Insecticide Resistance in Bed Bugs, lectularius and (: ), in Australia

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

By

David Lilly Student ID: 430446375

23rd May 2017

Supervisor: Dr Cameron Webb

Associate Supervisors: Dr Rogan Lee Mr Stephen Doggett

Department of

Sydney Medical School, University of Sydney Summary

Resistance to insecticides has limited the ability to manage pests, and in the urban environment this has resulted in the persistence and spread of a range of of public health and nuisance-biting significance. Bed bugs (Cimex spp.) have been one such , and from a position of relative obscurity over => years ago the number of has undergone perhaps the greatest global resurgence ever to involve an arthropod pest. The resurgence of bed bugs has affected virtually all sectors of society, and has imposed a multitude of negative health and economic impacts. Insecticide resistance is believed to have been a catalyst for the resurgence and a major limitation impacting on the safe and economical eradication of infestations. Understanding both the frequency of insecticide resistance in bed bugs across

Australia and the mechanisms that confer such resistance is thus vitally important. The findings of such research can be used to inform the development of new control strategies and to direct best practice.

This PhD thesis investigates the frequency of insecticide resistance in bed bugs, and C. hemipterus, in Australia to two groups of commonly used insecticides, the expression of mechanisms known to confer resistance to insecticides, and the impact of such mechanisms on the efficacy of desiccant dust insecticides. To assess the frequency of insecticide resistance in modern Australian field populations of bed bugs, a strategy to collect and successfully colonise bed bugs was undertaken. This resulted in JK field-strains from across

Australia being established for use in insecticide screening studies. Discriminating-dose assays of the strains indicated that resistance to pyrethroid insecticides was widespread, and that the susceptibility of several recently-collected strains to neonicotinoid insecticides was declining

(Chapter Two).

To examine the potential presence and action of metabolic detoxification (a major resistance mechanism that confers reduced insecticide susceptibility to a number of insecticide classes) in the expressed resistance, four field-strains that possessed high frequencies of

i resistance were selected for further investigation. Metabolic detoxification can be conferred by two major enzyme groups, cytochrome PNK> monooxygenases and/or hydrolytic esterases.

Commonly, the contribution of each enzyme type to resistance can be obscured by the non- specific action of synergists that inhibit enzyme action. This is the case with piperonyl butoxide

(PBO), a synergist commonly added by manufacturers to formulated insecticides, but that inhibits both cytochrome PNK> monooxygenases and hydrolytic esterases. However, with the development of a new, esterase-specific synergist, ENUV/K-U, a comparative experimental design with a non-specific synergists, such as PBO, could be employed to measure both the relative presence and contribution of each enzyme group to the observed resistance. The results of this investigation revealed that metabolic detoxification was largely responsible for the observed pyrethroid resistance in all four strains. Additionally, the action of the detoxifying enzymes was equally split between two strains with cytochrome PNK> monooxygenases dominance, and two strains with hydrolytic esterase dominance (Chapter Three). The screening of the four strains also revealed that the addition of detoxification-inhibiting synergists didn’t induce complete susceptibility, thereby suggesting that other resistance mechanisms were contributing to the observed resistance.

Thickening of the integument is an often-overlooked mechanism of resistance as it is believed to contribute only marginally to expressed resistance, especially in comparison to other mechanisms such as metabolic detoxification and target site insensitivity. However, cuticle thickening is also non-specific to insecticide type and thus may have an important function with the evolutionary development of resistance to new insecticide groups. It may also improve the function of other underlying mechanisms, as was suspected at the conclusion of

Chapter Three. Therefore, to further examine the potential presence and contribution of cuticle thickening, one highly pyrethroid-resistant strain from Chapter Three was selected and assayed according a to widely-used time-to-knockdown assay design. Examination of three response groups (‘intolerant’, ‘tolerant’, and ‘resistant’) under scanning electron microscope revealed that

ii cuticle thickness was positively correlated with time-to-knockdown. Comparison to an insecticide-susceptible strain also revealed that cuticle thickness was positively and significantly associated with increased resistance (Chapter Four).

The impact of resistance mechanisms within bed bugs on traditional insecticide efficacy has been extensively studied, and is often typified by the resistance expressed by the field-strains assayed in Chapters Two and Three. However, an area that had not been fully investigated is the cumulative impact of such resistance mechanisms on the performance of silica-gel and -based insecticide desiccant dusts. Desiccant dusts are a frequently recommended option for bed bug control, and have become particularly popular as a low-cost, long-term residual option for the treatment of low socio-economic multi-occupancy buildings in the USA. Therefore, to examine potential impacts on desiccant dust efficacy, the same C. lectularius strain from Chapter Four was utilised in a forced-exposure assay, which determined that tolerance is present to silica gel-based products when exposed to sub-label rates (Chapter

Five). Given the present-day popularity of these dusts, the finding of tolerance is a significant development that warrants further investigation.

Viewed in combination, the findings of my PhD have made an original contribution towards our knowledge and understanding of the presence of insecticide resistance in bed bugs, as well as some of the mechanisms that contribute to the expressed resistance. In particular, these findings increase our understanding of the evolutionary adaptations that have contributed to the modern resurgence of bed bugs. This information further clarifies both how and why unexpected bed bug treatment failures can occur presently, and are likely to occur into the future.

iii Declaration of Originality

I, David Gordon Lilly, declare that the contents of this thesis consist of original work carried out by the author unless otherwise stated and duly acknowledged. To the best of my knowledge no part of this thesis has been submitted in whole or in part for any other degree or diploma in any University.

......

David Lilly

=Jrd May =>U^

Structure of Thesis

This thesis has been written as a series of manuscripts that have been published. As such, there is redundancy in the introductions and methods of some chapters.

iv Acknowledgements

I would firstly like to thank my supervisors, Dr Cameron Webb, Mr Stephen Doggett, and Dr Rogan Lee, without who’s support, guidance and (when needed) gentle prodding, this thesis would be much poorer as a result.

To Cameron, your constant view towards the big picture, calm approach to the highs and lows of research and publishing, and frequent encouragement to simplify the science, have made me both a better scientist and communicator. I never really understood Twitter, but we were lucky enough to witness a Cronulla Sharks premiership during my candidature, so we’ll call it even, yeah?

To Stephen, your unsurpassed knowledge of bed bugs and medical entomology inspired me to learn and achieve as much as I could during the last four years. Your unceasing sense of inquiry meant that I was never short of new ideas, nor motivation when it was needed. You also taught me far more than I probably should know about the benefits of frequent flyer status – but either way, I’ll no doubt see you in the lounge!

To Rogan, thank you also the motivation, support, and fascinating glimpses into the world of parasitology. If ever I get bored of entomology, I know which door to knock on.

I am also deeply indebted to Merilyn Geary, Karen Willems, Cheryl Toi, John Haniotis,

John Clancy, and Kai Dang of the Department of Medical Entomology for the friendship, laughs, and encouragement during my time in the lab. You’ve taught me more about entomology than any university subject could, and your collective patience as the number of bed bug colonies grew ever larger will forever be appreciated.

Likewise, to the staff of the Department of Care, your diligent and attentive upkeep of animal stocks, and weekly assistance with maintenance of the bed bug colonies, meant that we always had the bed bugs that we needed. Your support has been greatly appreciated.

v

Enormous thanks are also due to Chris and Eva of the SciTech Library document delivery team for assiduously sourcing much of the literature associated with insecticide resistance in bed bugs from the mid-=>th century. The journals were often obscure, if not long-defunct, and the language invariably foreign, but the overwhelming success in finding so many articles speaks volumes to your collective expertise and the value of this service.

Immense thanks and appreciation to Naomi Hollier for holding the Westmead ship together. You do it almost single-handedly and it must, at times, feel like herding cats but your impact on the student experience is colossal and deserves greater recognition. Likewise, to the student committee of StResS (Student Research Society @ Westmead), your dedication and motivation in bringing some social interaction (and alcohol) to the Westmead precinct helped to diminish many of the negatives associated with being an “off-campus” student.

Acknowledgment is also due to Karen Byth for valuable advice with regard to the design of experiments, and the selection of appropriate statistical analysis. My understanding of the world of statistics has been significantly broadened as a result.

The collection of bed bugs from across Australia, and the subsequent results obtained in Chapter Two of this thesis, would not have been possible without the involvement of the following people (and organisations) who kindly collected and donated bed bug specimens: Mr

Garry Jones (formerly Ecolab Pest Elimination); Mark O’Brien (Termitedoc Pest Control); Kate

Fisher (Haynes Pest Management); Matthew McDonald (Acacia Pest Control); Maureen Koegel

(Sleep Tight Services); Mark Wilson (formerly Elders Pest Control); Nathan Jaffrey, Ron Van

Veen, Katy Pik and Andrew Fraser (Flick AntiCimex); Russell Smith (NT Department of Health); and David Watford.

Similarly, to Garry Jones and Dr Chris Orton, your encouragement for me to pursue my fledging interest in the world of bed bugs, their resurgence, and the potential for insecticide resistance was instrumental in making this thesis finally happen. I can only hope other people of a similar scientific bent are as fortunate as I have been to have had mentors like yourselves.

vi To two of the best friends I could ever have the good fortune to make, Kaitlin and

Andrea, it’s been one heck of a rollercoaster ride, but one that I wouldn’t have missed for the world. Your fascination with the beach, frankly though, continues to baffle me. Nevertheless, I hope that our shared love of good food, coffee, wine, craft beer, and the occasional J a.m. dance off at the Marly, stands our friendship in good stead as we move from the inner-west-life into the suburbs of the world.

And finally, to Cass, my love, what can I say? Your unwavering support and humour defines our marriage, and I’m eternally grateful for your patience, understanding, and culinary skills during these sometimes arduous last few years. Hopefully it was worth it, and I can’t wait to enter the next phase of our life together with ‘bubba’…

vii Declaration of Funding Sources

The study was largely funded by an Australian Postgraduate Award from the

Department of Industry, Innovation, Science, Research and Tertiary Education, Australian

Government and administered by the University of Sydney as a student stipend.

Additional funding through a postgraduate industry grant was received from Bayer

CropScience Pty Ltd, Australia (www.bayercropscience.com.au) and was administered by the

University of Sydney. The author gratefully acknowledges this contribution and industry support for this thesis.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscripts and thesis.

viii Publications Supporting Thesis

Chapter 2 Lilly, D. G., K. Dang, C. E. Webb, & S. L. Doggett. (=>UV). Are Australian field- collected strains of Cimex lectularius and Cimex hemipterus (Hemiptera: Cimicidae) resistant to deltamethrin and imidacloprid as revealed by topical assay? Austral Entomology, accepted article, U>.UUUU/aen.U==Vm.

Chapter 3 Lilly, D. G., K. Dang, C. E. Webb, & S. L. Doggett. (=>UV). Evidence for metabolic pyrethroid resistance in the common bed bug (Hemiptera: Cimicidae). Journal of Economic Entomology, ;<=(J), UJVN-UJVm.

Chapter 4 Lilly, D. G., S. L. Latham, C. E. Webb, & S. L. Doggett. (=>UV). Cuticle thickening in a pyrethroid-resistant strain of the common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae). PLOS ONE, ;;(N), e>UKJJ>=.

Chapter 5 Lilly, D. G., C. E. Webb, & S. L. Doggett. (=>UV). Evidence of tolerance to silica- based desiccant dusts in a pyrethroid-resistant strain of Cimex lectularius (Hemiptera: Cimicidae). Insects, D(^N), U-U=.

Authorship Attribution Statements

Chapters =-K of this thesis are published as cited above. For each publication, I designed the study, analysed the data, and wrote the manuscript, with input from the attributed co-authors.

...... David G. Lilly =Jrd May =>U^

As supervisor for the candidature upon which this thesis is based, I can confirm that the authorship attribution statement above is correct.

...... Cameron E. Webb =Jrd May =>U^

ix Conference Presentations Supporting Thesis

2016 Lilly, D. G., K. Dang, C. E. Webb, & S. L. Doggett. Insecticide resistance in bed bugs in Australia. Abstract presented at the XXV International Congress of Entomology, =K-J> September =>UV, Orlando, USA

2015 Lilly D. G., S. L. Doggett & C. E. Webb. Novel insecticide resistance mechanisms in the common bed bug, Cimex lectularius. Abstract presented at the Australian Entomological Society NVth Annual General Meeting and Scientific Conference, =^-J> September =>UK, Cairns, Australia

2014 Lilly D. G., C. E. Webb & S. L. Doggett. The importance of methodology and strain selection when determining efficacy of insecticides against bed bugs. Abstract presented at the Entomological Society of America V=nd Annual Meeting, UV-Uo November =>UN, Portland, USA

2014 Lilly, D. G., K. Dang, C. E. Webb, & S. L. Doggett. Protecting the future: Understanding insecticide resistance in field populations of bed bugs in order to preserve insecticidal control solutions. Abstract presented at the Australian Entomological Society NKth Annual General Meeting and Scientific Conference, =m September - J October =>UN, Canberra, Australia

2014 Lilly, D. G., K. Dang, C. E. Webb, & S. L. Doggett. Bed bugs: What strain do you have? The importance of resistance profiling for insecticide efficacy trials. Abstract presented at the mth International Conference on Urban Pests, =>-=J July =>UN, Zurich, Switzerland

2014 Lilly D. G., M. P. Zalucki, C. J. Orton, R. C. Russell, C. E. Webb & S. L. Doggett. Implications of insecticide resistance in Cimex lectularius (Hemiptera: Cimicidae), in Australia. Paper presented at the mth International Conference on Urban Pests, =>-=J July =>UN, Zurich, Switzerland

2013 Lilly D. G., M. P. Zalucki, S. L. Doggett, C. J. Orton, R. C. Russell & C. E. Webb. Insecticide resistance in bed bugs in Australia: are they getting a little too cosy in your bed? Abstract presented at the Australian Entomological Society NNth Annual General Meeting and Scientific Conference, =o August - = September =>UJ, Adelaide, Australia

x

Table of Contents

Summary ...... i

Declaration of Originality ...... iv

Structure of Thesis ...... iv

Acknowledgements ...... v

Declaration of Funding Sources ...... viii

Publications Supporting Thesis...... ix

Authorship Attribution Statements ...... ix

Conference Presentations Supporting Thesis ...... x

List of Figures ...... xiii

List of Tables ...... xv

Index of Insecticides in Thesis ...... xvi

Chapter 1 – General Introduction and Literature Review ...... 1

U.U Introduction ...... U

U.= The resurgence and impact of bed bugs ...... N

U.J Insecticide resistance in bed bugs: pre-resurgence ...... o

U.N Insecticide resistance in bed bugs: the modern resurgence ...... =V

U.K Mechanisms of insecticide resistance in modern bed bugs ...... J>

U.V Insecticide resistance in Australian bed bugs ...... J^

U.^ Thesis aims and structure ...... Jo

Chapter 2 – Are Australian Field-Collected Strains of Cimex lectularius and Cimex hemipterus (Hemiptera: Cimicidae) Resistant to Deltamethrin and Imidacloprid as Revealed by Topical Assay? ...... 43

=.U Introduction ...... NK

=.= Materials and methods ...... N^

=.J Results ...... K>

=.N Discussion ...... Km

=.K Conclusion ...... V=

xi Chapter 3 – Evidence for Metabolic Pyrethroid Resistance in the Common Bed Bug (Hemiptera: Cimicidae) ...... 63

J.U Introduction ...... VK

J.= Materials and methods ...... V^

J.J Results and discussion ...... ^>

J.N Conclusions ...... ^N

Chapter 4 – Cuticle Thickening in a Pyrethroid-Resistant Strain of the Common Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae) ...... 76

N.U Introduction ...... ^m

N.= Materials and methods ...... ^o

N.J Results ...... mN

N.N Discussion ...... mo

N.K Conclusions ...... oU

Chapter 5 – Evidence of Tolerance to Silica-Based Desiccant Dusts in a Pyrethroid- Resistant Strain of Cimex lectularius (Hemiptera: Cimicidae) ...... 92

K.U Introduction ...... oN

K.= Materials and methods ...... o^

K.J Results ...... U>U

K.N Discussion ...... U>K

K.K Conclusions ...... UU>

Chapter 6 – General Discussion ...... 111

V.U Overview ...... UU=

V.= Distribution of insecticide resistance in Australia ...... UUN

V.J The implications of multiple resistance mechanisms ...... UUV

V.N Future impacts of insecticide resistance ...... UUm

V.K Challenges and limitations ...... UUo

V.V Future research ...... U=U

V.^ Conclusion ...... U=J

References ...... 125

xii

List of Figures

Chapter 2 Figure =.U: Screening of Cimex lectularius field strains against deltamethrin ...... KU Figure =.=: Screening of Cimex hemipterus field strains against deltamethrin and imidacloprid ...... K= Figure =.J: Screening of Cimex lectularius field strains against imidacloprid ...... KJ Figure =.N: Linear regression analysis of Cimex lectularius field strains screened against deltamethrin ...... KN Figure =.K: Linear regression analysis of Cimex hemipterus field strains screened against deltamethrin ...... KK Figure =.V: Linear regression analysis of Cimex lectularius field strains screened against imidacloprid ...... KV Figure =.^: Linear regression analysis of Cimex hemipterus field strains screened against imidacloprid ...... K^

Chapter 3 Figure J.U: Synergistic effect of PBO and ENUV/K-U on deltamethrin efficacy against Parramatta strain Cimex lectularius ...... ^= Figure J.=: Synergistic effect of PBO and ENUV/K-U on deltamethrin efficacy against Alice Springs strain Cimex lectularius ...... ^= Figure J.J: Synergistic effect of PBO and ENUV/K-U on deltamethrin efficacy against Melbourne no.= strain Cimex lectularius ...... ^J Figure J.N: Synergistic effect of PBO and ENUV/K-U on deltamethrin efficacy against Melbourne no.N strain Cimex lectularius ...... ^J

Chapter 4 Figure N.U: Cuticle thickening measurement methodology used with Parramatta strain Cimex lectularius ...... mN Figure N.=: Time to knockdown of Parramatta strain Cimex lectularius ...... mK Figure N.J: Mean cuticle thickness of Parramatta strain Cimex lectularius ...... mV Figure N.N: Correlation between time to knockdown and cuticle thickness in Parramatta strain Cimex lectularius ...... m^ Figure N.K: Mean cuticle thickness between resistant and susceptible Cimex lectularius .. mo

xiii Chapter 5 Figure K.U: Kaplan-Meier analysis of the survival of resistant and susceptible Cimex lectularius exposed to label rate CimeXa Insecticide Dust ...... U>U Figure K.=: Kaplan-Meier analysis of the survival of resistant and susceptible Cimex lectularius exposed to half label rate CimeXa Insecticide Dust ...... U>= Figure K.J: Kaplan-Meier analysis of the survival of resistant and susceptible Cimex lectularius exposed to label rate Bed Bug Killer Powder ...... U>J

xiv

List of Tables

Chapter 1 Table U.U: Historical emergence of insecticide resistance in Cimex lectularius (UoN^ to UooU) ...... U= Table U.=: Historical emergence of insecticide resistance in Cimex hemipterus (UoKJ to Uom^) ...... UN Table U.J: Modern-day emergence of insecticide resistance in Cimex lectularius (Uoom to present) ...... =o Table U.N: Modern-day emergence of insecticide resistance in Cimex hemipterus (Uoom to present) ...... J>

Chapter 2 Table =.U: Field strains of Cimex lectularius collected in Australia between =>>m-=>UV ... Nm Table =.=: Field strains of Cimex hemipterus collected in Australia between =>>N-=>UV .. No

Chapter 3 Table J.U: Field strains of Cimex lectularius examined for the presence and type of metabolic detoxification resistance ...... Vm

Chapter 5 Table K.U: Calculated and applied doses of CimeXa Insecticide Dust (silica gel) and Bed Bug Killer Powder (diatomaceous earth) ...... U>> Table K.=: Lethal time determination for resistant and susceptible Cimex lectularius against CimeXa Insecticide Dust ...... U>N Table K.J: Lethal time determination for resistant and susceptible Cimex lectularius against Bed Bug Killer Powder ...... U>K

xv Index of Insecticides in Thesis

Insecticide IRAC Group Classification a-chlordane =A Cyclodiene organochlorine b-chlordane =A Cyclodiene organochlorine β-cyfluthrin JA Pyrethroid l-cyhalothrin JA Pyrethroid a-cypermethrin JA Pyrethroid g-HCH (syn. lindane) =A Cyclodiene organochlorine acetamiprid NA Neonicotinoid aldrin =A Cyclodiene organochlorine bendiocarb UA Carbamate bifenthrin JA Pyrethroid bioallethrin JA Pyrethroid bromophos UB Organophosphate carbaryl UA Carbamate chlorfenapyr UJ Pyrroles chlorpyrifos UB Organophosphate cyfluthrin JA Pyrethroid d-allethrin JA Pyrethroid DDT (dichlorodiphenyltrichloroethane) JB DDT group deltamethrin JA Pyrethroid diazinon UB Organophosphate dichlorvos UB Organophosphate dieldrin =A Cyclodiene organochlorine dinotefuran NA Neonicotinoid endrin =A Cyclodiene organochlorine fenitrothion UB Organophosphate fenpropathrin JA Pyrethroid fenthion UB Organophosphate fenvalerate JA Pyrethroid fipronil =B Phenylpyrazole imidacloprid NA Neonicotinoid isodrin =A Cyclodiene organochlorine Continued next page…

xvi

Index of Insecticides in Thesis (continued)

Insecticide IRAC Group Classification malathion UB Organophosphate methoxychlor JB DDT group metrifonate (syn. chlorophos) UB Organophosphate permethrin JA Pyrethroid perthane =A Cyclodiene organochlorine phenothrin (syn. sumithrin / d-phenothrin) JA Pyrethroid pirimiphos-methyl UB Organophosphate prolan =A Cyclodiene organochlorine propoxur UA Carbamate pyrethrin(s) JA Pyrethrin resmethrin JA Pyrethroid spinosad K Spinosyns temephos UB Organophosphate tetramethrin (syn. neopynamine) JA Pyrethroid thiamethoxam NA Neonicotinoid

xvii Chapter 1: General Introduction and Literature Review

Chapter 1 – General Introduction and Literature Review

1.1 Introduction

Arthropods pose many risks to humans, including nuisance biting and the transmission of pathogens (Russell et al., =>UJ), and managing these risks has required vast and varied approaches over millennia (Potter, =>UU). Whilst arthropod pests threaten the productivity of agriculture and human health (Whalon et al., =>>m; Yu, =>UKa; Zhu et al., =>UV), the nuisance impacts of insect pests in our dwellings can also substantially impact our quality of life (Russell et al., =>UJ). Few other insect pests cause as substantial nuisance impacts as bed bugs (Doggett et al., =>U=; Goddard & de Shazo, =>>oa; Potter et al., =>U>a) and, as has been required for many other arthropod pests, their control has relied substantively on the use of insecticides (Potter et al., =>U>a). However, the development of resistance to a wide range of insecticides is of significant concern for the future control of bed bugs.

The development of resistance to insecticides across a range of arthropod pests has, over the course of a century, become a serious international problem (Hemingway et al., =>>=;

Hemingway & Ranson, =>>>; Horowitz & Denholm, =>>U; Roush & Tabashnik, Uoo>; Yu, =>UKa).

Considered to be a Darwinian evolutionary response to human-induced environmental stress

(Feyereisen, UooK; ffrench-Constant et al., =>>N; Hoy, Uoom; Yu, =>UKa), resistance is, at a fundamental level, a genetic change in response to selection by toxicants that reduces an organism’s susceptibility and, in turn, impairs control in the field (Sawicki, Uom^). The definition of resistance has itself developed with advancements in the understanding of the physiological and behavioural mechanisms that may confer resistance (Whalon et al., =>>m). In an attempt to offer a practical definition interpretable by end-users of insecticides, the Insecticide

Resistance Action Committee (‘IRAC’) has defined resistance as “a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest

U Chapter 1: General Introduction and Literature Review species” (Elbert et al., =>>^). The emergence and development of resistance is initially influenced by a combination of factors, including, but not limited to, pre-exposure to similar insecticides, the frequency of resistant alleles, and the method of treatment by applicators

(Forgash, UomN; Georghiou & Taylor, Uo^V). However, once present, resistance is enhanced and becomes more frequent through the use of increasing doses and repeat applications of an insecticide in an attempt to kill individuals exhibiting reduced susceptibility, thus applying further selection pressure in favour of insects possessing resistant alleles (Casida & Quistad,

Uoom).

The earliest known case of resistance to insecticides was with the San Jose scale,

Quadrispidiotus perniciosus to lime sulphur (Melander, UoUN). Since then, more than KK> species of arthropod have developed resistance to over JJ> different insecticidal compounds, with cases covering virtually every insecticide mode of action (Whalon et al., =>>m). The consequences of this have been felt across agriculture, medical and veterinary entomology, parasitology, and urban pest management (Whalon et al., =>>m; Yu, =>UKa). The majority of resistant pests affect agriculture, with this sector accounting for between KK.J% and ^>% of the ‘most resistant’ worldwide (Whalon et al., =>>m; Yu, =>UKa; Zhu et al., =>UV). However, pests of medical and veterinary significance still account for a considerable J>% to JV.K% of resistant pests (Whalon et al., =>>m; Yu, =>UKa; Zhu et al., =>UV). This includes many pests afflicting urban environments including the (Musca domestica), German (Blattella germanica), yellow fever mosquito (Aedes aegypti), cat flea (Ctenocephalides felis), and bed bugs

(Cimex spp.) (Brogdon & McAllister, Uoom; Whalon et al., =>>m; Zhu et al., =>UV).

Populations of arthropod pests can develop resistance rapidly, even to new compounds with novel modes of action (Forgash, UomN; Georghiou & Taylor, Uo^V). In the absence of exposure and selection to pre-existing insecticides, the rate of emergence of resistance is, initially, dependent on the underlying frequency of genes that confer a degree of natural resistance within a population (Forgash, UomN). Once resistance has arisen, it may compromise

= Chapter 1: General Introduction and Literature Review the management of pests (Roush & Tabashnik, Uoo>) and, as it limits the availability of efficacious insecticidal control options, it increases reliance on alternatives that are either expensive, not readily available, or not particularly effective (Robinson & Boase, =>UU). Similarly, the ability for a resistance mechanism to confer reduced susceptibility to an insecticide to which the insect has not been exposed (defined as cross-resistance) can further limit the available control options (Yu, =>UKa). Consequently, a propensity exists for the preferential use of efficacious insecticides until resistance develops and product efficacy is compromised.

The potential for such rapid evolution in response to a new selection pressure was best exemplified by the development of resistance to dichlorodiphenyltrichloroethane (DDT) in a field population of house , M. domestica, a mere year after the compound was first registered for use (Lindquist & Wilson, UoNm; Sacca, UoN^). Similarly, after DDT was registered for mosquitoes in UoNV, resistance in Aedes tritaeniorhynchus and Ae. solicitans had emerged by

UoN^ (Brown, UomV). This pattern was repeated in subsequent years across a wide range of other pest insects, including bed bugs (Brown, UoKV). The rapid emergence of resistance continued from the mid-UoK>s until the Uom>s in other pests and against multiple insecticides, including against many of the organochlorines, carbamates, and pyrethroids. So much so that, during this three-decade period, approximately UK newly resistant species were, on average, emerging each year, with resistance taking between two to five years to develop depending on the specific compound, insecticide group, and target pest (Forgash, UomN; Metcalf, Uomo).

Insecticide resistance is, however, not always permanent in pest insect populations. It has been demonstrated that when selection pressure is completely removed (such as through the rotation or withdrawal of an insecticide) reversion to a susceptible state can also occur (Yu,

=>UKa). Reversion is driven by a benefit in returning to a natural state as a result of fitness costs, such as reduced fecundity or longevity, associated with the expression of resistance (Crow, UoK^;

Gordon et al., =>UK; Kliot & Ghanim, =>U=). The rate at which such reversion ensues is, however, dependent on several factors including the length of a pest’s life-cycle, the quantity of progeny

J Chapter 1: General Introduction and Literature Review produced, frequency of resistance alleles, insecticide exposure patterns, and the behaviour of resistant and susceptible pest populations (Georghiou & Taylor, Uo^V, Uo^^a, Uo^^b; Hemingway

& Ranson, =>>>). Reversion has been observed in several major pests, including the Australian sheep blowfly, Lucilia cuprina, which had developed resistance to dieldrin shortly after the product’s introduction in UoKK (Shanahan, UoKm). Following subsequent cessation of dieldrin- based treatments of sheep in UoKm, however, resistance was gradually lost over the next three to four decades (McKenzie, UooV). A similar phenomenon was observed in a highly temephos- resistant strain of Ae. aegypti from Brazil, following the application of three interventional methods in an attempt to achieve resistance reversion under laboratory conditions (Melo-

Santos et al., =>U>). Nonetheless, despite the above cases, even if resistance has reverted to a low level after a lengthy period of absence there remains the potential for it to be rapidly regained if a selection pressure is reintroduced (Yu, =>UKa). This has been demonstrated in a resistant strain of the peach-potato , Myzus persicae, which was able to be re-selected for esterase-mediated organophosphate resistance after becoming reversion-susceptible (Hick et al., UooV).

