Advances in the Biorational Control of Medical and Veterinary Pests 9780841233584, 0841233586, 9780841233591

Table of contents :
Content: PrefaceIntroduction1. Putting the Rational into BiorationalCurrent and Future Outlooks for Medical and Veterinary Pest Control2. Overcoming Insecticide Resistance: Proactive Detection and Management of Insecticide-Resistant Human Lice3. Current Evidence, New Insights, Challenges and Future Outlooks to the Use of Spatial Repellents for Public Health4. Present and Future Outlook: The Potential of Green Chemistry in Vector Control5. Attractive Toxic Sugar Baits (ATSB): A Novel Vector Management Tool6. Molecular and Nano-Scale Alternatives to Traditional Insecticides for in Situ Control of Mosquito Vectors7. Getting Them Where They Live-Semiochemical-Based Strategies To Address Major Gaps in Vector Control Programs: Vectrax, SPLAT BAC, Trojan Cow, and SPLAT TK8. G-Protein-Coupled Receptors: Their Expression, Function and Regulation in Insecticide ResistanceCharacterizing Molecular Targets and Pest Biology for the Development of New Technologies9. Insecticidal Activity and Physiological Actions of Matrine, a Plant Natural Product10. Development and Evaluation of Push-Pull Control Strategies against Aedes aegypti (Diptera: Culicidae)11. Monoterpenoid Isovalerate Esters as Long-Lasting Spatial Mosquito Repellents12. Plant Terpenoids Modulate ?-Adrenergic Type 1 Octopamine Receptor (PaOA1) Isolated from the American Cockroach (Periplaneta americana)13. Potential for Utilization of Spatial Repellents in Mosquito Control Interventions14. Determination and Comparison of the Cuticular Thickness Across Several Insecticide Resistant and Susceptible Populations of the Common Bed Bug, Cimex lectularius L.Editors' BiographiesAuthor IndexSubject Index

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Advances in the Biorational Control of Medical and Veterinary Pests

ACS SYMPOSIUM SERIES 1289

Advances in the Biorational Control of Medical and Veterinary Pests Edmund J. Norris, Editor Iowa State University Ames, Iowa

Joel R. Coats, Editor Iowa State University Ames, Iowa

Aaron D. Gross, Editor Virginia Tech University Blacksburg, Virginia

John M. Clark, Editor University of Massachusetts - Amherst Amherst, Massachusetts Sponsored by the ACS Division of Agrochemicals

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

Library of Congress Cataloging-in-Publication Data Names: Norris, Edmund (Edmund J.), editor. | Coats, Joel R., 1948- editor. | Gross, Aaron (Aaron D.), editor. | Clark, John Marshall, 1949- editor. Title: Advances in the biorational control of medical and veterinary pests / Edmund J. Norris (Iowa State University), Joel R. Coats (Iowa State University), Aaron D. Gross (Virginia Tech University), John M. Clark (University of Massachusetts - Amherst), [editors]. Description: Washington, DC : American Chemical Society, [2018] | Series: ACS symposium series ; 1289 | Includes bibliographical references and index. Identifiers: LCCN 2018035203 (print) | LCCN 2018046504 (ebook) | ISBN 9780841233584 | ISBN 9780841233591 Subjects: LCSH: Vector control--Biological control. | Biological pest control agents. Classification: LCC RA639.3 (ebook) | LCC RA639.3 .A39 2018 (print) | DDC 632/.96--dc23 LC record available at https://lccn.loc.gov/2018035203

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2018 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

Contents Preface .............................................................................................................................. ix

Introduction 1.

Introduction: Putting the Rational into Biorational ............................................. 3 Daniel Strickman

Current and Future Outlooks for Medical and Veterinary Pest Control 2.

Overcoming Insecticide Resistance: Proactive Detection and Management of Insecticide-Resistant Human Lice ...................................................................... 9 J. Marshall Clark, Ju Hyeon Kim, Kyong Sup Yoon, Barry R. Pittendrigh, and Si Hyeock Lee

3.

Current Evidence, New Insights, Challenges and Future Outlooks to the Use of Spatial Repellents for Public Health ......................................................... 25 Nicole L. Achee and John P. Grieco

4.

Present and Future Outlook: The Potential of Green Chemistry in Vector Control .................................................................................................................... 43 Edmund J. Norris, Lyric Bartholomay, and Joel Coats

5.

