University of Florida Thesis Or Dissertation Formatting Template

BIOLOGY AND CONTROL OF TWO SPECIES OF HUMAN PARASITIC BED BUGS: CIMEX LECTULARIUS LINNAEUS AND CIMEX HEMIPTERUS FABRICIUS

BRITTANY ELISE DELONG CAMPBELL

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2018

To my parents for their endless love and support

ACKNOWLEDGMENTS

I am extremely grateful for all the time and knowledge my committee members have dedicated to me over the previous few years. I am grateful to my committee chair,

Dr. Rebecca Baldwin, for her kind advice, support, and an ability to bring happiness at moments that I felt at a loss. Secondly, I am beyond grateful for my co-committee chair,

Dr. Phil Koehler. He believed in me and provided the support necessary to complete this degree with an unyielding optimistic attitude and quirky sense of humor. I thank my committee member, Dr. Salvador Gezan for his heaps of patience and guidance with statistical analyses. I am also very grateful for my committee member, Dr. Emma

Weeks. Emma is an academic saint and helped me tremendously with designing projects and science, but more importantly, with navigating PhD life and struggles.

Many more people were influential and helpful throughout this process. Thank you, members of the urban lab, Liz Pereira, Roberto Pereira, Casey Parker, Heather

Erskine, Kristen Stevens, Mark Mitola, Ben Hottel, Richard Murphy, Tanner Felbinger,

WinDI Sanchez, Tiny Willis, and Lettie Cronin. Heather and Casey, thank you for a wonderful friendship outside of work and your companionship; you are both one of my best friends. Many thanks to Mark Mitola for his help with experimental design and top- notch pranks. Liz, thank you for being a second mom in the lab and for your political views. Thank you Tiny, for your Louisiana tales and the millions of store errands for supplies.

There are too many people to thank outside of the urban laboratory, but a special thanks to all the friendships that I made with fellow graduate students at UF. I could not imagine doing this degree without the support, encouragement, and moaning fests that were beyond necessary for peace of mind. Just to name a few people that I owe my

sanity to, Mike Bentley, Oliver Keller, Haleigh Ray, Mike Vickers, and Chase Kimmel.

Thank you. You made graduate school so much more than earning a degree.

Many thanks to Amin Emanifar and Oliver Keller for their help with taxonomic and phylogenetic analyses. A tremendous thanks to Al Estep and Neil Sanscrainte at USDA

ARS in Gainesville for their help with Nano injection. I’ve met very few people who are as nice and witty as those two; they brought tremendous joy and enthusiasm daily for science.

Lastly, I thank my family for all of their support. I have been in school for most of my life now and I greatly appreciate the constant encouragement. Thanks to my husband, Josh Campbell, for dealing with all the stresses that this degree has brought and for always being a sense of calm in the storm. Thanks to my pup, BoJack. He sacrificed lots of walking and swimming because I had to be in the lab.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

ABSTRACT ...... 11

CHAPTER

1 INTRODUCTION ...... 13

2 LITERATURE REVIEW ...... 16

Bed Bug Biology ...... 16 Bed Bug Origins ...... 19 Cimex hemipterus and Cimex lectularius Distribution ...... 20 Medical Importance of Bed Bugs ...... 21 Bed Bug Control Methods ...... 22 Pyrethroid Resistance ...... 25 Juvenile Hormone Analogues and Chitin Synthesis Inhibitors ...... 26

3 RECENT DOCUMENTATION OF THE TROPICAL BED BUG IN FLORIDA SINCE THE COMMON BED BUG RESURGENCE (HEMIPTERA: CIMICIDAE) ... 29

Introduction ...... 29 Materials and Methods...... 30 Results and Discussion...... 31

4 MORPHOLOGICAL AND MITOCHONDRIAL DNA VARIATION BETWEEN COMMON BED BUGS (CIMEX LECTULARIUS) AND TROPICAL BED BUGS (CIMEX HEMIPTERUS) ...... 35

Materials and Methods...... 38 Bed Bug Strains ...... 38 Morphological Analyses...... 39 Statistical Analyses of Morphological Characteristics ...... 39 DNA Extraction, PCR Amplification, and Sequencing ...... 40 Phylogenetic Analyses ...... 41 Results ...... 42 Morphological Differentiation ...... 42 Genetic Diversity ...... 43 Phylogenetic Analyses and Haplotype Network ...... 44 Discussion ...... 44

5 LOCOMOTION INHIBITON OF CIMEX LECTULARIUS L., FOLLOWING TOPICAL, SUBLETHAL DOSE APPLICATION OF THE CHITIN SYNTHESIS INHIBITOR LUFENURON ...... 75

Materials and Methods...... 78 Insects ...... 78 Insecticide Dilutions ...... 78 Insect Growth Regulator Topical Application Bioassay ...... 79 Pulling Force Assay ...... 80 Statistical Analysis ...... 82 Results ...... 82 Discussion ...... 83

6 LUFENURON EFFECTS ON BED BUG FECUNDITY AND EGG DEVELOPMENT ...... 94

Material and Methods ...... 95 Insects ...... 95 Topically Treated Egg Dose Bioassay ...... 96 Topically Treated Egg Age Bioassay ...... 97 Topically Treated Adult Males and Females with Lufenuron ...... 97 Statistical Analysis ...... 98 Results ...... 99 Discussion ...... 100

7 EXTERNAL AND INTERNAL APPLICATION OF LUFENURON AGAINST TROPICAL BEDBUGS VIA TOPICAL APPLICATION, FEEDING, AND INJECTION BIOASSAYS ...... 106

Materials and Methods...... 108 Insects ...... 108 Insect Growth Regulator Topical Application Bioassay ...... 108 Insect Growth Regulator Feeding Bioassay ...... 109 Insect Growth Regulator Injection Bioassay ...... 110 Statistical Analyses ...... 110 Results ...... 111 Discussion ...... 112

8 CONCLUSION ...... 119

LIST OF REFERENCES ...... 121

BIOGRAPHICAL SKETCH ...... 133

LIST OF TABLES Table page

4-1 Sampling information, abbreviations, species status used in the present study...... 49

4-2 Bed bug species information for all bed bugs used in genetic analyses...... 51

4-3 Character measurements for adult male Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard)...... 55

4-4 Character measurements for adult female Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard)...... 56

4-5 Character measurements for 5th instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard)...... 57

4-6 Character measurements for 4th instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard)...... 58

4-7 Character measurements for 3rd instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard)...... 59

4-8 Character measurements for 2nd instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard)...... 60

4-9 Character measurements for 1st instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard)...... 61

4-10 Coefficients for canonical variables of the discriminant analysis for the first canonical variate...... 62

4-11 Coefficients for canonical variables of the discriminant analysis for the second canonical variate...... 63

4-12 Population genetic indices calculated based on 146 mtDNA-COI sequences of Cimex lectularius and C. hemipterus populations...... 64

5-1 Malformations and mortality of Harlan and Bradenton strain common bed bugs, Cimex lectularius, following topical application of lufenuron...... 88

7-1 Mortality of 5th instar tropical bed bugs, Cimex hemipterus, treated with lufenuron at different concentrations/doses between assays (i.e. topical application, feeding, and injection)...... 116

7-2 The amount of active ingredient (lufenuron) consumed by 5th instar tropical bed bugs, Cimex hemipterus, during the feeding assay...... 117

LIST OF FIGURES

Figure page

3-1 Auto-Montage photograph of an adult Cimex hemipterus male collected from Brevard County on the right and an adult Cimex lectularius male (Harlan strain) on the left...... 33

3-2 Auto-Montage photograph of a dissected pronotum from A) Cimex lectularius and B) Cimex hemipterus...... 34

4-1 Illustration of Cimex lectularius representing the characters that were measured for the morphological analysis of both species...... 50

4-2 Discriminant analysis plots of canonical variates 1 and 2 for A) males and B) females from Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard)...... 65

4-3 Discriminant analysis plots of canonical variates 1 and 2 for 5th instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard)...... 66

4-4 Discriminant analysis plots of canonical variates 1 and 2 for 4th instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard)...... 67

4-5 Discriminant analysis plots of canonical variates 1 and 2 for 3rd instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard)...... 68

4-6 Discriminant analysis plots of canonical variates 1 and 2 for 2nd instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard)...... 69

4-7 Discriminant analysis plots of canonical variates 1 and 2 for 1st instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard)...... 70

4-8 Median and distribution of Pronotum width (Pr_Width) values for three bed bug populations...... 71

4-9 Phylogenetic trees constructed for Cimex lectularius and C. hemipterus using the mtDNA-COI sequences obtained from 146 individuals...... 72

4-10 A minimum spanning network constructed using the eight mtDNA-COI haplotypes (H1-H8) identified in Cimex lectularius and C. hemipterus populations...... 73

4-13 Data matrix of 8 distinct haplotypes among 146 individual of C. lectularius and C. hemipterus populations using 463 bp of COI...... 74

5-1 Photograph of a pulling force assay on an analytical balance. A common bed bug, Cimex lectularius, is pictured gripping the rough sandpaper surface on the wooden platform...... 87

5-2 Photographs showing the lethal effects of lufenuron following ecdysis of treated 5th instar Harlan strain common bed bugs, Cimex lectularius, with a dose of 0.16% (w/v) lufenuron...... 89

5-3 Sublethal effect following topical application of 0.0016% (w/v) lufenuron on a Harlan strain common bed bug, Cimex lectularius...... 90

5-4 Pulling force (mN) over time (s) for one Harlan strain common bed bug, Cimex lectularius, that was non-treated (control) or treated with lufenuron (0.0016% w/v)...... 91

5-5 Average amount of force (mN) generated by Bradenton and Harlan strain common bed bugs, Cimex lectularius, when gripping a surface with tarsi following no exposure to lufenuron (Control) or exposure to sublethal doses of lufenuron (Treated)...... 92

5-6 Maximum amount of force (mN) generated by Bradenton and Harlan strain bed bugs when gripping a surface with tarsi following no exposure to lufenuron (Control) or exposure to sublethal doses of lufenuron (Treated)...... 93

6-1 Mortality (%) of Harlan strain common bed bugs, Cimex lectularius, following topical application of five concentrations (g/mL) of lufenuron...... 103

6-2 Number of dead common bed bug, Cimex lectularius, eggs after topical treatment of lufenuron at different ages (1 day, 3 day, and 5 day post- oviposition)...... 104

6-3 Number of common bed bug, Cimex lectularius, eggs laid by adults that were not treated with lufenuron (control) and adult females treated with lufenuron and adult males treated with lufenuron...... 105

7-1 The total amount of blood ingested by tropical bed bugs, Cimex hemipterus, containing lufenuron at different concentrations for the feeding assay...... 118

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

BIOLOGY AND CONTROL OF TWO SPECIES OF HUMAN PARASITIC BED BUGS: CIMEX LECTULARIUS LINNAEUS and CIMEX HEMIPTERUS FABRICIUS

Brittany Elise Delong Campbell

May 2018

Chair: Rebecca W. Baldwin Co-chair: Phillip G. Koehler Major: Entomology and Nematology

There are two primary species of bed bugs that torment and feed on humans, the common bed bug, Cimex lectularius L., and the tropical bed bug, Cimex hemipterus F.

To distinguish between these species, morphological and mitochondrial DNA assays were conducted. Pronotum width measurements for each feeding life stage were used to establish a discriminatory number or average that would differentiate between tropical and common bed bugs. The generated averages discriminated between each tested life stage, except for first instars that lacked well-developed pronotums for differentiation.

Mitochondrial DNA differentiated between common and tropical bed bugs, resulting in identification of 47 mutations between the populations that were tested. World-wide, both of these species are controlled using pesticides. While the pesticides used may vary globally, insecticide resistance has been documented in both common and tropical bed bugs throughout the world. Here, we evaluated the chitin synthesis inhibitor lufenuron, an insect growth regulator, to determine its efficacy in acute and sublethal studies against both common and tropical bed bugs. Topical application of a sublethal dose of lufenuron to common bed bugs resulted in significant morphological problems in

their legs and a large reduction in their ability to walk. Pulling force assays revealed that individuals from two strains of common bed bugs, Harlan and Bradenton, could barely grip a surface following sublethal lufenuron exposure, with control bugs generating approximately 0.35-4.50 mN force compared to almost 0.00 mN force for bed bugs that were treated. Topical assays confirmed that lufenuron had potent ovicidal activity and caused over 50% mortality of eggs or emergent nymphs. However, there was no evidence of transovarial transmission to offspring via treatment of adult males or females. When different toxicological assays were compared (topical, feeding, and injection) using lufenuron, feeding assays were the most effective method, indicated by the lowest doses required for mortality, followed by injection, and lastly topical application. These studies enhance knowledge on the biology of two bed bug species that feed on humans and presents data on the potential of lufenuron as a contact and ingestible insecticide for use on bed bugs.

CHAPTER 1 INTRODUCTION

Cimex lectularius Linnaeus and Cimex hemipterus Fabricius are obligate hematophagous ectoparasites of humans. Both species predominately feed on humans but are nest parasites of both birds and bats. Cimex lectularius, the common bed bug, is the prominent bed bug species found in temperate climates, whereas Cimex hemipterus, the tropical bed bug, is the dominant bed bug species in tropical and subtropical climates (Usinger 1966). Cimex lectularius is the primary bed bug species found in all 50 states in the United States; however, tropical bed bugs were discovered in Florida in 2015 (Campbell et al. 2015). This raises questions about the possibility of this species establishing and potentially spreading throughout the state of Florida and possibly into other states that remain within the tropical and sub-tropical latitudes that the tropical bed bug species dominates.

The recent presence of the tropical bed bug in Florida, which had not been documented since the 1930s-1940s (Hixson 1943), elicits interest in closely evaluating morphological differences between tropical bed bugs and common bed bugs to more easily differentiate the species. Previous studies relied on the ratio of the pronotum width to length for identification (Usinger 1966); however this methodology, while used often in the literature, has not been tested for accuracy and precision. The presence of an additional bed bug species in Florida could have important implications for controlling this newly introduced pest.

Multiple strategies have been implemented for controlling bed bugs with an integrated pest management approach. Non-chemical control methods are often used such as cold treatments, heat treatments, vacuuming, mattress exclusion, and trapping

(monitoring) (Gangloff-Kaufman et al. 2006). However, chemical treatments remain the most widely used method for bed bug control in the United States (Gangloff-Kaufman et al. 2006). Unfortunately, widespread documented resistance to pyrethroid insecticides in bed bugs has been reported (Moore and Miller 2006, Romero et al. 2007, Yoon et al.

2008, Adelman et al. 2011), as pyrethroids are one of the most commonly and widely used active ingredients for bed bug control (Gangloff-Kaufman et al. 2006).

This documented resistance necessitates alternative insecticidal modes of action for bed bug control and evaluation of their efficacy. There are other active ingredients available for bed bug control than pyrethroids, but one viable option is the use of insect growth regulators (IGRs). Insect growth regulators are an attractive option for bed bug control because of their low mammalian toxicity (Graf 1993), especially since bed bug control consists of insecticides being applied indoors, as well as their propensity to eliminate populations over time, subsequently reducing biting pressure. Insect growth regulators affect an entirely different physiological system within bed bugs than the neurotoxin pyrethroids; instead they affect the molting process. Thus IGRs are also advantageous because they can be used to circumvent resistance issues that have developed in bed bugs using pyrethroid insecticides (Graf 1993).

Thus far, there have been few studies evaluating insect growth regulators against bed bugs and these studies have focused on juvenile hormone analogs (Todd 2006,

Goodman et al. 2013), while neglecting alternative IGRs such as chitin synthesis inhibitors. Therefore, the premise of much of this dissertation is to evaluate the effects of chitin synthesis inhibitors against common bed bugs and tropical bed bugs. Overall, there were five research objectives addressed, as follows:

1. Recent documentation of the tropical bed bug in Florida since the common bed bug resurgence (Hemiptera: Cimicidae).

2. Morphological and mitochondrial DNA variation between common bed bugs (Cimex lectularius) and tropical bed bugs (Cimex hemipterus).

3. Locomotion inhibition of Cimex lectularius following topical, sublethal dose application of the chitin synthesis inhibitor lufenuron.

4. Lufenuron effects on bed bug reproduction and egg development.

5. External and internal application of lufenuron against tropical bed bugs via topical application, feeding, and injection bioassays.

CHAPTER 2 LITERATURE REVIEW

Bed Bug Biology

Both the common bed bug, Cimex lectularius Linnaeus, and the tropical bed bug,

Cimex hemipterus Fabricius, are taxonomically classified in the order Hemiptera and the family Cimicidae. The family Cimicidae comprises 22 genera with 74 species (Usinger

1966). Only three species within the family Cimicidae feed on humans: C. lectularius, C. hemipterus, and one species found in Africa, Leptocimex boueti Brumpt (Usinger 1966).

Most other species within Cimicidae are temporary nest parasites of bats or birds.

Cimex lectularius and C. hemipterus life cycles consist of an egg stage and five nymphal instars before reaching adulthood. Nymphal instars require a blood meal to molt into subsequent stages. Adult female bed bugs require a blood meal for oviposition and adult males require a blood meal for sperm production (Reinhardt and Siva-Jothy

2007).

Adult bed bugs will engage in mating after feeding by traumatic insemination, a process in which the male pierces the female’s abdomen and ejaculates sperm into a specialized organ, the spermalege or organ of Berlese. The sperm then migrates through the female’s paragenital system. Male bed bugs never use the genital tract for insemination although females possess a similar reproductive system compared to other insects (Reinhardt and Siva-Jothy 2007). Males select recently fed females for copulation, although it is unclear how males search or locate females (Reinhardt and

Siva-Jothy 2007). The general consensus is that males will attempt to mate any bed bug of the general size of a recently-fed female, including other males and larger nymphs (Reinhardt and Siva-Jothy 2007). On average, a female common bed bug

copulated approximately five times after the consumption of one blood meal in laboratory conditions (Stutt and Siva-Jothy 2001).

A blood meal is required for a female bed bug to begin ovipositing eggs. The number of eggs a female oviposits is dependent upon how many times she has been mated (Polanco et al. 2011). A female mated repeatedly versus just once has been shown to have a reduction the number of eggs laid by 27% over a six-week feeding cycle (Polanco et al. 2011). Approximately three days after feeding, a mated female will begin to oviposit eggs (Usinger 1966). If she has no access to an additional blood meal, oviposition will stop after approximately 11 days (Usinger 1966). A female bed bug will oviposit between 1 to 12 eggs per day (Krueger 2000). Polanco et al. (2011) found that female bed bugs, on average, lay between 132 and 156 eggs during their lifetime. Bed bug eggs have a high viability, and approximately 98% of eggs laid will hatch into live nymphs. However, lengthened time of bed bug feeding (15 minutes vs. 5 minutes) significantly decreased egg viability from approx. 96% to 90% viability, respectively

(Pereira et al. 2013). This was attributed to the environmental conditions that the eggs were held, especially the presence of adults in the containers potentially stepping on the eggs, thus consequently may have nothing to do with the length of bed bug feeding or nutritional status (Periera et al. 2013).

Bed bugs are attracted to carbon dioxide (CO2) and heat (Wang et al. 2009,

Anderson et al. 2009, Rivnay 1932) and use both of these cues for finding a host.

However, CO2 was found to be significantly more attractive to C. lectularius when both cues were baited inside of pitfall traps (Wang et al. 2009). In the presence of carbon dioxide and absence of heat, bed bugs were found to exhibit long range host-seeking

behavior, rather than localized host-seeking behavior (Suchy and Lewis 2011). After host location, bed bugs will probe the host using their piercing-sucking mouthparts until finding a blood vessel, then feed for several minutes on a human host until reaching engorgement (Reinhardt and Siva-Jothy 2007).

Both species exhibit cryptic behaviors; they will aggregate in harborages that are hidden in cracks and crevices with their conspecifics and produce aggregation and alarm pheromones as chemical signals (Siljander et al. 2008, Liedtke et al. 2011,

Weeks et al. 2011, Mendki et al. 2014). Gas chromatography mass spectrometry (GC-

MS) analysis in combination with olfactometry choice assays revealed that ten compounds (nonanal, decanal, (E)-2-hexenal, (E)-2-octenal, (2E, 4E)-octadienal, benzaldehyde, (+) - and (−)-limonene, sulcatone, benzyl alcohol) collected from bed bug harborages were all crucial components of the bed bug (C. lectularius) aggregation pheromone (Siljander et al. 2008). However, a later study determined that all of those compounds were present in exuvia and in feces (Gries et al. 2015). A step-wise elimination of each compound revealed that the aggregation pheromone was actually present in bed bug feces and was comprised of dimethyl disulfide, dimethyl trisulfide,

(E)-2-hexenal, (E)-2-octenal, 2-hexanone, which were highly volatile (Gries et al. 2015).

