds........................................................................... 87 N. Wakabayashi and R. M. Waters The Chemistry and Biology of Selected Insect Peptides....................................................... 153 W. Mordue and P. J. Morgan Index..............................................................................................................................................185 Part B Chemosterilants............................................................................................................................. 1 Alexej B. Borkovec Allelochemics Affecting Insect Growth and Development......................................................21 John C. Reese and Caleb W. Holyoke, Jr. Acute Insect Toxicants from Plants............................................................................................ 67 Caleb W. Holyoke, Jr. and John C. Reese Insect Growth-Disrupting and Fecundity-Reducing Ingredients from the Neem and Chinaberry Trees.................................................................................................................. 119 Heinrich Schmutterer Index.............................................................................................................................................. 171

Volume III: Insect Growth Regulators, Part A

1

INTRODUCTION TO THE INSECT NEUROENDOCRINE SYSTEM Geoffrey Richards and Jules A. Hoffmann

INTRODUCTION When the earliest insects diverged from other arthropods in the Devonian period some 300 million years ago, they were to found the most successful class of the phylum Arthropoda, which is itself the largest phylum of the animal kingdom. The insects are prolific with regard to speciation; current estimates suggest some 2 million extant species, and this has no doubt been an important factor in their adaptation to an impressive range of habitats. So versatile have they been in this colonization that it is perhaps as interesting to consider their relative failures, for example, why have so few insect species taken to the sea?, as to probe the means by which some species survive temperatures as low as - 5 0 °C , desiccation, or life in pools of crude petroleum. Salt water species such as Ephydra cinerae, which live in the Great Salt Lake of Utah, coping with a salinity that is five times that of sea water, suggest that insects could in principle be as successful in the oceans as they have been on land and in fresh water habitats. The Insecta are characterized by their respiratory tracheal system and the division of the adult body into head, thorax, and abdomen; the thorax carrying three pairs of legs and usually one or two pairs of wings (Pterygota), although a primitive wingless subclass (Apterygota) exists. In common with the other arthropods, immature insects undergo periodic moults of their rigid cuticles, the period between larval molts being termed an instar. The later developmental moults of insects include both the relatively simple changes of the hemimetabolous insects which show minor differences between larval (or nymphal) and adult stages, and the complex metamorphoses of the holometabolous insects, where the larval form bears little resemblance to the adult that emerges from the pupal stage. In the extreme, these two stages of the life cycle may exploit two distinct ecological niches and different substrates, or feeding may be entirely dispensed with in the adult stage. Among their strategies for coping with adverse conditions, many insects include diapause, a state of dormancy which may occur in embryonic, larval, pupal, or adult stages, depending on the species. Studies of the regulation of diapause suggest that it is mediated by the same hormones that control normal development. Our purpose is to summarize current knowledge on the neuroendocrine control of devel­ opment and reproduction. In presenting a simplified picture that derives from studies on a relatively small number of insect species we will describe what appears at first sight to be a uniform system. However, we should emphasize the differences that have been detected, as there is considerable variation even in this limited sample of insects. Insects are amenable to types of experimentation such as ligation, decapitation, brain removal, and the extirpation and insertion of endocrine glands to an extent that is not possible in the vertebrates. In addition, in recent years, a number of in vitro systems have been developed for the culture both of endocrine glands and tissues that respond to their hormones. In some instances it is the comparison of the in vivo and in vitro responses to hormones, their synthetic analogs or insect growth regulators that have revealed important differences in the peripheral me ­ tabolism of these substances in different species. This variation poses particular problems for the study of the endocrine control of development and reproduction and yet offers opportunities for specific control by insecticides. When considering points of attack on the neuroendocrine system there are practical aspects which may dictate the approach. Besides the obvious differences between terrestrial and aquatic insects, there are economically or medically important species such as Glossina, the

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CRC Handbook o f Natural Pesticides

Tsetse flies which carry trypanosomes, which are viviparous. In Glossina this viviparity is developed to the extent that mature larvae are released from the uterus and pupation follows soon after, there being no free-living feeding larval phase. Such species will require different methods for the presentation of insecticides than those suggested by the general scheme of development which we shall now present.

AN OVERVIEW OF THE NEUROENDOCRINE SYSTEM Development and Diapause The four elements of the neuroendocrine system that interact to regulate development are the neurosecretory cells of the brain, the corpora cardiaca, the corpora allata, and the prothoracic glands (Figure 1). The physical layout of these cell types varies between different insect species, although the functional connections are considered the same. In some species the paired organs are fused about the midline and in the larvae of the cyclorrhaphous Diptera the corpora allata, corpora cardiaca, and prothoracic glands all fuse into a structure known as the ring gland, the three types of glandular cells remaining distinct. In the brain there are two groups of neurosecretory cells (NSC), the median and the lateral NSC, respectively. Both groups have been proposed as the source of the neurohormone that stimulates the production of ecdysone, with recent in vitro analysis favoring the lateral NSC in lepidopterans. This hormone is variously known as brain hormone, ecdysiotropin, or prothoracicotropic hormone (PTTH). As ecdysone synthesis in the course of larval devel­ opment appears to be a property of the prothoracic glands, we will follow the current usage of PTTH. However, as ecdysone synthesis or release occurs from other tissues at different developmental stages, we advocate the use of ecdysiotropin as a generic noun for this class of neurohormones. Neurohormones are not released directly into the hemolymph but pass along the axons to the corpora cardiaca, where they may be released or stored. In this role the corpora cardiaca are termed neurohemal organs. These endocrine glands also have the capacity to synthesize hormones themselves, which as a rule are concerned with homeostasis. There are conflicting reports as to the neurohemal organs for PTTH. Earlier studies considered the corpora cardiaca the intermediary between the brain and the prothoracic gland, while more recent studies in lepidopterans, based on the capacity of corpora allata extracts to stimulate ecdysteroid production in isolated prothoracic glands in vitro, favor the corpora allata as the neurohemal organs for PTTH, at least in this order. The prothoracic glands synthesize and secrete ecdysone during larval development. The ecdysone is secreted, possibly associated with a carrier protein, and enters the hemolymph. Most of this ecdysone is converted into 20-hydroxy ecdysone, the Malpighian tubules and fat body in particular showing high levels of conversion. It is a common belief, derived from many in vitro studies, that 20-hydroxyecdysone is the active ecdy steroid in insects although the possibility should be left open as to whether or not ecdysone has a specific hormonal role in development. Ecdy steroids control the processes leading to larval molts and metamorphosis. In contrast to the vertebrates where steroids are conventionally associated with developmental activity in a limited number of tissues, ecdy steroids probably act on all insect tissues, although the responses they evoke are highly tissue-specific. We emphasize that the majority of our concepts of ecdysone synthesis and regulation derive from studies on larval prothoracic glands. There is increasing evidence that in embryonic and pupal development there are other sources of ecdy steroids, and in addition we will discuss the ovarian synthesis of ecdysone in adults below. The corpora allata are responsible for the synthesis and secretion of the juvenile hormones (JHs). These are a family of closely related sesquiterpenoid hormones, referred to as JHI, II, and III but which at this point, for the purpose of our general outline, we will consider

Volume 111: Insect Growth Regulators, Part A P hotoperiod

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CENTRAL NERVOUS SYSTEM

NEUROSECRETORY CELLS OF THE BRAIN

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CORPORA CARDIACA

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Neuroham al lo b e ^ j * X

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Glandular ________________ lobe

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PROTHORACIC GLANDS

CORPORA ALLATA

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Juvenile Horm ones

Peptidic Hormones

Peptidic Neurohorm ones

TARGET TISSUES

FIGURE 1. The elements of the neuroendocrine system that reg­ ulate development and homeostasis in insects.

together as JH. In studies of postembryonic development, JH is often referred to as the “ status quo” hormone as it appears to act to suppress the expression of adult characters. Differentiation to the adult stage requires a cessation of JH synthesis by the corpora allata. The regulation of JH synthesis in the corpora allata is not yet understood; it is presumed to be a complex balance between tropins and inhibins secreted by specialized neurosecretory cells of the brain. The corpora allata secrete JH into the hemolymph, where it is variously associated with binding proteins. JH acts on a variety of target tissues, the effects on epidermal cells being perhaps the best documented. Whereas the larval prothoracic glands histolyse during metamorphosis or after the larval-adult molt, the corpora allata resume activity in adult female insects, where they regulate reproduction (see below). In addition to these well-known elements of the neuroendocrine system, eclosion hormone, a neurohormone most extensively studied in the lepidoptera by Truman deserves closer attention. Originally characterized for its role in the specialized ecdy sis of emerging moths, it may be involved in all insect ecdyses. A neurosecretory hormone released in eclosing adults from the brain to the neurohemal organ, the corpora cardiaca, and the ventral chain of the segmental ganglia, it regulates the complex series of ecdysial motor programs. In larvae the synthesis or storage of a hormone with similar properties in bioassays is primarily in the ventral ganglia, although the chemical identity of the two neurohormones remains to be established. What is striking is the basic similarity of the body motions necessary for the eclosion of an adult lepidopteran and for a dipteran larva to shed its old larval cuticle.