Attractive Toxic Sugar Baits (ATSB): A Novel Vector Management Tool ....... 63 Daniel L. Kline, Gunter C. Muller, Amy Junnila, and Rui-de Xue

6.

Molecular and Nano-Scale Alternatives to Traditional Insecticides for in Situ Control of Mosquito Vectors ......................................................................... 75 Paul M. Airs and Lyric C. Bartholomay

7.

Getting Them Where They Live—Semiochemical-Based Strategies To Address Major Gaps in Vector Control Programs: Vectrax, SPLAT BAC, Trojan Cow, and SPLAT TK ............................................................................... 101 Agenor Mafra-Neto, Jesse Saroli, Rodrigo Oliveira da Silva, Leonard E. Mboera, Graham B. White, Woodbridge Foster, Kim Li Spencer, Babak Ebrahimi, Daniel E. Sonenshine, Thomas Daniels, Elison E. Kemibala, Rafael Borges, and Teun Dekker

vii

8.

G-Protein-Coupled Receptors: Their Expression, Function and Regulation in Insecticide Resistance ...................................................................................... 153 Ting Li and Nannan Liu

Characterizing Molecular Targets and Pest Biology for the Development of New Technologies 9.

Insecticidal Activity and Physiological Actions of Matrine, a Plant Natural Product .................................................................................................................. 175 Jeffrey R. Bloomquist, Shiyao Jiang, Jennina Taylor-Wells, Liu Yang, and Yu-Xin Li

10. Development and Evaluation of Push-Pull Control Strategies against Aedes aegypti (Diptera: Culicidae) ................................................................................ 187 Ulla Gordon, Joachim Ruther, Ulrich R. Bernier, Andreas Rose, and Martin Geier 11. Monoterpenoid Isovalerate Esters as Long-Lasting Spatial Mosquito Repellents .............................................................................................................. 205 James S. Klimavicz, Caleb L. Corona, Edmund J. Norris, and Joel R. Coats 12. Plant Terpenoids Modulate α-Adrenergic Type 1 Octopamine Receptor (PaOA1) Isolated from the American Cockroach (Periplaneta americana) .... 219 Edmund J. Norris, Aaron D. Gross, Michael J. Kimber, Lyric Bartholomay, and Joel Coats 13. Potential for Utilization of Spatial Repellents in Mosquito Control Interventions ......................................................................................................... 237 Daniel L. Kline and Joyce Urban 14. Determination and Comparison of the Cuticular Thickness Across Several Insecticide Resistant and Susceptible Populations of the Common Bed Bug, Cimex lectularius L. .............................................................................................. 249 Reina Koganemaru, Kristopher Patton, and Dini M. Miller Editors’ Biographies .................................................................................................... 277

Indexes Author Index ................................................................................................................ 281 Subject Index ................................................................................................................ 283

viii

Preface Insect vectors pose a significant threat to public health and veterinary medicine throughout the world. It is estimated that more than 700,000 people die annually from complications associated with mosquito-borne disease alone, without accounting for the impact caused by other arthropod pests of public health importance. As these pest insect populations become increasingly resistant to classical synthetic insecticides, new chemistries and control approaches need to be rapidly developed, characterized, and deployed. Biorational control methods are technologies derived from natural chemistry or exploit the physiology of the arthropod pest in question to limit potential harm to non-target organisms. As the global population grows and continues to encounter new and old arthropod-borne diseases, it is paramount that future arthropod control technologies are safe and sustainable to our communities and the environment around us. This book highlights prominent research investigating novel biorational control approaches and chemistries aimed at decreasing pest insect populations that transmit disease agents. This volume and the chapters contained within resulted from the “Biorational Control of Medical and Veterinary Pests” symposium held at the 254th ACS National Meeting in Washington, D.C., August 20-24, 2017. The symposium was comprised of 25 papers in four sessions: Novel Tools and Targets, Bringing New Products to Market, Characterization of Insecticide Resistance, and Development and Future Potential of Spatial Repellents. We gratefully thank the authors for contributing to this volume. We are also thankful to the Agrochemicals Division of ACS for supporting our symposium. Edmund J. Norris dedicates this book to his family and wife, Lauren. Joel R. Coats dedicates this book to his wife, Becky. Aaron D. Gross dedicates this book to his excellent mentors, family and friends. John M. Clark dedicates this book to Professor John D. Edman, Ph.D., Entomology, UC Davis (retired), who first introduced him to vector biology.