Once bed bugs approached a harborage, the less volatile chemical histamine caused bed bugs to stay (arrestment) (Gries et al. 2015). Therefore, the combination of the five volatile chemicals and one non-volatile chemical was important for attracting bed bugs and is being evaluated for its use in bed bug traps.

Male bed bugs have been shown to mount other males and also will attempt to mount late instar nymphs that are a similar size to adult females. This can have

negative effects because of the piercing action during mating and has been found to cause a reduction in male longevity after being pierced. Therefore, both male bed bugs and nymphs produce alarm pheromones in C. lectularius and C. hemipterus to limit this type of interaction (Ryne 2009, Harraca et al. 2010, Liedtke et al. 2011).

Bed Bug Origins

It has been suggested that C. lectularius originated from the Middle East and descended from ancestral bat bugs (Usinger 1966). As humans began to move into caves, it is presumed that bat bugs switched from feeding exclusively on bats and began to feed on humans (Usinger 1966). As humans moved out of caves and adopted a more hunter-gatherer lifestyle, they carried bed bugs with them, resulting in speciation of C. lectularius from bat bugs (Usinger 1966). Fossilized bed bugs and historical documents have provided evidence of the bed bug journey with humans. Archeologists

(Panagiotakopulu & Buckland 1999) reported that bed bug fossils have been found that were dated as being over 3,500 years old in the remains of an Egyptian workmen’s village.

Cimex hemipterus is thought to have originated in South Asia or Africa (Usinger

1966). Cimex hemipterus and C. lectularius are thought to have originated in the Old

World based on linguistics; there are many more names for a bed bug in the Old World, whereas there is no name for a bed bug in the Native American language (Usinger

1966). Morphological evidence also suggests that both species are from the Old World, because they both have a narrow cleft sinus, indicative of Cimex from the Old World

(Usinger 1966).

Newer genetic evidence supports host-associated divergence of C. lectularius

(Booth et al. 2015). Cimex lectularius were collected in numerous locations within

Europe from either bat nests or human dwellings and their genetic flow and structure were evaluated with mitochondrial DNA (mtDNA), microsatellite loci, and with knockdown resistance genes (Booth et al. 2015). There was no gene flow between human- associated and bat-associated C. lectularius. The human-associated bed bugs were found to have much lower genetic diversity compared to the C. lectularius that were associated with bats. This was most probably due to the fact that human infestations of bed bugs rarely have a re-introduction of bed bugs from another population, limiting the amount of genetic introductions, and consequently resulting in increased inbreeding

(Booth et al. 2015). A genetic study conducted previously of C. lectularius populations collected from multiple locations in the Eastern United States confirmed that C. lectularius populations had little genetic diversity, suggesting that populations could have been started from a single female mating with her offspring (Saenz et al. 2012).

Cimex hemipterus and Cimex lectularius Distribution

Cimex hemipterus and Cimex lectularius have both been found to occur sympatrically and will readily interbreed in natural settings. Female Cimex lectularius can easily be distinguished after being mated by C. hemipterus by the darkening mass present in the ectospermalage (Walpole and Newberry 1988). Nine different collection periods in two different houses in South Africa, representing a total of 16 C. lectularius and 68 C. hemipterus females, determined that 69% of C. lectularius females collected had been mated by C. hemipterus males, indicated by the dark mass in the spermalage.

Previous laboratory studies have shown that interspecific mating of C. lectularius females by C. hemipterus males results in sterility of eggs (Omori 1941). Therefore, the increased presence of C. hemipterus within both homes may have been due to the deleterious effects of interbreeding, allowing C. hemipterus to dominate areas where

both species overlap geographically (Walpole and Newberry 1988). Interestingly, C. hemipterus females mated with C. lectularius males also produce sterile offspring, however, out of 172 eggs laid by twenty-five different females in a laboratory setting, one egg was fertile that later hatched into a first instar nymph that morphologically resembled C. hemipterus. This nymph was found moribund and never took a blood meal.

These studies do not address why C. hemipterus is common in parts of the world where C. lectularius is absent, reported by Usinger (1966). Deleterious effects of cross- species matings to C. lectularius has been suggested, however, both species (C. lectularius and C. hemipterus) lay sterile eggs and are not killed by interspecific mating.

The more logical reasoning is most probably due to ecological constraints. Cimex lectularius is considered to be more dominant in temperate areas of the world, whereas

C. hemipterus is considered to be in tropical regions throughout the world, specifically remaining within 30° latitudinally north and south of the equator (Usinger 1966).

Medical Importance of Bed Bugs

The Centers for Disease Control and Prevention (CDC), Environmental

Protection Agency (EPA), and the United States Department of Agriculture (USDA) all consider bed bugs to be public health pests (EPA; Notice 2002-1). However, unlike many other public health pests, bed bugs are not currently known to transmit pathogens that cause diseases although they can carry multiple pathogenic organisms on their bodies and in their excrement (Burton 1963, Delaunay et al. 2010, Sabou et al. 2013).

Bed bugs are significant urban pests because their bites can cause allergic cutaneous reactions, potentially resulting in infections caused from scratching and introducing bacteria into the bite wound (Churchill 1930, Fletcher et al. 2002, Leverkus

et al. 2006, Reinhardt et al. 2009, Goddard and deShazo 2009, Goddard et al. 2011).

Not all humans have similar reactions to bed bug bites, thus bite reactions are dependent upon an individual’s immune system. Reactions to bites may worsen over time or develop more quickly after being bitten repeatedly by bed bugs (Reinhardt et al.

2009). Bed bug populations that become large, resulting in multiple bed bugs taking frequent blood meals, have been shown to cause anemia in humans (Pritchard and

Hwang 2009, Paulke-Korinek et al. 2011). Furthermore, many people experience anxiety and loss of sleep from the psychological stress that ensues from experiencing a bed bug infestation (Comack and Lyons 2011, Goddard and deShazo 2012, Susser et al. 2012).

Although bed bugs have not been found to currently transmit pathogens that cause disease in humans, a recent study determined that common bed bugs are capable of acquiring the parasite Trypansoma cruzi, the causal agent of Chagas disease (Salazar et al. 2014). This study determined that bed bugs could successfully acquire T. cruzi by feeding on infected mice and then subsequently infect pathogen-free mice by cohabitating with the mice or through T. cruzi infected bed bug feces contacting the broken skin of mice (Salazar et al. 2014). Although documentation of the transmission of the parasite that causes Chagas disease by bed bugs has not been found to naturally occur, the potential for pathogen transmission and other psychological problems that bed bugs can cause further establishes the need for more effective treatment methods to control this pest.

Bed Bug Control Methods

Methods for controlling bed bugs in the mid-1800s and early 1900s included using mercury chloride, pyrethrum, gasoline, kerosene, benzene, alcohol, sulfur and

hydrogen cyanide (Potter 2011). Many of these chemicals had to be directly applied to bed bugs to kill them and were extremely dangerous to the applicator. Dichloro-diphenyl trichloroethane (DDT) was first used for bed bug control in 1942 (Potter 2011). DDT was highly effective and bed bugs were essentially eliminated in the United States during the

1940’s-50 due to the widespread use of DDT. However, because of the widespread use of DDT, bed bugs soon became resistant to DDT (Usinger 1966). Malathion was the insecticide that replaced DDT for control of resistant bed bugs; however, within a decade bed bugs also became resistant to malathion (Feroz 1970).

Currently, pest control companies in the United States primarily rely on pyrethroid insecticide applications for bed bug treatments (Gangloff-Kaufman et al. 2006). The low cost and ease of applying pesticides compared to other methods (e.g. heat and fumigation) continues to drive this current trend of bed bug control. The large majority of pesticides labeled for indoor use are pyrethroids, consequently, bed bugs have been frequently exposed to pyrethroid insecticides. The frequent exposure of common bed bugs to pyrethroids has resulted in significant resistance to these active ingredients

(Moore and Miller 2006, Romero et al. 2007, Yoon et al. 2008, Adelman et al. 2011).

Pyrethroid resistance has created a need for alternative methods for control.

Non-chemical control methods include various dusts (e.g. diatomaceous earth and silica dust), laundering, spot cold treatments, and heat treatments. Heat treatments could arguably be the most effective non-chemical control method available (Kells and

Goblirsch 2011). If heat treatments are done properly, bed bug infestations can be eliminated. Pereira (2009) showed that elimination of bed bugs can be achieved using heat chambers constructed of polystyrene sheathing boards, with the addition of oil-

filled electric heaters and box fans to circulate the heat, to treat common household items. When the bed bugs were exposed to temperatures at or above 41 °C at times ranging from 2-7 hours, 100% mortality of bed bugs was achieved inside the chambers

(Pereira 2009). Mortality was not achieved if the bed bugs were placed into areas where the heat could not circulate (i.e. deep inside couches). Therefore, it is crucial to have proper air flow and to limit potential heat sinks in order for a heat treatment to be fully effective.

Desiccant dusts (i.e. diatomaceous earth and silica gels) have long been used for their insecticidal properties (Ebeling 1971). Diatomaceous earth and silica dust work essentially the same way, by absorbing cuticular lipids, thus causing the insects to dehydrate (Appel et al. 1999). Wang et al. (2009) determined that applying diatomaceous earth in combination with steam treatments caused over a 97% reduction in bed bug populations after ten weeks. Benoit et al. (2009) determined that combining desiccant dusts with alarm pheromones increased bed bug mortality. The alarm pheromone caused an increase in bed bug movement, therefore, increasing the amount of dust they walked in and accumulated on their bodies (Benoit et al. 2009).

Bed bug control efforts are extremely costly, primarily due to the fact that bed bug treatments are labor intensive. The cost of bed bug treatment for one bedroom was estimated at $400 US dollars (Harlan 2007). This treatment consisted of three hours of inspection, customer education, and a limited insecticide application by a pest control operator, all in a single visit (Harlan 2007). As is the case with almost all bed bug treatments, multiple visits are required for effective control. Therefore, the costs of bed bug control can quickly escalate.

Pyrethroid Resistance

The bed bug resurgence in the United States during the 1990s is primarily attributed to their resistance to pyrethroid insecticides, among other factors (Reinhardt and Siva-Jothy 2007). Bed bug resistance to deltamethrin, λ-cyhalothrin, bifenthrin and permethrin has been documented; each of these belonging to the same insecticidal class of pyrethroids (Moore and Miller 2006, Romero et al. 2007). Bed bugs have different physiological mechanisms as well as combinations of those mechanisms that attribute to their resistance to pyrethroid insecticides.

Three physiological mechanisms of resistance have been identified in pyrethroid resistant bed bug populations; these are knockdown (kdr) resistance, enhanced detoxification enzyme activity, and reduced cuticular penetration resistance. With regard to kdr resistance, two point mutations in the α-subunit gene in the voltage sensitive sodium channel were found in bed bugs resistant to deltamethrin (Yoon et al. 2008).

Frequencies of both of these mutations in a population were highly proportional to insecticide resistance, suggesting that these mutations are directly related to pyrethroid resistance in that population (Seong 2010). Adelman et al. (2011) discovered that certain genes were over expressed in bed bug pyrethroid resistant strains, suggesting an increase in metabolic resistance. Insecticide resistance also has been demonstrated in the egg and first instar stages of bed bugs, further demonstrating genetic components to pyrethroid resistance and their presence in early life stages (Campbell and Miller

2015). The overexpression of cuticle genes, contributing to reduced cuticular penetration has been demonstrated in bed bugs (Koganemaru et al. 2013). Additional morphological studies using scanning electron microscope photographs demonstrated that cuticle thickness was correlated with resistance to lambda-cyhalothrin in bed bugs

(Lilly et al. 2016). These studies established that bed bugs have various genetic mechanisms to enhance resistance to insecticides.

Repellency is a behavioral mechanism that can decrease the efficacy of insecticides because the insect actively avoids surfaces that are treated with insecticides. Repellency has been documented in many different pests, e.g. cockroaches, ants and termites (Ebeling et al. 1966, Knight and Rust 1990, Su and

Scheffran 1990). Moore and Miller (2006) found that C. lectularius were not repelled by pyrethroid insecticides (deltamethrin, lambda-cyhalothrin, and bifenthrin) and that these pesticides did not cause bed bug movement into untreated places. Romero et al. (2007) found that bed bugs did not avoid filter papers treated with deltamethrin but were unable to rest on the filter papers because they were too agitated by the resulting nerve excitation. However, bed bugs were still attracted to pyrethroid-treated harborages containing feces and eggs (Romero et al. 2007). Bed bug response to insecticides is influenced by a variety of factors, e.g., the amount of insecticide applied, insecticide susceptibility within a population, and stimuli within the environment (Romero et al.

2007).

Juvenile Hormone Analogues and Chitin Synthesis Inhibitors

Juvenile hormone analogs (JHAs) and chitin synthesis inhibitors (CSIs) collectively are the two main types of pesticides that are termed insect growth regulators

(IGRs). IGRs affect insect molting, often preventing insects from completing development. They may also cause sublethal effects that interfere with insect reproduction or an insect’s ability to consume food. JHAs essentially mimic insect juvenile hormone that is naturally present for larval growth. When abnormally high levels of JHAs are present during the last insect stadium, (e.g. following IGR treatment with

JHAs) it signals for the insect to remain in the immature form rather than molt into an adult.

Juvenile hormones (JHs) are naturally produced in insects and regulate many important physiological processes. JHs are secreted by the corpus allatum, an endocrine gland located behind the brain in the head. After secretion, JH binds to other molecules (juvenile hormone binding proteins) and is transported through hemolymph to reach target tissues (Klowden 2007). JHs have been found to regulate metamorphosis, reproduction, diapause, and behavior in insects (Klowden 2007). JHs moderate gene expression and are important for stage-specific expression of an insect for metamorphosis (Klowden 2007). Insects will not molt correctly when JH is applied at an incorrect stage of metamorphosis. The first indication of this was when an active corpus allatum was implanted into the last larval instar of Rhodnius prolixus Stal, and instead of molting into a nymph, the larval instar molted into a supernumery nymph (Wigglesworth

1940). Much later, the discovery of plant-produced juvenile hormone analogs (Slama and Williams 1965) resulted in vast research to identify and synthesize JH analogs for their potential in insect control.

Chitin synthesis inhibitors prevent an insect from molting correctly, often resulting in malformations in the new cuticle. Benzoylurea compounds have been documented to cause multiple effects directly related to chitin synthesis, however the mode of action of

CSIs has not been entirely determined thus far (Merzendorfer 2006). Chitin synthesis inhibitors have been shown to have an effect on species within the order Hemiptera.

The chitin synthesis inhibitor diflubenzuron has been shown to cause incomplete ecdysis of last instar milkweed bugs, Oncopeltus fasciatus Dallas, when topically

applied at 9 µg/last instar bug (Redfern et al. 1982). The milkweed bugs would initiate molting on the seventh day, however, death occurred within 24 hours and dissections revealed the formation of adult cuticle beneath the instar cuticle. In control treatments, all insects molted correctly by the eighth day. The predatory bug, Podisus maculiventris

Say, was not able to molt from the last larval instar to adult after preying on insects dipped in field rates of the chitin synthesis inhibitor novaluron (Cutler et al. 2006).

Insect growth regulators have been found to be highly effective against multiple urban pests, including flies (Geden and Devine 2012, Fulcher et al. 2014, Lohmeyer et al. 2014), stored product pests (Arthur and Fontenot 2012, Kavallieratos et al. 2012), and cockroaches (Kawada et al. 1989, Koehler and Patterson 1991, Reid et al. 1992,

Mosson et al. 1995, Ameen et al. 2005). However, their use has not been extensively investigated in bed bugs. The limited studies available on bed bugs and IGRs have mostly investigated JHAs and have largely neglected CSIs (Todd 2006, Goodman et al.

2013). This may be primarily because CSIs are known to be highly effective when ingested (e.g. as baits) but are not often used as contact insecticides. As of date, no baits have been developed or registered for bed bugs in the United States, although research has been conducted on active ingredients other than CSIs that could be potentially used in baits, as well as the other components necessary for a successful bait (feeding attractants and stimulants) (Sierras and Schal 2017). However, with the limited availability of highly effective insecticides against bed bugs due to resistance and the advantage of low mammalian toxicity, the addition of IGRs for an integrated pest management (IPM) program for bed bugs has potential.

CHAPTER 3 RECENT DOCUMENTATION OF THE TROPICAL BED BUG IN FLORIDA SINCE THE COMMON BED BUG RESURGENCE (HEMIPTERA: CIMICIDAE)

Introduction

Two bed bug species use human hosts for a blood meal: the common bed bug,

Cimex lectularius L. and the tropical bed bug, Cimex hemipterus (F.). The common bed bug is prevalent in the United States and is found throughout the entire country. This species has a world-wide distribution and is considered a pest in more temperate climates. In the USA, the common bed bug occurred only at low levels in the 50 years prior to the 1990s, mostly due to the widespread use of DDT and other chemical insecticides (Potter 2011). A resurgence of common bed bugs occurred in the 1990s in the United States and has been considered to be a result of multiple factors, including insecticide resistance, sale of second hand furniture, increased international travel, and changes in pest control practices.

Compared to C. lectularius, the tropical bed bug has a more subtropical and tropical distribution, specifically remaining within 30° latitudinally north and south of the equator (Usinger 1966). More recently, Cimex hemipterus was collected from Tanzania in 1995-1996 (Myamba et al. 2002), for the first time ever in Australia in 1998

(Bundaberg in 1998), and again in 2003 cohabitating with C. lectularius (Doggett et al.

2003), Sri Lanka in 2001-2003 (Karunaratne et. al. 2007), Brazil in 2005 (Araujo et al.

2009), Malaysia and Singapore in 2006 (How and Lee 2010), Bangladesh in 2007

(Khan & Rahman 2012), Rwanda in 2011 (Angelakis et al. 2013), and several provinces in Thailand in 2011 (Tawatsin et al. 2011). This list is indicative of the world-wide range of C. hemipterus and suggests that the geographic range of C. hemipterus remains within the 30° latitude lines north and south.

Consistent with the expected distribution of C. hemipterus in tropical and subtropical regions, this species has previously been documented in the state of

Florida, USA. Cimex hemipterus was first documented in Gainesville, Florida in 1938 with several other reports of this species across the state occurring in the 1930s and early 1940s, including in Alachua, Pinellas, Polk, and Sarasota counties (Hixson 1943).

However, there have been no recent published documentation of this species in the state of Florida since the resurgence of common bed bugs in the United States in the late 1990s.

Materials and Methods

Recently, a bed bug sample from a home in Brevard County was sent to the

Insect Identification Laboratory at the University of Florida and was identified as C. hemipterus using the taxonomic key in Usinger (1966), specifically the pronotum width to length ratio. The ratio recommended for identification of C. hemipterus is less than

2.5, and greater than 2.5 for C. lectularius (Usinger 1966). Also, C. lectularius has a more upturned lateral margin as compared to C. hemipterus (Ghauri 1973). The differential comparisons between the pronotum lateral margin of C. lectularius and C. hemipterus are pictured in Figures 1 and 2.

In October 2015, the homeowners in Brevard County were contacted and additional bed bug specimens were collected from their home for positive identification.

Pronotum measurements of C. hemipterus males and females were taken using a stereomicroscope system (Leica MZ12.5) and Auto-Montage Pro software (version

5.02, Syncroscopy, Frederick, MD). Mean ratios ± SE (pronotum width/pronotum length) of C. hemipterus for both male and female bed bugs combined were below 2.5 (2.30 ±

0.08, N = 10), confirming that they were tropical bed bugs. Mean (± SE) measurements of the C. hemipterus pronotum were 0.56 ± 0.02 mm for the length and 1.29 ± 0.04 mm for the width.

Results and Discussion

According to the homeowners, they believed a family member had unknowingly brought the bed bugs into the house and indicated that no one residing in the house had traveled outside of the state of Florida. Although the origin of the bed bug infestation could not be determined, the absence of international and national travel by the residents suggests that tropical bed bugs were present elsewhere in the state. Further studies are needed to determine the presence, establishment, and possible distribution of this species throughout the state.