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We are far from underderstanding the control of diapause. In general, factors such as photoperiod, temperature, and nutrition act on the brain which responds by neurosecretory control of the endocrine system. We can distinguish three types of facultative diapause. The first is best known from certain races of Bombyx mori where the conditions which the developing female encounters dictate whether the eggs she produces undergo embryonic diapause. In this case the neurosecretion is from the subesophageal ganglion and acts on the maternal ovary. The second type of diapause may be larval or pupal, and may be determined either in the same or earlier instars. The immediate cause for the cessation of development is a lack of PTTH secretion, and diapause may be broken by brain implants or ecdysteroid injections. In some species it is suggested that the corpora allata actively inhibit PTTH synthesis in the brain. The third type of diapause occurs in adults, e.g., Leptinotarsa decemlineata, where neurosecretions inhibit corpora allata activity. This is essentially a reproductive diapause where JH production, necessary for female fertility (see below), ceases, although general metabolism is also lowered in the case of overwintering. Reproduction The hormonal control of fertility is undoubtedly complex and there is probably more variation between species than is found in development. We will return to this point later. The first evidence for neuroendocrine regulation of female oogenesis came from studies of Wigglesworth, who showed that the corpora allata were necessary for egg production in Rhodnius, and suggested that this was a consequence of their synthesis of JH. It is now established that the production of JH by the corpora allata is required for vitellogenesis (Figure 2). The JH stimulates the fat body to synthesize vitellogenins, which are the pre­ cursors to the major yolk proteins, the vitellins, found in the insect egg. The corpora allata undergo cyclical changes in their synthesis of JH which are correlated with the egg maturation cycles. There are possibly two regulatory pathways of this activity. The most important of these is by way of neurosecretions, both allatotropins and allatohibins, the latter for example, repress JH synthesis during the period of sexual maturation of female Diploptera. In addition it is suggested that the corpora allata may be regulated by the ovary and/or ecdysteroids. Because the larval prothoracic gland cells do not persist in adult insects, it was assumed that the molting hormone was not present and therefore that ecdysteroids had no function in adult insects. Recently, titer studies have revealed non-negligible levels of ecdysteroids in both males and females, and in the latter they are concentrated in the ovaries. In Locusta, ecdysone is synthesized by the ovarian follicle cells and this synthesis is controlled by the medial NSC. We argue later that the bulk of this ecdysone and its derivatives are for transfer to the egg for the later regulation of embryonic development. However, we cannot exclude the possibility that in other insect species these ecdysteroids are released into the hemolymph where they may act on the brain, corpora allata, or fat body to provide a fine regulation of oogenesis. Embryonic Development The question has been repeatedly raised over the last 25 years whether certain aspects of embryonic development are under a neuroendocrine control similar to that of later stages of development. The data available so far actually point to a link between ecdysteroids and embryonic molting. In all cases investigated, the eggs of insects have been shown to contain peak concentrations of ecdysteroids at the stages of embryonic molting; in vitro experiments have demonstrated that exogenous ecdysteroids trigger anticipated cuticle depositions in explanted embryonic appendages. These results strongly suggest that ecdysteroids control embryonic molting. In some insects newly laid eggs contain, as mentioned earlier, a large supply of ecdy­ steroids of maternal origin. These molecules are present as complex conjugates. It is believed

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5

/

Exteroception

Propioception

CENTRAL NERVOUS SYSTEM

NEUROSECRETORY CELLS OF THE BRAIN

CORPORA CARDIACA

I CORPORA ALLATA

Juvenile Hormones

I FAT BODY

Vitellogenin

t OOCYTE (Vitellin)

FIGURE 2. insects.

Main axis of control of vitellogenesis in female

that the hydrolysis of the maternal conjugated ecdysteroids accounts for the peaks of free ecdysone monitored during embryogenesis (see Figure 3), at least during the earlier stages. It is not known whether or not the embryonic prothoracic glands engage in de novo ecdysone biosynthesis once they have differentiated. During advanced embryonic development, juvenile hormone is also present and apparently exhibits several peaks of concentration, namely at the last embryonic molt. It is probable that one of its functions at this stage is the control of the expression of larval characteristics, as is the case during later development. The origin of JH during embryogenesis awaits further investigation. How the titers of ecdysone and JH, which obviously fluctuate during embryogenesis, are regulated is unknown. The possible role of neurohormones in this regulation remains speculative.

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CRC Handbook o f Natural Pesticides CENTRAL NERVOUS SYSTEM NEUROSECRETORY CELLS OF THE BRAIN

____

CORPORA CARqiACA

i_____

FCTH

FOLLICLE CELLS AND OOCYTE I

l

■egg / laying • /

TRANSFER OF MATERNAL CONJUGATED ECDYSTEROIDS

OOCYTE MATURATION EMBRYOGENESIS

/ /

^ FREE ^ ECDYSTEROIDS

ACCUMULATION OF INACTIVATION CONJU­ GATES OF ECDY­ STEROIDS

CONTROL OF EMBRYONIC MOULTING

FIGURE 3. Proposed scheme for the control of embryonic events (e.g., early embryonic molting) by maternal ecdysteroids.

Homeostasis In addition to the neurosecretions that regulate development and reproduction, insects have a highly developed neurosecretory system. Among the functions they control are Malpighian tubule excretion, rectal absorption, lipid mobilization, blood sugar levels, myotropism, color change plasticication, tanning, etc. These hormones will be considered in detail in this volume by Mordue and Morgan; here we merely signal their existence. VULNERABILITY AND SPECIFICITY IN THE NEUROENDOCRINE SYSTEM Against the background of the previous section it may be helpful if we outline the general problems of insect control via the neuroendocrine system and speculate on the elements which may provide the means of designing more efficient, or more species-restricted, insect regulators. From our overview several strategies are suggested. They include the suppression of synthesis of ecdysiotropins, ecdysteroids, or JH, the premature presentation of the hor­ mones, or a persistent presence of hormones at stages in development when they would normally be eliminated. Among the consequences, we can imagine the blockage of embryonic development, the blockage or retardation of larval-adult development, and adult sterility. All of these effects have been realized by hormonally based treatments and have generally shown considerable variation between species. In particular the application of juvenoids (JHs or analogs) to larvae results variously in the production of supernumerary larval instars, or, in the case of the highter Diptera, in pupal and adult abnormalities. We should of course

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emphasize that the strategy to be adopted may depend on the particular threat posed by a given insect species. Thus, a migratory species, whose economic importance results from the damage to crops by a voracious larval form, should not be treated so as to produce supernumerary giant larval stages, even if this effectively prevents the majority of the population reaching the reproductive adult stage. It may also be possible to interfere with diapause, either to prevent it, for example, so as to severely reduce the overwintering population, or to induce permanent diapause. Finally, a more detailed knowledge of eclosion hormone may enable us to physically disrupt the molting process. Insects are perhaps most vulnerable at the molt when they expose the new flexible cuticle which must be expanded and hardened. Failure to discard the remains of the previous cuticle is often fatal in both the Hemimetabola and Holometabola. Practical considerations must include the method of presentation of the hormone or analog. Experimental treatments of insects in vivo consist essentially of topical applications, feeding, injections, and surgical procedures. While all four contribute to our understanding of the neuroendocrine system, only the first two are practical for insect regulation. The hormonal control of insect populations is not solely a human strategy. Many plant species appear to defend themselves by the production of ecdysteroids or juvenoids. The yews and ferns are a particularly rich source of ecdysteroids (usually termed phytoecdysteroids to distinguish them from the zooecdysteroids of insects and crustacea) some of which are hormonally very active in insect bioassays (e.g., cyasterone) and which appear more resistant to the degradative enzymes of insects than the endogenous zooecdysteroids. That ecdysteroids can be effective when fed to insects is perhaps surprising, given the hostile environment of the digestive tract containing inactivation enzymes, but has been demon ­ strated in ecdysteroid-deficient larval mutants of Drosophila which will commence gene activity related to pupariation very shortly after transfer from normal medium to medium supplemented with ecdysteroids. Similarly, the efficacy of natural juvenoids was shown by the now famous problem of Slama, who was unable to rear Pyrrhocoris apterus at Harvard University under apparently identical conditions to those used successfully in Czechoslo­ vakia. The cause of the problem lay in the balsam fir, used as a major component of the American-made paper towel in the rearing cage. This was subsequently shown by Bowers to contain a juvenile hormone analog, juvabione, which caused embryonic lethality and supernumerary larval instars in the Pyrrhocoris colony. Indeed it was the relative specificity of juvabione, when tested on a number of insect species, that encouraged the extensive synthesis and screening of juvenoids of the last 15 years. Feeding is mostly a chance effect, except when deliberately introduced into the insect diet, such as feeding cattle with juvenoids so as to control parasitic flies, or similarly, spraying plants to control aphids. In the main, it is necessary to rely on surface contact, and this requires that the applied compound is sufficiently stable in the environment to control a population in a number of developmental stages. When one considers that critical periods for hormone action studied in the laboratory are often very brief indeed, the im ­ portance of this latter point is apparent. If we first consider ecdysiotropins, then we must admit despite recent advances in bioassay, notably the in vitro stimulation of prothoracic glands to secrete ecdysteroids, we lack a good deal of basic knowledge. We can hope that PTTH will be successfully characterized in Manduca in the near future, and that this will be a prelude to a more general survey of PTTHs, at least in the major experimental species. Similarly, the ecdysiotropin of the median NSC that regulates ovarian ecdysone synthesis in Locusta is an obvious starting point for structural studies. Until these, and possibly other ecdysiotropins, are purified sufficiently for comparison, we will not know the extent of variation in the neurohormones between species or within a species at different developmental stages, nor possible points of inhibition of their synthesis by antihormones. One unintended interpretation of the in vitro stimulation