Edmund J. Norris Department of Entomology Iowa State University Ames, IA 50011

ix

Joel R. Coats Department of Entomology Iowa State University Ames, IA 50011

Aaron D. Gross Department of Entomology (MC 0319) Virginia Tech Price Hall, Room 216A 170 Drillfield Drive Blacksburg, VA 24061

John M. Clark Veterinary and Animal Sciences University of Massachusetts - Amherst Amherst, MA 01002-9301

x

Introduction

Chapter 1

Introduction: Putting the Rational into Biorational Daniel Strickman* Senior Program Officer, Bill & Melinda Gates Foundation, Seattle, Washington 98102, United States *E-mail: [email protected]

The biorational approach is usually defined as a series of negatives centered around avoidance of synthetic chemicals, minimizing environmental damage, and lowering the risk of unpredicted toxicity. Widely-accepted biorational products in vector control, like Bacillus thuringiensis israelensis based larvicides, have had a tremendous impact. It may be useful to broaden the definition of biorational to other rational approaches, like biological control, mass release of sterile or genetically-modified insects, and highly targeted use of pesticides. The invention of effective products is a challenge, and the scientific community has been very successful at finding innovative ways of performing vector control. Development of those inventions into widely-used tools can be even harder than their discovery. Researchers can do their part in the process by maintaining contact with the many kinds of disciplines required to get a product to market. This Introductory Chapter highlights the impacts of previous technologies, the current state of our field, and the possibilities for continuing progress in biorationals for vector control.

“Biorational” is one of those terms that has been interpreted diverse ways. The definitions ascribe natural origins, often botanical, to a biorational pesticide active ingredient. Some go further, qualifying those active ingredients as less harmful to humans, non-target organisms, or the environment. Taken together and thinking a little about the “rational” part of the term, it should be possible to agree © 2018 American Chemical Society

that biorational interventions are targeted at the problem, with as little collateral damage as possible to surrounding biology, whether human or not. The natural origin of any active ingredient is a more complicated part of the definition. Of course, the botanical or other natural origin of an active ingredient is no guarantee of safety, but there is a persistent feeling that it is less likely to have unpredicted harmful effects than a synthetic chemical. That conclusion is not entirely based on logic, but it influences opinions that affect regulation and development. The usual definitions of biorational active ingredients emphasize what they are not: they are not synthetic, they are not damaging to the environment, and they are not toxic to non-target organisms. Considering the history of biorational active ingredients in medical and veterinary entomology, it is worth thinking about what they are, as well. Pyrethrin powder, derived from a species of chrysanthemum, has been used for centuries and inspired the synthetic pyrethroids that are largely responsible for a 50% decrease in malaria in sub-Saharan Africa since 2000. Bacillus thuringiensis israelensis (Bti) was discovered in the field in the 1970s and has been the basis of a mosquito larvicide that is non-toxic to mammals and most aquatic insects. Quwenling was an old Chinese herbal mosquito repellent derived from lemon eucalyptus and found to contain para-menthane-3,8-diol, a very useful repellent active ingredient that is particularly good against vectors of malaria. Finally, ivermectin, derived from bacterial compounds, is an effective acaricide and nematicide in cattle and was probably responsible for the near-elimination of cattle lice and cattle bot flies as economic pests in the United States. Biological control can easily be considered biorational, as well. Widely accepted examples of biorational interventions like Bti, which is a preparation of peptides from sporulating bacteria, can be extended to living insect pathogens like Bacillus sphaericus or polyhedrosis viruses. Although pathogens can be easily considered biorational, it is less common to think this way about larger biocontrol agents. Possibly this is because the larger ones cannot be sprayed in imitation of synthetic pesticides. There is a rich menu of biological control agents that could be considered biorational in a broader definition. Outside of the two bacterial agents cited above, there is a wide variety of pathogens that affect vector species. Of the large variety of viruses, bacteria, fungi, protists, and nematodes that parasitize mosquitoes and other vectors, few have found a consistent role in intentional control. Larger predators like aquatic carnivorous plants, dragonflies, birds, etc. have sometimes been managed for vector control, but their more key role is presumably in the background of ecological services that reduce the reproductive potential of noxious species. Recently, there have been multiple efforts to use mosquito biology against itself through various forms of mass release of altered mosquitoes. One example using unnatural combinations of the endosymbiotic bacterium Wolbachia in Aedes albopictus was approved by the US Environmental Protection Agency as a biorational pesticide. Other ideas would seem to be biorational as well. Oxitec, a subsidiary of Intrexon, has developed a genetically-modified Aedes aegypti that produces only males when an antidote is withdrawn from the rearing medium. This strain can be reared normally in the presence of the antidote to produce male mosquitoes for release. When enough are distributed through a location, 4