While the issues associated with insecticide resistance have strongly influenced the management of arthropod pests of agricultural, veterinary and medical importance, a similar phenomenon of the emergence of resistance, partial-reversion, and re-emergence in the form of cross-resistance has likely contributed to the global resurgence of a critically important urban nuisance-biting pest, bed bugs.

1.2 The resurgence and impact of bed bugs

Bed bugs are small, flightless insects belonging to the Order Hemiptera and Family

Cimicidae (Usinger, UoVV). They are distinguished by being ovoid, dorsoventrally flattened when non-blood engorged, hematophagous ectoparasites that feed on , and, predominantly, humans (Carver et al., UooU). Of the more than o> species of Cimicidae only two,

N Chapter 1: General Introduction and Literature Review the common bed bug Cimex lectularius and tropical bed bug, Cimex hemipterus have established themselves as cosmopolitan nuisance-biting pests of humans (Usinger, UoVV). Bed bugs have been recognised as pests of humans for millennia but, while the relative importance of bed bugs declined in the decades following the development of cheap and widely-available synthetic insecticides in the mid-UoK>s, their resurgence in the past => years has again called for a refocusing of pest control efforts on this pest.

Bed bugs are hemimetabolous, with five nymphal stages, each requiring a blood meal for nutrition and development, before progressing to the next instar, or the adult stage.

Under optimum laboratory conditions of uninterrupted access to a host, suitable harbourage, and =^°C at ^K% relative humidity, development from hatched egg to adult takes approximately

=V-=^ days for C. lectularius and =m days for C. hemipterus (Johnson, UoNU; Omori, UoNU). Under similar conditions, insecticide susceptible females will produce, on average, five to eight eggs per day for Um weeks (Johnson, UoNU), although C. hemipterus will produce fewer (five or less per day) than C. lectularius (Omori, UoNU).

Resistance to both dehydration (Benoit et al., =>>^; Benoit et al., =>>oa) and starvation

(DeVries et al., =>UJ) enables bed bugs to withstand prolonged periods of quiescence, although susceptibility to insecticides eventually increases with the duration of starvation (DeVries et al.,

=>UKb). Starved bed bugs may live for between UJV-UoK days, although this is temperature dependent (Omori, UoNU; Polanco et al., =>UUb). There are some reports of bed bugs surviving for more than a year without feeding, albeit at very cold temperatures (Usinger, UoVV). Insecticide- resistant C. lectularius are more susceptible to starvation, with first instar nymphs being the most vulnerable, and fifth instar nymphs and adults the least (Polanco et al., =>UUb). Bed bugs are cryptic, thigmotactic, and largely photophobic, preferentially harbouring deep within available cracks and crevices until their peak host-seeking activity between U-K a.m. (Doggett et al., =>U=). Host-seeking behaviour in bed bugs is spontaneously initiated by the onset of darkness and, in the absence of any other host cues, both adult and nymphal bed bugs are more

K Chapter 1: General Introduction and Literature Review active in darkness than light (Romero et al., =>U>a). Aggregation in harbourages, such as cracks and crevices (or any other available space when crowding occurs), is driven by aggregation (Siljander et al., =>>m), and bed bugs that have successfully fed on a host will rapidly return to a harbourage to aggregate (Reis & Miller, =>UU). Unsuccessful host-seeking bed bugs also eventually return, albeit over a significantly longer period of time (Reis & Miller, =>UU).

The formation of aggregation clusters in harbourages aids in development and survival as a result of a water-conserving effect (Benoit et al., =>>^), with aggregating bugs surviving longer and undergoing faster rates of nymphal development (Saenz et al., =>UN). However, as bed bug density increases, or when the ratio of adult males to females exceeds U:U, females become more likely to disperse (Pfiester et al., =>>o). This can lead to the spread and establishment of new infestations (Cooper et al., =>UK; Wang et al., =>U>).

Unfortunately, the cryptic nature of bed bugs, when compounded by insecticide resistance, makes them one of the most difficult urban pests to control (Potter et al., =>UK; Potter et al., =>U>b). A thorough and time-consuming inspection is typically required to identify all bed bug activity prior to the treatment, and extensive client preparation may also be required to reduce or remove bed bug infested items that cannot be treated by insecticidal means

(Doggett, =>UJ). Additionally, most insecticides used for bed bug control have poor residual efficacy (Lilly et al., =>>ob) and so bed bugs must be sprayed directly in order to achieve an acceptable level of efficacy. As the insects tend to harbour in cracks and crevices, not all will receive direct insecticidal exposure during a treatment. Consequently, multiple treatments are required to achieve satisfactory control of all bed bugs (Doggett, =>UJ). Ineffective residual treatments may also encourage the dispersal of an (Cooper et al., =>UK; Romero et al., =>>ob; Wang et al., =>U>), further select for insecticide resistance (Gordon et al., =>UN), and will subject those inhabiting infesting dwellings to ongoing clinical impacts.

The reaction to, and clinical impact of, bed bug bites has been comprehensively reviewed by Doggett et al. (=>U=), Potter et al. (=>U>a), Goddard and de Shazo (=>>oa) and

V Chapter 1: General Introduction and Literature Review deShazo et al. (=>U=). Initially, the physical response to a bed bug bite is minimal and consists only of a “barely visible punctum at the location of the bite” (Goddard & de Shazo, =>>oa).

However, over time an immunological reaction develops in response to the salivary proteins injected by the bed bug during feeding (Doggett et al., =>U=; Goddard & de Shazo, =>>oa). Such a reaction may take anywhere from seconds to one or two weeks to develop (Reinhardt et al.,

=>>o; Sansom et al., Uoo=). The clinical progression often begins with macular lesions, leading to papular urticaria, ovoid wheals, and occasional bullous eruptions (deShazo et al., =>U=;

Doggett et al., =>U=). In lay terms, such reactions are most frequently described as “itchy red welts” (Potter et al., =>U>a). In severe cases systemic reactions may develop (Liebold et al., =>>J;

Minocha et al., =>UV) and, although reactions such as these appear to be rare, they highlight the potential significance of bed bug bites as a human health concern. It is also frequently reported that a percentage (usually cited as =>%) of people exposed to bed bug bites do not react (Potter et al., =>U>a). However it is now known that both the latency and frequency of not experiencing a reaction will decline significantly with repeated exposure (Reinhardt et al., =>>o), although a small proportion of the population may remain non-reactive despite repeated exposure

(Goddard & de Shazo, =>>ob). In addition to the immunological reaction to bites, people may experience psychological stress, with symptoms manifesting as insomnia, sleeplessness, emotional distress, hypervigilance, and anxiety (Potter et al., =>U>a). It has been consequently proposed that such symptoms are suggestive of a posttraumatic stress disorder (Goddard & de

Shazo, =>U=).

Various investigations have been made to link the bite of bed bugs to the transmission of various pathogens, such HIV (Webb et al., Uomo), MRSA (Barbarin et al., =>UN), hepatitis B

(Ogston & London, Uom>; Ogston et al., Uo^o), and the causative agent of Chagas’ disease,

Trypanosoma cruzi (Salazar et al., =>UK). However, any link appears tenuous, as while some laboratory trials have demonstrated pathogenic transmission by bed bugs, there is no evidence that transmission occurs in natural conditions (Goddard & de Shazo, =>>oa). The World Health

^ Chapter 1: General Introduction and Literature Review

Organization (WHO) has concluded that “although bugs from infestations have been found harbouring many parasites and diseases, it is generally accepted that the bed bug plays an insignificant role in the transmission of disease humans” (WHO, Uom=). In addition, there is no supporting epidemiological data of widespread increasing transmission of infectious agents correlated with the resurgence of bed bugs (Doggett et al., =>U=).

An association between bed bugs and humans can be dated back nearly four millennia, with early evidence of Cimex sp. discovered in mummified remains preserved from Pharaonic

Egypt (Panagiotakopulu & Buckland, Uooo) and ancient China (Wang, UooV). Bed bugs remained a common pest into the mid-=>th century but, with the development of synthetic insecticides, became increasingly rare (Busvine, UoVN; Potter, =>UU), particularly in developed countries.

However, over the past UK-=> years a resurgence of both C. lectularius and C. hemipterus infestations has occurred contemporaneously around the world (Doggett et al., =>U=), including in Europe (Birchard, Uoom; Boase, =>>U, =>>N, =>>m; Delaunay et al., =>>o; Giorda et al., =>UJ;

Kilpinen et al., =>>m; Levy Bencheton et al., =>UU; Masetti & Bruschi, =>>^; Papp et al., =>UN;

Richards et al., =>>o; Sadílek et al., =>UJ; Vander Pan et al., =>UN), Africa (Gbakima et al., =>>=;

Omudu & Kuse, =>U>), South America (Posso et al., =>UU), Asia (Hirao, =>U>; How & Lee, =>U>;

Komatsu et al., =>UV; Lee et al., =>>m; Tawatsin et al., =>UU; Wang & Wen, =>UU; Wang et al., =>UK;

Wang et al., =>UJc; Yaguchi & Kasai, =>U>), Australia (Doggett et al., =>>J; Doggett et al., =>>Nc;

Doggett & Russell, =>>m), and North America (Krueger, =>>>; Pinto, Uooo; Potter et al., =>>mb;

Potter et al., =>U>b). It is estimated that the resurgence of bed bugs has, conservatively, cost society billions of dollars (Curl, =>UU; Doggett & Russell, =>>m; Stuttaford, =>>V), including

AUD$=>> million in Australia since the resurgence commenced to the year =>UU (Doggett et al.,

=>UU). These costs include various factors such as eradication charges, lost revenue, and discarded infested items (Schaefer, =>>>).

Significantly, the resurgence of both C. lectularius and C. hemipterus has been unique when compared to the changing trends of other arthropod pests, as it occurred not just with

m Chapter 1: General Introduction and Literature Review two distinct species, but also simultaneously across multiple countries and continents. A combination of factors such as insecticide resistance, increased international travel, and poor control practices, are generally considered to have contributed to the resurgence (Boase, =>>m;

Davies et al., =>U=; Doggett et al., =>U=).

1.3 Insecticide resistance in bed bugs: pre-resurgence

1.3.1 DDT and organochlorine insecticides in bed bug control

From the moment of its first integration into control programs, DDT was heralded as a near-complete solution to the problem of bed bug infestations and, for a short period, it proved to be immensely successful. The potential of DDT for bed bug eradication was first demonstrated in a United States (USA) military base in Orlando, Florida, where bed bug control had previously been difficult to achieve. However, after treatments with DDT to UK> barracks,

UK warehouses, m,NVV beds, and m,JK= mattresses in April and May of UoNJ, complete eradication was achieved, and no further bed bugs were detected in the following six to seven months

(Knipling, UoK=; Madden et al., UoNK). A related military field trial also revealed the potential of

DDT as a preventative insecticide, with attempts at reintroducing a new infestation by way of adding =K bugs to DDT-treated beds each week proving completely unsuccessful for up to =mU days (Madden et al., UoNN). Laboratory trials with a mixed colony of C. lectularius and C. hemipterus were also successful, with high efficacy and “no particular species resistance” observed following treatments of infested bedding and furniture (Madden et al., UoNN).

In Europe, data gathered from the Danish Board of Health on infestation rates of dwellings similarly pointed to the potential for DDT to reduce the incidence of bed bugs on a district-wide scale. In this instance, the reported numbers of infested dwellings in Denmark dropped from a high of m,UJm in UoNK to a low of only =m= in UoKK (Busvine, UoK^b). Eradication of C. lectularius was, likewise, successfully obtained across Um hostels, eight apartments and one kindergarten in Pervomaysk, Ukraine [formerly the Union of Soviet Socialist Republics (USSR)]

o Chapter 1: General Introduction and Literature Review for up to three months after widespread application of U-K% DDT (Nabokov & Potikha, UoN^).

The cumulative success of such programs led to eradication of bed bug infestations becoming so routine that it was claimed that “the superiority of DDT over all other treatments for bedbugs

[sic] has now been amply demonstrated” (Bishopp, UoNV) and that “DDT is the most valuable weapon yet against bedbugs [sic]” (Leary et al., UoNV). However, as with mosquitoes and , bed bugs proved to be remarkably adaptable, and resistance to DDT soon emerged.

The first recorded case of resistance to DDT in C. lectularius was reported from a naval barrack in Pearl Harbour, Hawaii in UoN^. Bed bugs that had otherwise been controlled by DDT were found to be able to withstand both a K% DDT/kerosene mixture and a U>% DDT dust

(Johnson & Hill, UoNm). However, life expectancy of the resistant bed bugs was determined to be considerably shorter than that of susceptible strains. Furthermore, eggs laid on DDT- impregnated paper were unaffected and nymphs successfully emerged and fed to repletion.

Consequently, the use of DDT residual sprays was considered no longer an effective or reliable method to achieve control (Johnson & Hill, UoNm). Additional reports of resistance were made soon after in Greece (Livadas, UoKU) and Israel (Levinson, UoKJ). In Israel, complaints concerning the ineffectiveness of DDT escalated over the course of UoKU-UoK=, with evidence obtained that bed bugs in an agricultural settlement near to Tel Aviv were unaffected by applications of K%

DDT. Subsequent field trials of DDT and g-HCH (syn. ‘Lindane’, an organochlorine insecticide) were undertaken in a hotel with C. lectularius infested rooms. The results revealed that the bed bugs were resistant to DDT but susceptible to g-HCH, with eradication successfully achieved and then maintained during a KK-day monitoring period through the sole application of g-HCH

(Levinson, UoKJ).

It took several more years for the first reported case of resistance to emerge in C. hemipterus although, as with C. lectularius, it occurred on a military base (Chen et al., UoKV).

Over the course of three years between UoKJ and UoKK of antimalarial residual DDT applications in an army camp in Feng-Shan, Southern Taiwan, reports were received of such treatments

U> Chapter 1: General Introduction and Literature Review having a reduced impact against bed bugs. Investigations uncovered small numbers of C. hemipterus inhabiting areas on and around beds with visual DDT residues. Subsequent screening with K% DDT revealed only low levels of susceptibility, with a significant portion of the bed bugs surviving up to JJ days of continuous forced exposure. Comparison to a suspected susceptible field strain, also C. hemipterus, collected from a village never previously exposed to

DDT, confirmed the Feng-Shan bed bugs possessed discernible DDT resistance (Chen et al.,

UoKV).

Following these early reports of resistance to DDT in both C. lectularius and C. hemipterus, monitoring for the emergence of further resistance achieved a far higher level of attention and, subsequently, the number of newly reported detections and field observations of resistance in C. lectularius (Table U.U) and C. hemipterus (Table U.=) rapidly rose.

In the subsequent two decades between UoKJ-Uo^J, more than J> separate instances of resistance in C. lectularius were reported either directly by published research or through the personal communications and field observations compiled by the WHO technical report series.

Given its widespread use, the clear majority of these reports concerned resistance to DDT, however, resistance was also gradually detected to additional insecticides and insecticide groups. For instance, resistance to g-HCH was first reported in UoKN in Italy (WHO, UoVJ), and was then further confirmed by bioassay on a strain collected in Israel in UoKV (Cwilich et al.,

UoK^).

UU Chapter 1: General Introduction and Literature Review

Table 1.1: The emergence and spread of insecticide resistance in Cimex lectularius from UoN^ to UooU [modified from Dang et al. (=>U^a)]

Year Location(s) Insecticide(s) Principal Reference(s)

UoN^ USA (Hawaii) DDT Johnson and Hill (UoNm)

UoNo Greece DDT Livadas (UoKU)

UoKU Israel DDT Levinson (UoKJ)

UoK= Australasia & Pacific Islandsa DDT Hess (UoKJ) Congo, Korea, USAa DDT WHO (UoVJ)

UoKJ Belgian Congo, USAa DDT Simmons (UoKN) Italy DDT, dieldrin, methoxychlor Mosna (UoKN) Japana DDT WHO (UoVJ)

UoKN Italya g-HCH WHO (UoVJ)

UoKK Iran, USAa DDT WHO (UoVJ)

UoKV French Guiana DDT Floch (UoKV) Iran DDT Mofidi and Samimi (UoKV) Israel g-HCH Cwilich et al. (UoK^)

UoK^ Israel DDT Brown (UoKm) Italy DDT Busvine (UoK^a) Trinidad, Turkeya DDT WHO (UoVJ)

UoKm Middle Easta DDT Quarterman and Schoof (UoKm) Japan, Korea, USA DDT, methoxychlor Lofgren et al. (UoKm) Palestine dieldrin, DDT (“tolerance”) Konchady et al. (UoV=) Polanda DDT Bojanowska (UoKm) Israel (strain) DDT, g-HCH, dieldrin, pyrethrins Busvine (UoKm) - cf. London (susceptible) + various analogues of the above

UoKo Egypt g-HCH, dieldrin Guneidy and Busvine (UoVN) Bulgaria, Hungary, Polanda DDT Brown (UoKo) Israel DDT, g-HCH Gratz (UoKo)

UoV> Borneo, Indonesia, Colombiaa DDT WHO (UoVJ) Southern Rhodesia DDT, g-HCH, dieldrin Reid (UoV>a)

UoVU Englanda DDT, g-HCH, dieldrin Busvine (UoVU) Indiaa DDT, g-HCH WHO (UoVJ)

UoV= Palestine DDT, dieldrin Sharma (UoVJ) South Africa DDT, g-HCH, dieldrin Whitehead (UoV=) Tunisia DDT, dieldrin Juminer and Kchouk (UoV=)

UoVJ Israel malathion, fenthion Barkai (UoVN) a Reported field observation or personal communication. No supporting data provided

Table 1.1: Historical emergence of insecticide resistance in Cimex lectularius (1947 to 1991) Table ;.; continued next page…

U= Chapter 1: General Introduction and Literature Review

Table 1.1: Continued Year Location(s) Insecticide(s) Principal Reference(s)

UoVN USA DDT Mallis and Miller (UoVN) USSR DDT, g-HCH Vaškov et al. (UoVN)

UoVK Egypt DDT, dieldrin Kamel (UoVm) USSR DDT, metrifonate Kapanadze and Sukhova (UomU)

UoVm USSR metrifonate Gvozdeva et al. (Uo^=)

UoVo Egypt DDT, dieldrin Enan (UoVo), Gaaboub (Uo^U) Korea DDT, dieldrin Cha et al. (Uo^>)

Uo^> Libya DDT, g-HCH, dieldrin, pyrethrins Shalaby (Uo^>)

Uo^U Romania DDT, dieldrin Cristescu et al. (Uo^U)

Uo^= Egypt DDT, g-HCH, dieldrin Radwan et al. (Uo^=)

UomN Ethiopia DDT Mekuria and Tesfamariam (UomN)

UomK Brazil DDT Nagem and Williams (Uoo=b)

Uom^ Bulgaria DDT, permethrin, deltamethrin Zlatanova et al. (Uomo)

Uomm Iraq dieldrin Al-Barzangi et al. (Uomm)

UooU South Africa DDT, bendiocarb, deltamethrin Newberry (UooU)

Similarly, resistance to dieldrin was first detected in refugee camps in the Gaza Strip, Palestine, in UoKm-UoKo, before then being detected in Southern Rhodesia in UoV> (Reid, UoV>a), England in UoVU (Busvine, UoVU), and South Africa in UoV= (Whitehead, UoV=). Resistance to the organophosphates malathion and fenthion was confirmed in Israel in UoVJ (Barkai, UoVN), and to metrifonate (syn. chlorophos) in the USSR in UoVm (Gvozdeva et al., Uo^=).

A similar yet slightly less extensive incidence of resistance developed with C. hemipterus.

Nonetheless, between UoKJ and UoV=, more than => cases or observations of resistance were described or reported. As with C. lectularius, the development of resistance to DDT became frequent and widespread. Additionally, resistance to g-HCH was first reported in West India in

UoKV (WHO, UoVJ), to dieldrin in Nigeria in UoK^ (Hamon et al., UoK^), and to malathion in India in Uo^K (Shetty et al., Uo^K).

UJ Chapter 1: General Introduction and Literature Review

Table 1.2: The emergence and spread of insecticide resistance in Cimex hemipterus from UoKJ to Uom^ [modified from Dang et al. (=>U^a)]

Year Location(s) Insecticide(s) Principal Reference(s)

UoKJ Taiwan DDT Chen et al. (UoKV), Yokowo (UoKo)

UoKK West Indiaa DDT WHO (UoVJ)

UoKV West Indiaa g-HCH WHO (UoVJ)

India (Bombay) DDT Rao and Halgeri (UoKV)

UoK^ Hong Kong, Singapore DDT Busvine (UoK^a) India (Calcutta) DDT, g-HCH, dieldrin Sen (UoK^, UoKm, UoKo) Kenya, Somaliaa DDT, g-HCH WHO (UoVJ) Nigeria dieldrin Hamon et al. (UoK^)

Tanganyika (Tanzania) dieldrin Smith (UoKm)

UoKm Gambia, Hong Kong, DDT, g-HCH Busvine (UoKm) Mombasa, Somalia India (Pune) DDT, g-HCH Menon et al. (UoKm) Malaysia DDT, g-HCH, dieldrin Reid (UoV>b), Cheong (UoVN) Mombasa & Gambia (strains) DDT, g-HCH, methoxychlor, Busvine (UoKm) - cf. Tanganyika (susceptible) dieldrin, pyrethrins

UoKo Zanzibar DDT, dieldrin Gratz (UoVU)

UoV> Dahomey (Benin) dieldrin Holstein (UoV>) Gambiaa DDT, g-HCH WHO (UoVJ) Tanzania dieldrin Armstrong et al. (UoV=) Thailand DDT Notananda (UoV>)

UoVU Madagascar DDT, dieldrin Gruchet (UoVU) South Indiaa DDT, g-HCH WHO (UoVJ)

UoV= India (Chennai & Mysuru) DDT, dieldrin Sharma et al. (UoV=)

UoVK India (Dehli) DDT, g-HCH, dieldrin Kalra and Krishnamurthy (UoVK)

UoV^ India (Bihar) DDT, dieldrin Sinha (UoV^)

Uo^> Papua New Guinea DDT Bourke (Uo^J)

Uo^K India (Bangalore) malathion Shetty et al. (Uo^K)

Uo^^ India (Jawhar) DDT, g-HCH, malathion Bhatia and Deshpande (Uo^^)

Uom= Venezuela dieldrin Tonn et al. (Uom=)

Uom^ Israela carbaryl Rosen et al. (Uom^) a Reported field observation or personal communication. No supporting data provided Table 1.2: Historical emergence of insecticide resistance in Cimex hemipterus (1953 to 1987)

UN Chapter 1: General Introduction and Literature Review

In responding to the trend of mounting resistance, Busvine (UoKm) undertook the most comprehensive laboratory study of resistance in field-collected strains of both C. lectularius and

C. hemipterus. Importantly, these results were compared against those of two susceptible strains; the ‘London’ C. lectularius strain, and ‘Tanganyika’ C. hemipterus strain. These two strains provided a benchmark for the measurement of the magnitude of resistance and a consequent higher level of confidence to the results, compared with many of the field observations that had been reported previously. Busvine’s findings revealed that both species had developed extensive resistance to multiple insecticide groups and compounds. This included to; DDT, methoxychlor, perthane, prolan, g-HCH, dieldrin, aldrin, endrin, isodrin, a- chlordane, and b-chlordane. This was also the first record of cross-resistance in both species to pyrethrins which, albeit found to be at low levels, was nonetheless of particular significance as this had not been recorded or observed previously (Busvine, UoKm). The observed cross- resistance would have been due to DDT and pyrethrins sharing the same target site on the insect nerve voltage-gated sodium channel (VGSC) (Davies et al., =>>^; Yu, =>UKb). Additionally, the practice of often widespread and discriminant use of DDT against many insect pests, including bed bugs, is now thought to have predisposed bed bugs to cross-resistance to the pyrethrins and pyrethroids (Yoon et al., =>>m).

In continuing to record observations and cases of resistance, the WHO noted in Uo^> that, “resistance to DDT or the cyclodiene insecticides… are now frequent among bedbugs [sic],

Cimex hemipterus and C. lectularius, in most regions of the world” (WHO, Uo^>). Despite this, only one record existed of purported DDT resistance in the “Australasian and Pacific Islands” region (Hess, UoKJ), although the exact location is not described and, instead, may have been associated with the detection of DDT resistance in Hawaii (Johnson & Hill, UoNm). In addition to the issue of reduced product efficacy, the WHO also expressed concern that a perceived deficiency of control efforts against resistant bed bugs was also directly inhibiting the effectiveness of antimalarial treatment programs (Rafatjah, UoVm; WHO, Uo^>). This was based

UK Chapter 1: General Introduction and Literature Review on the tendency for DDT to irritate resistant bed bugs in treated homes and stimulate increased mobility, thereby leading to a perception of a lack of efficacy by householders in treatment zones. The perception resulted in up to m>% of houses slated for antimalarial treatments being left untreated due to refusal by the occupant (Rafatjah, UoVm). In causing suspicion and antagonism to householders when such spraying failed, the WHO subsequently noted that “the success of antimalarial campaigns can be jeopardized” (WHO, Uo^>).

1.3.2 Integration of organophosphate insecticides into bed bug control

Following the development of the organophosphate insecticides in the mid-UoK>s (Fest

& Schmidt, Uom=) and the identification of several highly insecticidal compounds such as diazinon, malathion, fenthion, and dichlorvos, a solution for the control of DDT-resistant bed bugs was possible. The organophosphates possessed demonstrable efficacy against both C. lectularius (Lofgren et al., UoKm; Whitehead, UoV=) and C. hemipterus (Cheong, UoVN; Kalra &

Krishnamurthy, UoVK; Menon et al., UoKm; Reid, UoV>b; Reid & Lim, UoKo; Sen, UoKm). They were successfully employed in a number of regions where DDT or dieldrin resistance had been detected (Cheong, UoVN; Lofgren et al., UoKm; Menon et al., UoKm; Reid, UoV>b; Reid & Lim, UoKo) or had otherwise limited the available options for control (Konchady et al., UoV=; Menon et al.,

UoKm; Sharma, UoVJ).

In contrast to DDT and the organochlorine insecticides, a key benefit of the organophosphates for use against bed bugs was an apparent decreased likelihood for resistance to become widespread. However, many of the organophosphates were also known to possess a high degree of mammalian toxicity and persistent odour (Fest & Schmidt, Uom=; Shetty et al.,

UoVK). An additional benefit was that when resistance did develop under some circumstances, it appeared only in a limited manner and to select compounds, and not via cross-resistance to the greater organophosphate group. This was evidenced with only limited records of resistance in field populations developing to diazinon in C. hemipterus in UoKm (Sen, UoKm), malathion and

UV Chapter 1: General Introduction and Literature Review fenthion in C. lectularius in Israel in UoVJ (Barkai, UoVN), malathion in India (Shetty et al., Uo^K), and metrifonate (chlorophos) in the USSR (Gvozdeva et al., Uo^=; Kapanadze & Sukhova, UomU).

In the case of the Israel strain of C. lectularius, variable levels of resistance were observed, with the highest against malathion and significantly less to fenthion. Despite this, eradication under field conditions was still able to be achieved after the pest control applicators rotated their product selection to diazinon (Barkai, UoVN). Similarly, despite the evidence of some organophosphate resistance to diazinon from C. hemipterus collected on the east coast of India

(Calcutta) (Sen, UoKm), the same compound was also found to be the most effective in field treatments on the west coast (Pune) (Menon et al., UoKm). Fenitrothion was, likewise, still effective despite some evidence for the emergence of resistance to malathion (Shetty et al., UoVK;

Shetty et al., Uo^K). Resistance to metrifonate also emerged in the USSR, but reportedly only as a side-effect after UU years of the compound being used continuously and repeatedly against house flies (Gvozdeva et al., Uo^=).