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of prothoracic glands by brain extracts is the idea that the neurohormones are synthesized and act immediately as a rapid trigger for ecdy steroid synthesis. There exists a considerable body of data accumulated on “ head critical” periods for molting, which shows that this often precedes the time of prothoracic gland stimulation — not by hours, but by several days. In addition, the discovery that the corpora allata act as the neurohemal organ for PTTH in Manduca suggests that there are further controls to the release from the brain, storage and subsequent release to stimulate the prothoracic gland, that merit attention. Similar remarks may be made regarding the eclosion hormone, in that chemical charac­ terization in a number of species and developmental stages, and the regulation of storage and release from the neurohemal organs will be required before this can be assessed for its potential in insect growth regulation. In both cases it would be encouraging if the molecules were specific to insects rather than related to vertebrate neurohormones. The ecdysteroids have rarely been considered as a means of insect control, largely because of the widespread distribution of steroids in the animal kingdom, and the fears that analogs might prove potent not only in arthropods, but also in other phyla. Indeed, there are a number of reports of ecdy steroid effects on cyclic AMP (cAMP) levels in mammalian tissues. Insects are unable to synthesize the steroid nucleus and therefore rely on dietary sterols. While many insects obtain cholesterol directly, others possess the necessary enzymes for modifying plant sterols. In the larvae of most insects so far studied, cholesterol is the apparent starting point for ecdy steroid synthesis. The conversion of cholesterol into ecdysone starts, according to present knowledge, by modifications on the steroid nucleus and proceeds by hydroxylation on the side chain. Within the prothoracic glands, there are postulated differences in intermediates between species, including the order of the terminal hydroxylations. Once it has been secreted into the blood, ecdysone is converted to a series of metabolites, namely via hydroxylation, epimerization, and conjugation which results in a network of related molecules. The distribution of the various metabolites may change according to developmental stage and species. Most of the enzymes of ecdysteroid metabolism so far characterized have their counterparts in vertebrates, suggesting a high degree of conservation of hormonal synthesis and degra­ dation. Thus, although azasterols have proved effective inhibitors of sterol metabolism in insects, and have considerable experimental importance, their potential range is too broad for use as insecticides. While differences exist in the ovarian synthesis of ecdysteroids, notably the accumulation of 2-deoxyecdysone as a storage product for transfer to the egg, we have no reason to believe that the basic enzymology differs in these two major ecdysteroid synthesizing tissues. Another aspect of exogenous ecdysteroids and their effects on insect development is the rapid degradation of applied hormones at times other than when the endogenous titers are high. It is possible to induce premature molting, or an abortive molt when not all tissues are competent to molt, but this requires the application of massive doses of hormone. Injected hormone, at least tenfold higher than the endogenous hormone, may have a halflife of minutes in noncompetent larval stages. In competent stages the molt is premature but normal. If the synthesis of ecdysone and its conversion to 20-hydroxy ecdysone are difficult targets, we may still hope that the newer studies on conjugation may reveal weaknesses in ecdysteroid regulation. The majority of structural and metabolic studies on ecdysteroids have used the larval prothoracic gland and have assumed that titers were a simple balance between synthesis and degradation followed by excretion. However, the literature is scattered with reports of the “ synthesis” of ecdysteroids in larvae and pupae without prothoracic glands and of the discovery of conjugated ecdysteroids in hormonal extracts. The complexity of the situation is slowly being revealed. We have discussed the importance of conjugation, release, and reconjugation of ecdysteroids in Locusta embryos, which appear to regulate embryonic cycles prior to the development of functional prothoracic glands (see Figure 3). We expect parallel

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progress in studies of holometabolous pupae where massive amounts of ecdysteroids are synthesized and degraded at a time when prothoracic gland cells are degenerating and, like the embryo, there is no possibility of excretion. Now that structural studies of these conjugates have started, we may hope to learn something of the enzymology involved and possibilities for inhibition, either to prevent the release of active ecdysteroids, or to prevent the reduction in bioactivity necessary for normal development. The JHs offer perhaps more scope for insect control, and we must consider in this respect the natural JHs, their analogs, both natural and synthetic, and the anti-JHs, typified by the precocenes. The list of JHs has recently been extended from three to five by the addition of JHO and iso-JHO, discovered initially in Manduca embryos and subsequently in other insect embryos. Stage and species analyses are still rather limited. An early observation that JHIII was usually the only JH detected in the Orthoptera and Isoptera, led to the hypothesis that this was the primitive JH and the only one to be found in the Hemimetabola. The presence of JHI, JHII, and JHIII in the Holometabola, notably in Lepidopteran species, was suggested to reflect an evolution and diversification of hormonal roles, JHI and II controlling metamorphosis and JHIII acting as the gonadotropin in adult females. This area is open to fresh speculation, and more importantly, new experimentation. It is not surprising that JHs should be finally chemically characterized in embryonic extracts, given the classical view that JH titers decrease throughout larval and pupal development. What will be interesting to determine is whether JH synthesis is restricted to functional embryonic corpora allata, or whether maternal transfer, with or without conjugation, is involved. As well as species variation in absolute amounts and proportions of JHs in different developmental stages, there are also differences in JH binding proteins. Two general classes may be distinguished although there are insufficient data to assess the variation within each class. The first group are the hemolymph lipoproteins, having a high molecular weight, high capacity, and low affinity for JHs. These are widespread and although JH may be recovered from lipoprotein extracts, many authors doubt the biological significance of this association. In certain species, a second class of binding protein has been detected, having a low molecular weight and high affinity. In these species it seems unlikely that much hormone remains with the lipoproteins as there is sufficient capacity to bind most of the circulating JH throughout development. These proteins are conventionally considered transport proteins, those of the second class are capable of protecting JH from degradation by general hemolymph esterases and, in vitro, they have been shown to have a synergistic effect on JH activity. The Manduca larval binding protein has an affinity for JHO that is higher than that for JHI or JHII and much higher than that for JHIII. This difference is important when considering the fate of injected hormones, as JHIII will be poorly protected from degradation compared to the other JHs. Evidently stage-specific binding proteins or different relative affinities for the JHs may act as a means of regulation of bioactivity. If the role of the binding proteins appears complex and differs between species, similar remarks apply to the JH specific esterases that degrade JHs even when associated with the binding proteins. Originally, studies concentrated on hemolymph esterases, following their titers with respect to JH titers. Depending upon the stage and insect studied, these esterases appear induced, repressed, or independent of increases in JH titer. Equally, studies in the Diptera have shown that while there is little or no hemolymph JH-specific esterase activity, the fat body and body wall, which are JH target tissues, have high JH esterase activity. The esterase type and titer may vary between tissues in a single insect stage, one following the JH titer, the other remaining constant. The importance of the binding proteins and esterases for our purposes lies in the potential of binding proteins to protect juvenoids from general esterase activity, and the variation in esterase activity between species. Elements similar to these must be involved when there are striking differences in the in vivo and in vitro activity of JH analogs or other insect

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growth regulators. Variation in such systems may also be reflected in the effective doses of juvenoids, which may differ by orders of magnitude even within the same suborder of insects. These binding proteins are presumably not involved in the responses of target tissues in vitro, although other JH binding proteins have been described in such tissues. Interestingly, very active JH analogs may show little competition with the natural hormones in displacement binding assays, so the role of these proteins is not clear. If they are cellular receptors, as has been proposed, then it seems that JH analogs may exert their effects via a different subcellular mechanism, despite their chemically similar structures. The action of precocenes now appears to depend very much upon their chemical similarities to JH. They are specifically metabolized in the corpora allata by the enzymes of JH bio­ synthesis into powerful cytotoxins, which results in the chemical allatectomy of sensitive species. Considerable variation exists, however, in their ability to penetrate to the target tissue. As with JH analogs, this probably reflects their binding to the hemolymph proteins present in different species, which may afford some protection against their degradation in the hemolymph. Studies of these binding proteins in different species, and their affinities for precocenes, may be an important step in designing efficient insect regulators, as will be studies of the specificities of the degradative enzymes in the insect defense. The variation described between species in the hormonal control of reproduction is po­ tentially of interest for the design of selective inhibitors. While the majority of the insect orders use JHs to regulate the synthesis of yolk proteins, according to several authors, the Diptera may rely on ecdysteroids for parts of this regulation. JH is nonetheless involved in the control of fertility in this insect order. It has been our intention in this introduction to present a broad overview of the major themes in insect neuroendocrinology, in both the holometabolous and hemimetabolous in­ sects. We have chosen to highlight some areas of research which may prove fruitful in the near future. We leave the reader the pleasure to discover the details in the chapters that follow. GLOSSARY Ablation: The surgical removal of a tissue, or its destruction by localized heat treatment. Allatectomy: The inactivation of the corpora allata by ablation or chemical treatments. Allatohibins and Allototropins: Generic nouns for inhibitors and stimulators respectively of corpora allata. Often used for uncharacterized factors. Apolysis: See under Molt. Apterygota: Primitive group of the Insecta, wingless, with slight or no metamorphosis. Chorion: The outer layer of an insect egg, separated from the vitelline membrane by a wax layer. The insect eggshell. Competence: A functional definition for a response to hormone. Different tissues acquire competence at different times in an instar. In some experimental systems the acquisition of competence is itself hormonally mediated. Corpora Allata: Endocrine glands of ectodermal origin in the retrocerebral gland system. The site of juvenile hormone synthesis and secretion. Active in larval and adult stages.

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GLOSSARY (continued)

Corpora Cardiaca: The corpora cardiaca lie between the brain and the corpora allata and are neurohemal organs for brain hormones. Critical Period: Experimental definition of the time of hormone synthesis and release. Ligatures applied after the critical period do not prevent development in both halves of the ligatured insect. Cyclorrhaphous Diptera: Also commonly known as the “ higher” Diptera. The last larval stage ends with pupariation when the larval cuticle is tanned to form a puparia. This puparia protects the insect during pupation and metamorphosis and is finally shed when the adult fly emerges. This group includes many species of experimental, medical, and economic importance, e.g., Dro­ sophila, Calliphora, Lucilia, Sarcophaga, Glossina (Tsetse), Stomoxys (stable flies), and Tephritidae (fruit flies). Cytotoxins: Chemicals which act to inhibit cellular metabolic processes, this inhibition leading to tissue breakdown. Diapause, Facultative, and Obligative: Diapause is a cessation of development which may occur at different stages in different insects. If facultative, it is usually triggered by adverse conditions and enables the insect to survive until there is an improvement in conditions. Obligative diapause is found in insects adjusted to regular seasonal changes in their environment which may, for example, require overwintering followed by a synchronized resumption of development in the spring. Ecdysis: See under Molt. Ecdysal Motor Programs: The series of movements mediated by the ganglia necessary for insect eclosion. Ecdysiotropins: Factors that elicit ecdysteroid synthesis. Used by some authors as equivalent to prothoracicotropic hormone (PTTH). Otherwise, a generic noun for such hormones and unchar­ acterized factors. Ecdysteroids: The preferred generic noun for ecdysone and its metabolites. In early literature, the term ecdysones is common in this sense. Current usage restricts ecdysone to a - ecdysone, (2(3, 3(3, 14a, 22R, 25-pentahydroxy-5(3-cholest,-7-en-6-one), the ecdysteroid secreted by pro­ thoracic glands. Eclosion: The act of hatching of a larvae from an egg or an adult insect from the pupa. Endopterygota: Insects carrying the wing buds internally during larval stages, generally synonymous with the Holometabola. Exopterygota: Insects carrying the wind buds externally during larval stages. With few exceptions, hemimetabolous insects. Fat Body: A complex tissue arranged in sheets or strands of cells. The site of synthesis and storage of major proteins and important in many metabolic processes, including steroid metab­ olism. In some insects there are two distinct fat bodies, the larval fat body, which is