they mate with wild-type females and the next generation is only male. This modification of the sterile insect technique (SIT) has resulted in excellent control of populations of this vector in some cities. The United Nations’ International Atomic Energy Agency has worked with Sudan and South Africa to develop programs for release of radiation-sterilized males of Anopheles arabiensis derived from a strain in which only males are resistant to dieldrin. In this case, the sterile males inseminate wild-type females with useless sperm and reduce the subsequent population. The male-only effect of dieldrin resistance is a means of sorting out females that are not needed for release. There are also attempts to develop genetically-modified mosquitoes that either suppress malaria transmission or that kill the subsequent generation, combined with a genetic construct that assures 100% transmission of the trait. In one vision for this technology, such “gene drive” mosquitoes could be released in small numbers and convert an entire wild population to the modified trait. It could be argued that genetically modified mosquitoes, especially those with gene drive, somehow violate the environmental safety aspect of biorationality, but others would point out that such methods accomplish control with great precision. Chemical delivery methods can also achieve very precise application of interventions, minimizing exposure of non-target organisms and minimizing the total amount of pesticide required. Autodissemination exposes female mosquitoes to the very powerful growth regulator, pyriproxyfen, which they carry to oviposition sites, poisoning their own larvae and the larvae of any other females that put their eggs there. Large scale demonstration of this technique has been achieved in Brazil and a commercial device is currently marketed and registered. Experimental devices take advantage of behavior by Anopheles gambiae and other malaria vectors to enter tropical homes through spaces between the roof and wall. By putting inserts with insecticide-coated screens into tubes embedded in this area of the house, a large proportion of the mosquito population is killed with very much less pesticide than would be required to spray the entire house. Baits for mosquitoes and other vectors offer the promise of even more targeted delivery. Sugar baits enclosed in membranes that allow feeding by mosquitoes and sand flies use a small amount of insecticide because the insect comes to the bait. The concentration of pesticide does not need to be great because each sugar meal is large in relation to the size of the target vector. Endectocides are drugs that make animal blood toxic to biting arthropods, familiar in the form of various products given orally to dogs and cats. New work suggests that ivermectin and possibly other drugs could be used by humans for the same purpose, targeting exactly those vectors that feed on people. Use of conventional pesticides in these new delivery methods may mean that they are not biorational, but they certainly achieve the same advantages. No matter what biorational method is used against vectors, the challenge of delivery is at least as great as the challenge of technical development. Efficacy of interventions against public health pests logically have a higher bar for proof than agricultural pesticides. The implication that an entomological intervention will reduce transmission of a pathogen contains a certain ethical requirement that the method works. Proving a reduction in pathogen transmission and disease is a large task, especially if it is shown with statistical rigor. A method like indoor 5

residual spraying (IRS) was developed based on common sense and direct effect on the insect target, but its real effect was not appreciated until it was used at scale in the late 1940s and 1950s. Similarly, insecticide-treated bed nets were invented in the late 1970s and refined in the 1980s and 1990s, but it was not until late in that process that the full public health value was shown in large scale studies. Once that finally happened, the use of bed nets became the backbone of malaria prevention in Africa during 2000-2015. In recognition of the poor evidence base for vector control, current practice is leaning toward requirements for rigorous demonstration of public health value before large-scale implementation. This is logical, but it also means that a potentially life-saving procedure will be delayed for years or, worse, not developed at all due to the expense of evidence generation. Application of biorational approaches face the same challenge as conventional ones. We have probably never seen such a rich mix of innovation in vector control, certainly not since the intense period of development in the late 1930s and early 1940s when DDT was invented, IRS was put in practice, and an invention as basic as the aerosol insecticide can was produced. Biorational vector control interventions are a part of that innovation. Continued efforts in innovative research and invention are certainly welcome as we try to find ways that are affordable, feasible, and safe for protection of all people – no matter what their circumstances – from disease. It is not easy to connect the two sides of research and development, especially since the experts in “R” are a different community from the experts in “D.” Certainly from the standpoint of those represented in this volume, the development side seems more challenging than the research. It does not matter whether that is because development is more difficult or because researchers have difficulty making the connection – it remains essential to get a product into wide scale use if it is ever going to have impact. Perhaps the most rational thing for those who work on biorational products is to maintain the multi-dimensional contacts with public health, industry, science, and program management. Our institutions can help, but at the end of the day it is up to us.