Attempts to induce organophosphate resistance in laboratory strain bed bugs also proved largely unsuccessful and supported the observation that the development of resistance to these compounds was not a common occurrence. In one such study, resistance to bromophos could not be developed despite over five years (=m generations) of continuous generational selection at LDm> levels. At the conclusion of the experiment, the strain was still not statistically different from an unselected susceptible strain (Cristescu et al., Uom>). This was in direct contrast to selection for dieldrin resistance, which was found to develop rapidly. Only UU generations of a C. lectularius field strain were needed before resistance emerged when selection pressure (also at levels equivalent to a baseline LDm>) was applied to each generation (Cristescu et al., Uom>; Duport et al., Uo^U). The authors concluded that “organophosphorus resistance is difficult to develop in bed bugs” (Cristescu et al., Uom>). Similarly, the expressed resistance to metrifonate in a strain of C. lectularius from the USSR was rapidly lost once the bed bugs were colonized under selection-free conditions and, thereafter, couldn’t be restored (Kapanadze &

U^ Chapter 1: General Introduction and Literature Review

Sukhova, UomU). Only one study successfully induced organophosphate resistance, with resistance to malathion achieved with an Indian C. hemipterus field strain after nine generations of selection at LDK> levels (Srivastava & Perti, Uo^=). However, given emerging organophosphate resistance to diazinon was noted elsewhere in the country in UoKm (Sen, UoKm), and again to malathion in Uo^K (Shetty et al., Uo^K), the possibility cannot be excluded that the collected field strain already possessed a higher frequency of resistance-associate alleles, thus potentially biasing the laboratory selection process. Additionally, the methodology employed in these studies in attempting to induce resistance from otherwise susceptible field strains has received some criticism (Roush & McKenzie, Uom^). It is now known that resistance-associated alleles may, initially, be present only at extremely low rates (requiring enormous numbers of insects initially for selection to occur) and any subsequent resistance may not reflect the selection processes that occurs under field conditions. Together, these factors may result in non- representative polygenic resistance or, potentially, misinterpretation as to the presence or mechanisms of resistance (Roush & McKenzie, Uom^).

Organophosphates continued to be effective against bed bugs throughout the Uom>s and early Uoo>s, nearly four decades after the compounds were first used for bed bug eradication.

Dieldrin-resistant C. lectularius from Romania were found to be completely susceptible to malathion and fenthion (Cristescu et al., Uo^U), and malathion was successfully used against suspected carbaryl-resistant (a carbamate) C. hemipterus in poultry sheds in Israel (Rosen et al.,

Uom^). Fenitrothion efficaciously controlled bendiocarb-, deltamethrin-, and DDT-resistant bed bugs in huts in Africa (Newberry, UooU), and was effective for over o> days when used to treat an infested army college in India (Varma & Dutta Gupta, UomJ). Similarly, pirimiphos-methyl successfully eradicated bed bugs in a heavily-infested apartment in Islington, London (King et al., Uomo). Malathion was also the most effective insecticide when assayed against a strain of C. lectularius collected from Belo Horizonte, Brazil, where organochlorine resistance had reportedly emerged as a result of treatments for other public health pests (Negromonte et al.,

Um Chapter 1: General Introduction and Literature Review

UooU). Malathion was, likewise, recommended as the insecticide of “first choice” when treating suspected DDT-resistant bed bugs in Tanzania (Kilama, UooU).

These results suggest that it was the extended efficacy of, and lack of widespread resistance to, the organophosphates that contributed to the sustained decline of bed bugs, particularly in the years beyond Uo^J, following the withdrawal of DDT (Turusov et al., =>>=).

Bed bugs had become such a minor pest by the Uom>s that the WHO’s fifth report on vector biology and control made only a cursory note that: “Cimex hemipterus, which earlier developed resistance to DDT, has been shown to be also resistant to malathion in India and Sri Lanka”

(WHO, Uom>). In the same report, C. lectularius failed to receive any mention (WHO, Uom>).

The Uoo= WHO report on vector resistance to pesticides similarly noted no further advancements in organophosphate resistance except for the cases noted above, namely in India

(UoKm), Israel (UoVJ), and the USSR (UoVm) (WHO, Uom=, Uoo=). This was in stark contrast to the status for resistance to DDT, which was reported as being present “almost everywhere” for C. lectularius and across multiple countries and continents for C. hemipterus (WHO, Uoo=).

However, despite the demonstrable and prolonged efficacy of organophosphate insecticides against bed bugs, several factors lead to the gradual withdrawal or replacement of many products. Principal amongst these was that fact that most organophosphates were highly toxic and target non-specific, affecting equally insects, birds, amphibians, and mammals (Fest

& Schmidt, Uom=; Roberts & Reigart, =>UJ). Toxicologically, the irreversible inhibition of acetylcholinesterase and significant lipid (fat) solubility also meant that people and exposed to organophosphate residues had a high potential of developing a delayed additive toxicity (Roberts & Reigart, =>UJ; Yu, =>UKb). Thus, despite some attempts to identify safer organophosphate compounds for bed bug control (Shetty et al., UoVK), only a limited number remain on the global insecticide market with newer-generation compounds, in particular pyrethroids and neonicotinoids, largely replacing their use against bed bugs and other urban pests.

Uo Chapter 1: General Introduction and Literature Review

1.3.3 DDT resistance and the future impact on pyrethroid insecticides

Pyrethroid insecticides are synthetic analogues of the natural pyrethrin esters derived from the chrysanthemum plant (Elliott & Janes, Uo^m). As is the case with DDT, both pyrethrins and pyrethroids bind to the VGSC on the membrane of the insect nerve. This leads to disruption of the nerve potential, which induces prolonged and excessive nerve excitation, resulting in hyperactivity, tremors, rigidity, and eventual death (Davies et al., =>>^; Yu, =>UKb).

Pyrethrum has been used in a crude, crushed flower or seed form for several thousand years but was first developed into a more refined ‘Persian powder’ around =>> years ago (Davies et al., =>>^). Following the biosynthesis of pyrethrins in Uo=N (Staudinger & Ruzicka, Uo=N), integration of pyrethrins into domestic insecticides, including for use against bed bugs, became widespread. Direct topical exposure of pyrethrins was necessary for effective control of bed bugs, as some repellency was observed to dust residues (Blacklock, UoU=). The addition of PBO was also found to improve pyrethrin efficacy (Dove, UoN^), however, the development of resistance to pyrethrins otherwise appeared rare in bed bugs and several other insect pests

(Busvine, UoV>b).

The importance and potential of pyrethroid insecticides to bed bug eradication efforts was first noted by Busvine (UoV>a) although, initially, environmental instability of the first- generation compounds in this group somewhat limited their widespread use (Soderlund &

Bloomquist, Uomo). However, with the development of permethrin, a compound that was between U>-U>>x more stable in light than the first-generation compounds (Elliott et al., Uo^J), the pyrethroids became the fourth major insecticide group for the control of various arthropod pests, including bed bugs.

Due to the highly favourable attributes of low mammalian toxicity, low volatility, high photostability, and extended residual efficacy, pyrethroids have come to represent one of the most important and widely-used insecticide groups developed over the last half century (Yu,

=>UKa). Presently, pyrethroids account for approximately U^% of the total global insecticide

=> Chapter 1: General Introduction and Literature Review market, second only to the neonicotinoids (Sparks, =>UJ), and pyrethroids are still widely used for the control of bed bugs (Doggett, =>UJ; Potter et al., =>UK), albeit with variable efficacy.

The first recorded instance of a modified response of DDT-resistant bed bugs to pyrethrins or pyrethroids was in UoKm, with a known DDT-resistant strain of C. lectularius found to express a degree of “tolerance up to ten times the normal” to pyrethrins (Busvine, UoKm,

UoV>b). Increased “tolerance” to pyrethrins in DDT-resistant was also observed in Libyan C. lectularius (Shalaby, Uo^>) and Indian C. hemipterus (Kalra & Krishnamurthy, UoVK). The reduced susceptibility reported by these studies likely resulted from an underlying mechanism that conferred such resistance to DDT, but also had the potential to confer cross-resistance to pyrethrins and other insecticides (Busvine, Uo^U). As DDT, pyrethrins, and pyrethroids all bind to the same sodium channel nerve receptor (Chang & Plapp Jr, UomJa), they share a common mode of action (Soderlund & Bloomquist, Uomo). As a consequence, the primary resistance mechanism that confers reduced susceptibility to DDT, a sodium channel target-site insensitivity mutation known as knockdown (‘kdr’) resistance, also confers cross-resistance to pyrethrin and the pyrethroids (Chang & Plapp Jr, UomJb). Thus, the prolonged persistence of

DDT resistance in bed bugs (WHO, Uoo=) had the potential to confer cross-resistance to the pyrethroids and pyrethrins, with this compounded by bed bugs being predisposed to such cross- resistance due to the often wholesale and injudicious use of DDT at the time (Yoon et al., =>>m).

Unfortunately, while DDT had been phased out many years earlier, there is evidence that reversion of DDT resistance in bed bugs was particularly slow to occur, and was likely a catalyst for the development of resistance to the pyrethroids.

The tendency for DDT resistance reversion to occur slowly was first documented in UoKm with a C. lectularius collected from Pittsburgh, Pennsylvania. Bed bugs from the colony, which had been maintained without any insecticide selection for nearly five years, had been repeatedly assayed by residual filter paper exposure to =>> mg/ft= DDT at two to three year intervals, with the results then compared to a susceptible strain. During this time, the resistant strain exhibited

=U Chapter 1: General Introduction and Literature Review no deviation in the expressed level of resistance, with ≤ K% mortality obtained despite six days of continuous exposure to the DDT treated surfaces. In comparison, the susceptible strain routinely succumbed completely within Nm-^= hours and maintained a consistent bioassay response over the course of the study (Mallis & Miller, UoVN). The authors noted that these results contradicted established theory that the stability of resistance would degrade and reversion occur as a result of release from insecticide pressure (Brown & Pal, Uo^U), and hypothesized that the bed bug strain tested may have become “fixed” in its degree of resistance, with the potential for this to also occur in other insect field strains (Mallis & Miller, UoVN).

Supporting this finding was a separate, considerably lengthier, study examining DDT and dieldrin-resistant C. lectularius, in which bed bugs were maintained for U= years (and an estimated V>-^> generations). Despite the complete lack of insecticide selection, the assayed strains only made minor shifts towards susceptibility, with resistance to DDT and dieldrin still discernibly maintained (Cristescu et al., Uom>). Similarly, studies on a DDT-resistant strain in the USSR over eight years (an estimated J> generations) found that the naturally-expressed resistance dropped only K.K-fold in the absence of selection but, in contrast, was then rapidly regained when selection was re-introduced, with the DDT-reselected bed bugs becoming resistant to such a magnitude that they were “practically insusceptible” to any further doses of the insecticide (Kapanadze & Sukhova, UomU).

Conversely, when DDT resistance reversion was found to occur, the efficacy of pyrethrins was also found to be improved, suggesting that any cross-resistance within a resistant strain could also be affected by resistance reversion. This was recognised in a Romanian strain of C. lectularius that was recorded as losing resistance to DDT and dieldrin (with mortality from residual assays dropping to between =>-^K% and JK-^>% respectively), although the period over which this had occurred was not recorded. Subsequent assays of the reverted bed bugs found a range of insecticides to be effective, with =% pyrethrin resulting in U>>% mortality (Nosec et al.,

Uo^m).

== Chapter 1: General Introduction and Literature Review

Given the above results, it was perhaps unsurprising that the efficacy of pyrethrins and various pyrethroids were found to be highly variable in subsequent years due to potential DDT- based cross-resistance within the strains being assayed. Several studies, though, reported permethrin to be effective at reducing C. lectularius infestations, albeit with this achieved incidentally through exposure to body lice treatments (Nassif et al., Uom>) and permethrin- impregnated mosquito nets (Charlwood & Dagoro, Uomo; Lindsay et al., Uomo). However, in the case of permethrin-impregnated mosquito nets, complete eradication wasn’t achieved in Papua

New Guinea, with bed bugs (species not recorded) returning after four months to bite inhabitants (Charlwood & Dagoro, Uomo). Eradication was purportedly achieved in Gambia, with the incidence of C. hemipterus around children’s beds reduced from J^.K% to nil over the course of four months after bed nets treated with K>> mg a.i./m= permethrin were provided (Lindsay et al., Uomo).

Conversely, the use of “domestic insecticides” in Natal, South Africa, was correlated with only slightly reduced rates of C. lectularius infestations, with an average of UoK bed bugs found in huts that used the insecticides versus JUm in those that did not (Newberry & Jansen, UomV).

Unfortunately, the authors did not define what constituted a “domestic insecticide” and thus it is impossible to determine, although pyrethroids such as bioallethrin and resmethrin were common ingredients in such products (Casida & Quistad, Uoom; Metcalf & Horowitz, =>>>).

Similarly, between Uom^-Uomm, C. lectularius from Bulgaria were also assayed against N% DDT,

>.>=K% deltamethrin, and >.=K% permethrin. Despite five days of continuous exposure, mortality values were predominantly low, with only UJ.J% of bed bugs affected by DDT and

=V.V% by permethrin. Deltamethrin was more efficacious with m>% mortality although, based on the discriminating dose used, the authors concluded that the result was nonetheless indicative of resistance being present to that compound as well. It was also noted that “the present results point out the…limitation of the use of piretroids [sic] in the struggle with them

[bed bugs]” (Zlatanova et al., Uomo).

=J Chapter 1: General Introduction and Literature Review

Deltamethrin, bendiocarb, and fenitrothion were also used in attempt to control DDT- resistant C. lectularius and C. hemipterus in South Africa in the early Uoo>s. However, of the three insecticides, only fenitrothion was found to be effective (Newberry, UooU). Inadequate residual activity, in particular, appeared to be an issue with both deltamethrin and bendiocarb, with the authors noting that “the other insecticides may have been affected by chemical, physical or bioactive properties of the mud surface, leading to the rapid breakdown or reduced availability to the target pest” (Newberry, UooU). It is now well known that the substrate can affect the efficacy of insecticides (Dang et al., =>U^b). However, given DDT resistance was present at high frequencies in both species when the field trial was conducted, the potential for cross-resistance cannot be excluded as a reason for the poor efficacy of deltamethrin. A later study assessing a similar pyrethroid, l-cyhalothrin, also found residual efficacy to be moderate against C. lectularius, lasting between four to ten weeks in several trial sites when applied to daub-substrate houses (Le Sueur et al., UooJ). Efficacy was extremely low in one treated area, with no mortality achieved despite five hours of continuous exposure to a surface treated ^> days prior. The study authors, however, do not factor in any potential for resistance into their analysis.

Equally, some evidence was available that pyrethroids could be effective against field infestations of C. lectularius. Residual bioassays of technical grade insecticides revealed that permethrin and l-cyhalothrin were efficacious against C. lectularius collected from a North

Carolina poultry shed (Fletcher & Axtell, UooJ). However, the observed efficacy was less than other non-pyrethroid insecticides such as dichlorvos, pirimiphos-methyl and bendiocarb.

Fenvalerate was found to be predominantly ineffective but, as with Newberry (UooU), it was assumed this was due to the method of exposure, as separate studies with fenvalerate by topical application had found it to be a highly efficacious pyrethroid (Dryomova & Tsetlin, Uomm;

Gleiberman et al., UomJ). However, a key limitation of the study was that the resistance status of the poultry shed strain was never determined, thereby making the overall conclusions difficult

=N Chapter 1: General Introduction and Literature Review to interpret in relation to other resistant or susceptible C. lectularius or C. hemipterus populations, and the subsequent effectiveness of pyrethroids such as fenvalerate. Given all insecticides, including representatives of the organophosphates, carbamates, and pyrethroids were all effective, it can be assumed that the strain employed by Fletcher and Axtell (UooJ) was likely to have been closer to insecticide-susceptible than resistant. This is supported by reports that permethrin was effective in poultry sheds during this period (Schmidtmann & Miller, UomN), with no documented cases of resistance (Axtell & Arends, Uoo>).

1.3.4 Potential reservoirs of resistant bed bugs

Despite the reported cases of resistance or reduced product efficacy, bed bugs had become rare by the mid-Uo^>s. This was most likely explained by the cumulative effect of having more than one effective insecticide group available for the treatment of infestations. In other regions, however, bed bugs remained a problem at varying levels, as recorded by several studies in Gambia (J^% of children’s bed infested) (Lindsay et al., Uomo), Tanzania (KV% of houses infested) (Temu et al., Uooo), India (VK% of houses infested) (King, Uoo>) and South Africa (up to oN.J% of huts infested) (Newberry, UooU; Newberry et al., Uom^). Bed bugs were also reported for the first time in U> out of UN counties in Brazil during this period (Nagem & Williams, Uoo=a).

It is similarly of note that, although rare, bed bugs were not entirely absent from western societies during the late Uom>s and early Uoo>s. In England and Wales, the Institution of

Environmental Health Officers reported ^,^^U premises as requiring treatment for bed bugs between the years UomK-mV, and further V,U^o between UomV-m^ (King et al., Uomo). A comparable trend was observed by the Danish Pest Infestation Laboratory, where the percentage of enquiries related to bed bugs rose from a low of approximately >.K% in the mid-UoK>s to a peak of =.K% in the mid-Uom>s (Kilpinen et al., =>>m). Thereafter, enquiries continued throughout the Uoo>s at an average of approximately U=>-UJ> cases per year (Kilpinen et al., =>>m). In the

UK, increases in demand for bed bug treatments from Uoo^-onwards were also reported both

=K Chapter 1: General Introduction and Literature Review from a private pest control company and two council municipalities (Boase, =>>U). Several anecdotal reports were made regarding the occurrence and early increase of bed bug infestations in the USA (Krueger, =>>>; Pinto, Uooo).

When also viewed in context with the historical development of insecticide resistance in bed bugs, this suggests that even during the period of ‘low’ infestation numbers, persistent bed bug populations (be they C. lectularius or C. hemipterus) may have been selected for pyrethroid resistance. This was likely exacerbated by the continued use of non-targeted insecticides such as permethrin-impregnated bed nets (Myamba et al., =>>=) and/or DDT- based vector control treatments (van den Berg, =>>o). Consequently, such populations were potentially primed for a resurgence when pest management practices or insecticides changed advantageously. However, this latent potential arguably went by predominantly unnoticed and unmanaged until a global resurgence was underway (Boase, =>>N, =>>^).

1.4 Insecticide resistance in bed bugs: the modern resurgence

The development, or re-emergence, of resistance amongst populations of bed bugs is one of the commonly cited theories for their resurgence internationally (Doggett et al., =>U=;

Doggett et al., =>>Nc; Myamba et al., =>>=; Romero et al., =>>^; Wang & Cooper, =>UU). The first anecdotal report of insecticides not being effective against ‘modern’ bed bugs was in Uoom

(Birchard, Uoom) and corresponded closely with the earliest reports of a increasing numbers of bed bug infestations (Boase, =>>U; Coghlan, =>>=; Doggett et al., =>>J; Koehler, =>>U; Krueger,

=>>>; Paul & Bates, =>>>; Pinto, Uooo). As discussed above, it is now also suspected that the genesis of the resurgence was likely much earlier in the Uoo>s (Boase, =>>m).

A report in =>>= from camps for internally displaced people in Sierra Leone, demonstrated that infestations of both C. lectularius (KV.U%) and C. hemipterus (NJ.o%) were present. The report also noted that “there has been mixed results when insecticides are applied to infested areas on a small scale, since reinfestation occurs rapidly” (Gbakima et al., =>>=).

=V Chapter 1: General Introduction and Literature Review

However, the exact nature of the insecticide treatments and the eradication protocols employed were not recorded.

The first confirmed case of insecticide resistance putatively associated with the modern resurgence was established in C. hemipterus in Tanzania (Myamba et al., =>>=). High levels of resistance to permethrin and a-cypermethrin were identified and proposed to be because of long-term exposure to insecticide treated bed nets. This was evidenced by a trend of mortality being lower where insecticide-treated bed nets were in use. Resistance also appeared to be higher to a-cypermethrin than permethrin, although the result may have been due to dose exposure differences in the chosen treated-net exposure methodology (Myamba et al., =>>=).

The first three confirmed cases of insecticide resistance in C. lectularius associated with the modern resurgence occurred, contemporaneously, in the UK and USA in =>>V. In the UK, two strains of C. lectularius, one collected from a private domestic home and the other an accommodation facility, were found to be highly resistant to a-cypermethrin and bendiocarb.

No mortality was recorded when the strains were assayed by residual exposure to doses equivalent to the mean LDoo of three susceptible strains (Boase et al., =>>V). A third strain, also collected from a private home, was intermediately resistant to the same compounds with =m% mortality recorded against bendiocarb, and U>% against a-cypermethrin (Boase et al., =>>V). In the US, assessment of the LTK> values achieved against deltamethrin with a C. lectularius strain collected from a multiple-occupancy apartment building in Arlington, Virginia, found that the field strain took nearly J>>x as long to succumb to the insecticide (Moore & Miller, =>>V). A subsequent USA-based study examining four recently collected field strains of C. lectularius from Lexington, Kentucky and Cincinnati, Ohio extended on the results of Moore and Miller

(=>>V), with further resistance identified through the use of discriminating doses to the pyrethroids deltamethrin and λ-cyhalothrin (Romero et al., =>>V; Romero et al., =>>^). A high deltamethrin residual dose of J.oV mg/cm= failed to induce any appreciable mortality (≤ K%) in the four field strains, in contrast to U>>% mortality for a comparison susceptible strain. This

=^ Chapter 1: General Introduction and Literature Review resulted in a resistance ratio of greater than U=,^VK-fold for each of the four strains. A repeat assay with one field strain (‘CINU’) against λ-cyhalothrin achieved similar results, with low field- strain mortality and a subsequent high (V,U=J-fold) resistance ratio (Romero et al., =>>V;

Romero et al., =>>^).

Further confirmed cases of resistance, predominantly to the pyrethroids, thereafter occurred more frequently and are summarised in Table U.J (C. lectularius) and Table U.N (C. hemipterus). Many of these studies concerned bed bugs collected from across the contiguous

USA, although cases from elsewhere in the world began to be reported as well. In particular, insecticide resistance was subsequently found in C. hemipterus collected from three districts in

Sri Lanka to DDT, malathion, propoxur, deltamethrin, and permethrin (Karunaratne et al.,

=>>^). In Thailand, C. hemipterus collected from six provinces with high tourism rates were similarly found to be resistant to DDT, dieldrin, bendiocarb, malathion, deltamethrin, and permethrin (Tawatsin et al., =>UU). In C. lectularius, resistance was also then confirmed in one

Australian strain to bendiocarb, permethrin, and deltamethrin (Lilly et al., =>>oc; Lilly et al.,

=>UK). For the same species, collected from two provinces in Thailand, resistance was reported to bifenthrin and a-cypermethrin (Suwannayod et al., =>U>). While, in Denmark, resistance to deltamethrin and permethrin (with low levels also to chlorpyrifos) were recorded in ten field- strains collected from across the country (Kilpinen et al., =>UU).

=m Chapter 1: General Introduction and Literature Review

Table 1.3: The emergence and spread of insecticide resistance in Cimex lectularius since Uoom (modified from Dang et al. (=>U^a))

Year Location(s)a Insecticide(s) Principal Reference(s)

Uoom UK bendiocarb, a-cypermethrin Birchard (Uoom); Boase (=>>U)b

=>>V UK bendiocarb, a-cypermethrin Boase et al. (=>>V)

USA (VA) deltamethrin Moore and Miller (=>>V)

=>>^ USA (KY, OH) deltamethrin, λ-cyhalothrin Romero et al. (=>>^)

=>>m USA (NY) deltamethrin Yoon et al. (=>>m)

USA (AR) DDT, diazinon, dichlorvos Steelman et al. (=>>m)

=>>o Australia bendiocarb, permethrin, Lilly et al. (=>>oc) deltamethrin

=>U> Japan permethrin, phenothrin Watanabe (=>U>)

USA (KY, MA, MI, NJ, NY, OH) deltamethrin Zhu et al. (=>U>) Thailand bifenthrin, a-cypermethrin Suwannayod et al. (=>U>)

=>UU USA (VA) deltamethrin, β-cyfluthrin Adelman et al. (=>UU)

USA (not stated) cyfluthrin, permethrin Cater et al. (=>UU) Denmark deltamethrin, permethrin, Kilpinen et al. (=>UU) chlorpyrifos Thailand DDT, dieldrin, bendiocarb, Tawatsin et al. (=>UU) malathion, deltamethrin, permethrin + various analogues of the above

=>U= USA (CT) permethrin, d-phenothrin, d- Anderson and Cowles (=>U=) allethrin, resmethrin, tetramethrin, fenpropathrin, fenvalerate, pyrethrins USA (OH) deltamethrin Mamidala et al. (=>U=)

France tetramethrin, phenothrin Durand et al. (=>U=)

=>UJ Russia deltamethrin, λ-cyhalothrin, Roslavtseva et al. (=>UJ) malathion, fenthion, bendiocarb

=>UN Germany deltamethrin Vander Pan et al. (=>UN)

=>UK Australia d-allethrin Dang et al. (=>UKb)

=>UV USA (MI, NJ, OH) imidacloprid, acetamiprid, Romero and Anderson (=>UV) thiamethoxam, dinotefuran Australia deltamethrin, Lilly et al. (=>UVb) imidacloprid (reduced susceptibility) a Multiple studies with the same resistant strain(s) have been excluded for the purposes of simplicity b Birchard (Uoom) and Boase (=>>U) cite only ‘pyrethroids and carbamates’. Ian Burgess (D. Lilly pers. comm.) confirmed that the insecticides tested were formulated products containing bendiocarb and a-cypermethrin Table 1.3: Modern-day emergence of insecticide resistance in Cimex lectularius (1998 to present)

=o Chapter 1: General Introduction and Literature Review

Table 1.4: The emergence and spread of insecticide resistance in Cimex hemipterus since Uoom (modified from Dang et al. (=>U^a))

Year Location(s)a Insecticide(s) Principal Reference(s)

=>>= Tanzania permethrin, a-cypermethrin Myamba et al. (=>>=)

=>>^ Sri Lanka DDT, malathion, propoxur, Karunaratne et al. (=>>^) deltamethrin, permethrin =>UU Thailand DDT, dieldrin, bendiocarb, Tawatsin et al. (=>UU) malathion, deltamethrin, permethrin + various analogues of the above =>UK Australia, India, Kenya, d-allethrin Dang et al. (=>UKc) Malaysia, Thailand =>UV Australia deltamethrin Lilly et al. (=>UVa) a Multiple studies with the same resistant strain(s) have been excluded for the purposes of simplicity Table 1.4: Modern-day emergence of insecticide resistance in Cimex hemipterus (1998 to present)

The spread and increased incidence of insecticide resistance was not a phenomenon restricted just to human dwellings, with evidence of bed bugs resistant to insecticides evolving in the poultry production industry as well (Steelman et al., =>>m). In particular, C. lectularius collected from a poultry facility in Arkansas, USA and assayed by a glass-vial exposure method were found to be resistant to DDT, diazinon, and dichlorvos. Susceptibility otherwise appeared to be maintained with the permethrin, bifenthrin, and λ-cyhalothrin. Screening of several new insecticides also indicated that imidacloprid and fipronil were effective and had potential for future integration as a treatment option. In contrast, chlorfenapyr and spinosyn were found to act slowly and thus had limited future potential (Steelman et al., =>>m)

1.5 Mechanisms of insecticide resistance in modern bed bugs

The evolved resistance of bed bugs to insecticides and general ineffectiveness of formulated insecticides to control a bed bug infestation may be as a result of numerous factors, including several resistance-conferring mechanisms. At a fundamental level, bed bugs have been reported to be repelled by pyrethroids and susceptible bed bugs will avoid resting on areas with residual levels of deltamethrin (Kramer, =>>N; Romero et al., =>>ob). However, resistant

J> Chapter 1: General Introduction and Literature Review populations may not be repelled from such pyrethroid residues in the presence of a heat source

(Moore & Miller, =>>V), and resistant bed bugs may still find harbourages that have been previously occupied by bed bugs attractive due to the presence of aggregation pheromones

(Romero et al., =>>ob). In the process, they may then be potentially exposed to further low insecticide doses (Romero et al., =>>ob; Wang et al., =>UJa). Some resistant bed bug strains will also exhibit increased activity upon exposure to a sub-lethal dose and may disperse or, at the very least, spend more time away from their harbourage area (Romero et al., =>>ob). The observed variation could relate to the degree of resistance and how effective the individual bed bugs are inactivating the insecticide, but this has yet to be proven. Additionally, residual efficacy of liquid-based insecticides has been found to be mostly poor with pyrethroids, and repeated applications of insecticide are usually required to achieve control (Lilly et al., =>>oa, =>>ob).

Together, a lack of topical insecticidal exposure, avoidance of residual deposits, or exposure to declining insecticides residues, may all further drive the evolution and enhancement of resistance mechanisms (Roush & Tabashnik, Uoo>). To date, the mechanisms conferring such resistance in bed bugs can be categorized as reduced penetration, metabolic detoxification, and target-site insensitivity.

1.5.1 Target site insensitivity

The presence of mutations in the VGSC that reduce the affinity of an insecticide to the receptor is a well-known resistance mechanism across a range insect pests (Davies et al., =>>^;

Yu, =>UKa). Target site insensitivity of this type is referred to as ‘knockdown’ (or kdr-type) resistance. It is named after a phenomenon first observed in houseflies that survived after they had been knocked down by DDT and which then subsequently exhibited cross-resistance to pyrethrins (Busvine, UoKU; Soderlund, =>>m). Kdr-type target-site insensitivity resistance has been extensively studied in both C. lectularius and C. hemipterus, and is near-ubiquitous within

JU Chapter 1: General Introduction and Literature Review pyrethroid-resistant field-strains, suggesting an important function within the expression of pyrethroid resistance.