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CRC Handbook o f Natural Pesticides

GLOSSARY (continued)

histolysed following metamorphosis, and the adult fat body which replaces it in the mature adult insect. Follicle Cells: See Ovarian Follicle Cells. Gonadotropin: A factor stimulating testis developments in males, or oogenesis or vitellogenesis in females. Hence, juvenile hormone in adult females is sometimes described as exerting a gonado ­ tropic role, distinct from its action in larval development. Hemolymph: Insect blood, often pigmentless, it fills the body cavity, bathing all tissues. Circulation is ensured by a tubular heart which pumps hemolymph toward the head. Its role in respiration is minor as this is provided mainly by the extensive tracheal system. It provides hydrostatic pressure to stabilize the exoskeleton, a reserve for sugars, proteins, and water, and carries hormones to their target tissues. Hemimetabola: Insects undergoing incomplete or gradual metamorphosis, the larval or nymphal stages resemble more or less the mature insect. Holometabola: Insects undergoing complete metamorphosis during a nonfeeding pupal stage. The larvae may bear no resemblance to the mature insect. Homeostasis: The means by which various vital processes (e.g., heartbeat, respiration, osmotic pressure of the blood, etc.) are maintained constant despite external changes (e.g., temperature, dessication, diet). Inhibins: Generic noun for uncharacterized factors inhibiting endocrine functions. Instar: The period between molts, most commonly used for larval stages. Juvenile Hormones (JHs): A related group of sesquiterpenoid hormones differing in the carbon skeleton as follows: JHIII — C 16, JHII — C 17, JHI — C 18, JHO — C 19, iso-JHI — C 19. Involved in the regulation of metamorphosis, reproduction, diapause, caste determination, etc. Juvenoids: Natural and synthetic compounds having JH activity in in vivo or in vitro bioassays. Ligature: A noose around an insect tied with hair or cotton to prevent hemolymph flowing between two body compartments. Particularly used to isolate abdomens from the head and thorax so as to study endocrine factors. Ligatures applied prior to hormone release may lead to development in the anterior compartment of the ligatured (or ligated) insect, while the posterior, deprived of hormone, does not develop. Malpighian Tubules: The major organ of excretion and ion balance. They are involved in uric acid extraction from the hemolymph, which they pass to the hind gut. In pharate stages they may accumulate uric acid, its salts, and other minerals as dry mass. Molt: An insect molt consists of two stages, apolysis and ecdy sis. In apolysis the epidermal cells detach from the old cuticle and synthesize a new cuticle, often reabsorbing com-

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GLOSSARY (continued)

ponents from the old cuticle. At ecdy sis the remains of the old cuticle are split and shed and the insect emerges to expand and harden its new cuticle. In certain insects embryos synthesize a number of embryonic cuticles before hatching, thus strictly undergoing cycles of apolysis, or embryonic cycles, rather than embryonic molts. A similar problem of terminology exists in connection with the cyclorrhaphous Dipteran puparia which is not shed until the emergence of the adult. Neurohemal Organs: An organ acting as the site of storage and/or release into the blood of neurosecretions. Oogenesis: Egg production. Insects generally produce a yolk-filled egg surrounded by a vitelline membrane and protected by a chorion. Ovarian Follicle Cells: The layer of somatic cells surrounding the developing oocyte. The site of synthesis of ecdysteroids for embryonic development in Locusta. The follicle cells synthesize the protective chorion or eggshell. Pharate: Between apolysis and ecdy sis (see Molt), an insect stage may be described as pharate, that is contained within the old cuticle. Precocenes: A class of antijuvenile hormones that disrupt the functioning of the corpora allata. Proprioreceptors (Propriorecptive Organs): Sense organs stimulated by body movement or muscle tension; includes “ stretch recep ­ tors” used in Rhodnius to initiate ecdysteroid secretion after a blood meal. Prothoracic Glands: Glands of the retrocerebral gland system producing ecdysteroids, principally ecdysone, during larval development. Generally they histolyse during metamorphosis and are absent in adult insects. Prothoracicotropic Hormone (PTTH): A brain neurohormone that stimulates ecdysteroid production in the prothoracic glands. Storage may occur between synthesis and release; it is a matter of debate as to whether the neurohemal organs are the corpora allata or corpora cardiaca. Pterygota: The main group of Insecta, consisting of two divisions, the exopterygota and the endopterygota. Ring Gland: In the Cyclorrhaphous Diptera the corpus allatum, corpus cardiacum, and the prothoracic gland fuse into a complex organ known as the ring gland or Weissman’s organ. Segmental Ganglia: In principle, each of the thoracic and abdominal segments has a ganglion in the ventral chain controlling movement. However, in some insects/stages such as adult Diptera, these may fuse into a single compound ganglion. Subesophageal Ganglion: First ventral nerve center after the brain, formed from the three posterior head segment ganglia. Involved in controlling insect movement via the thoracic and abdominal segmental ganglia. Supraesophageal Ganglia: The brain, formed from the fusion of ganglia of the three anterior head segments, the

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GLOSSARY (continued)

largest region being the protocerebrum containing neurosecretory cells regulating the neuroendocrine system, which is laterally expanded to form the optic lobes. Synergist: A compound or tissue having no hormonal activity, but enhancing the hormonal activity of other compounds. Fat body is often described as having a synergistic effect in in vitro assays, presumably as a result of its metabolism of ecdysteroids to more biologically active derivatives. Tropins (Trophins): Generic noun for factors stimulating endocrine functions. In contrast to other physiological systems, the convention in insects is to use tropin (tropos-directing toward) rather than trophin (trophikos-feeding). Vitelline Membrane: In general, a fragile membrane beneath the chorion, originating from the egg after yolk deposition is completed. In the higher Diptera, however, it receives components from the follicle cells so that dechorionated eggs kept in moist conditions will develop normally. Vitellogenesis: The deposition of yolk proteins in the maturing egg. Vitellogenins are synthesized in the ovary and fat body transported to the egg where they are modified and stored as vitellins. Viviparous: Viviparity is scattered throughout the Insecta. It takes various forms, from the retention of eggs until hatching up to the release of mature final stage postfeeding larvae. Especially important in the parthenogenetic generations of the Aphididae (aphids). Weissmann’s Ring or Organ: See Ring Gland.

REFERENCES 1. Wigglesworth, V. B., Insect Hormones, Oliver & Boyd, Edinburgh, 1970. 2. Gilbert, L. I. and Frieden, W ., Eds., Metamorphosis, A Problem in Developmental Biology, Plenum Press, New York, 1981. 3. Raabe, M ., Insect Neurohormones, Plenum Press, New York, 1982. 4. Hoffmann, J. A., Ed., Progress in Ecdysone Research, Developments in Endocrinology, Vol. 7, Elsevier/ North Holland, Amsterdam, 1980. 5. Gilbert, L. I., Ed., The Juvenile Hormones, Plenum Press, New York, 1976. 6. Pratt, G. F. and Brooks, G. T., Eds., Juvenile Hormone Biochemistry, Action, Agonism and Antagonism, Elsevier/North Holland, Amsterdam, 1982. General Reading in Insect Physiology and Endocrinology Chapman, R. F., The Insects: Structure and Function, 2nd ed., Hodder & Stoughton, London, 1971. Imms, A. D., A General Textbook o f Entomology. Including the Anatomy, Physiology and Development and Classification o f Insects, 9th ed., Methuen & Co., London, 1957. Richards, O. W. and Davies, R. G ., Imms' Outlines of Entomology, 6th ed., Chapman & Hall, London, 1978. Wigglesworth, V. B., The Principles o f Insect Physiology, 6th ed., Methuen & Co., London, 1968. Doane, W. W ., Role of hormones in insect development, in Development Systems: Insects, Vol. 2, Counce, S.T. and Waddington, C. H., Eds., Academic Press, New York, 291. Hagedorn, H. H. and Kunkel, J. G ., Vitellogenin and vitellin in insects, Annu. Rev. Entomol., 24, 475, 1979. Richards, G ., Insect hormones in development, Biol. Rev., 56, 501, 1981. Wyatt, G. R., Insect hormones, in Biochemical Actions o f Hormones, Vol. 2, Littwack, G., Ed., Academic Press, New York, 1972, 385.

Volume III: Insect Growth Regulators, Part A

15

THE ECDYSTEROIDS Ian D. Wilson

INTRODUCTION Arthropods are encased in a hard chitinous exoskeleton which both supports and protects the internal organs and muscles. Once hardened, the exoskeleton is incapable of growth or modification, and forms an unexpandable prison for the animal within. In order to grow and develop through the larval stages to the adult, insects (and crustaceans) must periodically shed this exoskeleton. This process is loosely termed molting and covers a number of events. These include the secretion of a new cuticle under the old, separation of old and new cuticles (apolysis), shedding of the old cuticle (ecdysis), and, following a rapid increase in size, the tanning of the new cuticle (sclerotization). Molting and the acquisition of adult characteristics are known to be under hormonal control, ecdysone and related compounds causing molting, and another hormone, juvenile hormone (JH), regulating the type of molt. Insects exhibit two distinct types of development. Holometabolous insects (moths, flies, bees, ants, etc.) pass through a series of larval stages (instars) before forming a pupa, emerging from this as an adult. Hemimetabolous insects (locusts, grasshoppers, aphids, roaches, etc.) develop regularly through a series of instars, each of which looks similar to the last, gradually acquiring more adult characteristics, until they finally molt directly to the adult, without an intermediary pupal stage. In the presence of JH both hemi- and holometabolous larva will molt to give another larva. In the absence of JH a hemimetabolous insect will molt to the adult. A holometabolous insect in the presence of a lesser amount of JH will give rise to a pupa, from which the adult will emerge on the next molt, which occurs in the absence of JH (Figure 1). That molting might be under endocrine control was suggested by the work of Kopec1 as long ago as 1922. He demonstrated that caterpillars of the gypsy moth Lymantria dispar surgically deprived of their brains did not molt. Severance of the nerve cord had no such effect. Further, to prevent molting, the operation had to be carried out before a “ critical period” had elapsed (7 days after the previous molt). From these and other experiments he concluded that molting was not under nervous control, but that the brain produced a hormone which caused molting. The significance of these experiments was not realized at the time and it was not until the advent of the experiments of Fraenkel2 on pupation in Calliphora and Wigglesworth’s3 research on Rhodnius that further advances were made. This work unambiguously demon­ strated the presence of circulating factors in the hemolymph which effected molting. Fraenkel was able to show that ligation of Calliphora larvae prevented the pupation of the portion posterior to the ligation. Blood from larvae which had just pupated, when injected into this unpupated portion of the ligated larva, could induce pupation. These experiments were to prove of great importance to the later isolation of the active principles involved, as they provided the basis for a bioassay by which their purification could be monitored. Wiggles­ worth showed that decapitation of Rhodnius could prevent molting if accomplished before a critical period following a blood meal had elapsed (10 to 20 days depending on the insect involved). Despite decapitation insects could survive for periods of over 1 year, but they did not molt. However, an insect decapitated after this critical period had elapsed, connected by a capillary tube to an insect decapitated shortly after feeding, would induce the second insect to molt. Wigglesworth was later able to show that certain neurosecretory cells would induce molting when implanted in the abdomen of brainless insects. The gland responsible for the production of the molting hormone was identified as the prothoracic gland by the work of Fukuda,4 and this was extended by the studies of Williams5 on the silkworm Hyalophora cecropia.