6

Current and Future Outlooks for Medical and Veterinary Pest Control

Chapter 2

Overcoming Insecticide Resistance: Proactive Detection and Management of Insecticide-Resistant Human Lice J. Marshall Clark,*,1 Ju Hyeon Kim,1 Kyong Sup Yoon,2 Barry R. Pittendrigh,3 and Si Hyeock Lee4 1Department

of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003, United States 2Department of Environmental Sciences, Southern Illinois University-Edwardsville, Edwardsville, Illinois 62026, United States 3Department of Entomology, Michigan State University, East Lansing, Michigan 48824, United States 4Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea *E-mail: [email protected]

New developments occurring over the past 10 years have impacted the control of human lice. In this time frame, the genomic and transcriptomic sequencing analyses of both the head and body louse have occurred. The development of an in vitro louse rearing system has allowed the colonization of reference insecticide-susceptible and -resistant strains and a more formal determination of resistance mechanisms. The use of functional genomics and reverse genetics has identified mutations in key insecticide target genes, which can be monitored for the extent and intensity of resistance in field populations. More recently, functional detoxification genes were identified during insecticide-induced tolerance in susceptible lice. Monitoring of the expression profiles of these genes should provide crucial information on resistance development, which then could be used in proactive resistance monitoring.

© 2018 American Chemical Society

Pediculosis, Pediculicides and Resistance Pediculosis Pediculosis is the infestation of humans by lice and is one of the most prevalent parasitic infestations of humans. The costs of pediculosis in the USA are estimated at >$367 million USD/yr (1) and infestation rates range from 6-12 million cases annually with 2.6 million households affected and 8% of all schoolchildren infested (2). Although not a vector of disease, head lice represent a major economic and social concern worldwide (3). Infestations can cause intense itching, which can injure skin allowing secondary infections (4). Unlike head lice, body lice pose a serious public health threat because they transmit several pathogenic bacteria (Rickettsia prowazekii, Borrelia recurrentis, and Bartonella quintana) that cause human diseases (epidemic typhus, louse-borne relapsing fever and trench fever, respectively) (5). Since the advent of antibiotics, outbreaks are sporadic but do occur particularly during times of war, famine and social unrest, and the body louse still serves as an important vector of re-emerging diseases in developed countries (6). Most people find lice intolerable and repeatedly and prophylactically apply pediculicides (insecticides) without realizing their harm and possible lethality. Misapplications affect children in particular due to their small size and higher sensitivity. There are two ways to combat pediculosis: 1) proactive prevention or 2) post-infestation treatment. Emphasis is increasingly on prevention (education) and physical removal (combing or shaving) because a crisis exists in the chemical management of pediculosis. Until quite recently, the pediculicide arsenal was limited and shrinking due to insecticide resistance. Effective management information was hard to find and few alternatives existed when standard treatments failed. Thus, a critical need existed for biological, biochemical and molecular information necessary in order to implement sustainable novel lice control strategies including the understanding of mechanisms of pediculicide resistance and monitoring of resistance, the use of genetic information to identify new and unique target sites, and the development of new and novel acting pediculicides, including their metabolic profiles leading to detoxification during insecticide-induced tolerance. The monitoring of such induced genes would be useful as a proactive resistance monitoring approach. Pediculicides and Resistance Over the past 70 years, the control of pediculosis has been largely dependent upon the availability of natural and synthetic insecticides starting with DDT (1943), natural pyrethrins (1945), the organochlorine lindane (1960), organophosphates (malathion, 1971), carbamates (carbaryl, 1977) and synthetic pyrethroids (permethrin, phenothrin, 1992) (7). In the USA, the pyrethrins/pyrethroids have dominated the over-the-counter (OTC) market (8). The pyrethrins/pyrethroids share a common target site in the nervous system, the voltage-sensitive sodium channel (VSSC), and act as agonistic neuroexcitants by increasing inward sodium current, leading to nerve 10