Interestingly, one other recent instance of C. hemipterus has occurred in Florida, however, this arrival went unnoticed. The Florida State Collection of Arthropods, housed in the Florida Department of Agriculture and Consumer Services Division of Plant

Industry Institution, Gainesville, Florida, has two adult female specimens of C. hemipterus (pronotum measurements: length = 0.52 mm, width = 1.07 mm, ratio = 2.08; length = 0.60 mm, width = 1.27 mm, ratio = 2.13) in their collection. The label identifier states that they were collected in Orange County, Florida on June 11, 1989 by T.

Nguyen in bedding and the database records state that the specimens were collected in a hotel in downtown Orlando. Whether this species has been present in Florida and never disappeared, or has been re-introduced and remains in small populations, is currently not known.

The recent discovery of C. hemipterus in Florida generates questions about the impact this species could potentially have compared to C. lectularius. Most people would have a difficult time distinguishing the difference between C. lectularius and C. hemipterus and many people most likely will not attempt to differentiate between the species once they positively identify a bed bug infestation.

Although C. hemipterus is biologically and behaviorally similar to C. lectularius, there is currently a dearth of knowledge pertaining to ecology, biology, and insecticide resistance of this species. Ecological and physiological differences between the species of bed bugs may require different management strategies for C. hemipterus than C. lectularius.

Figure 3-1. Auto-Montage photograph of an adult Cimex hemipterus male collected from Brevard County on the right and an adult Cimex lectularius male (Harlan strain) on the left. Arrows are pointing to the lateral pronotum margin on both species. Note the extended, flattened lateral margins of C. lectularius.

A B

Figure 3-2. Auto-Montage photograph of a dissected pronotum from A) Cimex lectularius and B) Cimex hemipterus. Cimex hemipterus is darker in color and does not have the flattened external margins of the pronotum indicative of C. lectularius.

CHAPTER 4 MORPHOLOGICAL AND MITOCHONDRIAL DNA VARIATION BETWEEN COMMON BED BUGS (CIMEX LECTULARIUS) AND TROPICAL BED BUGS (CIMEX HEMIPTERUS)

The family Cimicidae comprises 110 species that are primarily temporary nest parasites of bats or birds (Henry 2009). However, within Cimicidae, the common bed bug, Cimex lectularius Linnaeus, 1758, and the tropical bed bug, Cimex hemipterus

(Fabricius, 1803), are two of three species that are human ectoparasites, although they also feed on bats and poultry (Usinger 1966). Another species that is native to Africa,

Leptocimex boueti (Brumpt, 1910), also has been documented to be an ectoparasite of humans, as well as bats (Usinger 1966). Both C. lectularius and C. hemipterus exhibit similar biological behaviors; including hematophagy, aggregation, traumatic insemination, and a hemimetabolous life cycle consisting of an egg stage, five nymphal instars, and adults (Reinhardt et al. 2007).

Cimex lectularius has been suggested to originate from the Middle East, while C. hemipterus is native to South Asia or Africa. Both species are descendants of ancestral bat bugs that later evolved to feed on humans (Usinger 1966). Cimex lectularius and C. hemipterus are allopatric species that are divided by ecological barriers. Cimex lectularius is the dominate species in temperate areas, whereas C. hemipterus dominates in tropical and sub-tropical regions. However, there have been a few occurrences where both species overlap in the same geographic region and within the same human dwelling, probably attributed to passive movement by humans (Newberry et al. 1987).

The passive movement of bed bugs with humans over time has been widely documented in several historical accounts. Fossilized bed bugs and historical

documents have provided evidence of bed bug movement with humans

(Panagiotakopulu and Buckland 1999). Modern day human bed bug infestations are typically started by the movement of bed bugs in luggage, furniture, and other household belongings. The passive movement of bed bugs limits introductions among different populations into a human dwelling and consequently results in increased inbreeding within a bed bug population, limiting genetic flow and diversity (Booth et al.

2015).

Genetic discrimination between C. hemipterus and C. lectularius has been studied from a genus taxon perspective, however population discrimination between two species had not been previously studied to date. The use of the mitochondrial gene cytochrome oxidase subunit I (COI) has been widely used as a choice genetic marker for animal barcoding because it is a universal marker, species specific, and there is a high degree of genetic variation between species (Hebert et al. 2003). Sequencing of the COI gene has been accomplished in Hemiptera (Park et al. 2011, Raupach et al.

2014, Jung et al. 2010), and clustered the family Cimicidae within the infraorder

Cimicomorpha (Park et al. 2011). A phylogeny of the genus Cimex has been evaluated for the four previously proposed species groups and found that C. hemipterus and C. lectularius formed two clades, although the resolution of the generated tree was low for the C. lectularius group (Balvin et al. 2015). PCR-restriction fragment length polymorphism (RFLP analysis) has been used previously to differentiate between C. lectularius and C. hemipterus (Tawatsin et al. 2013). However, sequencing of the COI gene would provide increased taxonomic resolution compared to PCR-RFLP (Pereira et al. 2008).

Taxonomic differentiation between C. hemipterus and C. lectularius is currently based on the ratio of width to length of the pronotum. The ratio of pronotum width to length for C. lectularius has been suggested to be > 2.5, compared to < 2.5 for C. hemipterus. (Usinger 1966). Cimex hemipterus is also typically darker in color, however, this is not a diagnostic characteristic because of the phenotypic variation within species, as well as the darkening following blood meal consumption (Ghauri 1973).

Alternatively, the general shape of the pronotum also has been used for differentiation between the species. When both species are viewed dorsally, C. lectularius has pronotal margins that expand more laterally than C. hemipterus and also has more flattened pronotum margins (Ghauri 1973). While this characteristic allows for quick identification of adults, it is not easily distinguishable to the untrained eye and difficult when both species are not placed side-by-side for observation. Therefore, quantifiable measurements of distinguishable characteristics would provide a more accurate method for identifying the two species that commonly dwell in human domiciles. Since both of these species are associated with the same hosts (humans), occasionally overlap in the same geographic region and often need to be identified by non-entomologists (e.g. pest control operators, teachers or nurses), reliable identification procedures are needed.

The purpose of this study was to identify diagnostic morphological and genetic markers to differentiate C. lectularius and C. hemipterus. We measured morphological characteristics of adult bed bugs and all five nymphal instars, to determine a reliable diagnostic character that would be appropriate for identification across all blood-feeding life stages. We used COI to infer phylogenetic relationships regarding geographic

differences both intra- and inter-specifically and to determine the origin of the most recent introduction of tropical bed bugs into the United States.

Materials and Methods

Bed Bug Strains

Two strains of C. lectularius (Harlan and Bradenton) and one strain of C. hemipterus

(Brevard) were used for the morphological comparisons. Seven strains of C. lectularius

(Harlan, Bradenton, Kissimmee Valley strain [KVS], Cincinnati Ohio [COH], Poultry

House Tennessee [PHT], Lafayette Indiana [LIN], and Gainesville [GNV]), and two strains of C. hemipterus (Brevard and Malaysia) were used for mitochondrial gene analysis (Table 4-1).

The Malaysian C. hemipterus population was collected in Penang, Malaysia and stored in ethanol until DNA extraction. The Brevard C. hemipterus population was collected in Brevard Co., FL in 2015 (Campbell et al. 2016). The Harlan population was collected by Harold Harlan in 1973 in Ft. Dix, NJ. The Urban Entomology Laboratory, at the University of Florida, acquired this strain from Harold Harlan in the late 2000s. The

Bradenton population was collected by a pest control company in Bradenton, FL and was acquired in August 2013. The KVS strain was collected in a hotel in Kissimmee

Valley, FL. The GNV population was collected in Gainesville, FL in 2015 in a residential home. The COH population was collected in Cincinnati, OH. The PHT population was collected in a poultry house in Tennessee, and the original specimens were feeding on poultry at the time of collection. The LIN population was collected in Lafayette, IN. The

COH, PHT, and LIN populations were all acquired in 2015, sent from another laboratory, but the exact collection date is unknown.

All bed bug populations, except for the Malaysian population of C. hemipterus, were held in colony in the Urban Entomology Laboratory at the University of Florida.

These populations were fed bi-weekly on defibrinated rabbit blood (Hemostat, Dixon,

CA) using an artificial feeding system (Montes et al. 2002) and maintained at approx.

70% RH, 25 °C, and a 12:12 L:D photoperiod. All bed bug colonies were maintained in plastic jars, enclosed at one end with mesh for feeding, with accordion-style folded filter paper provided for harborage.

Morphological analyses

Two strains of C. lectularius (Harlan and Bradenton) and one strain of C. hemipterus (Brevard) were used in the morphological analyses. Ten specimens representing each blood feeding life stage and sex (all five instars, male adults, and female adults) were photographed and morphometrically measured using a stereomicroscope system (Leica MZ12.5) and Auto-Montage Pro software (version

5.02, Syncroscopy, Frederick, MD). Measurements (in mm) were taken of body length

(BL), clypeus length (CL), clypeus width (CW), pronotum length (PL), scutellum length

(SL), scutellum width (SW), distance between right and left pronotum flange margin

(PM), and pronotum area (PA). Averages were taken of the left and right pronotum flanges (PF) as well as the left and right wing pad width (HW) (Figure 4-1). All measurements were taken of bed bugs held in a standardized position with a dorsal body view in the photograph.

Statistical Analyses of Morphological Characteristics

Group means were compared between different populations of bed bugs using a multivariate analysis of variance (MANOVA) of all of the variables measured. Each

measured character between populations was tested for normality and homogeneity then analyzed with analysis of variance (ANOVA) and post-hoc comparisons were made using Tukey’s honest significant difference (HSD) tests. An ANOVA was conducted to compare mean differences of pronotum width to length ratios for adult males and females between species. The MANOVA and ANOVA were conducted using

JMP Pro 13 software (SAS Institute Inc., Cary, NC). Canonical discriminate analysis was conducted using SAS (v14.2, SAS Institute Inc., Cary, NC) to differentiate characters that were the most discriminating for identification between bed bug populations.

DNA Extraction, PCR Amplification, and Sequencing

Adult bed bugs were starved one week prior to genetic studies to eliminate DNA contamination from a blood meal. Three legs from the same side of each individual bed bug were removed and used for DNA extraction using the DNeasy Blood and Tissue kit

(Qiagen, Valencia, CA). DNA was extracted from the fresh specimens, as well as from specimens which had been held in 96% ethanol. The dissected legs were homogenized in tissue lysis buffer, followed by DNA extraction according to the manufacturer’s protocol.

The cytochrome oxidase subunit I (COI) gene fragment was amplified using forward and reverse primers designed for Lepidoptera (Balvin et al. 2012; Hajibabaei et al. 2006). This gene has been widely used to infer phylogenetic relationships of insects

(Hebert et al. 2003). PCR amplification was performed in a final reaction volume of 25 µl using a Mastercycler Gradient EP thermocycler (Eppendorf, Germany) with KlenTaq LA

DNA polymerase (DNA Polymerase Technology, Inc., St. Louis, MO). The PCR conditions used were 94 °C for 2 min. for the initial denaturation and then 34 cycles of

94 °C for 40 s, 49 °C for 40 s and 68 °C for 1 minute and 15 s. The final extension step was 68 °C for 1 minute. The PCR products of 658 base pairs (bp) were visualized using agarose gel electrophoresis. The PCR products were then purified using QIAquick PCR

Purification Kit (Qiagen, Valencia, CA) and submitted to the University of Florida

Interdisciplinary Center for Biotechnology Research for sequencing of both strands using the same primers as in the PCR amplification described previously. The complementary strands of mtDNA-COI gene were manually inspected and aligned using Geneious v 5.6.2 (http://www.geneious.com, Kearse et al. 2012), Evidence of premature stop codons in the COI sequences was checked using MEGA 7(Kumar et al.,

2016).

Phylogenetic Analyses

To assess the phylogenetic relationships between the two species, the dataset was expanded to include DNA sequences across different geographical regions of the two species available in GenBank (Table 4-2). The full dataset consisted of 60 sequences acquired from GenBank and 86 sequences acquired in this study.

Phylogenetic analyses were carried out by two different approaches including Maximum

Likelihood (ML) and Bayesian Inference (BI). The best-fit nucleotide substitution model was obtained using jModelTest V. 0.1.1 (Posada 2008) under the Akaike Information

Criterion (Akaike 1974). The HKY+G was selected as the best optimum model for construction of phylogenetic analyses. The ML tree was obtained using MEGA7 (Kumar et al., 2016) with complete deletion as the default setting. The BI tree was achieved using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Posterior probabilities were obtained by running two independent times using the Markov chain Monte Carlo

(MCMC) method for 10 million generations. Trees were sampled every 100 generations

followed by four simultaneous chains, one cold and three heated. The first 25% of the posterior samples were discarded as burn-in and the rest of the samples were used to generate a 50% majority-rule consensus tree. Cimex adjunctus (GenBank accession number KR035747.1) was used as an outgroup in the phylogenetic analyses. The DNA sequences were collapsed to the haplotypes using the PHASE algorithm (Stephens et al. 2001) implemented in DnaSP v5.0 (Librado and Rozas, 2009). The population genetic parameters were estimated including haplotype diversity and nucleotide diversity using DnaSP v5.0 (Librado and Rozas 2009). The median-joining spanning network was computed for all DNA haplotypes using the Network 4.6.1.0.

(http://www.fluxus-engineering.com/).

Results

Morphological Differentiation

Overall, there were significant differences between the bed bug populations for all life stages and the characteristics measured using the Wilks-lambda test (females:

th F22,38 = 4.34, P <0.0001, adult males: F22,12 = 3.7, P = 0.01, 5 instars: F18,66 = 13.23, , P

th rd nd <0.0001, 4 instars: F18,52 = 20.29, P <0.0001, 3 instars: F18,50 = 27.15, P <0.0001, 2

st instars: F18,50 = 25.90, P <0.0001, 1 instars: F18,50 = 5.66, P <0.0001). Univariate tests revealed that six characters were significantly different between populations for adult males (Table 4-3), six characters for adult females (Table 4-4), seven characters for 5th instars (Table 4-5), nine characters for 4th instars (Table 4-6), nine characters for 3rd instars (Table 4-7), nine characters for 2nd instars (Table 4-8), and only three characters for 1st instars (Table 4-9). The ratio of pronotum length to width was significantly different between the populations of C. lectularius and C. hemipterus for males (F2,25 =

24.84, P = <0.0001) and females (F2,27 = 17.94, P = < 0.0001) The population of C.

hemipterus (Brevard) was significantly smaller for many of the body measurements at all life stages (Tables [4-3] – [4-9]).

The canonical discriminant analysis explained 74% (CV1) and 26% (CV2) of the total variance for first instars, 92% (CV1) and 8% (CV2) for 2nd instars, 94% (CV1) and

6% (CV2) for 3rd instars, 95% (CV1) and 5% (CV2) for 4th instars, 84% (CV1) and 16%

(CV2) for 5th instars, 84% (CV1) and 16% (CV2) for adult females, and 86% (CV1) and

14% (CV2) for adult males (Tables 4-10 and 4-11).The contribution of each character to

CV1 varied between each life stage, with the pronotum width, pronotum length, flange length averages, hemelytra width averages, pronotum flange distance, and pronotum area contributing the most variation for adult females and males, with a combination of variables discriminating between the two species (Figure 4-2). Although many characteristics contributed to the overall morphological differences between species, pronotum width was significantly different between species across all life stages, except for first instars (Figures [4-3] – [4-7]). Box-and-whisker plots show that C. hemipterus

(Brevard) had shorter pronotum widths compared to both populations of C. lectularius for every life stage represented (Figure 4-8).

Genetic diversity

Multiple sequence alignment of 146 DNA sequences of the partial mtDNA-COI gene were generated of sequences 463 bp in length. Eight different haplotypes were identified of which the frequency of distribution was varied across geographical regions between the two species. The 51 nucleotide sequence of the COI gene for C. hemipterus revealed one polymorphic position, of which no position was parsimony informative. We found 14 polymorphic positions in 95 COI gene sequences of C. lectularius, of which seven were parsimony informative. We observed a higher

haplotype diversity (h) value in C. lectularius (0.32 ± 0.06) compared with C. hemipterus

(0.04 ± 0.04). The nucleotide diversity (π) value was higher in C. lectularius than C. hemipterus, estimated to be 0.0020 and 0.0002, respectively (Table 4-12).

Phylogenetic analyses and haplotype network

ML and BI phylogenetic analyses reconstructed using mtDNA-COI sequences produced monophyletic clades for each species with high support (Figure 4-9). The topologies of trees were consistent to each other.

The COI haplotype network with eight haplotypes showed that six haplotypes belonged to C. lectularius (H1, H4, H5, H6, H7 and H8), of which H1 was the most frequent (82/146 individuals) and was found in individuals from different geographical regions. We found two haplotypes in C. hemipterus species (H2 and H3), of which H2 was the most frequent haplotype (50/146) (Fig. 4-10, Table 4-13).

Discussion

This study provides a method for identification of the Cimex species that feed on humans. Our results show that two of the most prevalent human associated bed bugs can be differentiated using both morphological and genetic data. The combination of morphometric data from 13 measurements confirms that each species has a specific phenotype that can be used to accurately delimit species. The most reliable measurement for differentiation between the species was pronotum width. This characteristic was significantly different between both species for every life stage, except for the first nymphal instar stage. Alternatively, no other characteristics that were measured were significant across more life stages than pronotum width. Therefore, for ease in identification of these two species, pronotum width measurements would be the

best characteristic because it can be used as a ubiquitous characteristic across life stages for identification purposes.

Overall, Cimex hemipterus is smaller in size compared to C. lectularius, which also correlated to a smaller sized pronotum. There was no overlap between species in the variation of pronotum width for three of the life stages measured (3rd instars, 4th instars, and adult males), thus only one individual should be required to accurately identify the species for these life stages. Our study indicates that pronotum width measurements below 0.7 mm for 3rd instar and 0.8 mm 4th instar bed bugs would indicate C. hemipterus. A measurement ≤1.30 mm for adult males would indicate C. hemipterus. Although C. hemipterus is smaller, some overlap between species and their pronotum width in the 2nd instar stage, 5th instar stage, and adult females occurred. This suggests that an average pronotum width of several individuals would be necessary to correctly identify these life stages (mean <0.6mm for 2nd instars, <1.0 for 5th instars, and

<1.4 mm for adult females) to indicate C. hemipterus.

We tested both pronotum width measurements (our study) and ratio measurements (Usinger 1955) to see how accurate both methodologies were for species identification. Out of 70 individuals measured per strain and species

(Bradenton, Harlan, and Brevard; total 210), 42 individuals were misidentified by species using the ratios of pronotum width to length compared to six misidentified individuals using just the pronotum width measurements suggested by our study.

However, most misidentifications were in the nymphal stages – for instance, only four adults were misidentified by species using the pronotum ratio compared to two individuals that were misidentified using the pronotum width measurements out of a

total number of 30 adults (20 C. lectularius and 10 C. hemipterus). Although pronotum width is more accurate for identification of the species, it should be noted here that correct identification of the life stage would be important to distinguish between the species given the differences in widths and sizes across life stages.

Pronotum length was not statistically significant between species or populations for each life stage measured, thus should not be a standalone measurement for identification. Therefore, the recommended ratio from Usinger (1966) of pronotum width to pronotum length adds another variable (length) to complicate correct identification that is not necessary. This is the first robust morphological study of both species because we considered non-adult life stages, as well as phenotypic plasticity variation within a species (C. hemipterus) and population differences (C. lectularius) by using multiple specimens for measurements of 13 different characteristics. Increasing the number of measurements with numerous individuals enhances the ability to accurately identify the species.

We genetically sampled 146 bed bug specimens and revealed two distinct clades

(C. hemipterus and C. lectularius). The two clades represented the two different bed bug species, all collected from various geographical regions throughout the world. Each clade showed a genetically homogeneous group of populations within C. lectularius and

C. hemipterus species, supporting the lack of geographical structure between species.

Interestingly, the population of C. hemipterus collected in Florida, which is the first time this species had been documented in the US in over 70 years, was of the same haplotype and had little genetic variation compared to populations of C. hemipterus collected in Malaysia and Thailand. This suggests that this species may

have originated from a population in Asia and then was introduced into the United

States. Low genetic variation in the sequences of COI in C. hemipterus of Malaysia were found in our study, supporting that there is little genetic variation within this species from the 22 different populations sampled, similar to other studies of C. hemipterus collected in Malaysia (Masran and Ab Majid 2017).