16

CRC Handbook o f Natural Pesticides

A CD

egg

larva

JHjMH

JH^flH

JH*MH

imago

jh ^MH

MH

B egg

larva

pupa

imago

FIGURE 1. The involvement of juvenile hormone (JH) and molting hormone (MH) at various stages in the life cycle of holo- and hemimetabolous insects.

Our present understanding of molting suggests that it is initiated by neurosecretory cells in the brain. These cause the release of a proteinaceous prothoracicotropic hormone (brain hormone or PTTH) from the Corpus cardiacum.6 This stimulates the prothoracic glands or their equivalent (e.g., the ring gland in Diptera) to produce and release ecdysone. Ecdysone is transported by carrier proteins in the hemolymph to a variety of tissues, such as fat body and Malpighian tubules and there converted to 20-hydroxyecdysone. This, by its actions on the target tissues, sets in train the series of events which culminate in ecdy sis.

ISOLATION AND STRUCTURE DETERMINATION Once the existence of a molting hormone had been demonstrated, isolation and identifi­ cation could be attempted. Such studies began before World War II with the studies of Plagge and Becker7 in Berlin. They worked with extracts of Calliphora pupae, and their isolation attempts were only possible because of the development of a suitable bioassay, which allowed each stage of the purification to be monitored. This “ Calliphora bioassay” was based on the ability of extracts containing molting hormones, when injected into the posterior portion of ligated larvae of Calliphora erythrocephala, to cause puparium for­ mation. The quantity of hormone required to elicit 50 to 70% pupation was termed the “Calliphora unit” , and corresponds to approximately 0.01 |xg of pure ecdysone. These promising early studies were halted by Becker’s death in action in 1941. However, Butenandt and Karlson began further studies in 1943 on the isolation of the molting hormone. They found that pupae of Bombyx mori, the commercial silk moth, provided a more convenient starting material than Calliphora, being available in the huge quantities required. The first molting hormone to be isolated was ecdysone8 (in 1954). From 500 kg of silkworm pupae 25 mg of the pure hormone was obtained. Further fractionation of the residue from the extract yielded 2.5 mg of a second more polar compound. This was named P-ecdysone, in order to distinguish it from ecdysone (or a-ecdysone).9 The determination of the structure proved to be a long and complex task (reviewed in Reference 10). Initially from carefully dried samples, a molecular formulas of C l8H30O4 or C l8H3204 were suggested, while the UV absorbtion (241 to 242 nm in ethanol) and IR absorption (1657 cm -1) indicated that an a,p -unsaturated ketone was present. X-ray crys­ tallography provided a more reliable molecular mass of 464, also confirmed by mass spec-

Volume HI: Insect Growth Regulators, Part A

17

trometry. From these results the molecular formulas were revised to suggesting that the hormone might be a steroid. In the first instance the unsaturated en-one was assigned to the C-ring. However, Nuclear Magnetic Resonance (NMR) studies and work on the hydrogenation of model compounds implicated a 7-en-6-one grouping, combined with a labile hydroxyl at C-14. Further evidence for a hydroxyl at C-14 was provided by its ready elimination on treatment with acid (1%, 5 N HC1 in ethanol) to give the products shown in Figure 2, a reaction characteristic of 14 a - and p -hydroxyls. The structure of the side chain was deduced from the fragmentation of ecdysone in the mass spectrometer and the biosynthesis of ecdysone from cholesterol made a hydroxyl at C-3 likely. Oxidation of ecdysone using periodate provided evidence for a vicinal diol, which would be located at either C-2 or C-4. The complete structure and stereochemistry of ecdysone was finally determined using X-ray crystallography by Huber and Hoppe11 in 1965 (11 years after the first isolation), and shown to be 2p,3p,14a,22R,25S -pentahydroxy 5p-cholest-7 -en -6 -one (Figure 3). At about this time it was also demonstrated that extracts of crustaceans could elicit a positive response in the Calliphora bioassay. This led to the identification of a second molting hormone by Hampshire and Horn12 in 1966. From 1 ton of the marine crayfish Jasus lalandei, 2 mg of the pure active hormone, “ crustecdysone” , were isolated. This was identified as 20-hydroxyecdysone, and was shown to be identical with the P-ecdysone already isolated from Bombyx. Shortly after the isolation and identification of these hormones in arthopods came the exciting discovery of a range of related compounds in plants. This began with the report by Nakanishi et a l.13 that the Chinese antitumor remedy “ Pai-ju -chim” (derived from the leaves of Podocarpus nakaii Hay) contained a polyhydroxy steroid, struc­ turally related to ecdysone. This compound, ponasterone A, was the 25-deoxy-20-hydroxy analog of ecdysone. Shortly afterwards, Galbraith and Horn14 showed that another ecdysteroid, 20-hydroxyecdysone, was present in Podocarpus elatus. These discoveries were important to the development of ecdysone research, as concentrations of hormones in plants were many times higher than in arthropods, providing a much more convenient source of these hormones. The ready availability of material from plants greatly facilitated structure elucidation and the range of analogs provided a chance to study structure-activity relation­ ships. Since these early studies, over 70 structurally related compounds have been isolated from arthropods and plants (the great majority from plants). Arthropod-derived compounds are described as zooecdysteroids,15 while plant-derived compounds have been termed phytoecdysteroids.15 The isolation procedures used for ecdysteroids have been reviewed1016 and are also described in this handbook series (see CRC Handbook o f Natural Pesticides: Meth­ ods, Volume 2). Once the identity of ecdysone and 20-hydroxyecdysone had been established, attention was turned toward the biosynthesis, mode of action, and function of the molting hormones. The biosynthesis of ecdysone from cholesterol in isolated prothoracic glands was achieved in 1974.1719 However, no conversion of ecdysone to 20-hydroxyecdysone was seen with isolated PTGs although this conversion was rapid with other tissues. As the major circulating hormone in the hemolymph was 20-hydroxyecdysone, and as this compound appeared in - 1 felt that it represented certain bioassays to be more active than ecdysone, some workers202 the “ true” molting hormone. In this scheme of insect development, ecdysone was regulated to the status of a prohormone. In fact, as Karlson22 has pointed out, ecdysone, in the strict definition of the term, is not a prohormone, and 20-hydroxyecdysone is best described as an active metabolite. The mode of action of ecdysteroids appears to be at the level of gene transcription. As early as I96023 an effect of ecdysone on the puffing of the giant chro ­ mosomes of Chironomus tentans was observed. This observation was important in its own right, leading to the present concept of the mode of action of steroid hormones in general.

18

CRC Handbook o f Natural Pesticides

FIGURE 2.

The effect of acid in causing the dehydration of ecdysteroids possessing a 14a-hydroxyl group.

WAVENUMBER (CM - 1 )

FIGURE 3.

An IR spectrum of ecdysone obtained as a KBr disk.

The hypothesis of ecdysteroidal action is as follows: on entering the target cell the hormone combines with specific receptor molecules (proteins) and enters the nucleus. Here, the hormone-receptor complex combines with specific sites on the chromatin, resulting in the initiation of transcription. This leads to the production of mRNA, and the biosynthesis of the proteins required for molting and development. While it is clear that at least during embryonic development 20-hydroxyecdysone is quantitatively the most important ecdyster­ oid, a hormonal function for ecdysone cannot be excluded. Indeed the huge amounts of ecdysone found during embryonic development, compared to much lower levels of 20hydroxyecdysone, might be used as an argument for a hormonal function for ecdysone during this stage of development. Whether ecdysone or 20-hydroxyecdysone represents the “ true” molting hormone, the central role of these compounds in the cuticulogenesis and molting of arthropods is clear. For plants the situation is more complex. The large quantities present in some species suggest an important function for these compounds. In the absence of an unambiguous demonstration of “ plant hormone” activity, the use of these compounds for defense against phytophagous insects seems likely. Antifeedant, insecticidal, and insectistatic properties have been shown for ecdysteroids but the levels required are rather high,24 and it would seem that this area deserves more study in order to clarify the benefits which phytoecdysteroids confer on the plants which produce them. Since the isolation of the first compound, ecdysone, there has been a vast amount of research on the insect molting system and the role of ecdysteroids in insect and crustacean development. Clearly in a single chapter it is impossible to provide a detailed review of all the areas in which research has been performed and this has not been attempted. What has

Volume III: Insect Growth Regulators, Part A

19

been done is to provide comprehensive tables covering the available physical and chemical properties of the ecdysteroids, and briefly discuss their potential as pesticides. For accounts of the early work in this field the reader is directed to the reviews of Horn,10 Rees,25 Morgan and Poole,26 and Nakanishi.2 Much useful information is to be found in Invertebrate En­ docrinology and Hormonal Heterophylly, edited by Burdette,28 and in Progress in Ecdysone Research, edited by Hoffman,29 which provides a useful compilation of more recent work. This includes reviews on analysis, biosynthesis, structure and activity, and metabolism of ecdysteroids.