depolarization and hyperexcitation, followed by neuromuscular paralysis and death (9). Insecticide resistance to currently-used pediculicides, including permethrin, piperonyl butoxide-synergized pyrethrins and malathion, has occurred worldwide, is increasing (10–13), and certainly contributes to increased incidences of pediculosis (13–23). Current control and resistance problems underscore the need to understand the molecular mechanisms of insecticide resistance in lice. The identification of resistance mechanisms and novel target sites may allow the development of resistance-breaking compounds and specific non-toxic synergists useful in the implementation of novel control and resistance management strategies.

Development of the in Vitro Rearing System: Maintenance of Insecticide-Susceptible and -Resistant Strains and Determination of Resistance An improved in vitro rearing system was developed based on modifications to a manual prototype, which allowed for the first time the sustainable maintenance of head and body lice without human infestation (24). The improved system, based on a silicone-reinforced Parafilm® membrane, human hair tufts and reconstituted human blood, enabled the large-scale rearing of pediculicide-susceptible and -resistant strains of lice in a semi-sterile condition (25) (Figure 1).

Figure 1. Assembly of the in vitro rearing system for lice. Adapted with permission from reference (25). Copyright 2006 Elsevier. 11

Using the in vitro rearing system, the efficacies of three commerciallyavailable OTC formulations (Nix®, Rid®, Proto® Plus) were assessed by applying the products directly to the hair tufts with lice attached following the manufacturers’ instructions (25). All products were highly effective (100% mortality) in the pediculicide-susceptible strain but differentially efficacious (62-84% mortality) in the pediculicide-resistant strain, validating previous anecdotal reports of resistance to permethrin- and pyrethrin-based pediculicide formulations.

Sequencing of the Human Louse Genomes and Transcriptomes Estimation of nuclear DNA content by flow cytometry indicated that both head and body lice had small genomes compared to other insects, spanning only 108 Mb (26). Sequencing of the body louse genome validated this finding and revealed that despite its small size the genome retained a remarkably complete basal insect repertoire of 10,775 predicted genes (27). Evolutionary reduction of the genome size relative to other insects was accomplished by removing intergenic DNA, reducing the size and number of introns within genes and reducing the number of genes within large gene families, particularly those involved in environmental sensing and response, including odorant and gustatory receptor, detoxification enzyme and innate immune response genes (27–29). Comparison of the transcriptional profiles of body and head lice using expressed sequence tags identified 10,771 body and 10,770 head louse transcripts (30). Illumina sequence reads were mapped to the 10,775 body louse gene models and identified nine presence/absence differences between the two transcript sets. Only one gene difference between the two transcriptomes was determined, a hypothetical protein with no known function, indicating that these two organisms share virtually the same genome and are likely ecotypes of the same species. Interestingly, the numbers of detoxification genes involved in xenobiotic metabolism (e.g., cytochrome P450 monooxygenases, glutathione S-transferases, esterases) were dramatically reduced in both head and body lice compared with other insects, indicating that the decreased number of detoxification genes and small genome size would make human lice an efficient model to study insecticide resistance (28). With this information in hand, it became apparent that human lice could serve as an efficient model system to study: 1) the molecular mechanisms of insecticide resistance and the use of this information in monitoring of resistance and 2) how xenobiotic metabolism, which is involved in insecticide resistance, is induced and how this information might be used in ‘proactive’ resistance monitoring.