Recent studies have supported the evidence of host-associated divergence of C. lectularius (Balvin et al. 2012, Booth et al. 2015). A bottleneck effect has been suggested of bed bugs that had been eradicated from the US in human dwellings, yet survived in poultry facilities. These bed bugs that remained in poultry houses have been previously suggested as the source of bed bugs during the resurgence in the US beginning in the 1990s. However, no genetic divergence has been shown in bed bugs that had been feeding on poultry. Previous studies have shown that there is low genetic diversity between bed bugs collected in poultry houses and with human-associated bed bugs (Szalanski et al. 2008, Balvin et al. 2012, Saenz et al. 2012). The one population of C. lectularius collected in a poultry house in Tennessee was evaluated and had low genetic diversity and was in the same haplotype of many populations of C. lectularius collected in human dwellings.

The impact of the introduction of tropical bed bugs into the United States is unknown. The introduction of a new species could initiate or exacerbate a pest problem and the ability to limit the spread of an introduced species and manage risks associated with an introduction requires accurate recognition of that species. Species identification is a fundamental part of any integrated pest management program and usually requires trained taxonomists or technicians that have extensive experience. Therefore, a single,

reliable morphological characteristic across multiple life stages or a simple cost-effective molecular method, would increase the ease of differentiating between these two similar species that feed on humans.

Table 4-1. Sampling information, abbreviations, species status used in the present study. Locality, Province, City, Abbreviations Country Species County, or State

Bradenton, Manatee, CL_BRA_FLORIDA_USA_01-10 USA C. lectularius Florida Cincinnati CL_COH_OHIO_USA_01-10 USA C. lectularius Brevard, Florida CH_FLH_Florida_USA_01-10 USA C. hemipterus Gainesville, Alachua, CL_GNV_ FLORIDA_USA_01-10 USA C. lectularius Florida New Jersey CL_HAR_NEWJERSEY_USA_01-10 USA C. lectularius Osceola, Florida CL_KVS_FLORIDA_USA_01-10 USA C. lectularius Lafayette CL_LIN_ INDIANA_USA_01-10 USA C. lectularius Penang CH_MAH_PENANG_MALAYSIA_01- Malaysia C. hemipterus 10 Tennessee CL_PHT_TENNESSEE_01-10 USA C. lectularius

4 6 5 7 8 9

10 11

Figure 4-1. Illustration of Cimex lectularius representing the characters that were measured for the morphological analysis of both species. 1) Clypeus length. 2) Clypeus width. 3) Distance between left and right pronotum margin. 4) Left pronotum flange length. 5) Right pronotum flange length. 6) Pronotum width. 7) Pronotum length. 8) Scutellum width. 9) Scutellum length. 10) Left hemelytra width. 11) Right hemelytra width. 12) Body length.

Table 4-2. Bed bug species information for all bed bugs used in genetic analyses. Locality, GenBank Province, City, Abbreviation Country Species accession References County, or No State Bradenton, Manatee, CL_BRA_FLORIDA_USA_01 USA C. lectularius NA Current Study Florida CL_BRA_FLORIDA_USA_02 CL_BRA_FLORIDA_USA_03 CL_BRA_FLORIDA_USA_04 CL_BRA_FLORIDA_USA_05 CL_BRA_FLORIDA_USA_06 CL_BRA_FLORIDA_USA_07 CL_BRA_FLORIDA_USA_08 CL_BRA_FLORIDA_USA_09 CL_BRA_FLORIDA_USA_10 C. Chiang mai CH_CHIANG_MAI_THAILAND_01 Thailand JX826469 Jantanoi,P. et al. 2012 hemipterus

CH_CHIANG_MAI_THAILAND_02 JX826470

CH_CHIANG_MAI_THAILAND_03 JX826471

JX826472 CH_CHIANG_MAI_THAILAND_04 JX826473 CH_CHIANG_MAI_THAILAND_05 C. Phuket CH_PHUKET_THAILAND_01 Thailand JX826474 Jantanoi,P. et al. 2012 hemipterus C. Krabi CH_KRABI_THAILAND_01 Thailand JX826475 Jantanoi,P. et al. 2012 hemipterus C. Bangkok CH_BANGKOK_THAILAND_01 Thailand JX826476 Jantanoi,P. et al. 2012 hemipterus CH_BANGKOK_THAILAND_02 JX826477 CH_ BANGKOK_THAILAND_03 JX826479

Table 4-2. Continued Locality, GenBank Province, Abbreviation Country Species accession References City, County, No or State

C. Phitsanulok CH_PHITSANULOK_THAILAND_01 Thailand JX826478 Jantanoi,P. et al. 2012 hemipterus C. Tamil Nadu CH_INDIA_01 India KF018755 Balvin, O. et al. 2015 hemipterus C. Siti Nor Ain, S.M. and Abdul Hafiz, A.M. CH_MALAYSIA_01 Malaysia KT851503 hemipterus 2015 CH_MALAYSIA_02 KT851504 CH_MALAYSIA_03 KT851505 CH_MALAYSIA_04 KT851506 CH_MALAYSIA_05 KT851507 CH_MALAYSIA_06 KT851508 CH_MALAYSIA_07 KT851509 CH_MALAYSIA_08 KT851510 CH_MALAYSIA_09 KT851511 CH_MALAYSIA_10 KT851512 CH_MALAYSIA_11 KT851513 CH_MALAYSIA_12 KT851514 CH_MALAYSIA_13 KT851515 CH_MALAYSIA_14 KT851516 CH_MALAYSIA_15 KT851518 CH_MALAYSIA_16 KT851519 CH_MALAYSIA_17 KT851520 CH_MALAYSIA_18 KT851521 CH_MALAYSIA_19 KT851522 CH_MALAYSIA_20 KT851523 CH_MALAYSIA_21 KT851524 C. CH_MALAYSIA_22 Melaca KF01875 Balvin, O. et al. 2015 hemipterus HQ10555 Ontario CL_ ONTARIO_CANADA_01 Canada C. lectularius Park, D.S. et al. 2016 4

Table 4-2. Continued Locality, GenBank Province, Abbreviation Country Species accession References City, County, No or State

Ontario CL_ ONTARIO_CANADA_02 Canada C. lectularius KR035952 Gwiazdowski, R.A. et al. 2015 CL_ THAILAND_01 Thailand C. lectularius JX826480 Jantanoi,P. et al. 2012 CL_ THAILAND_02 JX826481 CL_ THAILAND_03 JX826482 Unknow CL_ UNKNOWN_02 C. lectularius KJ937982 Booth et al. 2015 n CL_ UNKNOWN_03 KJ937986

CL_ UNKNOWN_04 KJ937988 CL_ UNKNOWN_05 KJ937992 CL_UNKNOWN_06 KJ937989

CL_UNKNOWN_07 KJ937990 CL_ UNKNOWN_08 KJ937991 CL_ UNKNOWN_13 KR002576 Missouri CL_ MISSOURI_USA_01 USA C. lectularius Robison et al. 2015 KR002577 CL_ MISSOURI_USA_02 Tulsa CL_OKLAHOMA_USA_01 Oklahoma C. lectularius KR002575 Robison et al. 2015 Tulsa CL_ OKLAHOMA_USA_02 Muskogee CL_ OKLAHOMA_USA_03 KJ937979 Czech CL_ CZECH REPUBLIC_01 C. lectularius KJ937980 Booth et al. 2015 Republic KJ937983 CL_ CZECH REPUBLIC_02 CL_ CZECH REPUBLIC_03 CL_ GERMANY_01 Germany C. lectularius KJ937981 Booth et al. 2015 CL_ITALY_01 Italy C. lectularius KJ937984 Booth et al. 2015 CL_ HUNGARY_01 Hungary C. lectularius KJ937985 Booth et al. 2015

Table 4-2. Continued Locality, GenBank Province, Abbreviation Country Species accession References City, County, No or State

Deliblatská Pešcara, CL_ SERBIA_01 Serbia C. lectularius KJ937987 Booth et al. 2015 Rošiana CL_ SERBIA_02 Serbia C. lectularius KF018756 Balvin O. et al. 2015 CL_ Saskatchewan Canada KR044731 Gwiazdowski, R. A. et al. 2015 SASKATCHEWAN_CANADA_01

Table 4-3. Character measurements for adult male Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard). Mean ± SE and statistical parameters of each significant character (between populations). ANOVA (Fdf; P Character Strain N Size (mm) value Body Length Bradenton 9 5.26 ± 0.13 b 3.422,25; 0.05 Harlan 9 5.75 ± 0.13 a Brevard 10 5.50 ± 0.13 ab Pronotum Width Bradenton 9 1.47 ± 0.02 a 68.672,25; <0.0001 Harlan 9 1.38 ± 0.02 b Brevard 10 1.20 ± 0.02 c Pronotum Flange Bradenton 9 0.77 ± 0.02 a 11.472,25; 0.0003 Harlan 9 0.79 ± 0.02 a Brevard 10 0.68 ± 0.02 b Hemelytra Width Bradenton 9 0.94 ± 0.02 a 27.842,25; <0.0001 Harlan 9 0.91 ± 0.02 a Brevard 10 0.78 ± 0.02 b Pronotum Margin Distance Bradenton 9 1.07 ± 0.02 a 11.522,21; 0.03 Harlan 7 0.95 ± 0.02 b Brevard 8 0.94 ± 0.02 b Pronotum Area Bradenton 9 0.95 ± 0.03 a 7.912,25; 0.002 Harlan 9 0.98 ± 0.03 a Brevard 10 0.85 ± 0.02 b Means followed by different letters are significantly different.

Table 4-4. Character measurements for adult female Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard). Mean ± SE and statistical parameters of each significant character (between populations). ANOVA (F ; P Character Population N Mean ± SE df value)

Clypeus Width Bradenton 10 0.37 ± 0.01 a 3.742,29; 0.04

Harlan 12 0.33 ± 0.01 ab

Brevard 10 0.32 ± 0.01 b Pronotum Width Bradenton 10 1.61 ± 0.04 a 33.522,29;<0.0001 Harlan 12 1.60 ± 0.03 a Brevard 10 1.26 ± 0.04 b Pronotum Flange Bradenton 10 0.87 ± 0.02 a 14.642,29; <0.0001 Harlan 12 0.89 ± 0.02 a Brevard 10 0.76 ± 0.02 b Hemelytra Width Bradenton 10 1.04 ± 0.02 a 29.962,29; <0.0001 Harlan 12 1.04 ± 0.02 a Brevard 10 0.84 ± 0.02 b Pronotum Margin Distance Bradenton 10 1.14 ± 0.02 a 17.262,29; <0.0001 Harlan 12 1.17 ± 0.02 a Brevard 10 0.99 ± 0.02 b

Pronotum Area Bradenton 10 1.01 ± 0.03 a 10.022,29; 0.0005

Harlan 12 1.13 ± 0.03 b Brevard 10 0.95 ± 0.03 a Means followed by different letters are significantly different.

Table 4-5. Character measurements for 5th instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard). Mean ± SE and statistical parameters of each significant character (between populations). ANOVA (F ; P Character Population N Mean ± SE df value) Clypeus Length Bradenton 20 0.16 ± 0.01 a 9.752,41; 0.0003 Harlan 12 0.17 ± 0.01 a Brevard 12 0.13 ± 0.01 b

Clypeus Width Bradenton 20 0.33 ± 0.01 a 12.472,41; <0.0001

Harlan 12 0.28 ± 0.02 b

Brevard 12 0.24 ± 0.02 b Pronotum Width Bradenton 20 1.31 ± 0.02 a 45.182,41; <0.0001 Harlan 12 1.22 ± 0.03 a Brevard 12 0.94 ± 0.03 b Pronotum Flange Bradenton 20 0.65 ± 0.01 a 54.592,41;<0.0001 Harlan 12 0.68 ± 0.01 a Brevard 12 0.53 ± 0.01 b Hemelytra Width Bradenton 20 0.82 ± 0.01 a 36.452,41; <0.0001 Harlan 12 0.82 ± 0.01 a Brevard 12 0.67 ± 0.01 b Pronotum Margin Distance Bradenton 20 0.97 ± 0.01 a 37.82,41;<0.0001 Harlan 12 1.01 ± 0.02 a Brevard 12 0.82 ± 0.02 b

Pronotum Area Bradenton 20 0.84 ± 0.02 a 9.912,41; 0.0003

Harlan 12 0.71 ± 0.03 b Brevard 12 0.75 ± 0.03 b Means followed by different letters are significantly different.

Table 4-6. Character measurements for 4th instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard). Mean ± SE and statistical parameters of each significant character (between populations).

Character Population N Mean ± SE ANOVA (Fdf ; P value)

Body Length Bradenton 15 3.42 ± 0.06 a 97.712,34; <0.0001 Harlan 12 3.43 ± 0.06 a Brevard 10 2.31 ± 0.07 b Clypeus Length Bradenton 15 0.16 ± 0.01 a 11.422,34; 0.0002 Harlan 12 0.15 ± 0.01 a Brevard 10 0.11 ± 0.01 b Clypeus Width Bradenton 15 0.25 ± 0.01 a 14.492,34; <0.0001 Harlan 12 0.27 ± 0.01 a Brevard 10 0.20 ± 0.01 b Pronotum Length Bradenton 15 0.34 ± 0.01 a 19.062,34; <0.0001 Harlan 12 0.37 ± 0.01 a Brevard 10 0.30 ± 0.01 b

Pronotum Width Bradenton 15 0.96 ± 0.01 a 89.322,34; <0.0001

Harlan 12 0.91 ± 0.01 b

Brevard 10 0.76 ± 0.01 c

Pronotum Flange Bradenton 15 0.51 ± 0.01 a 61.162,34; <0.0001

Harlan 12 0.47 ± 0.01 b

Brevard 10 0.34 ± 0.01 c Hemelytra Width Bradenton 15 0.65 ± 0.01 a 131.072,34; <0.0001 Harlan 12 0.63 ± 0.01 a Brevard 10 0.43 ± 0.01 b Pronotum Margin Distance Bradenton 15 0.83 ± 0.02 a 36.102,34; <0.0001 Harlan 12 0.82 ± 0.02 a Brevard 10 0.60 ± 0.02 b Pronotum Area Bradenton 15 0.44 ± 0.01 a 102.742,34; <0.0001 Harlan 12 0.47 ± 0.01 a Brevard 10 0.23 ± 0.01 b Means followed by different letters are significantly different.

Table 4-7. Character measurements for 3rd instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard). Mean ± SE and statistical parameters of each significant character (between populations).

Character Population N Mean ± SE ANOVA (Fdf ; P value)

Body Length Bradenton 15 2.57 ± 0.02 a 110.292,33; <0.0001

Harlan 12 2.45 ± 0.03 b

Brevard 9 1.98 ± 0.03 c

Clypeus Length Bradenton 15 0.16 ± 0.01 a 21.252,33; <0.0001

Harlan 12 0.13 ± 0.01 b

Brevard 9 0.10 ± 0.01 c Clypeus Width Bradenton 15 0.20 ± 0.01 a 17.132,33; <0.0001 Harlan 12 0.21 ± 0.01 a Brevard 9 0.14 ± 0.01 b Pronotum Length Bradenton 15 0.29 ± 0.04 a 23.452,33; <0.0001 Harlan 12 0.28 ± 0.01 a Brevard 9 0.24 ± 0.01 b Pronotum Width Bradenton 15 0.81 ± 0.01 a 132.802,33; <0.0001 Harlan 12 0.84 ± 0.01 a Brevard 9 0.56 ± 0.01 b Pronotum Flange Bradenton 15 0.37 ± 0.01 a 126.682,33; <0.0001 Harlan 12 0.37 ± 0.01 a Brevard 9 0.25 ± 0.01 b Hemelytra Width Bradenton 15 0.50 ± 0.01 a 161.762,33; <0.0001 Harlan 12 0.48 ± 0.01 a Brevard 9 0.32 ± 0.01 b Pronotum Margin Distance Bradenton 15 0.68 ± 0.01 a 73.022,33; <0.0001 Harlan 12 0.67 ± 0.01 a Brevard 9 0.46 ± 0.02 b Pronotum Area Bradenton 15 0.24 ± 0.01 a 29.882,33; <0.0001 Harlan 12 0.26 ± 0.01 a Brevard 9 0.17 ± 0.01 b Means followed by different letters are significantly different.

Table 4-8. Character measurements for 2nd instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard). Mean ± SE and statistical parameters of each significant character (between populations). Character Population N Mean ± SE ANOVA (Fdf ; P value) Body Length Bradenton 15 1.61 ± 0.02 a 11.472,33; 0.0002 Harlan 12 1.71 ± 0.02 b Brevard 9 1.56 ± 0.03 a Clypeus Length Bradenton 15 0.11 ± 0.003 a 23.692,33; <0.0001 Harlan 12 0.09 ± 0.003 b Brevard 9 0.09 ± 0.004 b Clypeus Width Bradenton 15 0.15 ± 0.004 a 6.992,33; 0.0029 Harlan 12 0.14 ± 0.005 b Brevard 9 0.13 ± 0.005 b Pronotum Length Bradenton 15 0.24 ± 0.004 a 23.032,33; <0.0001 Harlan 12 0.20 ± 0.01 b Brevard 9 0.22 ± 0.01 b Pronotum Width Bradenton 15 0.65 ± 0.01 a 12.852,33; <0.0001 Harlan 12 0.65 ± 0.01 a Brevard 9 0.57 ± 0.01 b Pronotum Flange Bradenton 15 0.30 ± 0.01 a 66.722,33; <0.0001 Harlan 12 0.24 ± 0.01 b Brevard 9 0.22 ± 0.01 b Hemelytra Width Bradenton 15 0.37 ± 0.01 a 10.672,33; 0.0003 Harlan 12 0.34 ± 0.01 b Brevard 9 0.32 ± 0.01 b Pronotum Margin Bradenton 15 0.45 ± 0.01 a 18.27 ; <0.0001 Distance 2,33 Harlan 12 0.53 ±0.01 b Brevard 9 0.45 ± 0.01 a Pronotum Area Bradenton 15 0.17 ± 0.002 a 154.702,33; <0.0001 Harlan 12 0.12 ± 0.002 b Brevard 9 0.14 0.002 c Means followed by different letters are significantly different.

Table 4-9. Character measurements for 1st instar Cimex lectularius (Bradenton and Harlan) and Cimex hemipterus (Brevard). Mean ± SE and statistical parameters of each significant character (between populations). Character Population N Mean ± SE ANOVA (Fdf ; P value) Clypeus Bradenton 15 0.06 ± 0.002 a 25.31 ; <0.0001 Length 2,33 Harlan 13 0.06 ± 0.002 b Brevard 8 0.04 ± 0.002 c Clypeus Width Bradenton 15 0.12 ± 0.004 a 7.192,33; 0.0026 Harlan 13 0.10 ± 0.004 ab Brevard 8 0.09 ± 0.005 b Pronotum Margin Bradenton 15 0.34 ± 0.01 a 8.722,33; 0.0009 Distance Harlan 13 0.38 ± 0.01 b Brevard 8 0.36 ± 0.01 ab Means followed by different letters within the same character are significantly (P < 0.05) different according to Tukey’s HSD tests. N = number of individuals. Non-significant characters were not included.

Table 4-10. Coefficients for canonical variables of the discriminant analysis for the first canonical variate. 1st 2nd 3rd 4th 5th Variable Females Males Instars Instars Instars Instars Instars Body length 0.20 -0.28 0.72 -0.65 0.96 1.00 0.78 Clypeus Length 0.38 0.75 1.00 0.98 0.80 0.98 0.91 Clypeus Width 0.54 0.94 0.38 0.86 1.00 0.95 0.94 Pronotum Length -0.61 -1.00 -0.92 1.00 0.92 0.91 -0.30 Pronotum Width 1.00 1.00 0.47 0.16 1.00 0.98 1.00 Pronotum Flange 0.99 0.92 0.80 0.86 1.00 0.97 0.91 Scutellum Length 0.70 -0.50 * * * * * Scutellum Width -0.73 * * * * * * Hemelytra Width 1.00 0.99 0.64 0.71 0.99 1.00 0.98 Pronotum Margin Distance 1.00 * 0.50 -0.83 1.00 1.00 0.89 Pronotum Area 0.78 0.89 0.46 1.00 0.98 0.99 0.51 Total Proportion 0.84 0.85 0.74 0.92 0.94 0.95 0.84 *Instars do not develop a scutellum, thus scutellum length or width measurements are missing. The scutellum width and pronotum margin distance were measured for males and included in overall analyses,but excluded from this table because of a smaller sample size and missing values.