THE ECDYSTEROIDS Structures are given in Table 1 together with the molecular formulas, molecular mass, melting point, and the source from which they were first isolated, both plants and arthropods indicated by phyto and zoo, respectively. The first synthesis of these ecdysteroids, where applicable, is also given. Each compound is also numbered, and this numbering system is used in all subsequent tables on the physical and biological properties of the ecdysteroids. See Table 2 for UV absorption properties of ecdysteroids. These tables are complete for compounds discovered before January 1983. Compounds isolated after this date are included in the Appendix. No rigid definition of what constitutes an ecdysteroid exists, and indeed such a definition may not be possible. The selection of compounds included in the following tables is therefore necessarily arbitrary. In general, compounds not isolated from arthropods have been selected on the basis that they have structural similarities to ecdysteroids, even though in some cases they are not biologically active as molting hormones. The list also includes substances isolated from insects, which may represent intermediates in the biosynthesis of ecdysteroids from cholesterol. In the system of nomenclature adopted for naming ecdysteroids in Table 1, compounds such as biosynthetic intermediates are referred to as derivatives of ecdysone, rather than attempting to name them from cholestane. For example, 3p -hydroxy -5a-cholestan -7 -en -6 one becomes 2,14,22,25 -tetradeoxyecdysone. Where compounds were isolated simultane­ ously by a number of groups and given a variety of trivial names, the name in general use is given above the structure. While other trivial names are given below, these alternative names are not used in the other tables.

THE ULTRAVIOLET (UV) SPECTRA OF ECDYSTEROIDS Almost invariably ecdysteroids contain a 7-en-6-one group. This results in their UV spectra being characterized by a relatively strong absorption, \ max of approximately 243 nm, with an e of approximately 10 to 1600 (ecdysone \ max 242, e 12,400, in ethanol). In the absence of a 14 -a-hydroxyl, a hypsochromic effect is observed,10 whereby the \ max shifts to 248 nm. Some compounds, the cheilanthones for instance (and some biosynthetic intermediates), lack the 7-ene function, and thus have no appreciable absorption at 240 nm. The absence of an absorption in this region need not therefore rule out a compound as an ecdysteroid. Other ecdysteroids have UV absorptions in addition to that at 242 nm. This may be due to esterification to a UV absorbing molecule, as in the case of cinnamate72’73 and coumarate73 esters.This results in the 3-p-coumarate ester of 20-hydroxyecdysone having absorptions at 315, 300, 250, 238, and 212 nm. Alternatively additional conjugation, such as that found in kaladasterone80 and stachysterone B 100 due to the presence of a dien-one results in a shift in the \ max to 298 nm. As a final example, the unusual ecdysteroid calonysterone36 has a UV spectrum with peaks at 222, 244, and 294 nm resulting from a trien-one. While too unspecific for use in identification (the absorption is shared by too many classes of compounds) the UV absorption of ecdysteroids is useful in their isolation. This is because

1

No.

Acetylpinnasterol

Structure 30

31

32

33

Ajuga decumbens (phyto)

Ajuga incisa (phyto)

Ajuga japonica (phyto)

516 225— 235 C2i)H40O8

506 240 c 29h 460 v

480— C27H42O v

Ref.

Laurencia pinnata (phyto)

1st isolation & 1st synthesis

488 105— 107 C29H440 6

mol wt mp (°C) Composition

Table 1 THE ECDYSTEROIDS CRC Handbook of Natural Pesticides

8

7

6

5

Ho

----1 J If OH

j

HI

|j OH

I

o

F bh

H Ov^sl/^A L=J hoA A v ^ o OH

ir

OH HO 1

Calonysterone

T

OH

HO o1h nII

Amarasterone B

T

Amarasterone A

T

° H ,° y

Ajugasterone D

T°h

476 234— 235 C27H4CA

508 284— 285 ^29^4807

508 210—211 ^29^48^7

478 234— 237 ' ^27^42^7

(phyto)

(phyto)

(phyto)

(phyto)

I p o m o e a c a lo n y c tio n

C y a th u la c a p ita ta

C y a th u la c a p ita ta

A ju g a n ip p o n e n sis

36

35

35

34

Volum e III: In sec t G ro w th R e g u la to rs, P a r t A

21

9

No.

Capitasterone

Structure Cyathula capitata (phyto)

504 234— 235

39

39

Cheilanthes tenuifolia (phyto)

Cheilanthes tenuifolia (phyto)

466 235— 238 c 27h 46o 6

450 225— 228

^27^460.-5

38

Solatium xanthecarpum (phyto)

37

Ref.

562 251 c .„h mo 4

C29H4407

1st isolation & 1st synthesis

mol wt mp (°C) Composition

Table 1 (continued) THE ECDYSTEROIDS CRC Handbook of Natural Pesticides

16

15

14

13

5H

qh

T

h

T

r

0

y

3H

'

1

S

^

1

0

T

| OH

I

T

i

0

p

i

3H

1

3-Dehydroecdysone OH

H0 "

1

OH HO -

Dacrysterone

H0^

H0 -

1 F

0

roH

[uH

y -o

Cyasterone 22-acetate

y

H0w nX

T

hoideoxy-2 0 -hydroxyecdysone OH

" ^

j T j j P >H

X

/ t y OH HO* \ / K / Hs 2,22-Dideox}/ecdysone

2-Deoxy-20- tiydroxyecdysone OH HOJ: i T oh

T T foH HOM r0

22-Deoxyecdysone

496 149— 153 ^27^4408

448 — ^ 27^4405

432 — C27H4404

464 250—252 £-27^4406

448 — ^27H440 ' 5

M a n d u c a s e x ta

(zoo)

(phyto) (zoo)

(zoo)

P o d o c a r p u s e la tu s

B om byx m ori

L o c u s ta m ig r a to r ia

J a s u s la la n d e i

(phyto) (zoo)

(zoo)

B le ch n u m m in u s

M a n d u c a s e x ta

56 57

55

54

48 53

52

Volum e III: In sec t G ro w th R e g u la to rs, P a r t A

25

29

27

26

No.

Epicyasterone

H O

Ecdysone (a -ecdysone)

Structure

C29H4408

520 274— 275

C 27H 43O 9P

543 —

C29H 47O 7

Cyathula capitata (phyto)

Schistocerca gregaria (zoo)

51

62

Schistocerca gregaria (zoo) Synthesis (from ecdysone)

506 —

61

58 59,60

Bombyx mori (zoo) Polypodium vulgare (phyto) Synthesis

464 237— 239 (170 hydrate)

^•"27^44^6

Ref.

1st isolation & 1st synthesis

mol wt mp (°C) Composition

Table 1 (continued) THE ECDYSTEROIDS CRC Handbook of Natural Pesticides

34

33

32

31

30

|| OH

OH 'H PO

' roH

0

|| OH

6h

X^T0 ” oh

HO \ /

X

1\ O

[ | | OH

1

PO ' H

3-Epi-20,26-dihydroxyecdysone OH OHi

If

/ \ J1A1 s

11 0

HO^/s

3-Epi-26-Hydroxyecdysone OH

HO'”'

3-Epi-20-Hydroxyecdysone OH HO i! 'H 1X PO

"”v-—AH HO* V

1

I

ll °H

3-Epiecdysone

i 0

1 1

3-Epi-2-deoxyecdysone OH

496— ^27H440 8 '

480 — C27H44O7

480 — C27H44O7

464 — ^27^4406

488 264— 265 C27H44O5

Manduca sexta (zoo)

Manduca sexta (zoo)

Manduca sexta (zoo) Synthesis (from 20-hydroxyecdysone)

Manduca sexta (zoo) Synthesis (from ecdysone)

Schistocerca gregaria (zoo) Blechnum volcanicum (phyto)

69

68

66

67

66

65

63 64

V olum e III: In sec t G ro w th R e g u la to rs , P a r t A

27

20 -Hydroxy -5a -ecdysone (p -ecdysone, crustecdysone)

36

38

20 -Hydroxyecdysone (p -ecdysone, crustecdysone ecdysterone)

Structure

35

No.

C29H470 8

523 219— 220

C27H 44O 7

480 278

C 2 7H 4 4 O 7

480 241— 242.5 (150— 151 hydrate)

mol wt mp (°C) Composition

Dacrydium intermedium (phyto) Synthesis (from 20-Hydroxyecdysone)

Cyanothis arachnoidae (phyto)

72 72

34

71 71

Achyranthes fauriei (phyto) Synthesis

12

Ref.

48 70

1st isolation & 1st synthesis Jasus lalandei (zoo) Blechnum minus (phyto) Synthesis

Table 1 (continued) THE ECDYSTEROIDS CRC Handbook of Natural Pesticides

43

42

41

40

39

I

T

T

jr

HO

oh

T

M

T

V

y

6h

l A

V

o M | T

M

-

j

H

HO

/ ^ | T OH

HO

Integristerone B

-

H O ^ A L X /i

HO

H°

OH !

OH 1

|

OH i

Integristerone A

l T iT 6 h H O ^ s /T X ^ HX

Inokosterone

y

9H

26-Hydroxyecdysone

0

h

^O H

POH

\

n oh

V ° H

i X

HO

OH I X ° h

20-Hydroxyecdysone 3-/?-coumarate

o

244— 246

^

C 2 7 H 44 O 9

572 186— 190

4 96

c

480 225

C 2 7 H 44 O 7

480 252— 256

C 3 6 H 50O 9

626 265— 267

R h a p o n tic u m in te g r ifo liu m

R h a p o n tic u m in te g r ifo liu m

Synthesis

C a llin e c te s s a p id u s

(phyto)

(phyto)

(phyto)

(phyto) (zoo)

(zoo)

A c y r a n th e s f a u r ie i

M a n d u c a s e x ta

D a c r y d iu m in te r m e d iu m

79

78

75 76 77

74

73

V olum e III: In sec t G ro w th R e g u la to rs , P a r t A

29

47

46

45

44

No.

Isocyasterone

Structure

C28H46O7

494 172— 173

C28H46O7

494 263— 265

81 82

81

Podocarpus macrophyllus (phyto) Oncopeltus fasciatus (zoo)

Podocarpus macrophyllus (phyto)

80

Ipomea calonyction (phyto)

478 242— 243

C29H42O7

62

Ref.