Mechanisms of Resistance to Permethrin Lee et al. (31) first reported that head lice were resistant to permethrin and exhibited in vivo responses in behavioral bioassays that were consistent with the knockdown resistance (kdr) phenotype. Kdr is a heritable trait associated with nerve insensitivity to DDT, the pyrethrins and the pyrethroids (32) and point 12

mutations in VSSC genes are functionally responsible for the kdr, kdr-type and super-kdr traits (33). Three point mutations located in the domain IIS1-2 extracellular loop (M815I) and in the domain IIS5 transmembrane segment (T917I and L920F) of VSSC α-subunit (numbered according to the head louse amino acid sequence) were identified in permethrin-resistant head lice (34). Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology, Yoon et al. (35) assessed the impact of these mutations on permethrin sensitivity of the expressed channels (Figure 2). In the absence of the three mutations and their corresponding amino acid replacements, a dose-dependent increase in the late current seen during inactivation and a prolongation of the tail current seen during deactivation were apparent at increasing concentrations of permethrin (panel A). In the presence of the three amino acid replacements, superimposed current traces obtained at increasing concentrations of permethrin were indistinguishable from DMSO control traces, confirming that the MITILF haplotype (having all three mutations together) results in target site insensitivity of the VSSC and contributes to permethrin resistance in the head louse (panel B).

Figure 2. Comparative sodium current traces from the house fly VSSC variants with and without head louse mutations expressed in Xenopus oocytes before and after exposure to increasing concentrations of permethrin. Adapted with permission from reference (35). Copyright 2008 Elsevier.

Monitoring the Allele Frequency of kdr in North America The extent and frequency of a kdr-type resistance allele in North American populations of head lice were initially determined from lice collected from 32 locations in Canada and the USA (36). Using the serial invasive signal amplification (SISAR) technique to detect the frequency of the kdr-type T917I mutation (TI), it was found that TI occurs at high levels in North American lice (94.1%). The TI frequency in USA lice from 1999 to 2009 was 84.4%, increased to 99.6% from 2007 to 2009 and was 97.1% in Canadian lice in 2008. The authors 13

of the study cautioned, however, that their results were preliminary (based only on the TI mutation) and perhaps biased due to the small number of lice analyzed and because most of the lice were collected from metropolitan and urban collection sites. In response, a subsequent study expanded on the existing kdr-map (37). It utilized quantitative sequencing (QS) reactions to determine the kdr-type mutation frequency at each of the three alleles (MI, TI and LF). Lice from 138 geographical collection sites, ranging from rural to metropolitan areas, were collected from 48 US states (Figure 3). Mean percent resistance allele frequency values across the three mutation loci (mean % RAF) were determined from each collection site. The overall mean % RAF (± S.D.) for all analyzed lice was 98.3 ± 10%. 132 of the 138 sites (95.6%) had a mean % RAF of 100%, five sites (3.7%) had intermediate values and only a single site had no mutations (0.0%). Forty-two states (88%) had mean % RAF of 100%. The frequencies of kdr-type mutations did not differ regardless of the size of the human population from which the lice were collected, indicating a uniformly high level of resistant alleles. The loss of efficacy of the Nix® formulation from 1998 to 2013 was also correlated to the increase in kdr-type mutations. These data provide a plausible reason for the decrease in the effectiveness of permethrin in the Nix® formulation, which is the parallel increase of kdr-type mutations in lice over time.

Figure 3. Expansion of the knockdown resistance (kdr) allele frequency map for human head lice in the United States using quantitative sequencing. Reproduced with permission from reference (37). Copyright 2016 Oxford Univ. Press.

14

Thus, the frequency of kdr-type alleles in North American head louse populations was determined to be uniformly high, apparently due to the high selection pressure from the intensive and widespread use of the pyrethrins/pyrethroid-based pediculicides over many years, and is likely a main cause of increased pediculosis and failure of pyrethrins/permethrin-based products in Canada and the USA.

Optimization of the Non-Invasive Induction Assay To Identify Detoxification Genes Involved in Insecticide Tolerance as a Proactive Resistance Monitoring Approach Identifying xenobiotic detoxification genes based on insecticide-induced transcript profiles of insects has been suggested as a means of identifying metabolic pathways involved in insecticide resistance (28). Initial pilot studies using Drosophila melanogaster did identify a number of detoxification genes but most were not involved in insecticide metabolism (38). The ability to identify detoxification genes that metabolize/detoxify insecticides during the process of induced tolerance, prior to resistance evolving, would be a major step forward in resistance management because the expression of such genes could then be used proactively to monitor for metabolic resistance and/or increased detoxification such as monitoring for increased transcript levels and automated western blotting analyses. In a proof of principle experiment using the active ingredient in the Sklice® pediculicide formulation, ivermectin, the transcriptional profiling results using an “optimized” non-invasive induction assay [short exposure intervals (2-5 h) to sub-lethal amounts of insecticides (