Table 4-11. Coefficients for canonical variables of the discriminant analysis for the second canonical variate. 1st 2nd 3rd 4th 5th Variable Females Males Instars Instars Instars Instars Instars Body length 0.98 0.96 0.70 0.76 -0.27 0.049 -0.63 Clypeus Length -0.93 0.66 -0.01 0.21 -0.60 -0.207 0.41 Clypeus Width -0.84 -0.33 -0.93 0.51 -0.01 0.307 -0.35 Pronotum Length 0.79 0.07 0.39 0.03 -0.39 0.410 0.95 Pronotum Width -0.06 -0.08 0.88 0.99 0.03 -0.204 -0.03 Pronotum Flange 0.12 0.39 0.60 0.51 -0.02 -0.227 0.42 Scutellum Length 0.71 0.87 * * * * * Scutellum Width 0.69 * * * * * * Hemelytra Width -0.06 0.12 -0.77 0.70 -0.12 -0.031 0.17 Pronotum Margin Distance 0.08 * 0.86 0.56 -0.10 -0.028 0.45 Pronotum Area 0.63 0.45 0.89 0.00 0.18 0.159 -0.86 Total Proportion 0.16 0.14 0.26 0.08 0.06 0.05 0.16 *Instars do not develop a scutellum, thus scutellum length or width measurements are missing. The scutellum width and pronotum margin distance were measured for males and included in overall analyses but excluded from this table because of missing values and unproportionable sample sizes.

Table 4-12. Population genetic indices calculated based on 146 mtDNA-COI sequences of Cimex lectularius and C. hemipterus populations. Tajima Fu’s N V M H HD π K Species D Fs 0.32 ± 95 14 14 12 0.002 ± 0.002 0.61 -2.13 -10.33 C. lectularius 0.06 0.04 ± 0.0002 ± 51 1 1 2 27.1 -1.1 -1.65 C. hemipterus 0.04 0.0002 N = number of individuals; V = number of polymorphic sites; H = number of haplotypes; M = number of mutations; HD = haplotype diversity; π= nucleotide diversity; K = average number of nucleotide differences.

Figure 4-2. Discriminant analysis plots of canonical variates 1 and 2 for A) males and B) females from Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard). Cimex hemipterus clearly is distinguished by the green circles and is morphologically grouped into a clade alone from the two populations of C. lectularius. The means of each group are represented by +.

Figure 4-3. Discriminant analysis plots of canonical variates 1 and 2 for 5th instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard). Cimex hemipterus is distinguished by the green circles and is morphologically grouped into a clade alone from the two populations of C. lectularius. The means of each group are represented by +.

Figure 4-4. Discriminant analysis plots of canonical variates 1 and 2 for 4th instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard). Cimex hemipterus is distinguished by the green circles and is morphologically grouped into a clade alone from the two populations of C. lectularius. The means of each group are represented by +.

Figure 4-5. Discriminant analysis plots of canonical variates 1 and 2 for 3rd instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard). Cimex hemipterus is distinguished by the green circles and is morphologically grouped into a clade alone from the two populations of C. lectularius. The means of each group are represented by +.

Figure 4-6. Discriminant analysis plots of canonical variates 1 and 2 for 2nd instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard). The means of each group are represented by +.

Figure 4-7. Discriminant analysis plots of canonical variates 1 and 2 for 1st instar Cimex lectularius populations (Bradenton and Harlan) and one population of C. hemipterus (Brevard). The means of each group are represented by +.

A B

C D

E F E

Figure 4-8. Median and distribution of pronotum width (Pr_Width) values for three populations (Brevard [C.hemipterus], Harlan and Bradenton [C. lectularius]) of bed bugs. (A) 2nd instars, (B) 3rd instars, (C) 4th instars, (D) 5th instars, (E) adult males, and (F) adult females. 1st instars did not have significantly different pronotum widths between the three populations and are excluded, with C. hemipterus narrower in width across all represented life stages.

Cimex lectularius

Cimex hemipterus

Cimex adjunctus

Cimex lectularius

Cimex hemipterus

Cimex adjunctus

Figure 4-9. Phylogenetic trees constructed for Cimex lectularius and C. hemipterus using the mtDNA-COI sequences obtained from 146 individuals. A) Maximum Likelihood tree with bootstrap values behind the major nodes. B) Bayesian Inference tree with posterior probabilities behind the major nodes. H1−H9 denotes the major haplotypes found in whole data set. The distribution of individuals within each haplotypes are listed in Table 4-2.

Figure 4-10. A minimum spanning network constructed using the eight mtDNA-COI haplotypes (H1-H8) identified in Cimex lectularius and C. hemipterus populations. The designation of haplotypes are represented by circles. Distribution of geographical regions for each haplotype are given in Table 4-2. The sizes of the circles are proportional to the number of individuals represented. The numbers between circles are roughly proportional to the estimated number of mutational steps between the haplotypes.

Table 4-13. Data matrix of 8 distinct haplotypes among 146 individual of C. lectularius and C. hemipterus populations using 463 bp of COI. GenBank sequences are marked with bold.

Haplotypes Haplotype Frequency Individuals and Locations H1 82 CL_BRA_FLORIDA_USA_01-10 CL_ONTARIO_CANADA_01-02 CL_THAILAND_02 CL_SERBIA_01-02 CL_UNKNOWN_06-07 CL_OKLAHOMA_USA_01-03 CL_MISSOURI_USA_01-02 CL_SASKATCHEWAN_CANADA_01 CL_GNV_FLORIDA_USA_01-10 CL_COH_OHIO_USA_01-10 CL_HAR_NEWJERSEY_USA_01-10 CL_KVS_FLORIDA_USA_01-10 CL_LIN_INDIANA_01-10 CL_PHT_TENNESSEE_01-10 H2 51 CH_CHAING_MAI_THAILAD_01-05 CH_PHUKET_THAILAND_01 CH_KRABI_THAILAND_01 CH_BANGKOK_THAILAND_01-02 CH_PHITSANULOK_THAILAND_01 CH_BANGKOK_THAILAND_03 CH_MALAYSIA_22 CH_UNKNOWN_02 CH_MALAYSIA_01-19 CH_MALAYSIA_21 CH_FLH_FLORIDA_USA_01-10 CH_MAH_PENANG_MALAYSIA_01-04 CH_MAH_PENANG_MALAYSIA_06-07 CH_MAH_PENANG_MALAYSIA_10 H3 1 CH_ MALAYSIA_20 H4 4 CL_ THAILAND_01 CL_ THAILAND_03 CL_ UNKNOWN_05, CL_ UNKNOWN_08 H5 3 CL_ CZECH REPUBLIC_01-02 CL_ GERMANY_01 H6 4 CL_ UNKNOWN_02-03 CL_ CZECH REPUBLIC_03 CL_ HUNGARY_01 H7 1 CL_ITALY_01 H8 1 CL_UNKNOWN_04 H9 1 Cimex adjunctus

CHAPTER 5 LOCOMOTION INHIBITON OF CIMEX LECTULARIUS L., FOLLOWING TOPICAL, SUBLETHAL DOSE APPLICATION OF THE CHITIN SYNTHESIS INHIBITOR LUFENURON

Liquid chemical insecticide applications are advantageous for bed bug control because of their low cost and ease of application compared to other control methods

(e.g. heat and fumigation). Pest control companies in the United States rely heavily on pyrethroid insecticide applications for bed bug treatments (Gangloff-Kaufman et al.

2006). Furthermore, the majority of pesticides labeled for indoor use in the United

States contain pyrethroids as the active ingredient, consequently, bed bugs have been frequently exposed to these insecticides.

The frequent exposure of common bed bugs (Cimex lectularius) to pyrethroids has resulted in resistance to these active ingredients (Moore and Miller 2006, Romero et al. 2007, Yoon et al. 2008, Adelman et al. 2011, Koganemaru et al. 2013, Lilly et al.

2016). Resistance also has developed in bed bugs to neonicotinoid insecticides that are often combined and formulated with pyrethroids (Romero and Anderson 2016).

However, rotating and utilizing insecticides with different modes of action, as well as implementing other integrated pest management strategies, can circumvent insecticide resistance development. Unfortunately, there are limited products available with alternative modes of action to pyrethroids for a bed bug insecticide rotation program.

Insect growth regulators (IGRs) have an alternative mode of action to pyrethroid insecticides and affect insect growth, development, and reproduction. Insect growth regulators have been found to be highly effective against multiple urban insect pests, including flies (Arthur and Fontenot 2012, Geden and Devine 2012, Lohmeyer et al.

2014, Fulcher et al. 2014) fleas (Hinkle et al. 1995, Smith et al. 1996, Meola et al. 2000,

Kawada and Hirano 2014), termites (Jones 1984, Su and Scheffrahn 1993, Su 1994, Su et al. 1995) and cockroaches (Koehler and Patterson 1991, Reid et al. 1992, Mosson et al. 1995, Ameen et al. 2005). However, their use has not been extensively investigated in bed bugs. There is currently only one insect growth regulator that is labeled for bed bug control in the United States, manufactured under the trade name Gentrol® ((S)- hydroprene; Wellmark International; Schaumberg, IL).

Hydroprene is a juvenile hormone analog that affects multiple developmental processes (e.g. ecdysis and formation of reproductive organs) in insects that are regulated naturally by the presence of juvenile hormone during ecdysis. Although

Gentrol® is a registered insecticide in the United States for bed bug control, Gentrol® aerosol and Gentrol® concentrate ((S)-hydroprene; Wellmark International;

Schaumberg, IL) required application rates ≥ 3x the label rate to achieve 66-100% adult bed bug mortality (Todd 2006). Similar to Todd (2006), (S)-hydroprene was not effective against bed bugs except at elevated label rates (Goodman et al. 2013). Applications at

10x the label rate caused a 100% reduction in bed bug oviposition in one bed bug strain but only a 38% ovipositional reduction in another strain (Goodman et al. 2013).

The addition of insect growth regulators to an integrated pest management (IPM) program for bed bugs has potential because IGRs exhibit low mammalian toxicity

(Goodman et al. 2013), which would be advantageous for indoor use, as well as provide an alternative mode of action for rotation in a chemical program. The limited studies available on bed bugs and IGRs have mostly investigated juvenile hormone analogs

(JHAs) and have largely neglected another type of IGR, the chitin synthesis inhibitors

(CSIs). This may be primarily because CSIs are known to be highly effective when

ingested (e.g. as baits or foliar treatments) but are not often used as contact insecticides (Demark and Bennett 1989).

Chitin synthesis inhibitors impede the biosynthesis of chitin. As a result, the cuticle is usually malformed following ecdysis, causing morphological abnormalities or death. Few studies have investigated the efficacy of chitin synthesis inhibitors against bed bugs, and there are currently no insecticides labeled in the United States containing a chitin synthesis inhibitor for bed bug control. However, one insecticide, under the trade name Tenopa® (BASF, Ludwigshafen, Germany) is registered in Europe, South

America, and Mexico and contains a pyrethroid (alpha-cypermethrin) and a chitin synthesis inhibitor (flufenoxuron). Flufenoxuron has been evaluated against first and second instar bed bugs by exposure to Flufenoxuron impregnated filter papers (De

Andrade 2015). This exposure caused morphological abnormalities in the bed bugs following molting and approximately 82% mortality in bed bugs after 35 days.

The purpose of this study was to evaluate the lethal and sublethal effects of the chitin synthesis inhibitor lufenuron on bed bugs. Fifth instar bed bug ecdysis, morphological abnormalities, and mortality was evaluated following topical application of lufenuron to individual bed bugs.

The resultant bed bug adults that molted with leg malformations after sublethal exposure were used to quantify the effects of malformations on locomotion ability.

Locomotion ability was measured using pulling force assays to determine the force bed bugs generated when they attached their tarsae to a surface. Pulling force assays have been used previously to evaluate a bed bugs ability to climb different textured surfaces

(Hottel et al. 2015), as well as to measure the ability for tropical bed bugs, Cimex

hemipterus, to generate vertical friction and escape pitfall traps used for bed bug monitoring (Kim et al. 2017).

Materials and Methods

Insects

Two strains of common bed bug, Cimex lectularius L. (Harlan and Bradenton) were used for the topical application and pulling force assays. The Harlan strain was collected in 1973 in Ft. Dix, NJ and then was maintained in a laboratory on human blood. The University of Florida Urban Entomology Laboratory acquired this strain in the late 2000s. The Bradenton strain was collected by a pest control company in Bradenton,

FL in August 2013.

Bed bugs were fed weekly on defibrinated rabbit blood (Hemostat, Dixon, CA) using an artificial feeding system (Montes et al. 2002) and maintained at approx. 70%

RH, 25 °C, and a 12:12 L:D photoperiod. All bed bug colonies were maintained in plastic jars (Mold-Rite Plastics, Plattsburg, NY, 300 ml) enclosed at one end with mesh for feeding, with accordion-style folded filter paper (diam. = 9 cm, # 2, Whatman, GE

Healthcare UK Ltd., Buckinghamshire, UK) provided for harborage.

Insecticide Dilutions

Technical grade lufenuron (FMC Corporation, Philadelphia, PA) was weighed on an analytical balance then serially diluted 10-fold for Harlan strain bed bugs and 2-fold for Bradenton strain bed bugs with acetone into five concentrations. A control treatment consisted of acetone. The serially diluted concentrations of lufenuron for topical application to Harlan strain bed bugs were 0.000016, 0.00016, 0.0016, 0.016, and

0.16% (w/v), which were determined by preliminary exposure treatments. Topical

applications of lufenuron to Bradenton strain bed bugs were diluted to concentrations of

0.32, 0.64, 1.28, 2.56, and 5.1% (w/v).

Insect Growth Regulator Topical Application Bioassay

Individual bed bugs were topically treated with an experimental concentration of

1 µl of technical grade lufenuron (or acetone for control) to the ventral side of their abdomen using a Hamilton syringe mounted on a repeating dispenser (50 µl; Hamilton

Company, Reno, NV). The bed bugs were placed inside an aluminum weigh dish (6.4 cm diam., 1.7 cm ht., Fisher Scientific, Waltham, MA) and chilled on ice to restrict movement during the application of lufenuron. The bed bugs were individually treated with one dose of lufenuron or acetone (control) and then placed in a cohort of five bed bugs for feeding (5 bed bugs treated and fed = 1 replication). All doses and the control treatment had 11 replications of 5 (n = 55) treated bugs, except for the highest dose (17 replications of 5, n = 85), treated on individual days, for a total of 355 bugs treated for both Harlan and Bradenton strains (710 bugs total treated).

Fifth instar bed bugs were fed in a cohort for ease of feeding on rabbit blood

(Hemostat, Dixon, CA) 1 day after topical application of lufenuron. For feeding, bed bug cohorts were placed into a glass vial (20 ml, polypropylene caps, Wheaton, Millville, NJ,

USA) and the vial was enclosed with mesh (90 µm, nylon, Amazon supply, Seattle, WA) on the open end. The mesh was then covered with parafilm (“M”, 10.16 cm width,

Bemis, Neenah, WI, USA) and the vial was inverted directly onto defibrinated rabbit blood that was held in a soufflé cup (30 ml, DART, Mason, MI) placed on top of a hot plate (Isotemp, Fisher Scientific, Waltham, MA) maintained at ~40°C to simulate human body temperature. This technique differed from the methodology of colony

maintenance, to limit the amount of blood wasted for feeding small cohorts of bed bugs.

Furthermore, this feeding method encouraged bed bugs to feed more quickly, since they were placed directly on the blood.

Following topical application of the insecticide, the bed bugs were placed into a

Petri dish (Polystyrene, 6.0 x 1.5 cm; Fisher Scientific, Waltham, Massachusetts, USA) containing filter paper (#1, 4.3 cm diam.; Whatman, GE Healthcare UK Ltd.,

Buckinghamshire, UK). Bed bugs that did not feed to repletion following topical application of lufenuron were excluded from further analysis (<10%). Bed bug mortality, morbidity, and molting were recorded 14 days after treatment. Mortality was recorded as those insects that did not move when probed. Morbidity was recorded as insects that were still alive but exhibited restricted movement due to morphological deformities and had an extreme reduction in responsiveness following probing. Further analysis of leg abnormalities and locomotion inhibition was quantified with pulling force assays using bed bugs exposed to lufenuron that did not result in high mortality, but high levels of morphological abnormalities.

Pulling Force Assay

A dose that resulted in an approximate effective concentration that caused leg abnormalities in 25% of the population (EC25), determined by the topical application bioassay, was used to evaluate the effects of lufenuron on locomotion inhibition. The concentration selected for Harlan strain bed bugs was 0.0016% (w/v) and 0.64% (w/v) lufenuron for Bradenton strain bed bugs. Prior to the pulling force assay, individual bed bugs were treated with 1 µl acetone (control) or with lufenuron (0.0016% w/v for Harlan and 0.64% w/v for Bradenton) and allowed 9 days to molt. Following molting, the individual bed bugs were used for the pulling force assays. Twenty bed bug’s pulling

forces were individually measured for each treatment (control or treated with lufenuron) for both strains of bed bugs (Harlan and Bradenton). An individual bed bug was a replicate, totaling 80 bugs measured for the pulling force assays.

Methods similar to Hottel et al. (2015) were used to calculate the pulling force of bed bugs that were either exposed to lufenuron or to acetone alone (control). Briefly, individual bed bugs were tethered to a paint brush bristle using super glue (Loctite;

Henkel Corporation, Rocky Hill, CT, USA) attached at the first or second segment of the dorsal abdomen. Sandpaper (Aluminum Oxide, 60 grit; 3M, St. Paul, MN) was mounted to a glass microscope slide (Premium microscope slides plain; Fisher Scientific,

Pittsburgh, PA, USA) and then the slide with the sandpaper attached was mounted to a wooden platform. The end of the paintbrush bristle (4 cm long, polyester; Great

American Marketing, Valencia, CA, USA) that was not attached to the bed bug and loose was inserted into a ball of modeling clay (2.53 g; Van Aken International,

Charleston, SC, USA). The wooden platform with the attached sandpaper surface was placed outside of the weighing pan within an analytical balance (New Classic MF, Model

MS105D4; Mettler Toledo, Grietensee, Switzerland) and the tethered bed bug in the clay ball was placed directly onto the analytical balance. After the balance was tared to zero, the wooden platform with the sandpaper surface attached was moved forward, without directly contacting the weighing pan, until the bed bug could grip the surface

(Fig. 5-1).

Once all six bed bug tarsi contacted the sandpaper surface, negative mass changes (indicative of the mass pulled by the bed bug) were recorded directly from the analytical balance software for 240 s. The mass data was then converted to force using

the formula F = ma (F = force [mN], m = mass (g), and a = acceleration (m2/s

[acceleration due to gravity was a constant -9.81m2/s])). The maximum amount of force and the average amount of force (mean of several readings over 240 s) generated by each bed bug was then calculated after the four-minute duration.

Statistical analysis

Insecticide concentrations were chosen that resulted in mortality and malformations (leg abnormalities, cuticle abnormalities that reduced bed bug responsiveness) ranging from 10-80%. Effective concentrations (EC) were chosen instead of lethal concentrations (LC) because mortality did not reach 80%; however, significant morphological effects were observed that limited bed bug movement and responsiveness (recorded as malformed). The EC50 was calculated using a generalized linear model with a binomial distribution and probit link using JMP (JMP Pro 13; SAS institute, Cary, NC, USA). The maximum amount of force and average amount of force generated between bed bugs exposed and not exposed to the EC25 doses of lufenuron were evaluated using t-tests in JMP for both Harlan and Bradenton strains. Values of P

≤0.05 were used to indicate significance.

Results

The effective concentration that resulted in 50% malformations and mortality

(EC50) for Harlan strain bed bugs was 0.0081% (w/v) [95% CI = 0.0021-0.014] lufenuron. The EC50 of lufenuron for Bradenton strain bed bugs was much higher compared to Harlan strain bed bugs, with a value of 1.11% (w/v) [95% CI = 1.10-1.22].

Mortality from lufenuron did not result in a typical dose response, which is unlike characteristic responses of insects to neurotoxins (Table 5-1). However, in general, the sublethal effects increased to some extent with an increase in the concentration applied

(Table 5-1). As the concentration of lufenuron increased, the observed effects transitioned from sublethal to lethal (Table 5-1).