The presence of kdr-type mutations was first established in the USA with two target site point mutations (VNUoL and Lo=KI) identified from a C. lectularius strain collected in New York

(Yoon et al., =>>m). The presence of the mutations was correlated with expressed pyrethroid resistance within the strain based on LTK> responses to a residual exposure of a U% deltamethrin- treated surface. A subsequent study examining the distribution of kdr-type resistance in UU> field-strains of C. lectularius collected from across the USA, found mutations to be highly prevalent, being detected in mm% of the tested bed bugs (Zhu et al., =>U>). Several haplotypes were found to be present, with haplotype B (Lo=KI) and haplotype C (Lo=KI and VNUoL) present, respectively, in NU% and NJ% of the sampled bed bugs. A smaller percentage (=.^%), were found to possess the haplotype D (VNUoL) mutation only. However, the expression of resistance in response to exposure of >.UJ mg/cm= residual deltamethrin was somewhat variable across the detected haplotypes. Two strains, CINU and CINJ, even expressed discernible resistance despite possessing no kdr-type mutations (haplotype A). This indicated that other resistance-conferring mechanisms were likely present in those strains (Zhu et al., =>U>).

Additional studies have subsequently revealed that kdr-type mutations are frequently present in pyrethroid-resistant bed bugs around the world. In France, all specimens of C. lectularius collected from a single multi-occupancy high-rise apartment complex were found to homogeneously possess only haplotype B (Lo=KI) kdr-type mutations (Durand et al., =>U=).

Interestingly, the response of the bugs to a combined tetramethrin (syn. neopynamine) and phenothrin (syn. sumithrin) screening assay revealed only J^.m% of the tested bed bugs exhibited resistance to the two pyrethroids.

Quantitative sequencing of C. lectularius specimens collected from across Israel between =>UU-=>U= indicated that the majority (U> out of U=) were haplotype B, homogeneously possessing only the Lo=KI gene mutation (Palenchar et al., =>UK). One population was found to

J= Chapter 1: General Introduction and Literature Review be heterogeneously haplotype B (VU.J% Lo=KI), while another population was determined to be haplotype C, heterogeneously possessing the VNUoL mutation but homogeneous for the Lo=KI mutation (Palenchar et al., =>UK). The variable presence of both homogenous and heterogeneous kdr-type mutations likely indicates the action of ongoing evolutionary selection for these traits. In particular, given that kdr-type insensitivity is a recessive mechanism (Davies et al., =>>^), the evidence suggests that the Lo=KI mutation is particularly important, and advantageous, for conferring insensitivity to pyrethroid insecticides (Palenchar et al., =>UK;

Seong et al., =>U>; Yoon et al., =>>m; Zhu et al., =>U>).

Similarly, molecular analysis of C. lectularius in Korea revealed the presence of both

VNUoL and Lo=KI mutations, although the frequency of the mutations was changing with time

(Seong et al., =>U>). When first sampled in UooJ, no kdr-type resistance was detected, whereas by =>>^, populations were found to be homogeneously haplotype C (U>>% VNUoL and Lo=KI).

A further shift was detected in =>>m and =>>o, with the sampled bed bugs changing from heterogeneous haplotype C (m% VNUoL and U>>% Lo=KI) to homogeneous haplotype B (U>>%

Lo=KI only) respectively. The evolution of kdr-type mutations was theorised to be correlated with increasing resistance, however, no bioassay testing was undertaken with the Korean field strains as they were all preserved specimens.

A somewhat comparable trend was also observed in Australian C. lectularius, with specimens preserved at or before =>>= lacking both VNUoL and Lo=KI but possessing a novel mutation, IoJVF (Dang et al., =>UKb). As with the Korean study, the hypothesized evolution of pyrethroid resistance was also mirrored by changing kdr-type mutations in Australia. Local

‘modern’ C. lectularius strains (collected between =>>^-=>UJ) predominantly possessed the haplotype B Lo=KI mutation, with a small number also determined to be haplotype C (VNUoL and Lo=KI) (Dang et al., =>UKb). Related kdr-type analysis of C. hemipterus strains determined that the VNUoL, Lo=KI and IoJVF mutations were absent from the specimens investigated, although two other mutations, MoUmI and LU>UNF, were putative kdr-type mutations as they

JJ Chapter 1: General Introduction and Literature Review appear in other pyrethroid-resistant insects (Dang et al., =>UKc). Furthermore, two other mutations were identified in the VGSC gene; LmooV and MoUmI, but it is presently unknown if these confer kdr-type resistance (Dang et al., =>UKc).

1.5.2 Metabolic detoxification

The action of enhanced or over-expressed enzymes that metabolize or detoxify insecticides is a major mechanism of resistance across virtually all insecticide classes except for the non-DDT organochlorines (Soderlund & Bloomquist, Uoo>; Yu, =>UKa). The presence of metabolic-derived resistance to pyrethroids in bed bugs appears to be as a result of the action of two enzyme groups, cytochrome PNK> monooxygenases and hydrolytic esterases. As with kdr-type target-site insensitivity mutations, the presence of metabolic detoxification appears to be a manifestly important resistance mechanism in both C. lectularius (Adelman et al., =>UU;

Yoon et al., =>>m; Zhu et al., =>U=) and C. hemipterus (Karunaratne et al., =>>^).

The use of PBO, a known PNK> inhibitor, initially demonstrated that deltamethrin resistance could be significantly reduced with pre-application of PBO in three pyrethroid- resistant strains. However, the calculated synergistic ratios (ranging from N>- to U^V-fold) indicated that the response was variable and likely as a result of other resistance mechanisms also being present (Romero et al., =>>oc). This finding has been supported by research with formulated products on C. lectularius and C. hemipterus strains from Australia (Lilly et al.,

=>>oa, =>>ob), Malaysia, and Singapore (How & Lee, =>UU). In an Australian pyrethroid- resistant strain, PBO-synergized pyrethroids had improved efficacy compared with non- synergized pyrethroids, but not to the degree whereby complete control was achieved (Lilly et al., =>>oa, =>>ob). Similarly, PBO synergized several pyrethroids when assayed against

Malaysia and Singaporean C. hemipterus strains (How & Lee, =>UU). However, the effect was variable between different life stages and strains, as similarly observed by Romero et al. (=>>oc).

The use of gene silencing NADPH-dependent PNK> reductase against one deltamethrin-

JN Chapter 1: General Introduction and Literature Review resistant strain, CINU (Cincinnati, Ohio), determined that cytochrome PNK> monooxygenases were responsible for the observed pyrethroid resistance (Zhu et al., =>U>). Previously, this strain had been found not to possess kdr-type resistance (Zhu et al., =>U=). Similarly, RNA-sequencing of differential gene expression revealed the presence of cytochrome PNK> monooxygenases in three pyrethroid-resistant strains of C. lectularius from Columbus, Ohio. This resistance mechanism has also been identified in the insect’s cuticle (Mamidala et al., =>U=).

Molecular analysis of C. lectularius has also indicated that hydrolytic esterase-mediated detoxification enzymes may also contribute to the resistance observed in pyrethroid-resistant bed bugs (Adelman et al., =>UU; Zhu et al., =>U=). However, in the only two USA strains examined to date, the results appeared variable. In response to deltamethrin exposure, general esterase activity in a highly pyrethroid-resistant (resistance ratio > K,=>>-fold) strain of C. lectularius from Richmond, Virginia, increased by JK% compared with NU% for cytochrome PNK> monooxygenases (Adelman et al., =>UU). This suggested a potentially complex relationship of increased detoxification employing both enzyme groups. Separately, the expression of esterase- associated gene ClCE=UJJU was found to be highly strain dependent and variable in expression, but also potentially implicit in conferred resistance in a strain from Cincinnati, Ohio (Zhu et al.,

=>U=). More recently, analysis of enzyme activity in several recently-detected neonicotinoid- resistant C. lectularius field-strains from New Jersey, Michigan and Ohio, also found elevated esterases as being the most closely correlated with the observed resistance (Romero &

Anderson, =>UV).

Metabolic detoxification may also have an important function with expressed resistance, and cross-resistance, between pyrethroid and non-pyrethroid insecticides groups (Romero &

Anderson, =>UV). Resistance in C. hemipterus to deltamethrin, permethrin, DDT, malathion, and propoxur has been found to be the result of enhanced metabolic detoxification principally conferred by carboxylesterases and glutathione s-transferases (Karunaratne et al., =>>^).

JK Chapter 1: General Introduction and Literature Review

1.5.3 Reduced penetration

In addition to kdr-type target-site insensitivity and metabolic detoxification, supplementary mechanisms such as reduced penetration or cuticle thickening are likely to be present in bed bugs and acting as intensifiers of the other resistance mechanisms.

Reduced penetration of a toxicant through the cuticle has been known to be a resistance mechanism since it was first identified in the UoV>’s with pyrethrins (Fine et al., UoVJ). It has since been identified as a factor contributing to resistance across organophosphate (Forgash et al., UoV=; Matsumura & Brown, UoVU, UoVJ), carbamate (Georghiou et al., UoVU), organochlorine

(Plapp & Hoyer, UoVm; Vinson & Brazzel, UoVV), and pyrethroid insecticide groups (Ahmad et al.,

=>>V; Delorme et al., Uomm; DeVries & Georghiou, UomU; Gunning et al., UooU; Noppun et al., Uomo;

Valles et al., =>>>; Wood et al., =>U>). Changes in cuticle thickness and lipid composition may inhibit or prevent the passage of insecticides into the insect. When combined with detoxifying enzymes and target-site insensitivity, these factors will have important consequences for the use of formulated insecticides for the control of bed bug infestations.

Molecular analysis of several C. lectularius strains point toward changes in protein- expression and the manifestation of other resistance mechanisms in the cuticle being strongly indicative of reduced penetration and/or cuticle thickening (Koganemaru et al., =>UJ; Mamidala et al., =>U=; Zhu et al., =>UJ). In C. lectularius, cuticle-encoding DNA genome sequences have been found to be significantly over-expressed in a pyrethroid-resistant strain when compared to a pyrethroid-susceptible strain, suggesting the changes in the bed bug cuticle were involved in expressed resistance (Koganemaru et al., =>UJ). Similarly, pyrosequencing of the C. lectularius genome identified a high number of cuticular domains (Bai et al., =>UU). This finding was supported by further differential gene expression which identified the presence of complementary DNA sequences encoding for cuticular proteins in deltamethrin-resistant C. lectularius (Mamidala et al., =>U=).

JV Chapter 1: General Introduction and Literature Review

In addition to potential cuticle proteins, however, other resistance mechanisms may also contribute to perceived reduced-penetration resistance. Cytochrome PNK> monooxygenases, esterases, and ABC transporter genes have also been found to be overexpressed in the integument of pyrethroid-resistant C. lectularius strains (Zhu et al., =>UJ).

1.6 Insecticide resistance in Australian bed bugs

Early in the resurgence, insecticide resistance was proposed as a potential contributing or causative agent for the resurgence of bed bug infestations in Australia (Doggett et al., =>>Nb;

May, =>>K). This was based on anecdotal reports from professional pest managers of poor product efficacy and the observation of numerous treatment failures (Doggett et al., =>>Nb;

Doggett & Russell, =>>m). Other hypothesized contributing factors to the resurgence have been poor pest control practices (May, =>>K), increased international travel (Doggett et al., =>U=), and changes in pest management treatment methods away from broad-spectrum residual applications (Doggett et al., =>>J; Doggett et al., =>>Na). Additionally, pest managers may not have recognized control failures as being due to the presence of resistant bed bugs, but instead may have attributed the difficulty of bed bug infestation eradication to a myriad of other potential causes, including poor product efficacy (Doggett et al., =>>Nc).

The first studies on an Australian-collected field-strain of C. lectularius revealed generally poor efficacy of several formulated products when screened in laboratory trials (Lilly et al., =>>oa, =>>ob), and were suggestive of resistance being present. Eight products were initially assayed, encompassing the pyrethrins, several pyrethroids (permethrin, deltamethrin, b-cyfluthrin), a carbamate (bendiocarb), and two organophosphates (pirimiphos-methyl and diazinon). Testing was undertaken by both topical and residual application at recommended label rates, and only pirimiphos-methyl and diazinon achieved U>>% mortality after =N hours, regardless of the exposure method. A PBO-synergized deltamethrin and tetramethrin product was moderately efficacious, and more so than a non-synergized deltamethrin product. Yet this

J^ Chapter 1: General Introduction and Literature Review was only achieved when applied topically, directly onto the bed bugs. All remaining products failed to achieve greater than ^>% mortality despite continuous exposure to the treated surface for up to U> days (Lilly et al., =>>oa, =>>ob).

Extension of the investigation to technical grade insecticides, and comparison of dose- response data to an imported insecticide-susceptible C. lectularius strain, provided conclusive evidence of the presence of resistance to both pyrethroid and carbamate insecticides (Lilly et al., =>>oc; Lilly et al., =>UK). Calculation of resistance ratios between the susceptible strain and field-strain, based on estimated LDK> values, revealed a factor of U.J million for permethrin,

J^>,>>> for deltamethrin, and =K> for bendiocarb. Only pirimiphos-methyl and imidacloprid returned LDK> values comparable to susceptibility (Lilly et al., =>>oc; Lilly et al., =>UK).

As discussed previously, a subsequent study investigated the presence and distribution of kdr-type mutations in UJ C. lectularius field-strains from across the country that were held in colony, and a further UJ ‘modern’ strains preserved since =>>=. The majority were found to possess haplotype B (Lo=KI) target-site mutations, with a small number also determined to be haplotype C (VNUoL and Lo=KI). Preserved specimens collected at or pre-=>>= possessed either no mutation or a novel, putative, kdr mutation, haplotype IoJVF (found in only one field-strain)

(Dang et al., =>UKb). Similarly, in C. hemipterus, four novel mutations on the VGSC gene were discovered from colony and preserved specimens, of which two (MoUmI and LU>UNF) were deemed to be probable kdr-type mutations (Dang et al., =>UKc). When screened with a simplified d-allethrin assay, the response of the bed bugs possessing kdr-type mutations was moderately variable. However, they were all found to be statistically significantly different, regardless of the life stage, when compared to a susceptible strain (Dang et al., =>UKa). These studies, therefore, provided the first evidence that pyrethroid resistance conferred by kdr-type target site insensitivity was present in most field-strains across Australia.

Jm Chapter 1: General Introduction and Literature Review

1.6.1 Distribution of insecticide resistance in Australian bed bugs

Despite the above studies, the full extent of resistance in bed bugs in Australia remains unknown and further research is needed to elucidate additional mechanisms conferring resistance. The initial determination of the presence of insecticide resistance in C. lectularius was based on data from a field-strain established in =>>N from infestations in Sydney (Lilly et al., =>UK), and hence only provided a limited ‘snap-shot’ of the breadth of the problem.

Additionally, only a limited number of field-strains were assayed for kdr-type mutations (Dang et al., =>UKb; Dang et al., =>UKc) and thus, to date, no further investigation has been undertaken as to the potential presence of other resistance mechanisms, such as metabolic detoxification and reduced penetration. A further complicating factor is that Australia is one of the few countries to host both C. lectularius and C. hemipterus (Doggett et al., =>>J). As noted above, resistance to several insecticide groups has been previously confirmed in C. hemipterus, and

Australian strains are known to possess several putative kdr-type mutations with associated resistance demonstrated to d-allethrin (Dang et al., =>UKa). Hence, the susceptibility of C. hemipterus across northern Australia, and the efficacy of the products available for use against them, has not been fully investigated. Understanding the degree and mechanisms of insecticide resistance is critical to detecting when it evolves, along with monitoring of changes in resistance over time. This will assist in implementing timely and effective resistance mitigation programs, and the development of improved control techniques to manage the consequences of resistance field situations.

1.7 Thesis aims and structure

The primary aim of this thesis was to further elucidate the status of insecticide resistance in bed bugs in Australia and to continue the research into the potential mechanisms that confer such resistance. Understanding the presence and distribution of resistance, and monitoring for changes in any detected resistance, will help inform all stakeholders tasked with the eradication

Jo Chapter 1: General Introduction and Literature Review of resistant bed bugs. This includes pesticide manufacturers, registration bodies, and end-user professional pest managers. Therefore, this thesis presents the results of the most comprehensive insecticide resistance screening program of Australian bed bugs and investigations into resistance–conferring mechanisms.

The aim of Chapter Two of this thesis was to survey for C. lectularius and C. hemipterus strains from across Australia such that they could be successfully established as colonised field- strains. A range of both species were required to enable laboratory-based screening of the frequency of insecticide resistance to two common insecticides. This included the pyrethroid deltamethrin and neonicotinoid imidacloprid, with resistance defined by a precise topical discriminating-dose methodology. Based on each strain’s susceptibility to the assayed insecticides, field-strains of interest could then be identified and selected for further investigations with the aim to reveal any underlying resistance mechanisms. Additionally, the results provided from such a study would be invaluable in providing both a ‘snap-shot’ and longitudinal examination of the frequency of resistance across Australia. This will enable trends to be identified in any expressed resistance, with such data used for resistance management and the continual improvement of management programs.

The results of Chapter Two form the basis of later chapters, including Chapter Three.

Here, four C. lectularius strains that exhibited high frequencies of pyrethroid resistance in the field-strain screening with deltamethrin, were further examined for the presence and action of metabolic detoxification-derived resistance. This topic has not previously been investigated in

Australian bed bugs. This is despite evidence that metabolic detoxification is a particularly important mechanism that accounts for much of the reduced susceptibility in pyrethroid- resistant strains elsewhere in the world. To achieve this, two synergists, PBO and ENUV/K-U were included in bioassays with deltamethrin to act as comparative inhibitors of different enzyme groups. This method can determine whether cytochrome PNK> monooxygenases or hydrolytic esterases are putatively contributing to resistance. Identifying the type of metabolic

N> Chapter 1: General Introduction and Literature Review detoxification is pertinent to understanding how cross-resistance may develop in the future to insecticides with modes of action to which bed bugs have not evolved resistance. It may also provide further information on the types of synergist that could be used in insecticide formulations in order to minimize the effects of metabolically-derived resistance mechanisms.

Chapter Four further utilised a highly pyrethroid-resistant strain of C. lectularius from

Chapters Two and Three, with the aim of determining if cuticle thickening was present as a mechanism of resistance. Cuticle thickening was suspected to be present within pyrethroid- resistant bed bugs based on previous international studies employing molecular and gene- expression techniques. However, no attempts had been made to physically qualify this in any recently-collected pyrethroid-resistant field strains. This is despite cuticle thickening potentially conferring a low level of resistance across insecticides from more than one mode-of- action. It can also enhance other resistance mechanisms, such as metabolic detoxification, by delaying or slowing the penetration of insecticides. Previous results obtained with this field- strain, termed the ‘Parramatta’ strain, indicated that it was extremely resistant to pyrethroids, possessed a homogeneous profile of haplotype B kdr-type mutation, and esterase-derived metabolic detoxification. This strain was also chosen as it was found to include a proportion of bed bugs within the colony that were virtually invulnerable to even the highest doses of various pyrethroid insecticides, thus making it an ideal test strain for a study of this type.

Extending the findings of Chapter Four, Chapter Five investigates the potential consequences of a bed bug strain that possess multiple resistance mechanisms on the efficacy of insecticidal desiccant dusts. Desiccant dusts have become a frequently recommended option for bed bug control, and have become particularly popular as a low-cost, long-term residual option for the treatment of infestations. The efficacy of desiccant dusts against a pyrethroid- resistant strain was also an area of interest given the orthodox view that, due to the physical mode of action of silica gel and diatomaceous earth dusts upon the cuticle, that resistance would

NU Chapter 1: General Introduction and Literature Review be unlikely to develop. Thus, an assessment of efficacy against a pyrethroid-resistant colony such as the Parramatta C. lectularius field-strain was both timely and necessary.

Lastly, Chapter Six, reviews the findings from all previous chapters and summarises the status and mechanisms contributing to insecticide resistance in bed bugs in Australia. The implications for the presence of such resistance are discussed, both regarding immediate impacts on the Australian pest management industry, but also the global trend associated with the resurgence of bed bugs and proliferation of insecticide resistance. Overall, this thesis suggests that resistance to the pyrethroid insecticides in both C. lectularius and C. hemipterus is ubiquitous and is the result of multiple resistance mechanisms collectively degrading or inhibiting the action of such insecticides. Susceptibility to neonicotinoids also appears to be shifting negatively in Australian C. lectularius, a result which conforms to recent findings of emerging resistance in the USA. Additionally, two resistance mechanisms, esterase-derived metabolic detoxification and cuticle thickening, appear to be of greater importance than first thought. In particular, cuticle thickening, a novel resistance mechanism in bed bugs, has the potential to confer cross-resistance to more than one insecticide group. It is present to a significant degree in the strain tested, thus suggesting a strong selection pressure for this trait exists. It is also likely to be a consequence of evolutionary selection as a result of exposure to low doses of insecticides, and could therefore be developed in almost any insecticide-resistant field-strain. Finally, tolerance to desiccant dusts, a process thought not to be selected for in bed bugs, has been found in a highly pyrethroid-resistant strain of C. lectularius. This, along with the evolution of cuticle thickening, is an area particularly worthy of further monitoring and investigation.

N= Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Chapter 2 – Are Australian Field-Collected Strains of Cimex lectularius and Cimex hemipterus (Hemiptera: Cimicidae) Resistant to Deltamethrin and Imidacloprid as Revealed by Topical Assay?

Lilly, D. G., K. Dang, C. E. Webb, & S. L. Doggett

Austral Entomology (2016), published article (early view). doi: 10.1111/aen.12268

NJ Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Abstract

Resistance profiling of C. lectularius and C. hemipterus in Australia has previously revealed the existence of resistance to pyrethroid insecticides and that multiple resistant mechanisms exist in field populations. To further investigate insecticide resistance in modern field populations of bed bugs, a program to collect and screen field specimens was initiated.

Over K> bed bug strains (a mixture that included both C. lectularius and C. hemipterus) have been collected from across Australian, with JK subsequently colonized (thirty-one C. lectularius and four C. hemipterus) for laboratory-based insecticide resistance testing. Bed bugs were exposed topically to pyrethroids at a discriminating dose of =.K g/L deltamethrin in acetone applied at a rate of U μL/bug and, for neonicotinoids, a dose of >.U g/L of imidacloprid applied at a rate of U μL/bug. Mortality was recorded =N h post application. Twenty adult bed bugs of mixed sex and age in two replicates of ten were used for the bioassays, plus controls. Results indicated a broad spread in the frequency of resistance against deltamethrin in C. lectularius, represented by a range of mortality values between UK% and U>>%. Mortality was predominantly uniform with C. lectularius against imidacloprid at ≥ oK%, except for several recently collected strains that returned between ^>%-mK%, indicating that a degree of resistance may be developing in select strains. Three of four C. hemipterus strains returned mortality values at or below U>%, with the remainder recording ^K% mortality, thereby indicating pyrethroid resistance is similarly advanced in this species. Normal susceptibility (≥ oK% mortality) was otherwise evident in all four C. hemipterus strains to imidacloprid. These findings complement concurrent research, which indicates variable patterns of resistance are present across the country and around the world. The results have profound implications for product manufacturers and registration authorities in ensuring that a product is as effective at controlling bed bugs in the field as the laboratory.

NN Chapter 2: Insecticide Resistance in Bed Bugs in Australia

2.1 Introduction

Since the turn of the =Ust century a concurrent global resurgence of both the common bed bug, C. lectularius and tropical bed bug, C. hemipterus, has resulted in negative wellbeing and economic impacts for virtually all sectors of society (Doggett et al., =>U=; Minocha et al.,

=>UV). Insecticide resistance is now considered as one of the key drivers of the resurgence

(Doggett et al., =>U=; Doggett et al., =>>Nc; Myamba et al., =>>=; Romero et al., =>>^; Wang &

Cooper, =>UU) with resistance, particularly to the pyrethroid insecticides, well established in populations of both C. lectularius and C. hemipterus around the world (Adelman et al., =>UU;

Boase et al., =>>V; Dang et al., =>UN; Karunaratne et al., =>>^; Kilpinen et al., =>UU; Koganemaru et al., =>UJ; Moore & Miller, =>>V; Myamba et al., =>>=; Romero et al., =>>^; Tawatsin et al.,

=>UU; Yoon et al., =>>m). Most recently, resistance in C. lectularius was also detected to neonicotinoid insecticides in the USA (Romero & Anderson, =>UV), representing a likely further reduction in the number of insecticide groups that are efficacious against resistant strains. It is expected this will be widespread as both multiple- and cross-resistance mechanisms appear common (Adelman et al., =>UU; Zhu et al., =>UJ; Zhu et al., =>U=; Zhu et al., =>U>).

In Australia, resistance was suspected by the mid-=>>>s following anecdotal reports from professional pest managers of poor insecticide performance (Doggett et al., =>U=; Doggett

& Russell, =>>m) and, principally, mediocre efficacy with both pyrethroid and carbamate-based products when assessed by both topical and residual application under laboratory conditions

(Lilly et al., =>>oa, =>>ob). Subsequently, full examination of dose-responses and resistance ratios between a susceptible and suspected-resistant strain via topical application confirmed extremely high resistance to deltamethrin and permethrin, along with intermediate resistance to bendiocarb (Lilly et al., =>UK). No resistance to imidacloprid was detected at the time, although preliminary testing with a formulated product showed poor performance via residual application (Lilly & Doggett, unpublished data). Successive elucidation of kdr-type mutations determined that the majority of ‘modern’ strains (UJ colony strains and UJ preserved strains

NK Chapter 2: Insecticide Resistance in Bed Bugs in Australia dated post-=>>=) possessed the haplotype B (Lo=KI) target-site mutation, but that a smaller subset collected at or pre-=>>=, possessed either no mutation or a novel putative kdr mutation, haplotype IoJVF (Dang et al., =>UKb). This suggested that C. lectularius populations across

Australia were either rapidly evolving or new founders were introduced. Similarly, in C. hemipterus, four novel mutations on the voltage-gated sodium channel gene were discovered from colony and preserved specimens, of which two (MoUmI and LU>UNF) were deemed to be probable kdr-type mutations (Dang et al., =>UKc). A contingent link of the above kdr-type mutations to expressed resistance was determined in seven strains (three Australian, four international) by exposure to a simplified ‘mat-assay’ designed for rapid and economical field use by professional pest managers (Dang et al., =>UKa).

Despite the above Australian studies, there still remains a gap in our present understanding as to how widespread resistance to pyrethroids is in recently collected field strains and whether there is evidence for any change in resistance levels to pyrethroids and susceptibility to neonicotinoids. Additionally, uncertainty as to whether kdr-type target site mutations are wholly responsible for the observed resistance requires further investigation, as variability in the expressed resistance may otherwise point to multiple mechanisms being present. Moreover, no survey of field strains against neonicotinoid insecticides has been undertaken to date and, in lieu of the most recent detection in the USA, is thus urgently required. Consequently, it was the aim of this study to screen field strain bed bugs collected from across Australia using a laboratory topical bioassay employing discriminating doses of the pyrethroid deltamethrin and the neonicotinoid imidacloprid. A secondary aim was to assess if resistance or susceptibility was changing over time by plotting survival values against the collection date of each strain and analysing the data for trends using linear regression.

NV Chapter 2: Insecticide Resistance in Bed Bugs in Australia

2.2 Materials and methods

2.2.1 Bed bug strains

Thirty-one field strains of C. lectularius and four field strains C. hemipterus were derived from active bed bug infestations around Australia, collected either directly by the authors or submitted courtesy of professional pest managers, and state or territory environmental health officers (Tables =.U and =.=). Where possible, infestation details were recorded (such as date, location, provider/collector, and past treatment details) although, predominantly, the treatment histories from where the bed bugs were collected were not available or could not be disclosed due to confidentiality and privacy restrictions. After collection or receipt of field samples, all specimens were identified to species (Usinger, UoVV), and select strains tested for kdr-type mutations (Dang et al., =>UKa; Dang et al., =>UKb; Dang et al., =>UKc). Thereafter, colonies were established in the departmental insectary and maintained at =K°C (±U°C) and ^K%

(±U>%) RH with a photoperiod of U=:U= h (L:D). All bed bugs were offered a blood meal on specific-pathogen-free (SPF) anaesthetised rats seven days prior to being selected for use in bioassays. No insecticide selection was undertaken and all strains collected during or post-=>U= were tested for their susceptibility within twelve months of their collection in order to eliminate any bias as a result of potential resistance reversion.

LDoo values from an insecticide-susceptible strain were based on data obtained with the

‘Monheim’ C. lectularius strain (Lilly et al., =>UK), obtained from Bayer CropScience (Monheim,

Germany) and which has been maintained under laboratory conditions since the UoV>s. No equivalent insecticide-susceptible C. hemipterus strain exists, and thus data from the Monheim strain was regarded as indicative for C. hemipterus in selecting the same discriminating dose levels.