Cyathula capitata (phyto)

1st isolation & 1st synthesis

520 —

mol wt mp (°C) Composition

Table 1 (continued) THE ECDYSTEROIDS CRC Handbook of Natural Pesticides

52

51

50

49

48

O

J ° H

i

T

|

0

ii 0H

f^OH

^OH

xt5 ^

P odecd yson e B

0

X t^

Pinnasterol

T T ll ° H H o ^ \/r v H0T

OH HO I

M uristerone A

hoM

\

OH HO I 1

M akisterone D

T T

X

OH^ HO i 1

M akisterone c

^-'27^42^6

46 2 125 — 127

C 27H42 0 5

4 4 6 198 — 201

^27^44^8

4 9 6 238 — 244

^29^48^7

508 244 — 246 (R ef. 102)

^29^48^7

508 263 — 265

Podocarpus elatus (phyto)

Laurencia pinnata (phyto)

Ipomoea calonyction (phyto)

Podocarpus macrophyllus (phyto)

Podocarpus macrophyllus (phyto)

84

30

83

81

81

Volume III: Insect Growth Regulators, Part A 31

86

Polypodium aureum (phyto)

494 251— 253

Ponasterone A

56

^-27^4405

464 259— 260

^"28^46^7

Podocarpus nakaii (phyto) Callinectes sapidus (zoo) Gecarcinus lateralis (zoo) Synthesis

72 72

Dacrydium intermedium (phyto) Synthesis (from 20 -hydroxyecdysone)

626 268— 270

Polypodine B 2-cinnamate

54

C36H50O9

85

Ref.

Polypodium vulgare (phyto)

1st isolation & 1st synthesis

496 255— 257

mol wt mp (°C) Composition

Polypodine B

Structure

53

No.

Table 1 (continued) THE ECDYSTEROIDS CRC Handbook of Natural Pesticides

61

60

59

58

57

{

T

if 6

h

HO' ^ H

hoA

T

H0

mT

T

J

HOv/ nlA /

OH Poststerone

0

1OH

'V^ °

"

j £

?

HO«y/\l 0^\^4

HOs.

Ponasteroside A (Warabisterone)

0

if

3H

HO

0

OH

°H

H0 Ponasterone C 2-cinnamate OH OH H° i ,

HO.

OK 9H H° !

Ponasterone C

t

h° r im >H ho' ' M Vo

OH HO 1

Ponasterone B

362 234— 236 C2 »H3 0 O5

626 278— 279.5 C3 3 H5 4 On

572 — ^3 6 ^ 4 4 0 5

496 270—272

464 — ^-2 7 ^ 4 4 0 5

Cyathula capitata (phyto) Calliphora stygia (zoo) Synthesis

Pteridium aquiliuum (phyto)

Dacrydium intermedium (phyto)

Podocarpus nakaii (phyto)

Podocarpus nakaii (phyto)

91 92 93

90

72

89

89

Volum e III: In sec t G ro w th R e g u la to rs , P a r t A

33

536 159— 161

65

C27H44O9

Cyathula capitata (phyto)

Achyranthes rubrofusca (phyto) Synthesis

344 230 c 19h 26o 5

C27H44O7

64

63

Lastrea thelypteris and Onoclea sensibles (phyto)

1st isolation & 1st synthesis

480 228— 231

mol wt mp (°C) Composition Cyathula capitata (phyto)

Precyasterone

Structure 520 — C29H440 8

62

No.

Table 1 (continued) THE ECDYSTEROIDS

98

96 97

95

94

Ref.

CRC Handbook of Natural Pesticides

70

69

68

67

66

^ t t ^1----

V

(JOH

1

OH

!

OH

;

OH

T T y 6h HoM^'T's^V

OH ^ " Y

Stachysterone D

T

T

- M

HO

H0^ h r0 Stachysterone C

n o ^ o y

1Jf

HO

0 Stactiysterone B

hoV

A,

ho

I /KT Mm h HoM 0 Stachysterone A

HO.

OH

Ht> = \ IX MVVX

Sogdisterone

f^O H

POH

pOH

462 245— 250 C27H420 6

462 235— 240 C27H420 6

462 — c 27H42o 6

462 — c 27H42o 6

496 —

S ta c h y r u s p r a e c o x

S ta c h y r u s p r a e c o x

S ta c h y r u s p r a e c o x

S ta c h y r u s p r a e c o x

(phyto)

(phyto)

(phyto)

(phyto)

(phyto)

S e r r a tu la s o g id ia n a

101

101

100

100

99

Volum e III: In se c t G ro w th R e g u la to rs , P a r t A

35

Structure

2,14,22,25-T etradeoxy-5 a-ecdy sone (5a -Deoxyviperidone)

2,14,22,25 -T etradeoxy-5 0 -ecdy sone (5 0 -Deoxy viperidone)

2 ,14,22,25-Tetradeoxy-7,8dihydroecdysone

2,22,25 -Trideoxyecdysone

No.

71

72

73

74

Bombyx mori (zoo) Synthesis

Locusta migratoria (zoo) Synthesis

416 213— 234 C27H 44O 1

^"27^46^2 -

402 142— 144

54 103

105 106

104 54 103

Peniocereus greggiia (phyto) Locusta migratoria (zoo) Synthesis

C27H 44O 2

C27H 44O 2

400 —

Ref. 102 103

st isolation & 1 st synthesis

Wilcoxia viperina (phyto) Synthesis

1

400 195— 197

mol wt mp (°C) Composition

Table 1 (continued) THE ECDYSTEROIDS CRC Handbook of Natural Pesticides

Vipendinone

77

^'29^46^8 -

522 198— 199

C27H44O4

432 —

Vitex megapotamica (phyto)

Wilcoxia viperina (phyto)

Wilcoxia viperina (phyto)

416 208— 210 C27H 44O 3

Ajuga turkestanica (phyto)

496 —

108

102

102

107

a

Probably an artefact.

Note: In addition to these compounds a number of polar conjugates of some zooecdysteroids to sulfate, glucuronic acid, phosphate, and glucose have been identified in insects (on the basis of enzymic hydrolysis). This list updates that produced by Hetru and Horn109 in Progress in Ecdysone Research.

78

Turkesterone

75

Volume III: Insect Growth Regulators, Part A

38

CRC Handbook o f Natural Pesticides

it provides a means for their detection on thin-layer chromatography (TLC) (by fluorescence quenching), and after high pressure liquid chromatography (HPLC). As little as 10 ng of ecdysone can be detected using HPLC depending on the purity of the sample. Both UV absorption10 and the decrease in absorption seen following reduction with sodium borohydride10 have been used to determine the amount of ecdysteroids present in plant extracts. The ready dehydration of the 14-a -hydroxyl in the presence of acid10 is a useful test for its presence in a suspected ecdysteroid; the products have characteristic absorptions at 293 and 244 nm.10 FLUORESCENCE In the presence of sulfuric acid or aqueous ammonia, ecdysteroids can be induced to fluoresce.10 Typical values for excitation and emission wavelengths are in the region of 380 and 431 nm, respectively. However, variations in excitation and emission wavelength and fluorescence intensity are observed with structure. 112114 Thus, for ecdysone optimum ex ­ citation and emission wavelengths of 380 (ex) and 425 nm (em) are obtained, whereas 20hydroxyecdysone has values of 376 (ex) and 430 nm (em) respectively. In addition, the fluorescence yield of ecdysone is twice that of 20-hydroxyecdysone.114 Other factors which affect the fluorescence are the solvent, the final concentration of sulfuric acid, and the duration and temperature of the reaction.113 In a recent study114 it was concluded that the best solvent was ethanol, with maximal fluorescence obtained using 50% sulfuric acid (v/ v). Fluorescence has been selected as the basis of a number of quantitative assays for ecdysteroids113114 with limits of detection of approximately 5 ng for ecdysone and approx­ imately 10 ng for 20-hydroxyecdysone. The linear range of the fluorescence is narrow due to selfquenching, and blank values may be high (due to Rayleigh scattering and Raman emission of the solvent).116 The emission, excitation, and relative fluorescence of a number of ecdysteroids are given in Table 3. Other steroids do not interfere.114 OPTICAL ACTIVITY Ecdysteroids are chiral, and therefore rotate the plane of polarized light. Measurements of the optical activity of ecdysteroids have usually been made in methanolic solution using the sodium D line. These compounds usually display a modest specific optical rotation(a)D of between 60 and 80°. Little use has been made of this property except to determine purity and occasionally to show the identity of two compounds. Our limited understanding of the theory of chirality and optical activity has prevented its use in a predictive way. A number of factors are important when determining the optical activity of a compound including the temperature, solvent, and concentration. The available information for the ecdysteroids is summarized in Table 4. OPTICAL ROTATORY DISPERSION (ORD) When the optical rotation of a chiral substance is measured across the UV and visible spectrum an ORD curve is obtained. The optical rotation changes slowly with wavelength except near an absorption band (where a rapid change occurs which may result in a change of sign). These rapid changes in rotation observed near absorption bands are termed cotton effects. ORD may be expressed either as , the molecular rotation, or a, the molecular amplitude. The molecular amplitude, a, is the difference between the molecular rotation at the extremum of longer wavelength and the molecular rotation at the extremum of shorter wavelength divided by 100 (i.e., the vertical distance between the peak and trough in the ORD curve). 115 Both a and have been used in the construction of Table 4 which summarizes

Volume III: Insect Growth Regulators, Part A Table 2 THE UV ABSORPTION PROPERTIES OF ECDYSTEROIDS Compound Acetyl pinnasterol Ajugalactone Ajugasterone B Ajugasterone C Ajugasterone D Amarasterone A Amarasterone B Calonysterone