Lufenuron caused multiple morphological problems (Figures 5-2 & 5-3) that resulted in decreased locomotion of bed bugs following the molt from 5th instar to adult.

Lufenuron had a significant effect on bed bug locomotion following ecdysis for both

Harlan and Bradenton strain bed bugs. Most Harlan strain bed bugs that were treated could not generate any pulling force, compared to one representative non-treated bed bug that was able to grip and pull on the sandpaper surface at a maximum of 6 mN

(Figure 5-4).

Harlan strain bed bugs treated with lufenuron were significantly less able to grip the sandpaper surface, indicated by the reduction in the average force compared to non-treated bed bugs (F = 5.35, df = 1, 22, P <0.0001) (Figure 5-5), as well as the maximum amount of force generated across all readings over time (F = 6.8, df = 1,

32.75, P <0.0001) (Figure 5-6). Bradenton bed bugs treated with lufenuron also had a significant reduction in the average amount of force they could generate to grip onto a surface (F = 8.86, df = 1, 23.97, P < 0.0001) (Figure 5-5) as well as the maximum amount of force generated (F = 12.03, df = 1, 30.80, P < 0.0001) (Figure 5-6).

Discussion

The benzoylurea compounds, which include lufenuron and other CSIs that are chemical derivatives of benzoylurea, have been documented to cause multiple effects directly related to chitin synthesis, however, the mode of action of CSIs has not been entirely determined (Merzendorfer 2013). Studies have suggested that CSIs inhibit the action of chitin synthase, which is an integral protein that aids in the synthesis of N- acetylglucosamine (Merzendorfer 2006). Conversely, the mode of action of the CSI

diflubenzuron has been suggested to inhibit the incorporation of N-acetylglucosamine into insect chitin during the molting process (Matsumura 2010). Nevertheless, the external physiological ramifications of chitin synthesis inhibitors have been observed and reported in numerous insect taxa. Chitin synthesis inhibitors impede insect ecdysis, often resulting in malformations in the newly formed cuticle of an insect and can also affect the peritrophic matrix and digestive system (Merzendorfer 2013).

Previous studies have documented that chitin synthesis inhibitors have a broad range of efficacy against numerous insect pests, and they also interfere with hemipteran ecdysis. For example, the chitin synthesis inhibitor diflubenzuron caused incomplete ecdysis of last instar milkweed bugs, Oncopeltus fasciatus Dallas, when topically applied at the penultimate life stage (Redfern et al. 1982). The predatory bug, Podisus maculiventris Say, was not able to molt from the penultimate stage to adult after feeding on insects dipped in label rates for field application of the chitin synthesis inhibitor novaluron (Cutler et al. 2006).

In the current study, the chitin synthesis inhibitor lufenuron had a significant effect on ecdysis of fifth instar bed bugs to adult. Lufenuron caused mortality during, or immediately following ecdysis, resulting in insects with extreme cuticular deformities.

Bed bugs that died following treatment had multiple abnormalities associated with chitin biosynthesis inhibition. For instance, some bed bugs did not fully emerge from the previous exuvia during ecdysis, or their intestines ruptured within the cuticle causing hemolymph to spread to their extremities, or their gut intestines penetrated externally through the newly formed cuticle, causing death.

Higher doses of lufenuron were required for efficacy against Bradenton strain bed bugs compared to the Harlan strain that had been maintained in a laboratory for

>30 years. The Bradenton strain has exhibited levels of resistance to pyrethroid insecticides (Hottel et al. 2015); however, chitin synthesis inhibitors have an entirely different mode of action, acting on chitin synthesis rather than the nervous system.

Therefore, we hypothesize that the bradenton strain may have some cuticular resistance to pyrethroids that would also confer resistance from topical absorption of other insecticide types, including chitin synthesis inhibitors.

Most insecticidal efficacy studies report survival and mortality data, although sublethal effects may be equally as important in controlling or reducing a pest population (Haynes 1988). Sublethal doses of lufenuron to fifth instar bed bugs resulted in significant issues with cuticular integrity and structure, consequently causing leg malformations. Sublethal exposure of the chitin synthesis inhibitor novaluron to the

Colorado potato beetle, Leptinotarsa decemlineata Say, resulted in beetles with poor walking ability and caused them to fall off plants (Cutler et al. 2005).

Bed bugs exposed to sublethal doses of lufenuron in our study held their legs extended from their bodies and demonstrated limited walking ability (i.e. could not hold their body upright to walk, could not walk at all, or walked extremely slowly). Their ability to grip a rough surface was almost entirely impeded, exemplified by loss of generated force by treated bed bugs in the pulling force assays. In previous studies it has been demonstrated that bed bugs that encountered smooth surfaces with no insecticide application were not very successful at gripping those surfaces (Hottel et al. 2015) and, undoubtedly, bed bugs treated with a sublethal dose of lufenuron would not be able to

navigate smooth surfaces. Alternatively, we tested the pulling force of bed bugs on a rough sandpaper surface, and the treated bed bugs could not grip that surface and generated a minute amount of force. Therefore, in almost any environment with a multitude of surfaces, bed bugs affected by sublethal doses of lufenuron would be expected to not be mobile enough to navigate the environment and reach a host for a blood meal.

The documented widespread resistance to pyrethroid insecticides and the recently discovered resistance to neonicotinoids limits the effectiveness of products available for bed bug control. Juvenoids are currently used for bed bug control; however, the limited research available on these products suggests that the label rate of the one product currently used for bed bug control in the United States has limited efficacy. Therefore, given the lethal and sublethal effects demonstrated herein, chitin synthesis inhibitors would be a novel insecticide for rotational use in a bed bug integrated pest management program.

Figure 5-1. Photograph of a pulling force assay on an analytical balance. A common bed bug, Cimex lectularius (arrow) is pictured gripping the rough sandpaper surface on the wooden platform.

Table 5-1. Malformations and mortality of Harlan and Bradenton strain common bed bugs, Cimex lectularius, following topical application of lufenuron for each tested concentration (n=55 for all concentrations except highest concentration for each strain). Dose No. No. No.

Strain (% w/v) n Dead Malformed Affected1 % Affected2 % Malformed/Affected3 Harlan 0.000016 55 4 0 4 7 0 0.00016 55 1 0 1 2 0 0.0016 55 4 33 37 67 89 0.016 55 20 18 38 69 47 52 31 0.16 85 36 16 61 Bradenton 0.32 55 8 18 26 47 69 0.63 55 21 19 40 73 48 43 35 1.25 55 28 15 78 2.5 55 17 12 29 53 42 5.0 85 28 10 38 45 26 1Number Affected = (number dead + number malformed) 2% Affected = (number affected /n)*100 3%Malformed/Affected = (number malformed/number affected)*100

A B

Figure 5-2. Photographs showing the lethal effects of lufenuron following ecdysis of treated 5th instar Harlan strain common bed bugs, Cimex lectularius, with a dose of 0.16% (w/v) lufenuron. A) A fully molted adult bed bug that died shortly after emerging from the exuvia. B) A fifth instar that died during the process of molting with an extrusion of internal stomach structures. C) A fifth instar bed bug that died during the molting process and could not fully emerge from its exoskeleton.

Figure 5-3. Sublethal effect following topical application of 0.0016% (w/v) lufenuron on a Harlan strain common bed bug, Cimex lectularius. Complete ecdysis occurred; however, the bed bug could not properly walk and could not fold its legs underneath its body.

5 Control 4 Treated

1 Pulling Force (mN) Force Pulling

-1 0 20 40 60 80 100 120 140 160 180 200 220 240 Time (s)

Figure 5-4. Pulling force (mN) over time (s) for two individual Harlan strain common bed bugs, Cimex lectularius. One bed bug was not treated (control) and another bed bug was treated with lufenuron (0.0016% w/v).

0.6 A 0.5 A 0.4

0.3

0.2 Average force (mN) (mN) force Average 0.1 B B 0 Control Treated Bradenton Harlan

Figure 5-5. Average amount of force (mN) generated by Bradenton and Harlan strain common bed bugs, Cimex lectularius, when gripping a surface with tarsi following no exposure to lufenuron (Control) or exposure to sublethal doses of lufenuron (Treated, 0.0016% w/v). Bars indicate means ± SE (Harlan; F = 5.35, df = 1, 22, P < 0.0001; Bradenton; F= 8.86, df = 1, 23.97`, P < 0.0001).

3.5 A 3

2.5 A

1.5

1 Maximum Force (mN) Force Maximum 0.5 B B 0 Control Treated

Bradenton Harlan

Figure 5-6. Maximum amount of force (mN) generated by Bradenton and Harlan strain common bed bugs when gripping a surface with tarsi following no exposure to lufenuron (Control) or exposure to sub-lethal doses of lufenuron (Treated). Bars indicate means ± SE (Harlan; F = 6.8, df = 1, 32.75, P <0.0001; Bradenton; F = 12.03, df = 1, 30.80, P < 0.0001).

CHAPTER 6 LUFENURON EFFECTS ON BED BUG FECUNDITY AND EGG DEVELOPMENT

Insect growth regulators have been evaluated against numerous insect taxa to determine their effects on fecundity and overall reproduction. Both juvenile hormone analogs (JHAs) and chitin synthesis inhibitors (CSIs) can negatively impact egg development. Juvenile hormone analogues (JHAs) have been repeatedly shown to inhibit normal development of insect eggs (Kelly and Huebner 1986, Boase 2001,

Mojaver and Bandani 2010), including Cimex lectularius Linnaeus eggs (Shaarawi et al.

1982).

The application of JHAs topically to insect eggs has been shown to cause egg hatch failure. In Rhodnius prolixus Stal, the direct application of the JHA fenoxycarb to eggs affected katatrepsis (movement of an embryo from one pole to another), dorsal closure, and eclosion of embryos (Kelly and Huebner 1986). However, not all insect species are affected similarly by application of IGRs. The absence of broad spectrum activity has been documented as one of the problems associated with IGRs. For example, when the eggs of Podisus maculiventris Say were dipped into the CSI novaluron, there was no direct effect on egg hatching, however, there was a significant decrease in the ability of hatched nymphs to molt into subsequent stages (Cutler et al.

2006).

Not only does topical treatment of IGRs directly to eggs result in reduced egg viability, transovarial transport of IGRs also has been shown to occur (Medina et al.

2002). This was demonstrated by treating adult females with IGRs then subsequently observing the effects on their offspring. The chitin synthesis inhibitor diflubenzuron

caused all eggs of Chrysoperla carnea Stephens to die that were laid by females that were topically treated with the insecticide (Medina et al. 2002).

There have been limited studies on the effects of IGRs on bed bugs. To date, one study has been conducted to evaluate the effectiveness of a juvenile hormone analog against bed bugs (Shaarawi et al. 1982). In C. lectularius, a reduction in egg viability was achieved both by topical application to eggs and topical application to females prior to oviposition, however, the active ingredient of the JHA was not clarified in the study (Shaarawi et al. 1982).

The purpose of this research was to evaluate the CSI lufenuron on C. lectularius fecundity and egg development. Topical application of lufenuron to eggs was first conducted using five concentrations of lufenuron to determine a lethal concentration resulting in 50% mortality (LC50) against eggs at 5 days old. A further study was conducted using the predetermined LC50 to evaluate if lufenuron efficacy changed during egg development on 1 day, 3 day, and 5 day old eggs. To determine if lufenuron had transovarial potential, both adult male and female bed bugs were topically exposed to lufenuron and subsequent oviposition and egg hatch failure was recorded. This study provides relevant data for lufenuron, a novel insecticide against C. lectularius, as an ovicidal agent.

Material and Methods

Insects

The Harlan strain of common bed bugs were used for all assays. This strain was collected in 1973 by Dr. Harold Harlan in Ft. Dix, NJ and he maintained the colony by feeding them on himself. This strain was acquired from Dr. Harlan by the University of

Florida Urban Entomology Laboratory in 2000. Bed bugs were fed weekly on

defibrinated rabbit blood (Hemostat, Dixon, CA) using an artificial feeding system

(Montes et al. 2002) and maintained at approx. 70% RH, 25 °C, and a 12:12 L:D photoperiod. All bed bug colonies were maintained in plastic jars, enclosed at one end with mesh for feeding, with accordion-style folded filter paper provided for harborage.

Topically treated egg dose bioassay

Adult female and male Harlan strain common bed bugs, Cimex lectularius, (10 replications of five males and five females paired together = total 100) were removed from colony containers one week prior to application of insecticides, fed on defibrinated rabbit blood using an artificial feeding system (Montes et al. 2002), mated, and allowed to lay eggs in a Petri dish (Polystyrene, 6.0 x 1.5 cm; Fisher Scientific, Waltham,

Massachusetts, USA) on filter paper (Whatman, GE Healthcare Co., Buckinghamshire,

UK). Three hundred eggs (5 days old) were collected from filter papers by gently removing eggs using fine-tip forceps. The eggs were then separated into five replicates of 10 eggs per treatment into a new Petri dish. There were six treatments, one control and five lufenuron concentrations (0.0016, 0.0013, 0.0063, 0.0125, and 0.0250 g/ml).

Technical grade lufenuron (FMC Corporation, Philadelphia, PA, USA) was diluted into acetone for all insecticide doses and control treatments received only acetone.

Individual eggs were treated with 1 µl of solution using a Hamilton syringe mounted on a repeating dispenser (50 µl; Hamilton Company, Reno, NV, USA). Following application, the eggs were separated into Petri dishes into their replicates with clean filter paper and the number of eggs that hatched was recorded after 9 days. The number of eggs that did not hatch or died during partial hatch was recorded after 9 days.

Topically treated egg age bioassay

Adult female and male Harlan strain common bed bugs, Cimex lectularius, (10 replicates of 10 pairs [male and female]) were pulled from colony, fed on defibrinated rabbit blood (Hemostat, Dixon, CA) using an artificial feeding system (Montes et al.

2002), mated, and laid eggs. Eggs were collected at three different ages (1 day, 3 day, and 5 day post-oviposition) and treated topically with 1 µl of solution of lufenuron, using the previously determined LC50 from the egg dose bioassay (0.0125 g/ml), with a

Hamilton syringe mounted on a repeating dispenser. After application of lufenuron, the groups of eggs were placed into Petri dishes with filter papers (#1, Whatman, GE

Healthcare Co., Buckinghamshire, UK). There were 5 replicates of 10 eggs/treatment and 4 treatments (Control, 1d treated, 3d treated, and 5d treated). Controls consisted of eggs from each age treated with only acetone for a total of 300 eggs. Hatch rate and subsequent first instar mortality was recorded after 9 days, to allow enough time for all nymphs to hatch. First instars that survived treatment and hatched, also were fed and successful molting and mortality was recorded.

Topically treated adult males and females with lufenuron

Fifth instar Harlan strain common bed bugs, Cimex lectularius, were removed from the colony and fed on defibrinated rabbit blood (Hemostat, Dixon, CA) using an artificial feeding system (Montes et al. 2002), and then separated into individual wells in a 24-well polystyrene plate (Costar®, Corning Inc., Kennebunk, ME) to prevent mating.

Studies have shown that repeated mating can reduce egg production, therefore, we used recently eclosed virgin females so that the number of times a female was mated could be controlled. For the “female treatment”, recently eclosed virgin adult females were fed, treated with 1 µL of lufenuron on the ventral side of the abdomen, and allowed

to mate with non-treated males (paired individually into a single well of a well-plate).

Similarly, a “male treatment” consisted of males that were treated with 1 µl of lufenuron and paired with a female that was not treated. After treatment, the adult pairs were put into an individual well in a 24-well polystyrene plate (Costar®, Corning Inc., Kennebunk,

ME) provided with a filter paper (#1, 2.5 cm, Whatman, Buckinghamshire, UK). The number of eggs laid were counted after nine days and eggs were held to determine viability and survival of first instars.

Statistical Analysis

A generalized linear model with a binomial distribution and probit link was used to estimate the LC50 of lufenuron on bed bug eggs at one age (5 days old) for the dose bioassay (JMP Pro 13; SAS institute, Cary, NC, USA). This LC50 was then used as the discriminating concentration for the next egg age bioassay.

To determine if there were any differences between egg age and lufenuron effectiveness for the egg age bioassay, a two-way ANOVA (JMP Pro 13; SAS institute,

Cary, NC, USA) was used with treatment (control or treated) and age (1 day, 3 day, and

5 day post-oviposition) as the independent variables and total mortality as the response variable. Mortality was recorded separately as those eggs that did not hatch and died and then eggs that did hatch but died during the process or shortly thereafter as 1st instars (total mortality). Two separate one-way ANOVAs (JMP Pro 13; SAS institute,

Cary, NC, USA) were conducted to determine if there was a difference in egg age and the type of mortality (did not hatch or died during hatch).

For the last experiment, topically treated adult males and females with lufenuron, the effect of lufenuron on fecundity of both male and female bed bugs was evaluated with one-way ANOVAs (JMP Pro 13; SAS institute, Cary, NC, USA).The number of

eggs laid and the percentage of eggs that hatched after being laid was analyzed as the response variables by treatment (control, male treatment, or female treatment). The data were checked for normality and that they met the assumptions of ANOVA. Data were considered significant at α = 0.05. All experiments were analyzed using JMP software (JMP Pro 13; SAS institute, Cary, NC, USA).

Results

The generalized linear model was significant for all five lufenuron concentrations tested against Harlan strain eggs (P < 0.0001). Mortality increased as the concentration increased, then at the highest concentration, 0.025 g/ml, the mortality declined (Fig. 6-

1). The LC50 was calculated to be 0.01248 g/ml (CI = −0.060-0.088).

Mortality was significantly higher after application of lufenuron compared to the control regardless of the age of the egg (F = 5.9; df = 5,17; P = 0.006). However, egg age was a significant factor when the type of mortality was considered, described as (A) either eggs that did not hatch, or (B) eggs that partially hatched then died as first instars. More eggs at age 1 and 3 days old did not hatch (29 ± 0.29 and 21 ± 0.29) compared to those that were 5 days old (0 ± 0.00) (F = 8.45; df = 2,17; P = 0.0035) (Fig.

6-2). Conversely, significantly more 5-day old eggs died partially hatched or immediately after hatch (27 ± 0.08) compared to 1 day and 3 day old eggs (0 ± 0.00 and 6 ± 0.15) (F

= 15.95; df = 2,17; P = 0.0002) (Fig. 6-2).

Lufenuron had no significant effect on the number of eggs laid (F = 3.08, df =

2,29, P = 0.06) or the percentage of eggs that hatched successfully (F = 0.78, df = 2,29,

P = 0.47) between adult bed bugs that received no treatment, males that were treated, or females that were treated (Fig. 6-3).

Discussion

The direct application of lufenuron to bed bug eggs resulted in high mortality. The eggs continued to develop after application, but either did not hatch or died during the process of hatching. Studies have shown that chitin is a component of insect eggshells and can be affected by the application of chitin synthesis inhibitors (CSIs) (Moriera et al.

2007; Mansur et al. 2010). Chitin was found to be a component of oogenesis in

Rhodnius prolixus Stal, and the injection of the CSI lufenuron in females caused a 30-

50% reduction in the number of eggs laid, and < 1% of eggs that were laid hatched

(Mansur et al. 2010). The eggshell itself is a multi-layered protective barrier that is the first line of defense of the embryo against environmental dangers.

There was no difference in the number of eggs that died between eggs that were

1 day, 3 days, and 5 days old. Age had no significant impact on mortality; however, the time at which the bed bugs died was significantly different dependent upon age.

Younger eggs were more susceptible to lufenuron compared to older eggs and the former did not complete development and hatch. Older eggs did develop further, but they died during the process of hatching or immediately after hatch. Similarly, eggs of the chrysomelid beetle, Diabrotica undecimpunctata howardi Barber, were much more susceptible to insecticides earlier in development (Michaelides and Wright 1997).

Multiple studies have shown that CSIs are potent ovicides across insect species, but early age eggs are more susceptible (Michaelides and Wright 1996, Saenz-de-cabezon et al. 2006, Tiwari et al. 2012).

Transovarial transport of CSIs from mother to offspring has been shown in some insect species; however, most of these studies were conducted using feeding assays where the mother consumed a CSI mixed into food and then subsequent egg hatch and

100

mortality was recorded. For instance, the application of the CSI diflubenzuron to adult female Chrysoperla carnea Stephens resulted in 100% mortality of all eggs (Medina et al. 2002). A further radio-labeled isotope analysis revealed that diflubenzuron was absorbed slowly by the female’s body and was not excreted, resulting in high egg mortality. Alternatively, the application of pyriproxyfen to adult female C. carnea was found to have little effect on eggs because of rapid excretion by the mother. In bumblebees, Bombus sp., the addition of radioactive 14C-diflubenuzuron to pollen that worker bees fed on was transovarially transported to eggs and 1.89% of the diflubenzuron applied to the pollen was recovered in a batch of 13 eggs (Mommaerts et al. 2006).