N^ Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Table 2.1: Field strains of Cimex lectularius collected in Australia in the period 2008-2016

State City / Suburb Strain Code Year Founded Source kdr Haplotypea

New South Wales Abbotsford ABBF =>UU DMEb B New South Wales Ashfield ASHF =>UN DME - New South Wales Castle Hill CASTL =>U= Ecolabc - New South Wales Marsfield MARSF =>UJ DME - New South Wales Newcastle NEWC =>UJ TPCd B New South Wales Northbridge NOTHB =>UJ DME B New South Wales Parramatta PARRA =>U= DME B New South Wales Randwick RANDW =>>m DME - New South Wales Redfern REDF =>UU Ecolab B New South Wales Sydenham SYDH =>UJ Ecolab - New South Wales Westmead WESTM =>UN DME - New South Wales Tamworth TAMW =>UK HPMe - New South Wales Revesby REVB =>UV DME - Victoria Apollo Bay APOL-U =>UJ APCf - Victoria Apollo Bay APOL-= =>UJ APC - Victoria Apollo Bay APOL-J =>UJ APC - Victoria Armadale ARMA =>UJ STSg - Victoria Melbourne MELB-U =>UJ STS - Victoria Melbourne MELB-= =>UJ STS - Victoria Melbourne MELB-J =>UJ STS - Victoria Melbourne MELB-N =>UJ STS B Victoria Melbourne MELB-K =>UJ STS - Victoria Moonee Ponds MOON =>UJ STS B/C Victoria Ripponlea RIPP =>UJ STS B Victoria South Yarra YARRA =>UJ STS B Queensland Surfers Paradise SURF-U =>UJ EPCh - Queensland Surfers Paradise SURF-= =>UJ EPC - NT Northern Territory Alice Springs ALSP =>UJ B Healthi Northern Territory Winnellie WINN =>UK Flickj - South Australia Adelaide ADEL =>UJ - A (IoJVF) Western Australia Cottesloe COTTS =>UJ DWk B a As determined by Dang et al. (=>UKb) b Department of Medical Entomology, Westmead NSW =UNK c Ecolab Pest Elimination, Macquarie Park NSW =UUJ d Termitedoc Pest Control, Cardiff NSW ==mK e Haynes Pest Management, West Tamworth NSW =JNU f Acacia Pest Control, Grovedale VIC J=UV g Sleep Tight Services, Central Park VIC JUNK h Elders Pest Control, Oxenford QLD N=U> i Northern Territory Environmental Health, Alice Springs NT >m^U j Flick AntiCimex, Cairns QLD Nm^> and Darwin NT >m=> k David Watford, Cottesloe WA V>UU Table 2.1: Field strains of Cimex lectularius collected in Australia between 2008-2016

Nm Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Table 2.2: Field strains of Cimex hemipterus collected in Australia in the period =>>N-=>UV

State City / Suburb Strain Code Year Founded Source kdr Haplotypea

New South Wales Auburn AUBN =>UJ DME DoKJG / LU>UNF Queensland Surfers Paradise SURF-J =>UJ EPC - Queensland Cairns CAIRN =>UK Flick - Queensland Nth Queensland TROP =>>N DME MoUmI / LU>UNF a As determined by Dang et al. (=>UKc) Table 2.2: Field strains of Cimex hemipterus collected in Australia between 2004-2016

2.2.2 Insecticides

Technical grade deltamethrin (CAS Number K=oUm-VJ-K) and imidacloprid (CAS

Number UJm=VU-NU-J) were supplied by Bayer CropScience (Hawthorn East, Australia).

Analytical grade acetone (CAS Number V^-VN-U), used to dissolve the technical grade insecticides, was purchased from Sigma-Aldrich Co. LLC.

2.2.3 Discriminating dose bioassays

To determine topical resistance, for the pyrethroids, a dose of =.K g/L deltamethrin in acetone applied at a rate of U μL/bug was set as the discriminating value and, for the neonicotinoids, a dose of >.U g/L of imidacloprid was chosen, also applied topically at U μL/bug.

The deltamethrin dose was equivalent to approximately ten times the dose of many formulated insecticides registered for the treatment of bed bugs, and was otherwise > K>> times the LDoo of a C. lectularius susceptible strain. No equivalent direct relationship was available between imidacloprid and formulated products as, to date, only combination pyrethroid-neonicotinoid products are registered and a synergistic response occurs when the two such insecticide groups are combined. As no topical resistance to imidacloprid has ever been detected locally, the discriminating dose was selected based on it being approximately four times the LDoo of a C. lectularius susceptible strain. Controls received acetone only.

Two replicates of ten randomly selected adult bed bugs (mixed sex and age) per treatment plus controls were then affixed dorsally on double-sided tape to the lids of =K> mL

No Chapter 2: Insecticide Resistance in Bed Bugs in Australia plastic containers, and then U μL of solution was applied topically to the ventral side using a micro-applicator (Lilly et al., =>UK). This method is based on existing methodologies with tick species (Sabatini et al., =>>U; Tatchell, UoV^), and has extensively been used in our laboratory with minimal impacts on the bed bugs (Lilly et al., =>UVb; Lilly et al., =>UK). After exposure, the solution was allowed to dry for several minutes before the bugs were carefully removed with a fine pair of tweezers and placed in the base of the =K> mL container lined with øo> mm

Whatman number U qualitative filter papers (Catalogue number U>>U->o>, Buckinghamshire,

UK). Mortality was recorded at =N h and was defined as the bugs exhibiting no response to gentle pressure from a probe or, in the case of moribund vestigial nerve twitching, the inability to right themselves upon being inverted onto their dorsal side.

2.2.4 Statistical analysis

Mean mortality expressed inversely as the proportion of survivors per strain was analysed against the year of collection by linear regression using IBM SPSS Statistic v=J for Mac

(IBM Corp, =>UK).

2.3 Results

Discriminating topical dose assays with deltamethrin against JU field strains of C. lectularius resulted in a wide variety of mortality, ranging from UK% to U>>% (Fig. =.U). Mean mortality was Km% (S.E. ± N.VU%), indicating resistance, even to this high dose of pyrethroid, is widespread. A similar result was obtained against the four C. hemipterus strains (Fig. =.=), with three strains returning ≤ U>% mortality, while the other yielded ^K% mortality. Mean mortality was =J.^K% (S.E. ± U^.U=%) indicating an even higher level of pyrethroid resistance in this species compared to C. lectularius, albeit a result somewhat limited by a higher standard error due to the smaller sample size and one strain of C. hemipterus returning a much higher mortality result.

K> Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Figure 2.1: Screening of Cimex lectularius field strains against deltamethrin

Figure 2.1: Mean cumulative mortality (± S.E.) of Cimex lectularius field strains treated with =.K g/L deltamethrin via topical application.

KU Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Figure 2.2: Screening of Cimex hemipterus field strains against deltamethrin and imidacloprid

Figure 2.2: Mean cumulative mortality (± S.E.) of Cimex hemipterus field strains treated with =.K g/L deltamethrin and >.U g/L imidacloprid via topical application.

Repeat topical discriminating dose assays with imidacloprid against J> field strains of

C. lectularius (one strain had insufficient numbers for testing compared to deltamethrin assays) resulted in predominantly uniform levels of high mortality (Fig. =.J). Mean mortality was oN.N%

(S.E. U.=N%) although one strain, TAMW, was an exception in returning mortality of only ^>%

(n = =>, S.E. ± >%). Mortality was uniform with the four C. hemipterus strains, with o^% (S.E. ±

U.NN%) mean mortality (Fig. =.=) indicating imidacloprid remains a highly efficacious insecticide against current field strains of this species when applied topically. Mean control mortality across all strains was U.=K% (S.E. ± >.NK%) and thus Abbott’s correction was not performed.

K= Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Figure 2.3: Screening of Cimex lectularius field strains against imidacloprid

Figure 2.3: Mean cumulative mortality (± S.E.) of Cimex lectularius field strains treated with >.U g/L imidacloprid via topical application.

Linear regression analysis of mean survival values for each strain plotted against date of collection indicated no correlation for deltamethrin with C. lectularius (R= = >.>UJ, p = >.KJV)

(Fig. =.N). Conversely, however, a positive correlation against collection date was returned for imidacloprid with C. lectularius (R= = >.JK=, p < >.>>U) (Fig. =.K) with the newest colonized strains being less susceptible, suggesting a degree of reduced susceptibility to the insecticide is developing in field populations. No significant trend was determined for C. hemipterus either for deltamethrin (R= = >.>N^, p = >.^mN) (Fig. =.V) or imidacloprid (R= = >.UV>, p = >.V>>) (Fig.

=.^). The limited sample size for C. hemipterus makes the detection of any trend difficult to establish, with the confidence intervals correspondingly large in the above analysis for this species. The lack of a significant trend for both C. lectularius and C. hemipterus against deltamethrin suggests that, despite being inherently variable from strain to strain, there is no obvious positive or negative change evident in the levels of resistance in Australian bed bugs for the last eight years.

KJ Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Figure 2.4: Linear regression analysis of Cimex lectularius field strains screened against deltamethrin

Figure 2.4: Linear regression (± oK% C.I.) of mean cumulative survival (± S.E.) vs. time (year collected) of Cimex lectularius field strains treated with =.K g/L deltamethrin via topical application

KN Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Figure 2.5: Linear regression analysis of Cimex hemipterus field strains screened against deltamethrin

Figure 2.5: Linear regression (± oK% C.I.) of mean cumulative survival (± S.E.) vs. time (year collected) of Cimex hemipterus field strains treated with =.K g/L deltamethrin via topical application

KK Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Figure 2.6: Linear regression analysis of Cimex lectularius field strains screened against imidacloprid

Figure 2.6: Linear regression (± oK% C.I.) of mean cumulative survival (± S.E.) vs. time (year collected) of Cimex lectularius field strains treated with >.U g/L imidacloprid via topical application

KV Chapter 2: Insecticide Resistance in Bed Bugs in Australia

Figure 2.7: Linear regression analysis of Cimex hemipterus field strains screened against imidacloprid

Figure 2.7: Linear regression (± oK% C.I.) of mean cumulative survival (± S.E.) vs. time (year collected) of Cimex hemipterus field strains treated with >.U g/L imidacloprid via topical application

K^ Chapter 2: Insecticide Resistance in Bed Bugs in Australia

2.4 Discussion

This study represents the most comprehensive survey to date on the topical susceptibility of modern bed bug populations in Australia to commonly used pyrethroid and neonicotinoid insecticides.

The results from topical discriminating dose assays confirm that pyrethroid resistance in both C. lectularius and C. hemipterus is ubiquitous and present at an advanced level.

Susceptibility to neonicotinoids predominantly remains via topical application, although there may be evidence of reduced susceptibility developing in C. lectularius based on a slight, but significant, trend being evident when mean mortality values were compared against the year each strain was collected. This result is typified by the TAMW strain, collected in =>UK, which returned the lowest percentage mortality (^>%) upon exposure to the requisite dose of imidacloprid. Viewed alone, this result could arguably be dismissed as an exception or outlier; however, three of the four next lowest results are from strains collected in the three years up to

=>UV. This itself is perhaps the most concerning result from the study as the only neonicotinoid- based insecticide registered (as of February =>U^) on the Australian market, Temprid ^K (Bayer

CropScience, Australia) has been shown to be highly effective against pyrethroid-resistant strains (Gordon et al., =>UN).

The results of this study conform to other studies on bed bug field strains (both C. lectularius and C. hemipterus) in reinforcing a trend of pyrethroid resistance being widespread

(Durand et al., =>U=; Palenchar et al., =>UK; Seong et al., =>U>; Tawatsin et al., =>UU; Yoon et al.,

=>>m; Zhu et al., =>U>) and present at a level that could compromise the insecticide efficacy of some formulations (Boase et al., =>>V; Gordon et al., =>UN; Moore & Miller, =>>V, =>>o; Polanco et al., =>UUa), although we have found that aerosol based pyrethroids are still effective at killing even the most resistant strains when applied directly at the insect (Lilly & Doggett, unpublished). Furthermore, the results of imidacloprid topical discriminating dose assays against C. lectularius support the findings of the singular study to date, which determined via a

Km Chapter 2: Insecticide Resistance in Bed Bugs in Australia similar topical methodology that resistance had developed (to varying degrees) in three field strains from the USA (Romero & Anderson, =>UV). Evidence from this study suggests that the response of field strains to neonicotinoid insecticides is changing and, in the absence of a resistance management strategy, have the potential to develop into resistance in due course.

It is perhaps not unsurprising that susceptibility to neonicotinoids will change in

Australian field populations with time, as a common metabolic-derived mechanism of resistance between neonicotinoids and pyrethroids has been established in other insect pests

(Puinean et al., =>U>). Similarly, increased enzyme activity in the neonicotinoid-resistant USA bed bugs suggested a metabolic-derived mechanism (Romero & Anderson, =>UV). Several of the strains tested in this study have also been subsequently found to possess metabolic detoxification (Lilly et al., =>UVb), with two strains putatively possessing the same cytochrome

PNK> monooxygenases as was demonstrated to confer pyrethroid cross-resistance to neonicotinoids in the green peach , Myzus persicae (Puinean et al., =>U>). Development of cuticle thickening, as also discovered in the PARRA strain (Lilly et al., =>UVc), concentration of other resistance mechanisms in the integument (Zhu et al., =>UJ), and up-regulation of cuticle protein-encoding contigs (Koganemaru et al., =>UJ), may all also confer a degree of reduced susceptibility that could eventually lead to the development of neonicotinoid resistance. Such mechanisms can support the development of resistance by way of increasing the efficiency of metabolic detoxification (Ahmad et al., =>>V; Gunning et al., UooU; Mamidala et al., =>U=; Patil

& Guthrie, Uo^o; Sawicki, UoV=, Uo^>) or prolonging the onset of kdr-type derived knockdown

(Plapp & Hoyer, UoVm; Scott, Uoo>; Wood et al., =>U>).

Variability in the mortality achieved in both C. lectularius and C. hemipterus against deltamethrin could be due to a number of reasons and does not, per se, indicate for the absence of resistance in some strains. Notably, given kdr-type mutations have been found to be consistently present in field strains of both species collected post-=>>= both in Australia (Dang et al., =>UKb; Dang et al., =>UKc) and overseas (Durand et al., =>U=; Palenchar et al., =>UK; Seong

Ko Chapter 2: Insecticide Resistance in Bed Bugs in Australia et al., =>U>; Yoon et al., =>>m; Zhu et al., =>U>), variability in the mortality achieved against deltamethrin points to either the presence of some fitness-derived differences from strain to strain or, more likely, the activity of multiple resistance mechanisms. Evidence for multiple resistance mechanisms being present and widespread is supported by the fact that several C. lectularius strains, all with known haplotype B kdr-type resistance, returned cumulative mortality values between =>% and U>>%, with no apparent association to other facts (such as collection date). Subsequent research on a subset of these C. lectularius field strains has elucidated that metabolic detoxification and cuticle thickening contribute to the observed resistance (Lilly et al., =>UVb; Lilly et al., =>UVc). This is also reinforced by the findings of several international studies, where multiple resistance mechanisms have been found (Aak et al., =>UV;

Adelman et al., =>UU; Zhu et al., =>UJ; Zhu et al., =>U=; Zhu et al., =>U>).

The findings of this study, whilst perhaps unsurprising, nonetheless have important consequences both for the registration of insecticides in Australia and for professional pest managers undertaking bed bug eradication. In the first instance, the continued registration or use of exclusively pyrethroid-based non-aerosol insecticides for bed bug treatments must either be ceased or heavily curtailed. This is explained by the fact that, of the JK bed bug field strains tested in this study at the equivalent of approximately U> times the label rate of many deltamethrin-based insecticides (Anonymous, =>U>), U>>% mortality was only achieved in five

(UN%) strains. If applied to field situations and insecticides used at true label rate (and in the absence of complementary technologies) this likely represents a concerning lack of efficacy and a high-risk strategy prone to frequent eradication failures. Compounding this result is the well- established lack of residual efficacy of most pyrethroid-based products (Lilly et al., =>>oa,

=>>ob), although, efficacy of aerosol-based formulations appears unaffected (Lilly & Doggett, unpublished) suggesting that both formulation and product choice are integral both to infestation eradication and the potential selection of resistant bed bugs. Dispersal of the original infestation as a result of increased locomotor hyperactivity after residual pyrethroid exposure is

V> Chapter 2: Insecticide Resistance in Bed Bugs in Australia also an added risk (Romero et al., =>>ob), emphasising the need for a lethal topical dose at the time of treatment. Similarly, if resistance to neonicotinoids develops in bed bugs, behavioural avoidance of residual deposits may also need to be factored into treatments, as has been necessary in other neonicotinoid-resistant pests (Fray et al., =>UN). Preliminary evidence also suggests that combination pyrethroid/neonicotinoid products are mostly ineffective once an application has dried (Lilly & Doggett, unpublished), with only the pyrethroid component remaining active at a level that will affect exposed bed bugs (Gordon et al., =>UN). This further intimates that any reliance on residual efficacy with either pyrethroid-only and combination pyrethroid/neonicotinoid products is a particularly hazardous approach to bed bug eradication.

Secondly, with preliminary evidence that susceptibility to neonicotinoid insecticides is changing, a strategy to continually monitor field susceptibility needs to be established in order to detect the earliest instances of resistance developing. Evidence from the USA would suggest that this is imminent (Gordon et al., =>UN; Romero & Anderson, =>UV). There is a risk that, to some degree, the development of underlying resistance may initially be masked by purported field efficacy as a result of pyrethroids and neonicotinoids inducing a synergistic response when used together (Stanneck et al., =>U=). Nonetheless, given the potential for pyrethroids and neonicotinoids to develop cross-resistance through the presence of overexpressed cytochrome

PNK> monooxygenases (Puinean et al., =>U>), there is the potential for even combination pyrethroid/neonicotinoid products to become ineffective in due course. Addition of a synergist, such PBO, which inhibits the action of PNK> oxidases (Hodgson & Levi, Uoom) and may improve the efficacy of products in some bed bug strains (Lilly et al., =>UVb; Romero et al., =>>oc), should also be investigated. Finally, determination of more detailed dose-response data within targeted field strains against a range of current insecticides is urgently needed in order to measure and monitor the magnitude of the expressed resistance.

VU Chapter 2: Insecticide Resistance in Bed Bugs in Australia

2.5 Conclusion

The development of insecticide resistance in both C. lectularius and C. hemipterus is well established in Australia. This study confirms that field populations of C. lectularius are ubiquitously resistant to deltamethrin, and may be exhibiting the first signs of reduced susceptibility to neonicotinoids. Cimex hemipterus is even more resistant to deltamethrin, but not the neonicotinoids, although further collection and study of field specimens would be warranted beyond the four strains included in this study. Continued culturing and development of the collected field strain colonies, and subsequent elucidation of the resistance mechanisms, may provide further insights into the differences between strains. Ultimately, this will improve our understanding of how to effectively eradicate or prevent future infestations.

V= Chapter 3: Metabolic Detoxification as a Resistance Mechanism

Chapter 3 – Evidence for Metabolic Pyrethroid Resistance in the Common Bed Bug (Hemiptera: Cimicidae)

Lilly, D. G., K. Dang, C. E. Webb, & S. L. Doggett

Journal of Economic Entomology (2016), 109(3), 1364-68 doi: 10.1093/jee/tow041

VJ Chapter 3: Metabolic Detoxification as a Resistance Mechanism

Abstract

Resistance to insecticides, especially the pyrethroids, in the common bed bug, C. lectularius, has been well-documented. However, the presence and relative contribution of metabolic detoxifying microsomal oxidases and hydrolytic esterases to the observed resistance has yet to be fully elucidated. This is due, in part, to the absence of a simple bioassay procedure that appropriately isolates esterases from potentially competing oxidases. Recently, an analogue of PBO was developed, ENUV/K-U (V-[=-(=-butoxyethoxy)ethoxymethyl]-K-propyl-=,J- dihydrobenzofuranby), which inhibits esterases but has limited efficacy against the oxidases, whereas PBO inhibits both. The opportunity is now available to use both synergists via established bioassay methodologies and to screen for the potential presence of oxidase- or esterase-derived pyrethroid resistance in insecticide-resistant insects, including bed bugs. In the present study, ENUV/K-U and PBO were assayed in conjunction with deltamethrin against four field strains of C. lectularius collected from independent geographic locations across

Australia. All strains expressed a high degree of resistance to deltamethrin and significant inhibition of the observed resistance with pre-exposure to PBO. Non-significant differences between the cumulative mortality values for PBO and ENUV/K-U were then observed in two of the four bed bug strains, which indicate that detoxifying esterases are conferring substantially to the observed resistance in those strains. This study is the first to provide evidence that metabolic detoxification in the form of both hydrolytic esterases and microsomal oxidases is a major contributing factor to pyrethroid resistance in C. lectularius.

VN Chapter 3: Metabolic Detoxification as a Resistance Mechanism

3.1 Introduction

The resurgence of bed bugs, both with the common species, C. lectularius, and the tropical species, C. hemipterus, has been an international phenomenon, involving the resurgence of two public health pests of almost unprecedented magnitude and breadth

(Doggett et al., =>U=). One of the major drivers of the resurgence has been the development of insecticide resistance (Doggett et al., =>U=; Doggett et al., =>>Nc; Myamba et al., =>>=; Romero et al., =>>^; Wang & Cooper, =>UU) and, in particular, resistance to the pyrethroid insecticides.

Pyrethroid resistance exhibited in C. lectularius has become widespread (Adelman et al., =>UU;

Boase et al., =>>V; Dang et al., =>UKb; Kilpinen et al., =>UU; Koganemaru et al., =>UJ; Lilly et al.,

=>UK; Moore & Miller, =>>V; Romero et al., =>>^; Yoon et al., =>>m) and is the result of multiple resistance mechanisms (Adelman et al., =>UU; Zhu et al., =>UJ; Zhu et al., =>U=; Zhu et al., =>U>), including kdr-type target-site protein mutations (Dang et al., =>UKb; Yoon et al., =>>m; Zhu et al., =>U>), over expression of detoxifying cytochrome PNK> monooxygenases (Zhu et al., =>U=), and cuticular resistance (Koganemaru et al., =>UJ; Zhu et al., =>UJ). Molecular analysis of C. lectularius has indicated that esterase-mediated detoxification enzymes may also contribute to the resistance observed in pyrethroid-resistant bed bugs (Adelman et al., =>UU; Zhu et al., =>U=).

However, in the two North American strains examined to date the results appeared to be variable, with general esterases activity in a Richmond, VA, strain increased by JK% compared with NU% for cytochrome PNK> and, separately, the expression of esterase gene ClCE=UJJU in a

Cincinnati, OH, strain found to be highly strain dependent and variable in expression. More recently, analysis of enzyme expression in several newly detected neonicotinoid-resistant C. lectularius strains also found elevated esterases as being the most closely correlated with the observed resistance (Romero & Anderson, =>UV). These results may have important implications for future resistance management, as, in addition to resistance to pyrethroids, esterases have the potential to confer cross-resistance to arylpyrrole insecticides (Gunning & Moores, =>>=).

The arylpyrroles are a group to which bed bugs do not yet appear to have developed resistance,

VK Chapter 3: Metabolic Detoxification as a Resistance Mechanism but which if already present at low rates could explain the variable performance reported with this chemical class against C. lectularius (Doggett et al., =>U=). In addition, cytochrome PNK> monooxygenases could alternately confer cross-resistance to neonicotinoid insecticides

(Puinean et al., =>U>), and thus, there is the need to be able to distinguish between these enzyme systems in resistant bed bugs.

Studies screening for the potential of enzymatic detoxification in insecticide-resistant insects commonly use multiple synergists and insecticides in comparative treatments as a method to infer the relative synergism and toxicity of the various compounds. The results of such assays are then used to deduce the presence or absence of one or more detoxification systems (Cristina et al., =>UV; Demkovich et al., =>UK; He et al., =>U=; Liu & Yue, =>>>; Scott,

Uoo>; Yanola et al., =>UK; Zhao et al., UooK). PBO is routinely included in such studies due to its well-established capacity to inhibit cytochrome PNK> monooxygenase and, correspondingly, act as a synergist of pyrethroid-based insecticides (Hodgson & Levi, Uoom; Scott, Uoo>). The sole study to date of the effect of PBO on pyrethroid-resistant bed bugs found it acted as a variable synergist of deltamethrin depending on the insect strain tested (Romero et al., =>>oc). The researchers concluded that other resistance mechanisms might have been contributing to the underlying variation. It has been demonstrated, however, that PBO will equally inhibit esterases

(Gunning et al., Uoom; Moores et al., =>>o; Young et al., =>>K), and this consequently introduces ambiguity as to the relative presence and contribution of each enzyme group to any observed resistance. With the development of ENUV/K-U (V-[=-(=-butoxyethoxy)ethoxy-methyl]-K-propyl-

=,J-dihydrobenzofuranby) an analogue of PBO that inhibits only esterases (Moores et al., =>>o), a bioassay-based method for insecticide-resistant insects may now be available to screen for both cytochrome PNK> monooxygenase- and esterase-mediated metabolic resistance, and to further assess the relative performance of ENUV/K-U as a deltamethrin synergist in insect pests such as bed bugs. Under a comparative bioassay, PBO and ENUV/K- U will both act as inhibitors of esterases but, alternately, only PBO will also act as an inhibitor of cytochrome PNK>

VV Chapter 3: Metabolic Detoxification as a Resistance Mechanism monooxygenases. Thus, as complementary synergists, evidence can be obtained for the relative contribution of each enzyme group to underlying metabolic pyrethroid resistance in bed bugs, and the potential for the development of cross-resistance to other chemical classes.

Therefore, the aim of this study was to assess the performance of PBO and ENUV/K-U as synergists of deltamethrin against four modern C. lectularius field strains collected from across

Australia. Relative performance of the synergists can then be used to infer which enzyme group may be contributing to any observed resistance.

3.2 Materials and methods

Storage and culturing of the bed bug strains were conducted as approved by the

Westmead Hospital Animal Ethics Committee (WHAEC Protocol =>>=) and in accordance with

NSW Animal Research Review Panel (ARRP) Guidelines for the Housing of Rats in Scientific

Institutions.

3.2.1 Bed bug strains

Bed bugs were collected from disparate field infestations across New South Wales,

Victoria, and the Northern Territory in Australia, either by the authors, relevant state health department officers, or professional pest managers (Table J.U). Detailed treatment histories were not available for any of the collected field strains, although it is believed insecticide control had been attempted with the Parramatta and Alice Springs strains prior to their collection. All specimens were identified as C. lectularius (Usinger, UoVV), and kdr haplotype was determined

(Dang et al., =>UKb). Thereafter, the insects were used to establish colonies that were housed within the departmental insectary and maintained inside an insectary maintained at =K°C (±

U°C), ^K% (± U>%) relative humidity, and a photoperiod of U=:U= (L:D) h (Lilly et al., =>UK). An additional laboratory strain of C. lectularius, with known resistance to pyrethroids (and known kdr haplotype), designated the ‘Sydney’ strain (Lilly et al., =>UK) was also used in select pilot

V^ Chapter 3: Metabolic Detoxification as a Resistance Mechanism experiments. All bed bugs were offered a blood meal on specific-pathogen-free anaesthetized rats seven days prior to being selected for use in bioassays. No insecticide selection was undertaken on the field strains.

Table 3.1: Bed bug (Cimex lectularius) strain sources Bed bug strain Collection location Date kdr Maximum Source (Suburb, State) collected haplotypea generationsb

Sydney Sydney, New South Wales ca. =>>N A/B > U>> DMEc

Parramatta Parramatta, New South Wales UJ Dec. =>U= B ≈ Uo DMEc

Alice Springs Alice Springs, Northern Territory U July =>UJ B ≈ U= NT Healthd

Melbourne no.= Melbourne, Victoria K Mar. =>UJ B ≈ UV MKe

Melbourne no.N West Melbourne, Victoria UK Apr. =>UJ B ≈ UK MKe

a. As determined by Dang et al. (=>UKb) b. Based on minimum development period per generation of JU.J days at =^°C (Omori, UoNU) c. Department of Medical Entomology, University of Sydney and Pathology West d. Environmental Health – Central Australia, Northern Territory Department of Health e. Maureen Koegel – Sleep Tight Services, Central Park, Victoria Table 3.1: Field strains of Cimex lectularius examined for the presence and type of metabolic detoxification resistance

3.2.2 Chemicals

Technical grade deltamethrin (CAS Number K=oUm-VJ-K) and PBO (CAS Number KU->J-

V) were supplied by Bayer CropScience (Hawthorn East, Australia). The synergist ENUV/K-U was supplied by Endura SpA (Ravenna, Italy). Analytical grade acetone (CAS Number V^-VN-U), used to dissolve the technical grade insecticide, was purchased from Sigma-Aldrich Co. LLC.