Capitasterone Carpesterol Cheilanthone A Cheilanthone B Cyasterone Cyasterone 22-acetate Dacrysterone 3-Dehydroecdysone 3-Dehydro -20 -hydroxyecdysone 24(28)-Dehydromakisterone A 2-Deoxyecdysone 2-Deoxyecdysone 22-phosphate 22 -Deoxyecdysone 2 -Deoxy -20 -hydroxyecdysone 2,22 -Dideoxyecdy sone 2,22 -Dideoxy -20 -hy droxyecdy sone 20,26 -Dihydroxyecdy sone Ecdysone Ecdysone 3 -acetate Ecdysone 22 -phosphate Epicyasterone 3-Epi-2 -deoxyecdysone 3 -Epi-ecdysone 3 -Epi-20 -hydroxyecdysone 3-Epi-26 -hydroxyecdysone 3 -Epi-20,26 -hydroxyecdysone 20 -Hydroxyecdysone 20 -Hydroxy -5a -ecdysone 20-Hydroxyecdysone 2-acetate 20 -Hydroxyecdysone 2 -cinnamate

20-Hydroxyecdysone 3 -p-coumarate

26 -Hydroxyecdysone Inokosterone Integristerone A Integristerone B Isocyasterone Kaladasterone

\nax --­ 233 244 243 244— 246 244 244 222 224 294 242 — No strong No strong 243 240 240 242 242 245 244 — — 243 — 245 245 242 242 — 242 243 — 245 245 245 240 242 — 276 248sh,223 217 315 300 250sh,238 212 245 243 245 240 242 298

e — 15,700 10,675 10,320 11,800 — — 27,000 13,500 7,800 — —

Chromophore dien-one en-one en-one en-one en-one en-one en-one trien-one

en-one en -one benzoate UV absorption UV absorption 12,000 en-one 11,750 en-one 11,400 en-one 10,360 en-one en-one — 14,130 en-one 12,900 en-one — — en-one — 12,100 en-one en-one — 12,000 en-one 10,400 en -one 12,400 en -one — en-one — — — en -one 11,480 en -one — en-one 10,800 en-one en-one — en-one — 12,670 en-one en-one — — en-one 21,000 Cinnamate 19,200 20,100 22,500 Coumarate 19.000 19.000 12,800 11,600 en-one 12,100 en-one 12,670 en -one 12,590 en-one — en -one 10,800 dien -one

Solvent

rvci.

— Methanol Methanol Methanol Ethanol — — Methanol

30 31 32 33 34 35 35 36

— —

37 38

— — Ethanol — Ethanol Methanol Ethanol Ethanol Ethanol — — Ethanol — Ethanol Methanol Ethanol Methanol — — Ethanol — Ethanol Methanol Methanol Ethanol — — Ethanol

39 39 40 41 42 43

66 47 48 51 52 48 54 55 57 10 61 51 62 64 65 67 68

69 10

71 34 72

73

Methanol Ethanol Ethanol Ethanol — Methanol

74 10

78 79 62 80

40

CRC Handbook o f Natural Pesticides Table 2 (continued) THE UV ABSORPTION PROPERTIES OF ECDYSTEROIDS

No.

Compound

46 47 48 49 50 51 52 53

Makisterone A Makisterone B Makisterone C Makisterone D Muristerone A Pinnasterol Podecdysone B Polypodine B

54

Polypodine B 2 -cinnamate

55 56 57

Polypodoaurein Ponasterone A Ponasterone B

58 59

Ponasterone C Ponasterone C 2 -cinnamate

60 61 62 63 64 65 66 67 68 69 70 71 72 73

Ponasteroside A Poststerone Precyasterone Pterosterone Rubrosterone Sengosterone Sogdisterone Stachysterone A Stachysterone B Stachysterone C Stachysterone D 2,14,22,25-T etradeoxy -5a -ecdy sone 2,14,22,25-T etradeoxy-5 0 -ecdy sone 2 ,14,22,25 -Tetradeoxy -7,8 dihydroecdysone 2,22,25 -T rideoxyecdy sone Turkesterone Viperidone Vipendinone Viticosterone E

74 75 76 77 78

Xmax (nm)

c

243 243 243 244 236 — 244 243 317 216 248sh,223 217 244 244 241 320 244 276 250sh,224 218 245 240 244 243 239 240 242 248 298 242 243 245 — —

11,000 19.200 18,500 19.200 — 12,400 — 9,100 10,300 — 9,550 10,700 12,800 10,500 10,950 13,500 — —

240 244 237 229 —

13,000 8,913 10,780 10,800 —

12,400 11,000 14,800 — 8,900 — 13,200 11,750 160 21,000 19,200 20,100 10,000 12,400 —

Chromophore

Solvent

Ref.

en-one en-one en-one en-one en-one dien-one en-one en-one

Methanol Methanol Ethanol Methanol Methanol — Ethanol —

81 81 81 81 83 30 84 85

Cinnamate

Ethanol

72

en-one en-one en-one

Ethanol Methanol —

86 13 89

en-one Cinnamate

Methanol

89 72

en -one en-one en-one en-one en-one en-one en-one en-one dien-one en-one en-one en-one — —

— Ethanol — — — — Methanol Methanol Methanol Methanol Methanol Chloroform — —

90 93 94 95 96 98 99 100 100 101 101 103 — —

en-one en-one en-one en-one en-one

Ethanol Ethanol — Chloroform —

103 107 102 102 108

Note: sh is used to denote a shoulder on the side of a larger absorption.

the data for ecdysteroids. The UV absorption of the ecdysteroids, due to the unsaturated carbonyl in the B ring, results in two cotton effects.10 The amplitude of the positive cotton effect (for ecdysteroids, a is typically 40 to 80°, when measured in dioxan) can be used to obtain structural information about the A/B ring fusion. The values of a 40 to 80° are typical of 5|3-ecdysteroids (cis fused), while 5a-ecdysteroids exhibit much larger effects (see 5a 20-hy droxyecdy sone). In the absence of the A7 double bond a large negative cotton effect is observed (of the order of —160 to —190° for the chielanthones). The ORD curves of most ecdysteroids are superimposable, since the majority of ecdysteroids differ in regions distant from the 6-one.

Volume III: Insect Growth Regulators, Part A

41

Table 3 FLUORESCENCE PROPERTIES OF ECDYSTEROIDS AFTER TREATM ENT W ITH SULFURIC ACID IN ETHANOL (50% v/v)114

No. 13 19 22 26 35 41 46 50 56 61

Compound Cyasterone 2 -Deoxyecdysone 2-Deoxy -20 -hydroxyecdysone Ecdysone 20-Hydroxyecdysone Inokosterone Makisterone A Muristerone Ponasterone A Poststerone

Excitation wavelength (nm)

Emission wavelength (nm)

380 389 380 380 376 378 378 410 382 380

432 440 431 440 430 432 430 460 432 430

Relative intensity 40 79 40 100 47 46 45 9 40 56

CIRCU LA R DICHROISM (CD) CD is the unequal absorption of right and left circularly polarized light plotted as a function of wavelength. CD can be expressed as molecular elliplicity (0), specific elliplicity (i|/), and differential dichroic absorption Ae.115 In the same way as ORD, the technique may be used to obtain structural information about the A/B ring junction. The amplitude of the cotton effects for trans ring-fused ecdysteroids is greater than that for the corresponding cis fused compounds.28 Additionally 5p -OH compounds differ from 5p -H compounds. In a further development of the technique Harada and Nakanishi116 have devised a method for determining the absolute configuration of vicinal hydroxyls using the “ dibenzoate chirality rule” . The ecdysteroid is converted to a 2,3-dibenzoate and the shape of the CD curve used to obtain the absolute configuration, (e.g., the 2,3-dibenzoate of ponasterone A). This approach has been extended to determine the spatial dispositions of nonadjacent hydroxyls117 and applied to the 2,3,11-tribenzoate of ajugasterone C. The increasing and widely applicable use of high resolution NMR and mass spectrometry (MS) as tools for structure elucidation has overshadowed the more limited usefulness of ORD and CD studies. In addition few laboratories are equipped for ORD and CD studies, further limiting the application and usefulness of the technique for ecdysteroids. The available CD data for ecdysteroids are given in Table 4 and IR spectra are given in Table 5. TH E IN FR A R ED (IR) SPECTRA OF ECDYSTEROIDS The presence of a number of hydroxyl groups on most ecdysteroids ensures a strong absorption in the IR spectrum in the region of 3340 to 3500 cm - 1. The a,p - unsaturated ketone results in a characteristic cyclohexenone absorption at 1640 to 1670 cm - 1, with a weaker alkene stretch at approximately 1612 cm -1 usually present. In the 5p-hydroxyecdysteroids, such as polypodine p, the en-one absorption is shifted to about 1690 cm - 1. Loss of the 7-ene (seen with the chielanthones) has a similar effect with the absorption occurring at 1684 c m " 1. Some of the more unusual phytoecdysteroids contain either lactones, absorbing at between 1700 and 1780 cm - 1, or additional double bonds. Thus, kaladasterone with its dien-one has absorptions at 1650 and 1605 cm - 1. The 2-cinnamates, and 3-/?-coumarate esters show an ester carbonyl absorption at 1720 c m " 1, and an additional carbonyl absorption

Acetyl pinnasterol Ajugalactone Ajugasterone B Ajugasterone C Ajugasterone D Amarasterone A Amarasterone B Calonysterone

Capitasterone Carpesterol Cheilanthone A Cheilanthone B Cyasterone Cyasterone 22-acetate

Dacrysterone

3-Dehydroecdysone 3-Dehydro -20 -hydroxy ecdysone 24(28)Dehydromakisterone A

2-Deoxyecdysone 2 -Deoxyecdysone 22-phosphate 22-Deoxyecdysone

9 10 11 12 13 14

15

16 17 18

19 20 21

Compound

1 2 3 4 5 6 7 8

No.

—

—

----­

—

20

— — [m] + 43 x 102 At 340 + 1-27 A* 288 — 0.66 [0]37O 0 9,282 [0] 328 + [0] 275,285 0 [0]253 16,700 [ 0 ]240 0

—

— — —

—

----­

±

—

—

a

a a a a

a

a a

+

—

—

43 dioxan

—

—

_

— 167 dioxan - 192 dioxan + 65 dioxan + 53

—

+ 75 dioxan + 82 dioxan — + 54 dioxan

—

— — — —

a , mol amplitude , mol rotation

At nmM absorptivity [0 ], mol ellipticity — Ae 244 + 11.6 dioxan —

Optical rotatory dispersion

Circular dichroism

—

[a]2D° + 54.4 MeOH

—

_

—

[a]D -1- 64.5 pyridine

—

—

—

—

[