Our results showed that using topical assays to either adult females, or adult male bed bugs, did not result in significant differential mortality of bed bug eggs. This may be due to the application method, whereas most of the studies showed positive results when females were fed the CSI; however, it was more relevant for this study to use topical assays because CSIs would be unlikely to be fed to bed bugs for control strategies. To date, no bait that can be used for bed bug control has been developed.

Further studies should be conducted to determine if the uptake of lufenuron occurs in adults using topical assays and to determine the absence or occurrence of rapid excretion.

Although transovarial transport did not occur in this study, lufenuron still had a significant effect on bed bug eggs. Topical treatment to eggs caused significant mortality, even for more developed eggs that died during or directly after hatch. These findings suggest that lufenuron could be an alternative mode of action that would

101

provide ovicidal activity to bed bug populations. Targeting the egg stage would be critical in helping eliminate a bed bug infestation over time and reducing the overall population.

102

30 % Mortality %

0 0.0016 0.003125 0.00625 0.0125 0.025 Concentration g/ml

Figure 6-1. Mortality (%) and standard error bars of Harlan strain common bed bugs, Cimex lectularius, following topical application of five concentrations (g/mL) of lufenuron.

103

30 A A A C 25 C C No Hatch

20 Hatched and died 15

5 Total number number dead Total 0 5d 3d 1d Age

Figure 6-2. Number of dead common bed bug, Cimex lectularius, eggs after topical treatment of lufenuron at different ages (1 day, 3 day, and 5 day post- oviposition). Some eggs died and did not complete development (no hatch) while other eggs died either during, or immediately after, hatching (hatched and died). Letters that differ indicate significant differences (α = 0.05).

104

Figure 6-3. Number of common bed bug, Cimex lectularius, eggs laid by adults that were not treated with lufenuron (control) and adult females treated with lufenuron and adult males treated with lufenuron.

105

CHAPTER 7 EXTERNAL AND INTERNAL APPLICATION OF LUFENURON AGAINST TROPICAL BED BUGS VIA TOPICAL APPLICATION, FEEDING, AND INJECTION BIOASSAYS

The tropical bed bug, Cimex hemipterus, is the dominant bed bug species found throughout tropical and subtropical regions of the world. This species feeds primarily on humans and is considered a nuisance pest like the common bed bug, which is found in temperate climates. Control of this species is primarily with the use of liquid chemical insecticides. However, the tropical bed bug has been found to be resistant to pyrethroid insecticides (Tawatsin et al. 2011), creating a need for efficacy trials of insecticides alternative to pyrethroid insecticides.

There are few liquid insecticides specifically labeled for bed bugs that do not incorporate a pyrethroid as the active ingredient. Subsequently, high levels of insecticide resistance to pyrethroid insecticides have developed in common bed bugs

(Moore and Miller 2006, Romero et al. 2007, Yoon et al. 2008, Adelman et al. 2011) as well as tropical bed bugs (Myamba et al. 2002, Karunaratne et al. 2007). However, rotating and utilizing insecticides with different modes of action in addition to incorporating other integrated pest management strategies can circumvent insecticide resistance problems. Unfortunately, there are limited products available with alternative modes of action to pyrethroids available for a bed bug rotation program.

There are currently combination products on the market (e.g. pyrethroid plus neonicotinoid) that incorporate two different modes of action to increase effectiveness

(Potter et al. 2012). Alternatively, the active ingredient chlorfenapyr has been found to be effective against pyrethroid resistant bed bugs because it has a different mode of action to the pyrethroids. Unlike pyrethroid insecticides that target sodium channels,

106

chlorfenapyr disrupts the oxidative phosphorylation of mitochondria (Romero et al.

2010). However, it is a slow acting insecticide that takes several days to cause bed bug mortality thus, it is not ideal for quickly eliminating a population (Romero et al. 2010). An additional alternative mode of action to pyrethroid insecticides employed for bed bug control are IGRs. However, there is currently only one insect growth regulator that is labeled for bed bug control in the United States, manufactured under the trade name

Gentrol® ((S)-hydroprene; Wellmark International; Schaumberg, IL).

Gentrol® aerosol and Gentrol® concentrate ([S]-hydroprene; Wellmark

International; Schaumberg, IL) was found to cause 66-100% adult bed bug mortality but only at application rates ≥3x the label rate (Todd 2006). There was little mortality in nymphal bed bugs that were treated with either Gentrol® product (Todd 2006). This is expected because JHAs only affect the last stadium of insects before molting to an adult when naturally occurring juvenile hormones are absent. Similar to Todd 2006, (S)- hydroprene was found to have little effect on bed bugs except at elevated label rates, at

10 times the label rate the IGR caused 100% reduction in bed bug oviposition in one bed bug strain, but only a 38% ovipositional reduction in another strain (Goodman et al.

2013).

Evaluation of dried residues of technical grade (S)-methoprene applied to filter papers, resulted in a reduction of bed bug fecundity, caused morphological abnormalities, and resulted in supernumerary nymphs (Naylor et al. 2008). Adults that were treated with (S)-methoprene were not affected, however, there was a significant reduction in the number of eggs laid (1.9 daily in control to 1.3 daily in treatment) and hatching rate (99% in control to 94% in treatment) at the highest dose tested (16 mg/m2)

107

compared to the control. Alternatively, a formulated product, Precor® ([S]-methoprene;

Wellmark International; Schaumberg, IL) had no effect on bed bug eggs, nymphs, or adults at the label rate or even 2x the label rate (Goodman et al. 2013) and is not labeled for bed bugs.

Although these two IGR products have not been found to be highly efficacious against bed bugs, these studies evaluated formulated products rather than evaluating technical grade efficacy. Furthermore, they did not focus on the life stage that these products would work most effectively (i.e. the penultimate or 5th instar). Currently, no studies have evaluated chitin synthesis inhibitors (CSIs) against tropical bed bugs.

Therefore, the objective of this study was to evaluate different bioassay methods (i.e. topical, injection, and feeding) using the CSI lufenuron on tropical bed bugs for a holistic evaluation of its potential use as an insecticide for tropical bed bug control.

Materials and Methods

Insects

The Brevard strain of tropical bed bugs, Cimex hemipterus Fabricius, were used for all assays. This strain was collected in Brevard County, Florida in 2015 in a single- story home. Bed bugs were fed weekly on defibrinated rabbit blood (Hemostat, Dixon,

CA) using an artificial feeding system (Montes et al. 2002) and maintained at approx.

Insect Growth Regulator Topical Application Bioassay

Five replications of five 5th instar bed bugs per insecticide concentration (five concentrations; 1.60, 3.13, 6.25, 12.50, 25.00 mg/ml) were removed from colony

108

containers. Individual bed bugs were topically applied with 1 µl of five different concentrations of lufenuron (FMC Corporation, Philadelphia, PA, USA), to the ventral side of their abdomen using a Hamilton syringe mounted on a repeating dispenser (50

µl; Hamilton Company, Reno, NV, USA). The bed bugs were then allowed to feed on defibrinated rabbit blood (Hemostat, Dixon, CA) one day post-insecticide application using a hot plate. For feeding, the blood was put into a plastic soufflé cup, covered with plastic film (Parafilm M®; Bemis Flexible Packaging, Oshkosh, WI) so that the blood was touching the film, and heated on a hot plate to 35-40 °C . The insecticides were formulated with acetone and serially diluted, therefore, a control treatment consisted of bed bugs that were treated with only acetone.

Following topical application of the insecticide, the groups of bed bugs were be placed into a Petri dish containing filter paper. All bed bugs were monitored daily for 14 days, mortality and molting were recorded.

Insect Growth Regulator Feeding Bioassay

Five groups of ten 5th instar bed bugs were removed from the colony and starved for one week prior to the assay. After one week, groups of bed bugs were weighed and then fed on five concentrations of lufenuron mixed into defibrinated rabbit blood

(Hemostat, Dixon, CA). The blood was put into a plastic soufflé cup, covered with plastic film (Parafilm M®; Bemis Flexible Packaging, Oshkosh, WI) so that the blood was touching the film, and heated on a hot plate to 35-40 °C to simulate human blood temperatures. Bed bugs were put into a glass vials in their groups with a mesh covering secured at one end with a rubber band. The vials containing bed bugs were then inverted onto the film-covered blood. A control treatment of five groups of ten 5th instars were fed on rabbit blood mixed with acetone. After feeding, the groups of bed bugs

109

were re-weighed and then put into Petri-dishes with filter papers. After one week, the bed bugs were checked for mortality and morphological deformities. The volume of blood ingested (as a function of weight gain) was used to calculate the consumed dose of lufenuron.

Insect Growth Regulator Injection Bioassay

Ten starved 5th instar bed bugs per dose were injected with 0.5 µl lufenuron, with a 10% rhodamine B dye solution for visualization, into the intersegmental membrane between the first and second coxa on the thorax using a Nanoliter 2000 microinjection system (World Precision Instruments, Inc., Sarasota, FL). Bed bugs were starved prior to the experiment to limit blood from leaking from the abdominal cavity during injection.

The insecticide was formulated with acetone, therefore, a control treatment consisted of bed bugs that were injected with acetone and dye.

Following injection, the bed bugs were placed indvidually into a 24 well multi- plate containing filter paper circles in each well. The bed bugs were allowed to feed on rabbit blood and then removed and placed into a new Petri dish. All injected bed bugs were monitored daily for 14 days and then mortality, molting, and any morphological differences were recorded.

Statistical Analyses

Topical, feeding, and injection bioassays were analyzed using a generalized linear model with a binomial distribution and probit link. The concentration or dose was log-transformed for all analyses. Values of P ≤ 0.05 were used to indicate significance.

The amount of blood ingested by bed bugs was calculated by weighing bed bugs before and after the feeding assay and subtracting the difference. An ANOVA was conducted to compare the amount of blood ingested between treatments and means were

110

separated using a Tukey’s HSD test. All analyses were completed using JMP (JMP Pro

13; SAS institute, Cary, NC, USA).

Results

The topical assay did not result in a dose response as concentrations increased.

Mortality was high for each concentration (>50%) (Table 7-1); however, the model was not significant, and mortality did not significantly differ between doses (GLM, df = 1,

Pearson χ2 = 0.71, P = 0.28).

Mortality increased for the feeding assays as concentrations increased.

Concentration had a significant effect on mortality (GLM, df = 1, Pearson χ2 < 0.0001, P

< 0.0001) but the two lowest concentrations resulted in only 0-2% mortality, with a large increase between the 10-fold concentration of 0.0001 mg/ml to 0.001 mg/ml, that resulted in 78% mortality at the 0.001 mg/ml dose. The LC50 for the feeding assay was calculated to be 0.0026 mg/ml (CL = 0.0022 - 0.0032). The concentration of lufenuron had a significant effect on the amount of blood ingested (F = 3.87, df = 5,29, P = 0.01).

However, the difference was not due to the concentration of insecticide dissolved into the blood and more likely due to other factors such as hunger and random variation (i.e. there was no significant reduction in ingestion as insecticide concentration increased).

There was no significant difference between the amount of blood ingested by bed bugs fed the highest concentration (0.1 mg/ml) compared to the control (mean blood ingested

= 0.044 and 0.046 g, respectively) (Figure 1).

The injection method resulted in a range of mortality from 30-60%. Although, it should be noted that this was a small sample size (10 individual bed bugs injected per dose) due to the high mortality from the injection process itself. Only bed bugs that did not die from the injection process were used for data collection. The ability of 5th instars

111

to molt into adults was significantly affected by the injected dose of lufenuron. At the lowest dose, 100% of 5th instars were able to molt compared to lower than or equal to

20% successfully molting when the dose was increased (Table 7-1). The dose injected into individual bed bugs did not significantly effect mortality (GLM, df = 1, Pearsons χ2 =

0.56, P = 0.55).

Discussion

Several factors are key for selection of a toxicological assay for insecticide efficacy evaluations. Topical assays are primarily used for evaluation of technical grade products because of their reproducibility and the use of a large number of insects for evaluation (Yu 2008). Primarily, these assays are conducted using a repeating dispenser microsyringe with an insecticide dissolved into acetone, and a small amount, typically 1 µl, is applied to the outer surface of the insect abdomen. Injection assays are used so that the exact amount of toxicant is known that is injected into the insect body

(i.e. the dose). These injections are typically completed with glass needles and a microinjection system, and the needle is injected between the intersegmental membranes on the ventral abdomen non-centrally to avoid hitting the nerve cord along the central longitudinal line (Yu 2008). Feeding assays are often chosen because of their practicality and field application, such as insecticides that are intended to be ingested (Yu 2008). It is for these reasons that these assays were chosen for a broad analysis of lufenuron activity against tropical bed bugs, C. hemipterus.

The range of doses chosen for the topical assay were effective; however, did not result in a dose response typical of similar insecticidal assays with a resultant 10-90% range in mortality. Interestingly, the smallest dose (1.6 mg) resulted in almost the same mortality as the selected highest dose (25 mg) (76 and 84% mortality, respectively). All

112

doses had a significant effect on the mortality of 5th instars once they began the molting process. Many of the 5th instar bed bugs were not able to escape their exuvia and died during the molting process. Similarly, topical application of the CSI diflubenzuron to the predatory bug, Orius laevigatus, resulted in 5th instars that did not shed their exuvia.

They remained alive but did not feed and died 24 hours after attempting to emerge from their exuvia (Delbeke et al. 1997). Furthermore, those adults that did emerge had morphological problems with their wings, including incomplete formation and indentations (Delbeke et al. 1997).

The injection process resulted in mortality to 5th instars. The highest percentage of mortality was at 1.0-2.0 mg lufenuron/insect but there was a decrease in mortality at the highest tested dose of 4.0 mg ai/insect. Injecting bed bugs was not a practical application method for treating large numbers of bed bugs due to the high percentage of mortality from injecting the needle. The advantage of injecting lufenuron to circumvent the cuticle may not be advantageous for this CSI against bed bugs because much higher concentrations of insecticide were required than expected (i.e. 0.25-4.0 mg ai/insect) to have an effect. Thus, the cuticle does not appear to be as significant of a barrier to lufenuron within bed bugs compared to other toxicants that are used as contact insecticides.

Bed bugs readily fed on blood with dissolved lufenuron to the point of engorgement. The two lowest concentrations that were administered (0.00001 and

0.0001 mg/ml) were ineffective; however, the three highest concentrations caused substantial mortality. Similarly, an evaluation of four insecticides: abamectin, clothianidin, fipronil, and indoxacarb, have been evaluated via ingestion against bed

113

bugs (Sierras and Schal 2016). The ingestion of abamectin, clothianidin, and fipronil caused significant mortality in 1st instar nymphs and adult male bed bugs. Not surprisingly, fipronil was much more effective when ingested (43x) than when topically applied (Sierras and Schal 2016). Indoxacarb, which is a pro-insecticide and must be metabolized for effectiveness, was not effective when ingested even at the highest doses (Sierras and Schal 2016). The development of a bed bug bait, discussed in

Sierras and Schal (2016) would be advantageous because it would specifically target bed bugs and would be minimally toxic because the active ingredient would be in very small amounts housed within a bait matrix. This study shows that lufenuron could be another viable option for an insecticide in a bait for bed bugs because it was highly palatable and effective when ingested. Chitin synthesis inhibitors are commonly used in termite baiting stations because of their slow action and ability to cause colony elimination. However, a bed bug bait would need significant further study to find a matrix

(formulation) that would be suitable for indoors, have a feeding stimulant as well as heating source, and that would be more attractive than a human or animal host for bed bug feeding.

High activity of lufenuron was demonstrated in tropical bed bugs, regardless of the application method. These assays were conducted with 5th instar bed bugs, but further studies evaluating other instars would be beneficial to determine if activity is similar across nymphal life stages. Other species tested (Lepidoptera) using lufenuron have demonstrated activity in several larval stages, with similar LC50 values calculated regardless of the age of the larvae (Butter et al. 2003, Sáenz-de-cabezón et al. 2006).

Feeding assays resulted in effectiveness at the lowest concentration tested, followed by

114

injection and then topical assays. This is not surprising considering that feeding would allow the toxicant to enter the blood stream quickly and injection allows for potentially more uptake of the insecticide without the cuticle barrier. This study demonstrated that the CSI lufenuron does have contact toxicity and that it could potentially be used as a liquid insecticide for bed bug control, as well as having potential as an active ingredient in a baiting system.

115

Table 7-1. Mortality of 5th instar tropical bed bugs, Cimex hemipterus, treated with lufenuron at different concentrations/doses between assays (i.e. topical application, feeding, and injection). Assay Treatment n % Mortality ± SE % Molted ± SE Topical mg/ml 0 25 4 ± 4.0 72 ± 8.0 1.6 25 76 ± 7.5 84 ± 4.0 3.13 25 68 ± 10.2 16 ± 11.7 6.25 25 88 ± 4.9 0 ± 0.0 12.5 25 80 ± 6.3 20 ± 12.6 25 25 84 ± 7.5 4 ± 4.0 Feeding mg/ml 0 49 2 ± 2.0 86 ± 9.8 0.00001 50 0 ± 0.0 44 ± 11.2 0.0001 50 2 ± 2.0 90 ± 3.2 0.001 50 78 ± 4.9 78 ± 4.9 0.01 50 58 ± 11.6 0 ± 0.0 0.1 50 80 ± 5.5 0 ± 0.0 Injection mg 0 10 10 ± 0.10 80 ± 0.13 0.25 10 50 ± 0.17 100 ± 0 0.5 10 50 ± 0.17 20 ± 0.13 1.0 10 60 ± 0.16 10 ± 0.10 2.0 10 60 ± 0.16 0 ± 0.0 4.0 10 30 ± 0.15 0 ± 0.0

116

Table 7-2. The amount of active ingredient (lufenuron) consumed by 5th instar tropical bed bugs, Cimex hemipterus, during the feeding assay. Concentration (µg/ml) Weight (g) Before Weight (g) After AI Ingested (µg) Control (0) 0.0157 ± 0.0004 0.061 ± 0.005 0.00000 ± 0.00000 0.01 0.0151 ± 0.0006 0.040 ± 0.006 0.00025 ± 0.00006 0.1 0.0161 ± 0.0009 0.061 ± 0.002 0.00450 ± 0.00030 1.0 0.0165 ± 0.0004 0.054 ± 0.005 0.03700 ± 0.00500 10 0.0171 ± 0.0006 0.046 ± 0.004 0.29000 ± 0.04000 100 0.0172 ± 0.0056 0.061 ± 0.003 4.37000 ± 0.35000

117

0.06 A 0.05 A A AB 0.04 AB B 0.03

0.02 Blood Ingested (g) Ingested Blood 0.01

0 0 0.00001 0.0001 0.001 0.01 0.1 Dose (mg)

Figure 7-1. The total amount of blood ingested by tropical bed bugs (means ± SE), Cimex hemipterus, containing lufenuron at different concentrations for the feeding assay. There was a significant difference (indicated by the different letters) in the amount of blood ingested between doses, but the concentration of insecticide did not influence blood ingestion in a concentration dependent manner.

118

CHAPTER 8 CONCLUSION

The dominate bed bug species found throughout the United States is Cimex lectularius, the common bed bug. In 2015, the occurrence of the tropical bed bug in

Brevard County, Florida, last seen in the state in the 1940s (Hixson 1943), raised awareness of this species nationwide and initiated questions regarding its propensity to spread, it’s origin, as well as identification of the species (Campbell et al. 2016). A description regarding finding the species, as well as previous history of C. hemipterus in

FL is documented in Chapter 3.

Both the common bed bug and the tropical bed bug are important pests of humans. They are both hematophagous and have similar biological and ecological requirements. They are also morphologically very similar and furthermore, the signs of a bed bug infestation (i.e. live bugs, exuvia, and fecal spots) would not help with distinguishing the species because they are identical. Under a microscope, the pronotum margin in the common bed bug is more laterally extended towards the eye compared to the tropical bed bug. Identification of tropical bed bugs has previously been established as the ratio of the pronotum width to length being <2.5 compared to the common bed bug (Usinger 1966). However, this identification method had not been validated for accuracy with multiple tropical bed bugs (i.e. no methods were established that verified how this ratio was determined) and no ratio was established for other life stages than adult. Therefore, in Chapter 4, data is presented with pronotum width numbers for all life stages of bed bugs, except eggs, as well as mitochondrial DNA data to differentiate tropical bed bugs from common bed bugs.