3.2.3 Bioassays

3.2.3.1 Deltamethrin screening of field strains

Pilot assays with deltamethrin were undertaken to determine if the bed bug field strains exhibited an appreciable degree of pyrethroid resistance. Deltamethrin was dissolved in analytical grade acetone to form a top dose of =K> g/litre. Log dilutions were then performed to establish the remaining dose levels down to a lowest dose of >.>=K g/litre (which represented approximately five times the LDoo of a known susceptible laboratory strain). Controls received

Vm Chapter 3: Metabolic Detoxification as a Resistance Mechanism acetone only. Two replicates of ten randomly selected mixed sex and age adult bed bugs were then topically treated with U µL of solution using an established method shown to have minimal impact on bed bug control survival (Lilly et al., =>UK). Briefly, bed bugs were lightly secured, dorsal side down, to small strips of double-sided tape affixed to the plastic lid of a =K> mL plastic container. After topical exposure, the solution was allowed to dry for several minutes and then the bugs removed with fine tweezers into a =K> mL plastic recovery container lined with o> mm diameter Whatman number U qualitative filter papers (Catalogue number U>>U->o>,

Buckinghamshire, UK) placed in the bottom. Mortality was then recorded =N h post application.

3.2.3.2 Toxicity pilot assays of EN16/5-1 and PBO

Additional assays were conducted to determine the relative toxicity of ENUV/K-U and PBO in the absence of insecticides to ensure suitability for the next stage of the study. The ‘Sydney’ strain was used in this instance in order to preserve field strain bed bugs for latter bioassays.

Technical grade samples of both synergists were prepared as described above, and log doses ranging from K>–>.>K g/litre were applied as U mL doses to two replicates of ten randomly selected mixed sex and age adult bed bugs. Controls received acetone only. Mortality was recorded =N h post application.

3.2.3.3 Synergism assays

Dose levels for the synergism study were selected from the pilot assays as being =.K g/litre deltamethrin and either K> g/litre ENUV/K-U or K> g/litre PBO. Six treatments were prepared for each field strain: U) acetone control, =) K> g/litre PBO control, J) K> g/litre ENUV/K-

U control, N) =.K g/litre deltamethrin, K) K> g/litre PBO + =.K g/litre deltamethrin, and V) K> g/litre ENUV/K-U + =.K g/litre deltamethrin. Four replicates of ten randomly selected mixed sex and age adult bed bugs were used per treatment; thus, =N> total bed bugs were assayed per field strain. Pre-treatment with PBO or ENUV/K-U occurred = h prior to exposure to deltamethrin with the bed bugs left secured to the restraining double-sided tape for the intervening period. In

Vo Chapter 3: Metabolic Detoxification as a Resistance Mechanism treatments without a synergist, bed bugs were left secured to the restraining double-sided tape for = h post acetone or deltamethrin exposure to mimic any potential stress-induced mortality that may have influenced the observed results.

3.2.4 Statistical analysis

Counts of mortality were examined for significance using a generalized linear model for binomial distribution with a log-link in IBM SPSS Statistic v== for Mac (IBM Corp. =>UJ).

3.3 Results and discussion

3.3.1 Deltamethrin screening of field strains

Initial resistance screening demonstrated that all four field bed bug strains possessed a high level of resistance to deltamethrin, with a non-dose-dependent response exhibited despite extremely high quantities of insecticide being administered. At the highest dose of =K> g/ litre deltamethrin, the Parramatta, Melbourne no.=, and Melbourne no.N strains returned a maximum N>% mortality, whereas the Alice Springs strain was not statistically significantly different from the acetone-only control. This result conforms to other published research that has demonstrated pyrethroid resistance has become widespread in C. lectularius field populations (Adelman et al., =>UU; Boase, =>UJ; Dang et al., =>UKb; Kilpinen et al., =>UU; Moore &

Miller, =>>V; Romero et al., =>>^; Yoon et al., =>>m; Zhu et al., =>U>). Previous work on these bed bug strains in Australia has shown that the Parramatta strain exhibits a pyrethroid-resistant response to a simplified field-screening assay (Dang et al., =>UKa) and, separately, that the

Melbourne no.=, Melbourne no.N, and Alice Springs strains also possess the haplotype ‘B’ (Lo=KI only) kdr-type protein mutation that is known to confer pyrethroid resistance (Dang et al.,

=>UKb; Zhu et al., =>U>).

^> Chapter 3: Metabolic Detoxification as a Resistance Mechanism

3.3.2 Toxicity pilot assays of EN16/5-1 and PBO

The toxicological screening bioassays with PBO or ENUV/K-U utilizing the Sydney laboratory strain also demonstrated that there was no appreciable mortality with either synergist when applied in isolation (no insecticide), with mortality not exceeding K% at any dose level. PBO is often included in formulated insecticide products in Australia (Doggett,

=>UJ), and research with the technical grade form of the chemical on bed bugs has also demonstrated that no lethal effect was observed up to a dose of K> mg/mL (Romero et al.,

=>>oc). This information was the basis of the selected dose of K> g/ litre for the synergism studies using PBO or ENUV/K-U in comparative treatments.

3.3.3 Synergism assays

A single = h pre-treatment with PBO resulted in a significant improvement in deltamethrin efficacy as measured by mean cumulative mortality in the Parramatta (Fig. J.U, p

= >.>>m), Alice Springs (Fig. J.=, p < >.>>U), Melbourne no.= (Fig. J.J, p < >.>>U), and Melbourne no.N (Fig. J.N, p < >.>>U) bed bugs, with all strains achieving > o>% mortality after the inclusion of the synergist.

^U Chapter 3: Metabolic Detoxification as a Resistance Mechanism

Figure 3.1: Synergistic effect of PBO and EN16/5-1 on deltamethrin efficacy against Parramatta strain Cimex lectularius

Figure 3.1: Effect of synergists ENUV/K-U and PBO on cumulative mortality of Parramatta strain of Cimex lectularius to deltamethrin. Letters indicate significant difference at p < >.>K.

Figure 3.2: Synergistic effect of PBO and EN16/5-1 on deltamethrin efficacy against Alice Springs strain Cimex lectularius

Figure 3.2: Effect of synergists ENUV/K-U and PBO on cumulative mortality of Alice Spring strain of Cimex lectularius to deltamethrin. Letters indicate significant difference at p < >.>K.

^= Chapter 3: Metabolic Detoxification as a Resistance Mechanism

Figure 3.3: Synergistic effect of PBO and EN16/5-1 on deltamethrin efficacy against Melbourne no.2 strain Cimex lectularius

Figure 3.3: Effect of synergists ENUV/K-U and PBO on cumulative mortality of Melbourne no.= strain of Cimex lectularius to deltamethrin. Letters indicate significant difference at p < >.>K.

Figure 3.4: Synergistic effect of PBO and EN16/5-1 on deltamethrin efficacy against Melbourne no.4 strain Cimex lectularius

Figure 3.4: Effect of synergists ENUV/K-U and PBO on cumulative mortality of Melbourne no.N strain of Cimex lectularius to deltamethrin. Letters indicate significant difference at p < >.>K.

^J Chapter 3: Metabolic Detoxification as a Resistance Mechanism

Further comparison between the relative performance of PBO or ENUV/K-U at synergizing deltamethrin showed that only two strains, Parramatta (p = >.oU) and Melbourne no.= (p =

>.=m^), exhibited equivalent mean mortality values regardless of the presence of either PBO or

ENUV/K-U as the synergist of deltamethrin. PBO as a deltamethrin synergist otherwise resulted in higher mean mortality than ENUV/K-U in the Alice Springs (p = >.>Nm) and Melbourne no.N (p

= >.>U^) strains.

These results, when considering each strain possessed the same kdr-type mutation, clearly suggest that metabolic detoxifying enzymes are a contributing mechanism toward the magnitude of pyrethroid resistance observed in these field strains. PBO effectively synergized deltamethrin efficacy in all field strains, and competitively outperformed ENUV/K-U in the Alice

Springs and Melbourne no.N bed bugs. This thereby implies that cytochrome PNK> monooxygenases may be responsible for much of the pyrethroid resistance observed in these strains. Conversely, non-significant differences in the performance of ENUV/K-U as a deltamethrin synergist in the Parramatta and Melbourne no.= bed bugs imply that esterases are responsible for the pyrethroid resistance observed in these strains.

Future research should expand testing to multiple dose applications against each strain to explore any effect of varying insecticide and synergist ratios and the determination of an effective synergistic ratio based on a complete dose-response assay. However, given all strains in this study failed to respond in a dose-response manner to deltamethrin in the pilot trials and that large doses of each synergist were used, it can be assumed that in this instance any synergistic ratio would have been correspondingly large.

3.4 Conclusions

This is the first study to investigate the potential presence of esterase-derived pyrethroid resistance in C. lectularius via a bioassay method using two synergists, PBO and ENUV/K-U. All four recently collected field strains of bed bugs were highly resistant to deltamethrin but, with

^N Chapter 3: Metabolic Detoxification as a Resistance Mechanism pre-exposure of PBO, such resistance could be effectively inhibited, thereby suggesting a significant contribution from metabolic detoxifying microsomal oxidases or hydrolytic esterases. Further analysis has revealed that in two such strains, collected from Parramatta,

NSW, and Melbourne, Victoria, the observed pyrethroid resistance is potentially derived from hydrolytic esterases. The remaining two strains, collected from Alice Springs, NT, and West

Melbourne, Victoria, were not synergized by the esterase-specific synergist ENUV/K-U, thus suggesting the observed pyrethroid resistance is due to cytochrome PNK> monooxygenases.

Additional research to determine respective enzyme expression levels and the molecular basis for pyrethroid resistance in these bed bug strains is currently underway.

^K Chapter 4: Cuticle Thickening as a Resistance Mechanism

Chapter 4 – Cuticle Thickening in a Pyrethroid-Resistant Strain of the Common Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae)

Lilly, D. G., S. L. Latham, C. E. Webb, & S. L. Doggett

PLOS ONE (2016), 11(4), e0153302 doi: 10.1371/journal.pone.0153302

^V Chapter 4: Cuticle Thickening as a Resistance Mechanism

Abstract

Thickening of the integument as a mechanism of resistance to insecticides is a well- recognised phenomenon in the insect world and, in recent times, has been found in insects exhibiting pyrethroid-resistance. Resistance to pyrethroid insecticides in the common bed bug,

C. lectularius, is widespread and has been frequently inferred as a reason for the pest’s resurgence. Overexpression of cuticle depositing proteins has been demonstrated in pyrethroid- resistant bed bugs although, to date, no morphological analysis of the cuticle has been undertaken in order to confirm a phenotypic link. This paper describes examination of the cuticle thickness of a highly pyrethroid-resistant field strain collected in Sydney, Australia, in response to time-to-knockdown upon forced exposure to a pyrethroid insecticide. Mean cuticle thickness was positively correlated to time-to-knockdown, with significant differences observed between bugs knocked-down at two hours, four hours, and those still unaffected at =N hours.

Further analysis also demonstrated that the =N hour survivors possessed a statistically significantly thicker cuticle when compared to a pyrethroid-susceptible strain of C. lectularius.

This study demonstrates that cuticle thickening is present within a pyrethroid-resistant strain of C. lectularius and that, even within a stable resistant strain, cuticle thickness will vary according to time-to-knockdown upon exposure to an insecticide. This response should thus be considered in future studies on the cuticle of insecticide-resistant bed bugs and, potentially, other insects.

^^ Chapter 4: Cuticle Thickening as a Resistance Mechanism

4.1 Introduction

Since the turn of the century there has been a dramatic resurgence across many countries in the number of infestations of bed bugs, both the common, C. lectularius, and tropical C. hemipterus species (Doggett et al., =>U=). Insecticide resistance has been commonly inferred as a key driver of the bed bug resurgence (Doggett et al., =>U=; Doggett et al., =>>Nc;

Myamba et al., =>>=; Romero et al., =>>^; Wang & Cooper, =>UU) as pyrethroid resistance has been found to be concomitantly widespread with the growth and spread of infestations

(Adelman et al., =>UU; Boase et al., =>>V; Dang et al., =>UKb; Dang et al., =>UKc; Kilpinen et al.,

=>UU; Koganemaru et al., =>UJ; Lilly et al., =>UK; Moore & Miller, =>>V; Romero et al., =>>^;

Tawatsin et al., =>UU; Yoon et al., =>>m; Zhu et al., =>U>). Several mechanisms have been discovered that contribute to the observed resistance, including kdr-type target-site protein mutations (Dang et al., =>UKb; Dang et al., =>UKc; Yoon et al., =>>m; Zhu et al., =>U>), overexpression of detoxifying enzymes (Yoon et al., =>>m; Zhu et al., =>U=), and reduced penetration (Koganemaru et al., =>UJ; Zhu et al., =>UJ).

Reduced penetration in other insecticide-resistant insects has been known to be a resistance mechanism since it was first established in the UoV>s with pyrethrin (Fine et al., UoVJ), organophosphates (Forgash et al., UoV=; Matsumura & Brown, UoVU, UoVJ), carbamates

(Georghiou et al., UoVU; McDonald & Cochran, UoVm), and organochlorines (Plapp & Hoyer, UoVm;

Sawicki & Lord, Uo^>; Sellers et al., Uo^J; Vinson & Brazzel, UoVV). It can occur via multiple mechanisms, including enhanced expression of metabolic resistance mechanisms in the integument (Zhu et al., =>UJ), the increased presence of binding proteins, lipids, and/or sclerotization that trap insecticides (Patil & Guthrie, Uo^m; Vinson & Law, Uo^U), a measurably thicker cuticle (Wiesmann, UoN^; Wood et al., =>U>), or a combination of some or all of these mechanisms together (Patil & Guthrie, Uo^m, Uo^o; Terriere, Uom>; Yu, =>>m). Several of these mechanisms have been found in bed bugs with, to date, the exception of cuticle thickening.

^m Chapter 4: Cuticle Thickening as a Resistance Mechanism

Ordinarily, reduced penetration does not, by itself, impart a high degree of resistance

(Gunning et al., UooU; Plapp & Hoyer, UoVm; Yu, =>>m), although it may nonetheless have importance by way of conferring a level of cross- resistance to a wider variety of insecticides

(Oppenoorth, UomN; Plapp & Hoyer, UoVm; Yu, =>>m), increasing the efficiency of metabolic detoxification (Ahmad et al., =>>V; Mamidala et al., =>U=; Sawicki, Uo^>; Sawicki & Lord, Uo^>), or delaying the onset of knockdown (Plapp & Hoyer, UoVm; Scott, Uoo>; Wood et al., =>U>).

However, the expression of one or more resistance mechanisms does not necessarily predicate a corresponding change in expression of cuticular proteins (Yu & Nguyen, UooV) although it appears that, conversely, reduced penetration is typically found only when other mechanisms are present (Alyokhin et al., =>>m; Argentine et al., UooN; Valles et al., =>>>; Wood et al., =>U>).

No measurable comparison of the cuticle thickness has thus far been undertaken in bed bugs, although evidence from very recent molecular analysis of several C. lectularius strains point toward changes in protein expression (Koganemaru et al., =>UJ; Mamidala et al., =>U=).

The manifestation and intensification of other resistance mechanisms in the cuticle is strongly indicative of reduced penetration and/or cuticle thickening (Zhu et al., =>UJ) and this has most recently been successfully established in the South African malaria vector mosquito Anopheles funestus (Wood et al., =>U>). Thus, in adapting the experimental design used with An. funestus, it is the aim of this paper to investigate the potential presence of cuticle thickening in a highly pyrethroid-resistant strain of C. lectularius and to examine the relationship of any such thickening to time-to-knockdown upon exposure to an insecticide.

4.2 Materials and methods

4.2.1 Chemicals

Demand Insecticide (=K g/L l-cyhalothrin) was supplied free of charge by Syngenta Crop

Protection Pty Limited (Level U, =–N Lyonpark Road, Macquarie Park NSW =UUJ).

^o Chapter 4: Cuticle Thickening as a Resistance Mechanism

4.2.2 Bed bug strains

Storage and culturing of the bed bug strains were conducted as approved by the

Westmead Hospital Animal Ethics Committee (WHAEC Protocol No.=>>=) and in accordance with NSW Animal Research Review Panel (ARRP) Guidelines for the Housing of Rats in

Scientific Institutions.

Cimex lectularius specimens were collected from a single, domestic, field infestation in the suburb of Parramatta, NSW, Australia, in December =>U=. A detailed treatment history was not available for this strain, although it is believed insecticide control had been attempted prior to its collection. The specimens were examined for species (Usinger, UoVV), kdr haplotype (Dang et al., =>UKb) and, thereafter, used to establish a colony in the departmental insectary maintained at =K°C (±U°C) and ^K% (±U>%) RH with a photoperiod of U=:U= (L:D) h (Lilly et al.,

=>UK). All bed bugs were offered a blood meal on SPF anaesthetised rats seven days prior to being selected for use in bioassays. Rat anaesthesia was achieved with an intraperitoneal injection of ketamine and xylazine, and recovery monitored until complete. No insecticide selection was undertaken. Resistance profiling indicated this strain homogeneously possessed haplotype B (Lo=KI only) kdr-type resistance (Dang et al., =>UKb). Exposure to a d-allethrin based rapid resistance field assay also failed to elicit any statistically significant levels of knockdown (Dang et al., =>UKa). Given the lack of efficacy with d-allethrin (a first generation, type I pyrethroid), a second-generation type II pyrethroid, l-cyhalothrin, was preferentially selected for use as the screening agent in this study.

An additional laboratory strain of C. lectularius kept in colonies since the UoV>s with known susceptibility to pyrethroids (designated the ‘Monheim’ strain (Lilly et al., =>UK)) was also used selectively to assess for any effect due to variations in insect body size. The Monheim strain lacks any kdr-type mutation (haplotype A) (Dang et al., =>UKb).

To ensure all bed bugs used in the experiments were of equal age, cohorts of fifth instar bed bugs were isolated from the stock colonies, fed to repletion, and monitored daily for

m> Chapter 4: Cuticle Thickening as a Resistance Mechanism appearance as adults. Once matured the bugs were immediately separated by sex to limit the potential for breeding, and aged for a further eight or nine days (depending on their intended use) without any further blood meal.

4.2.3 Knockdown bioassay

A => mL/L solution of Demand Insecticide was mixed according to label directions and applied at the rate of U.= mL (to the point of run-off) to o> mm Ø WhatmanTM No. U qualitative filter papers (Cat. No. U>>U–>o>) held in petri dishes. Sixteen replicates of ten male Parramatta strain nine-day-old bed bugs were then immediately placed onto the treated surface (no drying period) and knockdown was recorded at ten minute intervals up to four hours. Knockdown was defined as the bugs not being able to right themselves when inverted onto their dorsal side.

Affected bugs were immediately transferred to labelled specimen vials containing ^>% ethanol.

Bed bugs that were knocked-down within the first two hours were marked as the ‘intolerant’ group, and unaffected bugs at four hours were marked as the ‘tolerant’ group. The experiment was repeated until at least ten bugs were collected for each response group.

In order to control for the additional time required in selecting for the most resistant bed bugs, the above experiment was repeated with a cohort of eight-day-old Parramatta strain male bugs, and the experiment (nine replicates of ten bugs) allowed to run for =N hours of continuous forced exposure. Any bed bugs unaffected at the conclusion of =N hours were then marked as the ‘resistant’ group, and were thus nine days old at the point of sampling as per the

‘intolerant’ and ‘tolerant’ response groups.

Exposure of the Monheim strain bugs to the above regimen was attempted, however, all bugs rapidly succumbed to the insecticide within a single ten minute period and thus no separation into groups of varying tolerance was possible. Consequently, the Monheim strain was excluded from the inter-group cuticle thickness and time-to-knockdown analysis, but a

mU Chapter 4: Cuticle Thickening as a Resistance Mechanism random sample of nine-day-old bugs were nonetheless processed for cuticle measurements in order to provide for a comparison between ‘susceptible’ and ‘resistant’ bugs.

4.2.4 Preparation for scanning electron microscope

Specimens preserved in ^>% ethanol were examined under the dissecting microscope in fresh ^>% ethanol. The middle legs of each specimen were dissected from the body at the apical region of the femur. A subsequent severance was made at the tibia midpoint whilst the legs were suspended in a small drop of ^>% ethanol. The remaining specimen bodies were stored in ^>% ethanol for later analysis. Legs were moved to Eppendorf tubes, washed twice more in ^>% ethanol to remove sectioning debris and processed in an ascending ethanol series as follows: m>%, o>%, oK% and U>>%, twice for five minutes each, followed by three washes for ten minutes each in ultrapure ethanol. Samples were chemically dried for three minutes in hexamethyldisilazane (Proscitech), transferred to new Eppendorf tubes and air-dried for five minutes. SEM specimen stubs (Ted Pella) were modified with a diamond saw to expose a flattened surface. Legs were mounted to this surface with carbon tape and carefully positioned to ensure the tibia was vertically positioned and the severed midpoint was exposed at the stubs apical surface. Carbon paint was applied to secure samples to the specimen stubs. Specimens were sputter coated with gold for two minutes at =K mA with the EMITECH KKK>X and were stored in a desiccator.

4.2.5 Electron microscope imaging

Secondary electron micrographs were obtained with a Zeiss EVO K> SEM at U> kV. Each sample was individually rotated and tilted towards the secondary electron detector, varying the working distance between o and =K mm. Image focus and scale calibrations were corrected with the dynamic focus and tilt correction tools. Integrated line averaging was utilised to minimise charging artefacts during image acquisition.

m= Chapter 4: Cuticle Thickening as a Resistance Mechanism

4.2.6 Image analysis

Raw and processed images were analysed in ImageJ (version U.NVr) (Rasband, Uoo^–=>UU).

To assess cuticle width, the known scale in microns from the processed imaged was measured according to the number of pixels, and this value transferred to the raw image. Thereafter, U= point-to-point measurements were made, roughly equidistantly, from the outermost to innermost visible sections of the cuticle, whilst avoiding obvious debris, damage, or setae beds

(Fig. N.U). Measurement of the various specimens was made while blinded to the allocated response group (‘intolerant’, ‘tolerant’, ‘resistant’ and ‘susceptible’) in order to limit the potential for bias.

Sample sizes were balanced by random selection in IBM SPSS Statistic v== for Mac (IBM

Corp., =>UJ) with mean cuticle thickness values of the three Parramatta response groups com- pared via one-way ANOVA and Fisher’s Least Significant Difference (LSD) post-hoc tests.

Cuticle thickness vs. time-to-knockdown was analysed by linear regression. Cuticle thickness between ‘susceptible’ and ‘resistant’ bugs was compared via a one-way ANOVA.

4.2.7 Insect body measurements

Additional body measurements were made of the specimens from all four response groups used for cuticle analysis in order to check for any effect of varying body or appendage size on the resultant mean cuticle values. Several target measurements were adapted from previously published research (Balvin et al., =>U=), and included the pronotum width (pw), pronotum length (pl), head width (hw), length of the Ust antennal segment (after the scape–aQl), hind leg tibia length (tRl), and hind leg tibia width (tRw). Measurements were made under dissecting microscope using stage and/or optical micrometers and evaluated for statistical significance and effect sizes (Partial Eta= values) using general linear model multivariate analysis and Bonferroni post-hoc comparisons.

mJ Chapter 4: Cuticle Thickening as a Resistance Mechanism

Figure 4.1: Cuticle thickening measurement methodology used with Parramatta strain Cimex lectularius

Figure 4.1: Transverse section of Cimex lectularius (Parramatta strain) middle leg tibia and example of twelve point-to-point cuticle measurements methodology. Letters denote measurements of: a = o.UK μm, b = o.m= μm, c = U>.=K μm, d = ^.oU μm, e = ^.UV μm, f = m.N= μm, g = m.>= μm, h = m.NU μm, k = U>.U= μm, m = m.m= μm, n = m.JU μm, o = m.>o μm [i, j and l not included for purpose of legibility in the published manuscript].

4.3 Results

4.3.1 Knockdown

Knockdown of the exposed nine-day-old Parramatta-strain bed bugs occurred asymptotically up to a value of approximately ^V% (S.E. ± U.m%) over the course of four hours, with the first bugs affected after K> minutes (Fig. N.=) and increasing up to Jm% (S.E. ± V.K%) after two hours (the arbitrary end-point of ‘intolerant’ response group). Bed bugs aged to eight days and continually force-exposed for =N hours (to an equivalent age of nine-days-old) had knockdown of m=% (S.E. ± =.>%), leaving a further Um% of bugs that were unaffected and thereafter classed as the ‘resistant’ group.

mN Chapter 4: Cuticle Thickening as a Resistance Mechanism

Figure 4.2: Time to knockdown of Parramatta strain Cimex lectularius

Figure 4.2: Knockdown (% ± S.E, n = UV>) over time (minutes) of Parramatta strain Cimex lectularius upon forced exposure to wet residues of => mL/L Demand InsecticideU (=K g/L l- cyhalothrin).

4.3.2 Imaging and cuticle thickness

Several specimens had to be excluded from analysis at the imaging stage due primarily to damage that had occurred during sectioning and mounting, or as a result of internal tissues obscuring the cuticle. Where this affected overall parity of sample size, excess specimens were excluded via random sampling in SPSS such that, in total, measurements from ten specimens were eventually analysed from each response group.

Mean cuticle thickness was found to be significantly different (p < >.>>U) between the three Parramatta-strain response groups (Fig. N.J). ‘Resistant’ bed bugs had a mean cuticle thickness of U>.UJ μm (S.E. ± >.UK μm), ‘tolerant’ bugs o.KU μm (S.E. ± >.=V μm) and ‘intolerant’ bugs m.^J μm (S.E. ± >.Um μm). ‘Resistant’ bugs thus had a significantly different mean cuticle

mK Chapter 4: Cuticle Thickening as a Resistance Mechanism thickness of +>.V= μm and +U.N μm compared to the ‘tolerant’ (p < >.Jm) and ‘intolerant’ (p <

>.>>U) bugs respectively, and the ‘tolerant’ bugs were also found to have a significantly different mean cuticle thickness of +>.^m μm compared to the ‘intolerant’ bugs (p < >.>U=). Linear regression analysis of the time-to-knockdown and mean cuticle thickness (Fig. N.N) revealed a significant trend (p < >.>U), with cuticle thickness positively correlated (R= adjusted = >.JJ^) with increasing time-to-knockdown. Given the relatively limited sample sizes and length between time intervals the otherwise modest adjusted R= value is, in context, a valid result.

Figure 4.3: Mean cuticle thickness of Parramatta strain Cimex lectularius

Figure 4.3: Mean cuticle thickness (μm ± S.E) of intolerant (n = U>), tolerant (n = U>) and resistant (n = U>) response group Parramatta strain Cimex lectularius. Asterisks (*) indicate statistical significance (p < >.>K).

mV Chapter 4: Cuticle Thickening as a Resistance Mechanism

Figure 4.4: Correlation between time to knockdown and cuticle thickness in Parramatta strain Cimex lectularius

Figure 4.4: Positive correlation of time-to-knockdown to mean cuticle thickness of Parramatta strain Cimex lectularius (n = U> per response group) upon continuous forced-exposure to wet residues of => mL/L Demand InsecticideU (=K g/L l-cyhalothrin).

4.3.3 Insect measurements

No statistically significant differences existed within the Parramatta strain between the three response groups for all the various body measurements (pronotum width, pronotum length, head width, length of the first antennal segment, hind leg tibia length, hind leg tibia width and ratio of tibia width to cuticle thickness), except cuticle thickness as noted separately above.

Monheim strain pyrethroid-susceptible bugs were found to be statistically significantly larger than the Parramatta strain bugs in pronotum width (all groups p < >.>>U), pronotum

m^ Chapter 4: Cuticle Thickening as a Resistance Mechanism length (‘intolerant’ p < >.>>=; ‘tolerant’ p = >.>N=), head width (all groups p < >.>>U), antennal length (all groups p < >.>>=), and tibia length (all groups p < >.>>=). No statistical significant differences were observed for tibia width (all groups p > >.>K), and pronotum length (‘resistant’ p > >.>K).

Previously, head width in C. lectularius has been established as correlated both to differences in the size of specimens between strains and differences in body extremities relative to overall body size (Balvin et al., =>U=). In this study, examination of Partial Eta= (corrected) values similarly indicated that both pronotum width (>.^J>) and head width (>.^Ko) were also prominent factors in accounting for the proportion of variance associated with each of the aspects measured. Consequently, head width was selected as the most appropriate feature to correct for size differences between the Monheim and Parramatta bed bug strains in order to make an appropriate comparison of mean cuticle thickness. To this effect, the observed difference in mean head width between the Monheim strain and Parramatta ‘resistant’ group bugs was found to be +o.o%. When this strain size difference is adjusted and factored into the cuticle thickness comparison, ‘resistant’ bugs had a significantly different (p < >.>>U) mean cuticle thickness of +U.JK μm compared to the ‘susceptible’ bugs (Fig. N.K).

mm Chapter 4: Cuticle Thickening as a Resistance Mechanism

Figure 4.5: Mean cuticle thickness between resistant and susceptible Cimex lectularius

Figure 4.5: Mean cuticle thickness (μm ± S.E) of Monheim susceptible (n = U>) and Parramatta resistant (n = U>) Cimex lectularius. Asterisks (*) indicate statistical significance (p < >.>K).

4.4 Discussion

Insecticide resistance is a genetic change in response to selection by toxicants that impairs control in the field (Sawicki, Uom^) and is considered to be a natural evolutionary response to human-induced environmental stress (Feyereisen, UooK; ffrench-Constant et al.,

=>>N; Hoy, Uoom; Yu, =>>m). In the case of bed bugs, this would be failure of pest management attempts at infestation eradication through insecticidal means. Development or enhancement of the cuticle as the primary barrier to insecticides is integral to this process with an obvious advantage (in the absence of any other fitness costs) conferred to the bed bug phenotype of higher tolerance to forced insecticide exposure. Using a well-established time-to-knockdown method enables identification and separation of bed bugs of varying insecticide resistance. In

mo Chapter 4: Cuticle Thickening as a Resistance Mechanism this study, the most resistant bed bugs (themselves separated from a highly resistant field strain) were found to have cuticle thickness UV% greater than the least resistant (‘intolerant’) bugs and

V.K% from the intermediately resistant (‘tolerant’) bugs. Further analysis (after correcting for strain size differences) indicated the ‘resistant’ bugs possessed cuticle thickness UK.J% greater than that of a susceptible strain, thereby confirming a link between increased cuticle thickness and observed insecticide resistance.

Under normal field circumstance it would be expected that some variation in cuticle phenotype within a strain could be apportioned to factors such as diet or age. However, both diet and age were controlled prior to forced-selection of the bugs based on research that has shown bed bugs are appreciably resistant and express a stable metabolism at nine days (DeVries et al., =>UKa; DeVries et al., =>UKb), and that no difference in efficacy has been observed regardless of whether bugs have been fed previously (e.g. at day one) or starved for up to nine days (Choe & Campbell, =>UN). This aligns with the practices of other laboratories employing bed bugs in insecticide-based bioassays, with most selectively using male bed bugs, aging between seven to ten days after emergence, and not feeding for five to eight days prior to the experiment(s), if at all (Adelman et al., =>UU; Feldlaufer et al., =>UJ; Koganemaru et al., =>UJ;

Moore & Miller, =>>V; Romero et al., =>>oc, =>U>b).

A difference in cuticle thickness between strains of varying insecticide susceptibility was established in this study, after corrections were made for the increased size of the susceptible strain over the resistant strain. Given the Monheim strain bed bugs were found to be larger than the Parramatta strain bugs across a range of physical features (pronotum width, pronotum length, head width, antennal length, and tibia length) it is evident that overall insect size is a factor that should be considered in any future studies targeting morphological features if more than one field strain is being examined. Use of morphological features, and particularly measurement of head width, has been used to analyse evolutionary aspects of bed bugs (Balvin

o> Chapter 4: Cuticle Thickening as a Resistance Mechanism et al., =>U=) and our results suggest a similar approach may be warranted when examining other physical mechanisms that may confer reduced insecticide susceptibility in C. lectularius.

Thickening of the cuticle in resistant bed bugs may have important consequences for field control as the delivery of insecticides becomes potentially less efficient. However, further study of the cuticle layers, the relative contribution of each to the overall thickness, and the process of cuticle development and deposition in resistant bed bugs may be required in order to under- stand to what degree the cuticle thickening is reducing penetration (either alone or by enhancement with other resistance mechanisms). This, in turn, may influence advancements in insecticides and formulations in an effort to overcome the increased resistance of bed bugs.

Ideally, this research should be repeated with more field strains of both the common and tropical species of bed bug, however, the high cost of SEM research (currently USD$K>>> per strain) was a limiting factor in this study. Nonetheless, further work is ongoing on a full examination of other resistance mechanisms within this and other field strains from Australia.

4.5 Conclusions

This study demonstrates that the most highly resistant bed bugs from a pyrethroid- resistant field strain possess a thicker cuticle that enables those bugs to survive exposure to insecticides when compared both to less resistant insects from the same strain and also those from a susceptible strain. This may help to explain why failures in the control of field infestations are so common, and further emphasizes the need for an integrated approach in the control of bed bugs to prevent the further spread of highly resistant insects. Changes in cuticle thickness, the lipid composition and passage of insecticides, if combined with expression of detoxifying enzymes or target-site insensitivity, may also have important consequences for the use of formulated insecticides for the field control of bed bugs.

oU Chapter 5: Tolerance to Silica-Based Desiccant Dusts

Chapter 5 – Evidence of Tolerance to Silica-Based Desiccant Dusts in a Pyrethroid-Resistant Strain of Cimex lectularius (Hemiptera: Cimicidae)

Lilly, D. G., C. E. Webb, & S. L. Doggett

Insects (2016), 7(74): 1-12 doi: 10.3390/insects7040074

o= Chapter 5: Tolerance to Silica-Based Desiccant Dusts

Abstract

Insecticide resistance in bed bugs (C. lectularius and C. hemipterus) has become widespread, which has necessitated the development of new integrated pest management (IPM) strategies and products for the eradication of infestations. Two promising options are the diatomaceous earth and silica gel-based desiccant dusts, both of which induce dehydration and eventual death upon bed bugs exposed to these products. However, the impact of underlying mechanisms that confer resistance to insecticides, such as cuticle thickening, on the performance of these dusts has yet to be determined. In the present study, two desiccant dusts,

CimeXa Insecticide Dust (silica gel) and Bed Bug Killer Powder (diatomaceous earth) were evaluated against two strains of C. lectularius; one highly pyrethroid-resistant and one insecticide-susceptible. Label-rate doses of both products produced U>>% mortality in both strains, albeit over dissimilar time-frames (J-N days with CimeXa vs. UN days with Bed Bug

Killer). Sub-label rate exposure to CimeXa indicated that the pyrethroid-resistant strain possessed a degree of tolerance to this product, surviving K>% longer than the susceptible strain. This is the first study to suggest that mechanisms conferring resistance to pyrethroids, such as cuticular thickening, may have potential secondary impacts on non-synthetic insecticides, including desiccant dusts, which target the bed bug’s cuticle.

oJ Chapter 5: Tolerance to Silica-Based Desiccant Dusts

5.1 Introduction

The re-emergence of bed bugs, namely both the common species, C. lectularius, and the tropical species, C. hemipterus, as public health pests over the last UK years has presented a unique challenge from both a clinical and management perspective (Doggett et al., =>U=;

Minocha et al., =>UV), and has necessitated a multi-disciplinary approach in an attempt to develop best practice control options (Doggett, =>UJ; Doggett et al., =>UU). Key amongst these has been a response to the growing global evidence that most field bed bug populations have developed resistance to multiple insecticide groups (Karunaratne et al., =>>^; Kilpinen et al.,

=>UU; Lilly et al., =>UK; Romero & Anderson, =>UV; Suwannayod et al., =>U>; Tawatsin et al., =>UU) and, in particular, that they have become highly resistant to pyrethroid insecticides (Adelman et al., =>UU; Anderson & Cowles, =>U=; Campbell & Miller, =>UK; Dang et al., =>UKb; Dang et al.,

=>UN; Gordon et al., =>UN; Koganemaru et al., =>UJ; Moore & Miller, =>>V; Myamba et al., =>>=;

Romero et al., =>>^; Zhu et al., =>UJ; Zhu et al., =>U=; Zhu et al., =>U>). Despite this, and due to a general scarcity of alternative control options (Doggett & Russell, =>>m), when faced with a bed bug infestation both pest management professionals and residents of infested properties continue to apply pyrethroid insecticides in attempts to achieve control (Doggett & Russell,

=>>m; Potter et al., =>UK; Potter et al., =>>ma; Singh et al., =>UJ; Wang et al., =>U>). Several different formulations of insecticides are available, although liquid sprays (oV%) and dusts

(oJ%) were found to be to most commonly selected insecticidal formulations used by USA- based pest management professionals when surveyed in =>UK (Potter et al., =>UK).

Insecticidal dusts would normally be recommended for only limited application to areas such as voids, cavities, behind electrical plates, under carpet or skirting boards, and other non- obvious locations. This is typically in an effort to both control the infestation but also to eliminate harbourage options and the potential spread of the infestation (Doggett, =>UJ), as it is well known that bed bugs can migrate from one unit or apartment to the adjacent dwellings in multi-occupancy buildings (Doggett & Russell, =>>m; Wang et al., =>U>). However, in recent

oN Chapter 5: Tolerance to Silica-Based Desiccant Dusts times, dusts have also been proposed for, and used, around the interior of properties (such as around the perimeter of bedrooms and lounge rooms, on bed frames and sofas, or as a fabric- treated ‘dust band’ on furniture legs) as a low-cost, long-term residual treatment option applicable particularly in properties housing the financially or socially disadvantaged (Singh et al., =>UJ; Wang et al., =>>o; Wang et al., =>U=; Wang et al., =>UJb). Such a modified application of dusts for bed bug control has the potential to achieve high levels of population reduction

(typically in the vicinity of oK-om%), but rarely complete elimination (Wang et al., =>>o; Wang et al., =>U=; Wang et al., =>UJb).

Despite being in use for many decades against , drywood , mites, and stored product and (Anderson & Cowles, =>U=; Ebeling, Uo^U, Uo^K; Golob,

Uoo^; Quarles & Winn, UooV; Subramanyam & Roesli, =>>>), silicon dioxide-based (SiO=) desiccant dusts (both naturally-occurring diatomaceous earth and synthetically-manufactured silica gel) are a relatively new option available to pest management professionals for bed bug control. These dusts are regarded as a popular treatment option due to characteristics of low mammalian toxicity, long residual life (in dry environments), and an orthodox view that as a result of the physical mode of action they are non-selective for physiological-derived resistance

(Doggett & Russell, =>>m; Golob, Uoo^; McLaughlin, UooN). Both diatomaceous earth and silica gel-based dusts will absorb lipids from the insect’s cuticle, leading to eventual dehydration and death (Ebeling, Uo^U; Korunic, Uoom; Prasantha et al., =>UK; Quarles & Winn, UooV).

Diatomaceous earth is both abrasive and sorptive (Ebeling, Uo^U; Malia et al., =>UV), however, against various stored product pests it has been found to be significantly less effective than non- abrasive silica gels, even when used in higher doses (Ebeling, Uo^U).

Both forms of desiccant dust possess discernible efficacy against bed bugs (Akhtar &

Isman, =>UV; Benoit et al., =>>ob; Doggett & Russell, =>>m; Donahue Jr. et al., =>UK; Goddard,

=>UN; Potter et al., =>UN; Romero et al., =>>oa; Singh et al., =>UV) although results have been variable depending on the product tested and bed bug strain(s) used. Diatomaceous earth has

oK Chapter 5: Tolerance to Silica-Based Desiccant Dusts been found to be effective, under laboratory conditions, at application rates between U-m g/m=, achieving U>>% mortality against adults over the course of o-UK days and oo% mortality against nymphs after two days (Doggett & Russell, =>>m). Contrastingly, two studies (one field trial- based study and a second, double-blind laboratory study) have otherwise presented evidence that diatomaceous earth may be largely ineffective over longer monitoring periods (Goddard,

=>UN; Potter et al., =>UJ). This may be due to environmental factors such as humidity or the physical properties of the diatoms and how the product has been manufactured (Prasantha et al., =>UK; Quarles & Winn, UooV). When compared, silica gel appears faster-acting with U>>% mortality achieved typically within =N-^= hours compared to >o>% mortality after ten days for diatomaceous earth (Donahue Jr. et al., =>UK; Potter et al., =>UN; Romero et al., =>>oa). The addition of bed bug alarm pheromones improves efficacy (Benoit et al., =>>ob), as does employing CO= as an attractant to increase contact with dust residues (Aak et al., =>UV).

To date, the effect of any insecticide resistance conferring mechanisms on dust performance has only been superficially investigated. Most notably, two studies have reported that pyrethroid-resistant strains of C. lectularius took longer than susceptible strains to succumb to desiccant-based dusts (Anderson & Cowles, =>U=; Romero et al., =>>oa). Given both studies employed dusts with an insecticide component, the authors postulate that differences in each strain’s response to the insecticide component most likely explained the observed variation. However, Anderson and Cowles (=>U=) also noted that “pyrethroid-resistant bed bugs have adaptive changes to their integument that could provide some protection against desiccation”.

Cuticle thickening has most recently been confirmed as a mechanism conferring insecticide resistance in an Australian strain of C. lectularius, with the most resistant bugs

(themselves selected from a highly pyrethroid-resistant strain) possessing a cuticle that is up to

UK.J% thicker than that of a susceptible C. lectularius strain and UV% thicker than less-resistant bugs from within the same strain when measured under scanning electron microscope (Lilly et

oV Chapter 5: Tolerance to Silica-Based Desiccant Dusts al., =>UVc). In addition, research on C. lectularius strains collected in the USA has determined that pyrethroid-resistant bed bugs over-express genes responsible for cuticle protein development (Koganemaru et al., =>UJ), and that other mechanisms of insecticide resistance

(such as metabolic detoxification enzymes and kdr-type target site mutations) are concentrated in the epidermal layer of the bed bug’s integument (Zhu et al., =>UJ). The impact of the concentration of such resistance mechanisms all within the insect’s integument against insecticides has yet to be fully quantified, and any impact on the efficacy of products containing desiccant dusts is wholly unknown. Thus, in lieu of these research findings, it is the aim of this paper to assess the efficacy of two desiccant dusts (one diatomaceous earth-based, one silica gel-based) against a highly pyrethroid-resistant strain of C. lectularius that possesses a known profile of kdr-type target-site insensitivity, esterase-derived metabolic detoxification, and thickened cuticle, compared with a susceptible C. lectularius strain.

5.2 Materials and methods

Storage and culturing of the bed bug strains were conducted as approved by the

Westmead Hospital Animal Ethics Committee (WHAEC Protocol No. =>>=) and in accordance with NSW Animal Research Review Panel (ARRP) Guidelines for the Housing of Rats in Scientific

Institutions.

5.2.1 Experimental insects

Resistant strain C. lectularius specimens were collected from a single, field infestation from a domestic house in the suburb of Parramatta, New South Wales, Australia, in December

=>U=. The specimens were examined for species (Usinger, UoVV) and, thereafter, used to establish a colony in the departmental insectary maintained at =K°C (±U°C) and ^K% (±U>%) RH with a photoperiod of U=:U= h (L:D) (Lilly et al., =>UK). No insecticide selection was undertaken.

This strain has been extensively profiled for potential mechanisms conferring pyrethroid resistance, and has been found to possess haplotype B (homogeneous Lo=KI) kdr-type resistance

o^ Chapter 5: Tolerance to Silica-Based Desiccant Dusts

(Dang et al., =>UKb), hydrolytic esterase-derived metabolic detoxification (Lilly et al., =>UVb), and cuticle thickening (Lilly et al., =>UVc). Exposure to a d-allethrin based rapid resistance field assay elicits no statistically significant levels of knockdown when compared to an untreated control (Dang et al., =>UKa).

An additional laboratory strain of C. lectularius maintained in colony since the UoV>s with known susceptibility to pyrethroids (designated the ‘Monheim’ strain (Lilly et al., =>UK)) was also used. The Monheim strain lacks any kdr-type mutation (homogeneous haplotype A)

(Dang et al., =>UKb).

To ensure all bugs used in the experiments were of equal age, cohorts of fifth instar bugs were isolated from the stock colonies, fed to repletion, and monitored daily for appearance as adults. Once matured, the bed bugs were immediately separated by sex to limit the potential for breeding, and aged for a further o days without any further blood meal. Only o-day-old, male, adult bed bugs from either strain were used in the dust exposure assays.

5.2.2. Products

Commercial samples of CimeXa Insecticide Dust (‘CimeXa’, o=.U% SiO= as amorphous silica, Rockwell Labs Ltd, North Kansas City, MO, USA) and Bed Bug Killer Powder (‘Bed Bug

Killer’, oV% diatomaceous earth, Bed Bug Barrier Pty Ltd, Saint Kilda, VIC, Australia) were purchased through Do It Yourself Pest Control Supplies (www.doyourownpestcontrol.com) and

Globe Pest Solutions (www.globepestsolutions.com.au) respectively.

5.2.3. Desiccant dust assays

Filter paper discs (Advantac No.= o>mm diameter, Advantec MFS Inc., Dublin, CA,

USA) were placed in ocm diameter Petri dishes and weighed on an analytical balance (sensitivity

>.U mg) to determine pre-application weights. The amount of dust required was then calculated using the published label application rates of V.U g/m= (= oz./U>> ft=) for CimeXa and N g/m= for

Bed Bug Killer, with each value proportioned to the area of the Petri dish (VJ.V= cm=). An

om Chapter 5: Tolerance to Silica-Based Desiccant Dusts additional treatment of CimeXa applied at K>% of the recommended label rate (J.>K g/m=) was included for the purposes of this study only in an attempt to prolong the advancement of mortality and was informed by pilot assay results that indicated the onset of mortality was rapid.

Due to previously published research with diatomaceous earth indicating a slow mode of action even at high doses, no further doses either above or below recommended label rate were included for this product. Each dust was then applied by hand directly from the product containers according to the calculated rate and weighed to ensure the dose applied did not exceed ± K% (Table K.U). Any excess dust was removed with a flat-headed probe. No spreading or brushing of the dust was undertaken, with dusts allowed to spread and/or clump according to how it was dispensed from the product package. Controls consisted of untreated filter papers in o cm diameter Petri dishes.

Four replicates of ten male adult bed bugs that had been aged to nine days old were added to each petri dish and were continuously maintained on the treated surfaces for the duration of the study. Mortality was recorded hourly for =N hours, again at JV hours, then daily for UN days or until all bed bugs had died. Mortality was defined as the bugs not responding to gentle pressure through probing.

LTK> values were estimated using a Logit generalized linear model in IBM SPSS Statistic v=J for Mac (IBM Corp, =>UK). Cumulative mortality values at each time point were then also used to create Kaplan-Meier survival curves, with differences between the Parramatta and

Monheim strain bugs within treatment groups compared by Log-rank tests.

oo Chapter 5: Tolerance to Silica-Based Desiccant Dusts

Table 5.1: Calculated and applied rates of CimeXa Insecticide Dust and Bed Bug Killer Powder

Strain Treatment Label Replicate Label Rate Actual Deviation Deviation Rate Dose (g) Dose (g) (g) (%) Parramatta CimeXa >.Kx U >.>UoN >.>UoK >.>>>U >.K=%

= >.>UoN >.>UoK >.>>>U >.K=%

J >.>UoN >.>Uo^ >.>>>J U.KK%

N >.>UoN >.>Uo^ >.>>>J U.KK%

CimeXa U.>x U >.>Jmm >.>Jm^ ->.>>>U ->.=V%

= >.>Jmm >.>Jo> >.>>>= >.K=%

J >.>Jmm >.>Jo> >.>>>= >.K=%

N >.>Jmm >.>JoN >.>>>V U.KK%

Bed Bug Killer U.>x U >.>=KN >.>=KJ ->.>>>U ->.Jo%

= >.>=KN >.>=KJ ->.>>>U ->.Jo%

J >.>=KN >.>=No ->.>>>K -U.o^%

N >.>=KN >.>=KN >.>>>> >.>>%

Monheim CimeXa >.Kx U >.>UoN >.>UoN >.>>>> >.>>%

= >.>UoN >.>UoK >.>>>U >.K=%

J >.>UoN >.>Uo= ->.>>>= -U.>J%

N >.>UoN >.>Uo> ->.>>>N -=.>V%

CimeXa U.>x U >.>Jmm >.>JoJ >.>>>K U.=o%

= >.>Jmm >.>JmV ->.>>>= ->.K=%

J >.>Jmm >.>Jo> >.>>>= >.K=%

N >.>Jmm >.>JmJ ->.>>>K -U.=o%

Bed Bug Killer U.>x U >.>=KN >.>=KK >.>>>U >.Jo%

= >.>=KN >.>=KN >.>>>> >.>>%

J >.>=KN >.>=KK >.>>>U >.Jo%

N >.>=KN >.>=K^ >.>>>J U.Um%

Table 5.1: Calculated and applied doses of CimeXa Insecticide Dust (silica gel) and Bed Bug Killer Powder (diatomaceous earth)

U>> Chapter 5: Tolerance to Silica-Based Desiccant Dusts

5.3 Results

Adult, male bed bugs exposed to both applications rates of CimeXa dust began to succumb after more than six hours of continuous exposure, with survival decreasing along a broadly inverse-sigmoidal manner over the resulting JV-^= hours. At the prescribed label rate of CimeXa, complete mortality was achieved after JV hours for both strains of bed bugs, with no statistically significant difference (p = >.moo) existing between the Parramatta (resistant) and

Monheim (susceptible) bugs in the rate of diminishing survival over the course of the experiment (Figure K.U). No control mortality was recorded within JV hours on the untreated filter papers for either strain of C. lectularius.

Figure 5.1: Kaplan-Meier analysis of the survival of resistant and susceptible Cimex lectularius exposed to label rate CimeXa Insecticide Dust

Figure 5.1: Proportional cumulative survival in response to continuous exposure to the prescribed label rate of CimeXa Insecticide Dust of Parramatta (resistant) and Monheim (susceptible) strains of Cimex lectularius (n = N> bugs per strain, control mortality = >%)

U>U Chapter 5: Tolerance to Silica-Based Desiccant Dusts

At half of the prescribed label rate of CimeXa dust, onset of mortality began to occur after approximately U= hours of continuous exposure, however, the Parramatta (resistant) bugs ultimately took longer to succumb (Figure K.=) with a statistically significantly lower rate of decrease (p < >.>>U) compared to the Monheim (susceptible) bugs. Interestingly, Monheim

(susceptible) strain bugs succumbed after JV hours regardless of the dose being applied, whereas Parramatta (resistant) strain bugs took » K>% longer to die at half label rate (^= hours) than at the prescribed label rate (JV hours). No control mortality was recorded within JV hours on the untreated filter papers for the Monheim (susceptible) bed bugs, and only =.K% was recorded with the Parramatta (resistant) bed bugs at the final ^=-hour monitoring point.

Figure 5.2: Kaplan-Meier analysis of the survival of resistant and susceptible Cimex lectularius exposed to half label rate CimeXa Insecticide Dust

Figure 5.2: Proportional cumulative survival in response to continuous exposure to the half (>.Kx) prescribed label rate of CimeXa Insecticide Dust of Parramatta (resistant) and Monheim (susceptible) strains of Cimex lectularius (n = N> bugs per strain, Monheim (susceptible) control mortality = >%, Parramatta (resistant) control mortality at ^= hours = =.K%)

U>= Chapter 5: Tolerance to Silica-Based Desiccant Dusts

Bed Bug Killer proved to be a much slower acting product compared to CimeXa, with a full two weeks (UN days) required before U>>% mortality was achieved in either strain (Figure

K.J). No statistically significant difference was observed in mortality or the rate of kill (p = >.UJU).

Control mortality in both strains did not exceed =.K% for the first UU days, and thereafter was ≤

K% for the remainder of the experiment. Thus no Abbott’s correction was made to the treatment values (WHO, Uoom).

Figure 5.3: Kaplan-Meier analysis of the survival of resistant and susceptible Cimex lectularius exposed to label rate Bed Bug Killer Powder

Figure 5.3: Proportional cumulative survival in response to continuous exposure to the prescribed label rate of Bed Bug Killer Powder of Parramatta (resistant) and Monheim (susceptible) strains of Cimex lectularius (n = N> bugs per strain, control mortality at UN days (both strains) ≤ K%)

U>J Chapter 5: Tolerance to Silica-Based Desiccant Dusts

Estimates of the LTK> values for CimeXa (Table K.=) also indicated no statistically significant difference between the Parramatta (resistant) and Monheim (susceptible) bugs when exposed to the prescribed label rate, with K>% mortality expected after ≈ => hours of continuous exposure. However, at half label rate of CimeXa the estimated LTK> value for Parramatta

(resistant) bugs (J> hours), was approximately K>% more than that of the Monheim

(susceptible) bugs (=>.N^ hours). Failure of the oK% confidence intervals to overlap for these values indicates that this is, putatively, a statistically significant difference.

Table 5.2: LTK> values in hours (± oK% confidence intervals) for Parramatta (resistant) and Monheim (susceptible) Cimex lectularius when assayed against CimeXa Insecticide Dust (at U.>x and >.Kx label rate) LT MK% CI MK% CI Treatment Label Rate Strain KL (Hours) (Lower) (Upper)

CimeXa >.Kx Parramatta J>.>>* =K.>^ JN.oJ

>.Kx Monheim =>.N^ Um.N> ==.KK

U.>x Parramatta =>.U^ U^.VK ==.^>

U.>x Monheim =>.>> U^.mV ==.UN

* Indicates statistically significant difference as determined by failure of the oK% confidence intervals to overlap

Table 5.2: Lethal time determination for resistant and susceptible Cimex lectularius against CimeXa Insecticide Dust

Estimates of the LTK> values for Bed Bug Killer (Table K.J) also indicated no statistically significant difference between the Parramatta (resistant) and Monheim (susceptible) bugs when exposed to the prescribed label rate, with K>% mortality expected after ^.K-m days of continuous exposure.

U>N Chapter 5: Tolerance to Silica-Based Desiccant Dusts

Table 5.3: LTK> values in days (± oK% confidence intervals) for Parramatta (resistant) and Monheim (susceptible) Cimex lectularius when assayed against Bed Bug Killer Powder (only tested at U.>x label rate) LT MK% CI MK% CI Treatment Label Rate Strain KL (Days) (Lower) (Upper)

Bed Bug Killer U.>x Parramatta ^.N= V.KN m.J>

U.>x Monheim m.U= V.oo o.=K

Table 5.3: Lethal time determination for resistant and susceptible Cimex lectularius against Bed Bug Killer Powder

5.4 Discussion

Insecticide tolerance is defined as the ‘the natural ability of a population to withstand the toxic effect of a particular insecticide’ (Yu, =>UKa) and in contrast to resistance is not, per se, a genetic change as a result of selection pressure. However, it may nonetheless be a by-product of an insect’s inherent physiological ability to detoxify an insecticide or resist penetration through the thickening of the integument. As far as current knowledge exists, desiccant dusts act exclusively on the cuticle, and the results of this study suggest that bed bugs that possess cuticle thickening as a mechanism to resist insecticides also then exhibit tolerance to desiccant dusts if exposed to sub-label rates.

Upon exposure to a silica gel product, CimeXa Insecticide Dust, applied at half the prescribed label rate, the Parramatta (resistant) bed bugs were able to survive an additional ten hours compared to the Monheim (susceptible) strain. The estimated LTK> for the resistant bugs was also found to be statistically significantly different, at approximately K>% more than the

LTK> of the susceptible strain. Despite this, label rate application of the product achieved U>>% mortality after JV hours, with near identical estimated LTK> values suggesting the product remains efficacious against resistant strains when used according to label directions. The diatomaceous earth product, Bed Bug Killer Powder, was found to be extremely slow acting compared to CimeXa, but was ultimately efficacious over the long term with no statistically significant differences observed after UN days.

U>K Chapter 5: Tolerance to Silica-Based Desiccant Dusts

The results of this study conform to the findings of several laboratory and field studies into the effectiveness of various dust products against resistant and susceptible C. lectularius strains. In the USA, assays employing CimeXa against C. lectularius found that it was the only product of eight tested (including several other insecticide-only products, one insecticide + oK% diatomaceous earth product, and one U>>% diatomaceous earth product) that resulted in U>>% mortality regardless of whether bed bugs were temporarily exposed, forcibly and continuously exposed, or offered a treated/untreated harborage choice (Singh et al., =>UV). CimeXa was also found to transfer and induce mortality between treated and untreated bed bugs effectively at ratios of both U:K and N:V (Singh et al., =>UV). Separately, CimeXa was found to be highly efficacious against two pyrethroid-resistant strains and one susceptible strain of C. lectularius, with mortality in a laboratory-based assessment typically reaching U>>% after only =N-Nm hours

(Potter et al., =>UN), as was predominantly achieved in this study.

A combination desiccant and insecticide, Drione Insecticide (U% pyrethrins, U>% PBO,

N>% silica gel, Bayer Environmental Science, Research Triangle Park, NC, USA), was also found to work quickly (K>% survival < =N hours) on hardboard and mattress-fabric surfaces, while an insecticide only dust, DeltaDust (>.>K% deltamethrin, Bayer Environmental Science, Research

Triangle Park, NC, USA), took significantly longer (K>% survival = J.K days) to achieve an equivalent degree of mortality (Anderson & Cowles, =>U=). Interestingly, when tested in the same experiment, the excipient of Drione, Syloid ==N (Grace Davison, Columbia, MD, USA) performed better than Drione itself, a result explained by the fact that the end-product of

Drione consists of N>% less silica that Syloid ==N. The authors concluded that desiccant dusts

“appear to be superior to sprayable pyrethroid products for killing bed bugs” (Anderson &

Cowles, =>U=).

In a similar comparative study of three desiccant dust products against USA-sourced pyrethroid susceptible and resistant strains, Drione again proved efficacious against the resistant bed bugs, achieving U>>% mortality in ^= hours (Romero et al., =>>oa). Drione also

U>V Chapter 5: Tolerance to Silica-Based Desiccant Dusts worked faster compared to a diatomaceous earth-based formulation, Mother Earth D

(Whitmire Micro-Gen Research Laboratories Inc., St Louis, MO, USA), which resulted in >o>% mortality in the first four days, and an eventual U>>% after ten days (Romero et al., =>>oa). A third, limestone-based product, NIC J=K (ACM-Texas, Fort Collins, CO, USA), proved to be ineffective against both susceptible and resistant strains. Interestingly, in the same study,

Tempo Dust (U% cyfluthrin, Bayer Environmental Science, Research Triangle Park, NC, USA) proved to be the most efficacious product, killing all strains (including pyrethroid resistant strains) within =N hours. The authors theorized this could be as a result of other inert products in the formulation improving uptake of the insecticide.

In an Australian study employing diatomaceous earth, U>>% mortality was also achieved with the highest dose after nine days, and UK days for all other doses against a pyrethroid- resistant strain of C. lectularius (Doggett & Russell, =>>m). In the same study, nymphs were found to be significantly more susceptible, with oo% mortality achieved after only two days, although this result was unsurprising as it is known bed bug nymphs can lose water rapidly and thus are more susceptible to water stress (Benoit et al., =>>^). The response of nymphs and adult females was not assessed in this study but may be an area worthy of further investigation.

Several studies have also been undertaken examining, in a mostly indirect manner, the efficaciousness of desiccant dusts as part of treatments for C. lectularius control in low economic or social housing (Singh et al., =>UJ; Wang et al., =>>o; Wang et al., =>U=). When either Tempo

Dust or Mother Earth D were included as part of an ‘Insecticides Only’ or ‘IPM’ program for the treatment of a low-income housing building in Indianapolis, IN, reductions of JJ% and NN% respectively were seen after ten weeks (Wang et al., =>U=). Both dusts were applied to many areas (such as cracks and seams around sofas and sleeping areas), although no indication was recorded by the authors as to the rationale for selecting Tempo or Mother Earth D for use, thus making direct inferences about the performance of each treatment regime and dust against each other difficult. Nonetheless, in a separate study employing a similar methodology, use of Mother

U>^ Chapter 5: Tolerance to Silica-Based Desiccant Dusts

Earth D, bed bug interceptor traps, and steam treatments, achieved a o^.V% (± U.V%) reduction in the number of bed bugs after ten weeks suggesting some efficacy at population reduction over the long term (Wang et al., =>>o), although again the relative contribution of each of the program components to the overall reduction is impossible to gauge. Additionally, despite the promising levels of bed bug population reduction, complete eradication was ultimately only achieved in K>% of the treated apartments, suggesting that a refinement in non-chemical control options or a secondary insecticide treatment method is needed to make the final step to elimination.

The main contrasting study to the above results achieved with diatomaceous earth concerns a field trial run in Kentucky, USA, where Mother Earth D was used exclusively in six domestic apartments, with neither chemical or non-chemical complementary treatments undertaken over the course of an initially planned U=-week trial (Potter et al., =>UJ). However, five of the six apartments had to be withdrawn prematurely from the study due to tenant dissatisfaction, and despite the intensive inspection and treatment with diatomaceous earth, average bed bug counts rose by U%. The only apartment successfully treated involved a minor infestation, and a tenant that travelled frequently and thus was away for periods of the trial. A suspected reason for the failure was that bed bugs were only receiving “abbreviated” exposure to the dusts and were thus not receiving a lethal dose (Potter et al., =>UJ).

Similarly, Alpine Dust Insecticide (>.=K% dinotefuran, oK% diatomaceous earth dust,

Whitmire Micro-Gen Research Laboratories Inc., St Louis, MO, USA) was evaluated in combination with Alpine Aerosol (>.K% dinotefuran, Whitmire Micro-Gen Research

Laboratories, St. Louis, MO, USA) across nine C. lectularius infested apartments. Despite dramatically reducing the amount of insecticide applied in the course of a routine bed bug treatment, after six months bed bugs had only been eliminated from three apartments (as measured by visual inspection and monitoring devices), suggesting that exclusively employing a combination of neonicotinoid plus diatomaceous earth based treatments also is not, by itself,

U>m Chapter 5: Tolerance to Silica-Based Desiccant Dusts an effective solution (Singh et al., =>UJ). This may be due to the recent emergence of neonicotinoid resistance in the USA (Romero & Anderson, =>UV) or, as the authors suggest, the consequence of a generally scattered bed bug distribution as a result of resident’s relocating infested belongings. However, a second study examining V-month-old aged residues of Alpine

Dust Insecticide found the product resulted in only N>% mortality when tested against susceptible C. lectularius, and negligible mortality against resistant C. lectularius, further suggesting this product should only be included as part of a completely integrated program of other control methods for long-term reduction or control against modern field strains of bed bugs (Goddard, =>UN).

The results of this study have important implications for future use of such desiccant dust products. Any enhancement of underlying insecticide resistance mechanisms (such as cuticle thickening) that potentially reduce the efficacy of desiccant dusts may, eventually, lead to an increased risk of treatment failures. Evidence from stored product pests has shown that, with continued exposure to desiccant dusts, some species have developed tolerance to the point of compromising product efficacy, and are thus at the threshold of evolving from tolerant to resistant (Prasantha et al., =>UK). For example, tolerant Tribolium castaneum beetles have been found to both avoid diatomaceous earth dust residues down to levels as low as >.>^K g/L and, when forced into a treated area, will have decreased movements compared to non-tolerant strains (Rigaux et al., =>>U). Similarly, silica gels are known to be highly repellent to Blatella germanica (Ebeling, UooK). Such adaptions, if translated to bed bugs, may complicate the development of devices such as some lethal harbourage traps which rely on the ability of bed bugs to enter traps and/or horizontally transfer small amounts of insecticidal dusts to other bed bugs, most likely during contact in a harbourage (Akhtar & Isman, =>UJ).

The result is also concerning when considering evidence that, under normal circumstances, pyrethroid-resistant bed bugs are more vulnerable to starvation than susceptible bugs (Polanco et al., =>UUb), a response that is likely due to a combination of factors such as

U>o Chapter 5: Tolerance to Silica-Based Desiccant Dusts differences in size to volume ratios between resistant and susceptible bugs, and increased susceptibility to dehydration with decreasing size. Of note, however, is that the Parramatta

(resistant) strain of C. lectularius used in this study has been found to be significantly smaller than the comparison Monheim (susceptible) strain bugs, on some body features up to ten percent (Lilly et al., =>UVc). Conventional theory would dictate that the smaller the insect, the more susceptible to dehydration it becomes (Gibbs & Rajpurohit, =>U>). However, as water-loss through the cuticle accounts for >m>% of overall water-loss (Chown et al., =>>V; Quinlan &

Gibbs, =>>V), the potential for a thickened cuticle layer to potentially provide greater tolerance to dehydration and, thus indirectly, increased tolerance to desiccant dusts warrants further monitoring and investigation.

5.5 Conclusions

This study demonstrates that a pyrethroid-resistant strain of C. lectularius that possesses multiple resistance mechanisms, including cuticular thickening, is tolerant to sub- label rates of silica gel-based dusts. Despite this, the development and inclusion of desiccant dusts, both diatomaceous earth and silica gel-based, for the control of bed bugs, presents as one of the more promising developments since the bed bug resurgence began. Nonetheless, preliminary evidence that resistance mechanisms derived from underlying pyrethroid- resistance may confer a natural level of tolerance to low doses of such dusts provides an early warning that any new or existing artificial selection in response to the use of insecticides will, inevitably, lead to a genetic change and the eventual manifestation of resistance.

UU> Chapter 6: General Discussion

Chapter 6 – General Discussion

UUU Chapter 6: General Discussion

6.1 Overview

The findings of this research project have made a considerable contribution towards understanding the presence of insecticide resistance in Australian bed bugs, and the mechanisms that contribute to the expressed resistance. In particular, these findings increase our understanding as to the mechanisms which may have driven the resurgence of bed bugs in

Australia. The results also further clarifying both how and why poor product efficacy can be an evolving challenge to end-user professional pest managers.

Establishing over thirty field-collected strains of both C. lectularius and C. hemipterus as laboratory-based colonies enabled investigations into the frequency of resistance both spatially and temporally. Strains are now held with founder specimens originating between the years =>>N-=>UV, from all Australian states and territories, excluding Tasmania and the

Australian Capital Territory (ACT). Additionally, continued colonization and access to an insecticide-susceptible strain of C. lectularius provided for an effective baseline with which to compare the response of the various field-collected strains.

Laboratory-based topical assays with discriminating doses of deltamethrin and imidacloprid (based on data derived from the insecticide-susceptible strain) revealed that a high frequency of resistance existed to deltamethrin in both C. lectularius and C. hemipterus, and to imidacloprid in C. hemipterus. There was no obvious trend evident with regard to increasing or decreasing frequencies based on strains tested between =>>m-=>UV (Chapter Two). However, a key finding of these experiments was that the frequency of response of C. lectularius to discriminating doses of imidacloprid appeared to changing, with a positive trend of reduced susceptibility identified in bed bugs collected post-=>UK.

The results of Chapter Two thereafter formed the basis of the later chapters, including

Chapter Three. Here, four C. lectularius strains that exhibited some of the highest frequency of pyrethroid resistance to the field-strain screening with deltamethrin were further examined for the presence and action of metabolic detoxification-derived resistance. This topic had not

UU= Chapter 6: General Discussion previously been investigated in detail in Australian bed bugs, despite anecdotal evidence that metabolic detoxification was a particularly important mechanism that potentially accounted for much of the reduced susceptibility in pyrethroid-resistant strains. The two synergists, PBO and

ENUV/K-U, were assayed in conjunction with deltamethrin to identify different forms of metabolic resistance to the pyrethroids in C. lectularius. PBO inhibits both cytochrome PNK> monooxygenases and hydrolytic esterases, whereas ENUV/K-U inhibits only hydrolytic esterases.

The results indicated that the level of synergism with deltamethrin was large, with metabolic detoxification evidently a mechanism that confers much of the expressed resistance to deltamethrin. Additionally, two strains each were putatively identified as possessing cytochrome PNK> monooxygenases-conferred metabolic resistance and hydrolytic esterase- conferred resistance. It was also apparent that the use of high doses of enzyme inhibiting synergists restored previously pyrethroid-resistant bed bugs near, but not completely, to susceptibility. Such a result was suggestive of further mechanisms of insecticide resistance being present within the assayed strains.

Chapter Four further utilised one strain of C. lectularius from chapters two and three, the ‘Parramatta’ field strain that was identified as being highly pyrethroid-resistant. It was determined that cuticle thickening was present as a resistance mechanism and was positively correlated with time to knockdown upon forced-exposure to a l-cyhalothrin residual bioassay.

The findings of this chapter represented the first time such a mechanism had been demonstrated within C. lectularius. This research also supported the findings of several molecular and gene-expression studies, which had identified that the bed bug integument was an important factor in conferring resistance to insecticides. Additionally, the findings of this chapter demonstrated that the Parramatta-strain exhibits all three of the major physiological resistance mechanisms, these being: esterase-mediated metabolic detoxification, homogeneous haplotype B kdr-type target site insensitivity and, now, cuticle thickening.

UUJ Chapter 6: General Discussion

Extending on the findings of Chapter Four, Chapter Five investigated the potential consequences of a bed bug strain that possess multiple resistance mechanisms on the efficacy of insecticidal desiccant dusts. It was determined that tolerance is present to silica gel-based products when exposed to sub-label rates. Desiccant dusts have become a frequently recommended option for bed bug control. They have become particularly popular as a low-cost, long-term residual option for the treatment of low socio-economic multi-occupancy buildings, particularly in the USA. Thus the finding of tolerance is a significant development that warrants further investigation.

6.2 Distribution of insecticide resistance in Australia

The results of this investigation have increased our understanding of the current distribution of insecticide resistance in Australian bed bugs. The findings suggest that resistance to pyrethroid insecticides is ubiquitous within field populations of both C. lectularius and C. hemipterus. Although no specimens were obtained from the ACT, the territory’s geographical position being completely bounded within the state of New South Wales (NSW) would, logically, predicate that the resistance otherwise expressed within NSW bed bugs would be similarly established in ACT bed bugs. However, the status of bed bugs in Tasmania remains unknown, and being a small island state, perhaps there could be differences in resistance responses in the local bed bug populations due to the state’s isolation and modest population.

However, the inability to source specimens in this study was similarly reflective of the lack of responses from the state to the most comprehensive bed bug-related surveys of Australian pest managers in =>>m and =>UV (Doggett, =>UV; Doggett & Russell, =>>m). Nonetheless, the finding that resistance occurs in every field strain tested to date has important implications for the future development and registration of insecticides for bed bug control. The presence of resistance should influence product selection by insecticide end-users, namely professional pest managers engaged in bed bug eradication services.

UUN Chapter 6: General Discussion

The findings conform to, and extend, previous research on Australian bed bugs that had previously detected pyrethroid and carbamate resistance in a Sydney-collected field strain of C. lectularius (Lilly et al., =>>oc; Lilly et al., =>UK). They also contribute to the various explanations of the generally poor level of efficacy of several formulated products registered for bed bug control (Lilly et al., =>>oa, =>>ob). The research herein also further expands upon the previous resistance mechanisms identified in Australian bed bugs, such as the presence of kdr-type target site insensitivity in both C. lectularius and C. hemipterus (Dang et al., =>UKa; Dang et al., =>UKb;

Dang et al., =>UKc). Importantly, in the screening study in Chapter Two, it was discovered that of JK field-collected bed bug strains tested, only K (UN%) were killed when exposed to a dose of deltamethrin equivalent to approximately ten times to concentration of the active ingredient in a formulated product. This represents a concerning high frequency of resistance across the country, and a result that is likely to be translated into eradication failures if professional pest managers are relying on liquid residual pyrethroid-only products for effective control.

Additionally, evidence was gathered from several field strains recently collected during

=>UK and =>UV that the frequency of susceptibility to neonicotinoid insecticides is also changing.

The evolution of resistance to the neonicotinoids has been very recently demonstrated in the

USA (Romero & Anderson, =>UV), and the data presented in Chapter Two supports the notion that this will inevitably also emerge in Australia bed bugs and wherever else this insecticide group is employed.

Similarly, there is evidence that the response of pyrethroid-resistant C. lectularius when exposed to sub-label rates of silica-gel desiccant dusts may be different from susceptible strains.

This is highly suggestive of reduced susceptibility being conferred by the accumulation and expression of one or more resistance mechanisms. It is apparent, however, that these products are still demonstrably effective when used according to the prescribed label rate, and thus remain the most promising option for both bed bug prevention and eradication.

UUK Chapter 6: General Discussion

6.3 The implications of multiple resistance mechanisms

Based on the cumulative results of this thesis, it is now apparent that strains of C. lectularius exist in Australia that possess multiple resistance mechanisms, including kdr-type mutations, metabolic detoxification, and cuticle thickening. The manifestation of such mechanisms confers a high frequency of resistance to pyrethroid insecticides and is likely central to the reduced susceptibility observed to neonicotinoids and silica-based desiccant dusts.

However, variability in the mechanisms present within each strain may still occur, and should be considered both for further monitoring and when developing insecticide resistance management strategies. Such variability in the strains examined in this thesis was epitomized by the variable mortality when exposed to a high dose of deltamethrin (Chapter Two), and the presence of different enzyme groups responsible for conferring metabolic detoxification resistance (Chapter Three). In response to deltamethrin discriminating-dose screening, mortality responses across JK strains (thirty-one C. lectularius and four C. hemipterus) ranged from UK% to U>>%. This was in spite of the majority of the strains tested possessing homogeneous kdr-type target site insensitivity. Such a result suggests that multiple resistance mechanisms are present, but may vary in their expression from strain to strain. Evidence supporting such an explanation was obtained when four C. lectularius strains were examined for metabolic detoxification-derived resistance. Two strains were found to be likely hydrolytic esterase-dominated, and another two, cytochrome PNK> monooxygenase-dominated.

Additional studies internationally have obtained comparable findings, variously indicating the presence and function of both oxidases and esterases as a mechanism of pyrethroid (Adelman et al., =>UU; Mamidala et al., =>U=; Zhu et al., =>U=) and neonicotinoid (Romero & Anderson,

=>UV) resistance. As a practical response to the presence of metabolic detoxification resistance, the addition of an enzyme inhibiting synergist, such as PBO, has been found to improve product performance when assayed against C. lectularius (Lilly et al., =>>oa) and C. hemipterus (How &

UUV Chapter 6: General Discussion

Lee, =>UU). However, other research has indicated the results obtained may be variable in not entirely overcoming expressed resistance (Romero et al., =>>oc). The presence of a resistance mechanism like cuticle thickening provides one explanation for the variable efficacy observed in such studies. Thickening of the integument is known to enhance other resistance mechanisms such as kdr-type target-site insensitivity and metabolic detoxification. This is imparted by slowing the penetration of insecticides, enhancing the action metabolic detoxification, and delaying the onset of knockdown.

The development of compounding resistance mechanisms and the subsequent negative effect across more than one insecticide mode of action is perhaps not an unsurprising result given the history of similar mechanisms emerging in other arthropod pests. However, in bed bugs, it is of particular importance given the relatively few (and diminishing) options available for the safe and economical treatment of infestations in the human environment. The combined findings from this thesis demonstrates that a resistant strain of C. lectularius may not be completely killed despite continuous forced exposure to a pyrethroid insecticide. This is further compounded by the fact that bed bugs were found to be tolerant to low doses of a silica-gel dust. This suggests that in any given infestation, the manifestation of resistance mechanisms will lead to a small percentage of bed bugs that are extremely difficult to eradicate by traditional control methods, and that enhanced resistance will continue to evolve. Indeed, this has become evident by the changing status of susceptibility in the field to the neonicotinoid insecticides.

Additionally, it should be expected that the perceived efficacy of insecticide-based treatments by professional pest managers will, in fact, vary from infestation to infestation as a direct result of mechanism-based variability between bed bug strains and the evolution of resistance.

Nonetheless, a key communication to end-users should be that every infestation, regardless of species, comprises bed bugs that will be highly resistant pyrethroids, potentially tolerant to neonicotinoids, and with a reduced susceptibility to desiccant dusts if not carefully and precisely applied according to label directions.

UU^ Chapter 6: General Discussion

6.4 Future impacts of insecticide resistance

Viewed historically, the development of resistance to insecticides in both C. lectularius and C. hemipterus associated with the modern resurgence was simply the continuation of a trend dating back over V> years. Resistance to DDT, the organochlorines, selected organophosphates, carbamates, pyrethrins, and some pyrethroids had been extensively reported up to UooU in C. lectularius, and to a lesser extent, in C. hemipterus up to Uom^ (Chapter

One). Given the early cases of resistance associated with the modern resurgence (to bendiocarb and a-cypermethrin in C. lectularius in Uoom, and to permethrin and a-cypermethrin in C. hemipterus in =>>=), suggests that bed bugs retained the ability to evolve resistance rapidly.

This was despite an intervening decade of bed bugs being rare in western society, but nonetheless still present to some degree in other, often developing, regions of the world.

Additionally, there is evidence that resistance-reversion occurred slowly in C. lectularius, and that insecticide selection pressure was continually applied against bed bugs in regions of the world where infestations didn’t decline. Both factors likely contributed significantly to the rapid resurgence.

Such historical trends also provide significant forewarning as to the future development and impact of insecticide resistance in bed bugs. This includes the discovery of resistance in C. lectularius to neonicotinoid insecticides in the USA (Romero & Anderson, =>UV), and the reduced susceptibility evident in Australian C. lectularius (Chapter Two). Along with the detection of tolerance to silica-gel desiccant dusts (Chapter Five), this suggests that bed bugs will continue to evolve rapidly to most forms of insecticidal control. This is arguably epitomized by the discovery of a resistance-conferring mechanism such as cuticle thickening (Chapter

Four), which is non-specific in nature. Importantly, cuticle thickening has the ability to both confer a degree of resistance (or tolerance) to a wide range of insecticides types, whilst also enhancing other resistance mechanisms present within the insect. Consequently, the cumulative presence of other metabolic-derived resistance mechanisms (Chapter Three) and

UUm Chapter 6: General Discussion kdr-type target site insensitivity, all within a single strain, further emphasize the challenge faced by professional pest managers if undertaking solely insecticide-based treatment procedures.

Unfortunately, the diversity of mechanisms now known to be expressed within resistant bed bugs populations would appear to predicate that, even in the presence of a global resistance management strategy, efficacy will inevitably be compromised to the few remaining insecticides with demonstrable efficacy against resistant strains. As a result, insecticide regulators would be wise to consider a more active function in the ongoing assessment of insecticides registered for use against bed bugs. As discussed in Chapter Two, for example, the continual registration of non-aerosolized, non-synergized pyrethroid insecticides for bed bug control is patently untenable given the resistance detected in this, and several other, studies.

Similarly, the role of professional pest management associations in continuing to define and encourage best practice is as critical as ever. Many of the resistance mechanisms expressed with bed bugs have a complex biological basis, and the subsequent impact on present and future insecticide efficacy may not be immediately apparent to those at the forefront of bed bug eradication. Thus, the development and continual review of guidelines, industry standards, and codes of practice, is vital. These documents must aim to distil each advancement in our understanding of the bed bug biology into meaningful and practical recommendations for bed bug eradication procedures, and will be integral to curbing the incidence of bed bugs across society.

6.5 Challenges and limitations

A key challenge that limited this thesis was the number of strains of C. hemipterus that were available for bioassay and mechanism-based experiments. Over the course of three years of actively collecting and sourcing bed bug strains, only five C. hemipterus strains were obtained, with only three established as colonies. However, even with the successful establishment of a

UUo Chapter 6: General Discussion colony, most strains failed to flourish despite intensive and repeated attempts to accumulate sufficient numbers of bed bugs that would permit more extensive bioassay investigations.

It is likely that the inability to raise sufficient colony numbers of C. hemipterus was related to a known aspect of this species possessing both a slower rate of colony development and lower egg-depositing rate compared to C. lectularius (Omori, UoNU) when maintained under the same environmental conditions. However, an interesting aspect was also that as many strains of C. lectularius as C. hemipterus were collected or received from regions of the country nominally belonging to the expected distribution of C. hemipterus (Doggett et al., =>>J).

Hypothetically, this may be due to a number of factors, including the initial source of infestations in Australia. However, it also potentially suggests that it is, in fact, C. lectularius which continues to be the predominant species of bed bug in Australia, with infestations of C. hemipterus being the exception even within the expected geographic range of the species.

The cumulative effect of few colonies of C. hemipterus being either collected or established is that our understanding of the potential mechanisms present within this species in Australia has not advanced as far or at the same rate as for C. lectularius. Nonetheless, preliminary screening with discriminating doses of deltamethrin and imidacloprid has confirmed that resistance to pyrethroids is as frequent in C. hemipterus as with C. lectularius.

Additionally, of the four strains assayed, susceptibility appeared to be maintained with imidacloprid, suggesting that neonicotinoid-based products should still be effective under field conditions. Nonetheless, collection of additional C. hemipterus specimens and the establishment of colony strains for the purposes of both insecticidal bioassay screening and elucidation of resistance-conferring mechanisms would be a potentially fruitful area of future research.

U=> Chapter 6: General Discussion

6.6 Future research

The results of the studies described in this thesis pose a number of questions which could not be answered within the timeframe of this candidature, but which are worthy of future research.

6.6.1 Changes in the bed bug integument

In Chapter Four, it was determined that a proportion of a C. lectularius strain possessed cuticle thickening in comparison to a susceptible strain. Furthermore, the thickness was positively correlated with time-to-knockdown when the resistant strain was forcibly exposed to a freshly treated pyrethroid surface (Lilly et al., =>UVc). Several international studies have also examined the molecular and genetic basis of genes responsible for cuticle-depositing proteins.

These also have indicated that the bed bugs integument is important to the expression of resistance (Bai et al., =>UU; Koganemaru et al., =>UJ; Mamidala et al., =>U=; Zhu et al., =>UJ).

An area that requires further investigation is a finer elucidation as to any relative changes to the composition of the cuticle, both chemically and physically, in an effort to understand precisely what is contributing to the measurably thicker cuticle in some pyrethroid- resistant bed bugs. Additionally, a fuller examination of the bed bug cuticle should focus on how such changes impact on the rate and degree of penetration of insecticides. Such a study should similarly consider the very recent finding bed bugs may possess more than one cuticular barrier to the penetration of hydrophilic molecules (Wang et al., =>U^), especially given that a strain of C. lectularius with known cuticle thickening was also found to express a degree of tolerance to silica-gel-based desiccant dusts (Lilly et al., =>UVd). The extension of such an investigation as to the mode of action of desiccant dusts on the bed bug’s cuticle, and the impact of any compositional changes, may also be a particularly valuable research with implications for professional pest managers.

U=U Chapter 6: General Discussion

6.6.2 Action of type-I and type-II pyrethroids

Given the importance of pyrethroids to continued efforts of bed bug infestation eradication, a fuller understanding is needed as to the impact of mechanisms such as metabolic detoxification and kdr-type target site insensitivity on pyrethroid mode of action. The magnitude of pyrethroid resistance has been demonstrated to vary according to the class of the compound assayed, with type-I ‘non-cyano’ pyrethroids (such as permethrin, d-phenothrin etc.) resulting in lower mortality compared to type II ‘α-cyano’ pyrethroids (such as deltamethrin, cyfuthrin, cypermethrin and fenvalerate etc.) when applied to a suspected-resistant field strains

(Anderson & Cowles, =>U=). Such a result conforms with evidence that type-I and type-II pyrethroids may bind to separate receptor sites (Breckenridge et al., =>>o).

The impact of such differences between pyrethroid types has not been fully examined against Australian field-strain bed bugs, with screening predominantly undertaken using either d-phenothrin or deltamethrin. As yet with both compounds have not been used together in a comparative bioassay. This is despite some evidence, based on dose response data with the

‘Sydney’ C. lectularius laboratory strain, indicating that resistance to permethrin (a type-I pyrethroid) was significantly greater than to that of deltamethrin (a type-II pyrethroid) (Lilly et al., =>UK).

Future studies should therefore focus on determining the impact of target site mutations, such as kdr-type resistance, that are known to be present within field-strain bed bugs on the biochemical action of type-I and type-II pyrethroids. This may assist in prolonging the effective life of some pyrethroid insecticides, and can be used to inform a resistance management strategy for bed bugs.

6.6.3 Behavioural resistance

A final mechanism, behavioural resistance, similarly needs to be considered as it may enable resistant bed bugs to avoid a lethal dose through stimulus-dependent hypersensitivity

U== Chapter 6: General Discussion or hyperirritability (Georghiou, Uo^=). As a resistance mechanism, behavioural resistance is less well understood than other physiological forms of resistance. However, it has been demonstrated that behaviourally resistant insects may respond differently to lower concentrations of insecticide than resistant strains (Silverman & Bieman, UooJ; Silverman &

Ross, UooN; Young & McMillian, Uo^o). This indicates a potential change in receptor sensitivity, and thus that behavioural resistance may have a physiological basis.

With regard to bed bugs, C. lectularius have been found to be repelled and irritated by pyrethroid residues in some studies, but not all. However, any perceived repellency can be overcome as a result of resistance, the presence of a host, the availability of a suitable harbourage, or the presence of an aggregation (Romero et al., =>>ob; Wang et al.,

=>UJa). Thus, there is the potential that if bed bugs have attained the tendency to be hypersensitive to such residues, that such a behavioural response may also intensify other pre- existing physiological resistance mechanisms.

6.7 Conclusion

The evolution of insecticide resistance in bed bugs is the expected result of our concerted efforts over several decades to eradicate them through the reliance on insecticides. A high frequency of resistance in C. lectularius and C. hemipterus has now been confirmed in

Australia. An early warning has been provided that reduced susceptibility to neonicotinoids may be emerging in C. lectularius, along with tolerance to what is often thought to be one of the most viable alternatives to insecticides, silica-gel desiccant dusts.

The inability to control bed bugs with existing products and insecticides will necessitate a rethinking of control methodologies employed against this resurgent pest. A better understanding of the ecology, biology, risks and effective treatment options associated with bed bugs will be required for successful eradication of future infestations. Products that employ new insecticide modes of action, or the re-labelling and reformulation of existing products will be

U=J Chapter 6: General Discussion needed. Also required is the greater development and utilisation of efficacious non-chemical methods such as mattress encasements, heat, vacuuming, and monitoring devices. Adherence by professional pest managers to industry standards or codes of practice on bed bug management will similarly improve the likelihood of successful infestation eradication. Further susceptibility research and exploration of resistance mechanisms, combined with a renewed focus at including C. hemipterus in such testing, is fundamental to both a better understanding of the causes of the resurgence and our ability to effectively counter infestations. Without an integrated approach, control of bed bugs will continue to be challenging.

U=N References

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