119

The last three research chapters of this dissertation address chemical control of bed bugs with the chitin synthesis inhibitor lufenuron using different populations of common bed bugs as well as one population of tropical bed bugs. To date, no studies have examined the use of chitin synthesis inhibitors against bed bugs in the United

States. These insecticides would be advantageous for indoor bed bug control because of their low mammalian toxicity and because they are a novel insecticide to bed bugs and would circumvent resistance issues that are prevalent in bed bug populations across the United States. Therefore, multiple studies were conducted to determine the effectiveness of lufenuron and to evaluate it as a “proof of concept” insecticide for bed bug control. Lufenuron was effective and caused high rates of mortality in bed bugs but also caused significant sublethal effects, such as reduced ability to walk and grip a surface (Chapter 5), potent ovicidal activity (Chapter 6), and had enhanced insecticidal activity when it was fed in a blood solution to bed bugs (Chapter 7).

120

LIST OF REFERENCES

Adelman, Z.N., Kilcullen, K.A., Koganemaru, R., Anderson, M.A., Anderson, T.D. and Miller, D.M. (2011) Deep sequencing of pyrethroid-resistant bed bugs reveals multiple mechanisms of resistance within a single population. PLoS One, 6, e26228.

Akaike, H. (1974) A new look at the statistical model identification. IEEE Transactions on Automatic Control 19, 716-723.

Ameen, A., Wang, C., Kaakeh, W., Bennett, G.W., King, J.E., Karr, L.L. and Xie, J. (2005) Residual activity and population effects of noviflumuron for german cockroach (Dictyoptera: Blattellidae) control. J Econ Entomol 98, 899-905.

Anderson, J., Ferrandino, F., McKnight, S., Nolen, J. and Miller, J. (2009) A carbon dioxide, heat and chemical lure trap for the bedbug, Cimex lectularius. Med Vet Entomol 23, 99-105.

Angelakis, E., Socolovschi, C. and Raoult, D. (2013) Bartonella quintana in Cimex hemipterus, Rwanda. Am J Trop Med Hyg 89, 986-987.

Appel, A., Moar, W. and Tanley, M. (1999) Water loss and mortality of adult cowpea weevils (Coleoptera: Bruchidae) exposed to desiccants and desiccating environments. Environ Entomol 28, 979-982.

Araujo, R.N., Costa, F.S., Gontijo, N.F., Gonçalves, T.C. and Pereira, M.H. (2009) The feeding process of Cimex lectularius (Linnaeus 1758) and Cimex hemipterus (Fabricius 1803) on different bloodmeal sources. J Insect Physiol 55, 1151-1157.

Arthur, F.H. and Fontenot, E.A. (2012) Residual activity of methoprene and novaluron as surface treatments to manage the flour beetles, Tribolium castaneum and Tribolium confusum. J Insect Sci 12, 1-11.

Balvín, O., Roth, S. and Vilimova, J. (2015) Molecular evidence places the swallow bug genus Oeciacus Stål within the bat and bed bug genus Cimex linnaeus (Heteroptera: Cimicidae). Syst Entomol 40, 652-665.

Balvín, O., Munclinger, P., Kratochvíl, L. & Vilímová, J. (2012) Mitochondrial DNA and morphology show independent evolutionary histories of bedbug Cimex lectularius (Heteroptera: Cimicidae) on bats and humans. Parasitol Res 111, 457-469.

Benoit, J., Lopez‐Martinez, G., Teets, N., Phillips, S. and Denlinger, D. (2009) Responses of the bed bug, Cimex lectularius, to temperature extremes and dehydration: levels of tolerance, rapid cold hardening and expression of heat shock proteins. Med Vet Entomol 23, 418-425.

Boase, C. (2001) Bedbugs-back from the brink. Pestic Outlook 12, 159-162.

121

Booth, W., Balvín, O., Vargo, E.L., Vilímová, J. and Schal, C. (2015) Host association drives genetic divergence in the bed bug, Cimex lectularius. Mol Ecol 24, 980-992.

Burton, G.J. (1963) Bedbugs in relation to transmission of human diseases: review of the literature. Public Health Reports (Washington, D.C.: 1896), 78, 513-524.

Butter, N., Singh, G. and Dhawan, A. (2003) Laboratory evaluation of the insect growth regulator lufenuron against Helicoverpa armigera on cotton. Phytoparasitica 31, 200- 203.

Campbell, B.E. and Miller, D.M. (2015) Insecticide resistance in eggs and first instars of the bed bug, Cimex lectularius (Hemiptera: Cimicidae). Insects 6, 122-132.

Campbell, B.E., Koehler, P.G., Buss, L.J. and Baldwin, R.W. (2016) Recent documentation of the tropical bed bug (Hemiptera: Cimicidae) in Florida since the common bed bug resurgence. Fla Entomol 99, 549-551.

Churchill, T.P. (1930) Urticaria due to bedbug bites. J Am Med Assoc 95, 1975-1975.

Comack, E. and Lyons, J. (2011) What Happens when the Bed Bugs do Bite?: The Social Impacts of a Bed Bug Infestation on Winnipeg's Inner-City Residents. Canadian Centre for Policy Alternatives, 1-29.

Cutler, G.C., Scott-Dupree, C., Tolman, J. and Harris, C. (2006) Toxicity of the insect growth regulator novaluron to the non-target predatory bug Podisus maculiventris (Heteroptera: Pentatomidae). Biol Control 38, 196-204.

Cutler, G.C., Tolman, J.H., Scott-Dupree, C.D. and Harris, C.R. (2005) Resistance potential of colorado potato beetle (Coleoptera: Chrysomelidae) to novaluron. J Econ Entomol 98, 1685-1693.

De Andrade, J. (2015) Effect of Flufenoxurom Growth Regulator in Cimex lectularius L. Ph.D. Thesis, Universidade Estadual Paulista, Sao Paulo, Brazil.

Delaunay, P., Blanc, V., Del Giudice, P., Levy-Bencheton, A., Chosidow, O., Marty, P. and Brouqui, P. (2011) Bedbugs and infectious diseases. Clin Infect Dis 52, 200-210.

Delbeke, F., Vercruysse, P., Tirry, L., De Clercq, P. and Degheele, D. (1997) Toxicity of diflubenzuron, pyriproxyfen, imidacloprid and diafenthurion to the predatory bug, Orius laevigatus (Heteroptera: Anthocoridae). Entomophaga 42, 349-358.

Demark, J. and Bennett, G. (1989) Efficacy of chitin synthesis inhibitors on nymphal German cockroaches (Dictyoptera: Blattellidae). J Econ Entomol 82, 1633-1637.

Doggett, S.L., Geary, M.J. and Russell, R.C. (2004) The resurgence of bed bugs in Australia: with notes on their ecology and control. Environ. Health 4, 30.

122

Ebeling, W. (1971) Sorptive dusts for pest control. Annu Rev Entomol 16, 123-158.

Ebeling, W., Wagner, R. and Reierson, D.A. (1966) Influence of repellency on the efficacy of blatticides. I. learned modification of behavior of the German cockroach. J Econ Entomol 59, 1374-1388.

Feroz, M. (1970) Toxicological and Genetical Studies of Organophosphorus Resistance in the Bed-Bug, Cimex Lectularius L. Doctoral Dissertation, University of London.

Fletcher, C., Ardern‐Jones, M. and Hay, R. (2002) Widespread bullous eruption due to multiple bed bug bites. Clin Exp Dermatol 27, 74-75.

Fulcher, A., Scott, J.M., Qualls, W.A., Müller, G.C. and Xue, R. (2014) Attractive toxic sugar baits mixed with pyriproxyfen sprayed on plants against adult and larval Aedes albopictus (Diptera: Culicidae). J Med Entomol 51, 896-899.

Gangloff-Kaufmann, J., Hollingsworth, C., Hahn, J., Hansen, L., Kard, B. & Waldvogel, M. (2006) Bed bugs in America: a pest management industry survey. Am Entomol 52, 105-106.

Geden, C.J. and Devine, G.J. (2012) Pyriproxyfen and house flies (Diptera: Muscidae): Effects of direct exposure and autodissemination to larval habitats. J Med Entomol 49, 606-613.

Ghauri, M.S.K. (1973) Hemiptera (bugs). Insects and Other Arthropods of Medical Importance (ed K. Smith), pp. 373. British Museum of Natural History and John Wiley & Sons Ltd.

Goddard, J. and deShazo R. (2012) Psychological effects of bed bug attacks (Cimex lectularius L.) Am. J. Med. 125, 101-103.

Goddard, J., Edwards, K. and de Shazo, R. (2011) Observations on development of cutaneous lesions from bites by the common bed bug, Cimex lectularius L. Midsouth Entomol 4, 49-52.

Goddard, J. (2009) Bed bugs (Cimex lectularius) and clinical consequences of their bites. Jama 301, 1358-1366.

Goodman, M.H., Potter, M.F. and Haynes, K.F. (2013) Effects of juvenile hormone analog formulations on development and reproduction in the bed bug Cimex lectularius (Hemiptera: Cimicidae). Pest Manag Sci 69, 240-244.

Graf, J. (1993) The role of insect growth regulators in arthropod control. Parasitol Today 9, 471-474.

123

Gries, R., Britton, R., Holmes, M., Zhai, H., Draper, J. and Gries, G. (2015) Bed bug aggregation pheromone finally identified. Angewandte Chemie International Edition, 54, 1135-1138.

Hajibabaei, M., Smith, M., Janzen, D.H., Rodriguez, J.J., Whitfield, J.B. and Hebert, P.D. (2006) A minimalist barcode can identify a specimen whose DNA is degraded. Mol Ecol Resour 6, 959-964.

Harlan, H.J. (2007) Bed bug control: challenging and still evolving. Pestic Outlook 18, 57.

Harraca, V., Ryne, C. and Ignell, R. (2010) Nymphs of the common bed bug (Cimex lectularius) produce anti-aphrodisiac defence against conspecific males. BMC Biology, 8, 121.

Haynes, K.F. (1988) Sublethal effects of neurotoxic insecticides on insect behavior. Annu Rev Entomol 33, 149-168.

Hebert, P.D., Ratnasingham, S. and deWaard, J.R. (2003) Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc Biol Sci Ser B, 270, S96-S99.

Henry, T.J. (2009) Biodiversity of Heteroptera. Insect Biodiversity: Science and Society, 1, 223-263.

Hinkle, N.C., Koehler, P.G. and Patterson, R.S. (1995) Residual effectiveness of insect growth regulators applied to carpet for control of cat flea (Siphonaptera: Pulicidae) larvae. J Econ Entomol 88, 903-906.

Hixson, H. (1943) The tropical bedbug established in Florida. Fla Entomol 26.

Hottel, B., Pereira, R., Gezan, S., Qing, R., Sigmund, W. and Koehler, P. (2015) Climbing ability of the common bed bug (Hemiptera: Cimicidae). J Med Entomol 52, 289-295.

How, Y. and Lee, C. (2010) Effects of life stages and feeding regimes on active movement behavior of the tropical bed bug, Cimex hemipterus (Hemiptera: Cimicidae). J Med Entomol 47, 305-312.

Jones, S.C. (1984) Evaluation of two insect growth regulators for the bait-block method of subterranean termite (Isoptera: Rhinotermitidae) control. J Econ Entomol 77, 1086- 1091.

Jung, S., Duwal, R.K. and Lee, S. (2011) COI barcoding of true bugs (Insecta, Heteroptera). Mol Ecol Resour 11, 266-270.

124

Karunaratne, S., Damayanthi, B., Fareena, M., Imbuldeniya, V. and Hemingway, J. (2007) Insecticide resistance in the tropical bedbug Cimex hemipterus. Pestic Biochem Physiol 88, 102-107.

Kavallieratos, N.G., Athanassiou, C.G., Vayias, B.J. and Tomanović, Ž. (2012) Efficacy of insect growth regulators as grain protectants against two stored-product pests in wheat and maize. J Food Prot 75, 942-950.

Kawada, H. and Hirano, M. (2014) Insecticidal effects of the insect growth regulators methoprene and pyriproxyfen on the cat flea (Siphonaptera: Pulicidae). J Med Entomol 33, 819-822.

Kawada, H., KojiMA, I. and Shinjo, G. (1989) Laboratory evaluation of a new insect growth regulator pyriproxyfen, as a cockroach control agent. Med Entomol Zool 40, 195- 201.

Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S. and Duran, C. (2012) Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 1647-1649.

Kells, S.A. and Goblirsch, M.J. (2011) Temperature and time requirements for controlling bed bugs (Cimex lectularius) under commercial heat treatment conditions. Insects, 2, 412-422.

Khan, H.R. and Rahman, M.M. (2012) Morphology and biology of the bedbug, Cimex hemipterus (Hemiptera: Cimicidae) in the laboratory. Dhaka University Journal of Biological Sciences, 21, 125-130.

Kim, D., Billen, J., Doggett, S.L. and Lee, C. (2017) Differences in climbing ability of Cimex lectularius and Cimex hemipterus (Hemiptera: Cimicidae). J Econ Entomol 110, 1179-1186.

Klowden, M. (2007) Physiological Systems in Insects, London, UK, Elsevier.

Knight, R.L. and Rust, M.K. (1990) Repellency and efficacy of insecticides against foraging workers in laboratory colonies of argentine ants (Hymenoptera: Formicidae). J Econ Entomol 83, 1402-1408.

Koehler, P.G. and Patterson, R.S. (1991) Incorporation of pyriproxyfen in a German cockroach (Dictyoptera: Blattellidae) management program. J Econ Entomol 84, 917- 921.

125

Koganemaru, R., Miller, D.M. and Adelman, Z.N. (2013) Robust cuticular penetration resistance in the common bed bug (Cimex lectularius L.) correlates with increased steady-state transcript levels of CPR-type cuticle protein genes. Pestic Biochem Physiol 106, 190-197.

Krueger, L. (2000) Features-don't get bitten by the resurgence of bed bugs-properly identifying a bed bug infestation is the key to quick control. Pest Control, 68, 58-64.

Kumar, S., Stecher, G. and Tamura, K. (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33, 1870-1874.

Leverkus, M., Jochim, R.C., Schäd, S., Bröcker, E., Andersen, J.F., Valenzuela, J.G. and Trautmann, A. (2006) Bullous allergic hypersensitivity to bed bug bites mediated by IgE against salivary nitrophorin. J Investig Dermatol 126, 91-96.

Librado, P. and Rozas, J. (2009) DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451-1452.

Liedtke, H.C., Åbjörnsson, K., Harraca, V., Knudsen, J.T., Wallin, E.A., Hedenström, E. and Ryne, C. (2011) Alarm pheromones and chemical communication in nymphs of the tropical bed bug Cimex hemipterus (Hemiptera: Cimicidae). PLoS One, 6, e18156.

Lilly, D.G., Latham, S.L., Webb, C.E. and Doggett, S.L. (2016) Cuticle thickening in a pyrethroid-resistant strain of the common bed bug, Cimex lectularius L.(Hemiptera: Cimicidae). PloS One, 11, e0153302.

Lohmeyer, K., Pound, J., Yeater, K. and May, M. (2014) Efficacy of novaluron as a feed- through for control of immature horn flies, house flies, and stable flies (Diptera: Muscidae) developing in cow manure. J Med Entomol 51, 873-877.

Mansur, J.F., Figueira-Mansur, J., Santos, A.S., Santos-Junior, H., Ramos, I.B., de Medeiros, M.N., Machado, E.A., Kaiser, C.R., Muthukrishnan, S. and Masuda, H. (2010) The effect of lufenuron, a chitin synthesis inhibitor, on oogenesis of Rhodnius prolixus. Pestic Biochem Physiol 98, 59-67.

Matsumura, F. (2010) Studies on the action mechanism of benzoylurea insecticides to inhibit the process of chitin synthesis in insects: a review on the status of research activities in the past, the present and the future prospects. Pestic Biochem Physiol 97, 133-139.

Medina, P., Smagghe, G., Budia, F., del Estal, P., Tirry, L. and Viñuela, E. (2002) Significance of penetration, excretion, and transovarial uptake to toxicity of three insect growth regulators in predatory lacewing adults. Arch Insect Biochem Physiol 51, 91-101.

126

Mendki, M.J., Ganesan, K., Parashar, B., Sukumaran, D. and Prakash, S. (2014) Aggregation responses of Cimex hemipterus F. to semiochemicals identified from their excreta. J Vector Borne Dis 51, 224.

Meola, R., Meier, K., Dean, S. and Bhaskaran, G. (2000) Effect of pyriproxyfen in the blood diet of cat fleas on adult survival, egg viability, and larval development. J Med Entomol 37, 503-506.

Merzendorfer, H. (2013) Chitin synthesis inhibitors: old molecules and new developments. Insect Science 20, 121-138.

Merzendorfer, H. (2006) Insect chitin synthases: a review. J Comp Physiol 176, 1-15.

Michaelides, P.K. and Wright, D.J. (1997) Sub-lethal effects of tefluthrin on Diabrotica undecimpunctata howardi, Barber: Adult emergence weights, fecundity and fertility. Crop Protection 16, 431-438.

Mojaver, M. and Bandani, A. (2010) Effects of the insect growth regulator pyriproxyfen on immature stages of sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae). Munis Entomol Zool 5, 187-197.

Mommaerts, V., Sterk, G. and Smagghe, G. (2006) Hazards and uptake of chitin synthesis inhibitors in bumblebees Bombus terrestris. Pest Manag Sci 62, 752-758.

Montes, C., Cuadrillero, C. and Vilella, D. (2002) Maintenance of a laboratory colony of Cimex lectularius (Hemiptera: Cimicidae) using an artificial feeding technique. J Med Entomol 39, 675-679.

Moore, D.J. and Miller, D.M. (2006) Laboratory evaluations of insecticide product efficacy for control of Cimex lectularius. J Econ Entomol 99, 2080-2086.

Moreira, M.F., dos Santos, A.S., Marotta, H.R., Mansur, J.F., Ramos, I.B., Machado, E.A., Souza, G.H., Eberlin, M.N., Kaiser, C.R. and Kramer, K.J. (2007) A chitin-like component in Aedes aegypti eggshells, eggs and ovaries. Insect Biochem Mol Biol 37, 1249-1261.

Mosson, H.J., Short, J.E., Schenkerb, R. and Edwardsa, J.P. (1995) The effects of the insect growth regulator lufenuron on oriental cockroach, Blatta orientalis, and German cockroach, Blattella germanica, populations in simulated domestic environments. Pest Manag Sci 45, 237-246.

Myamba, J., Maxwell, C., Asidi, A. and Curtis, C. (2002) Pyrethroid resistance in tropical bedbugs, Cimex hemipterus, associated with use of treated bednets. Med Vet Entomol 16, 448-451.

127

Naylor, R., Bajomi, D. & Boase, C. (2008) Efficacy of (S)-methoprene against Cimex lectularius (Hemiptera: Cimicidae). Proceedings of the Sixth International Conference on Urban Pests, 115-121.

Newberry, K., Jansen, E. and Thibaud, G. (1987) The occurrence of the bedbugs Cimex hemipterus and Cimex lectularius in northern Natal and KwaZulu, South Africa. Trans R Soc Trop Med Hyg 81, 431-433.

Omori, N. (1941) Comparative studies on the ecology and physiology of common and tropical bed bugs, with special reference to the reactions to temperature and moisture. Journal of the Medical Association of Formosa 40, 55-636.

Panagiotakopulu, E. and Buckland, P.C. (1999) Cimex lectularius L., the common bed bug from pharaonic Egypt. Antiquity 73, 908-911.

Park, D., Foottit, R., Maw, E. and Hebert, P.D. (2011) Barcoding bugs: DNA-based identification of the true bugs (Insecta: Hemiptera: Heteroptera). PLoS One, 6, e18749.

Paulke-Korinek, M., Széll, M., Laferl, H., Auer, H. and Wenisch, C. (2012) Bed bugs can cause severe anaemia in adults. Parasitol Res 110, 2577-2579.

Pereira, F., Carneiro, J. and Amorim, A. (2008) Identification of species with DNA-based technology: Current progress and challenges. Recent Pat DNA Gene Seq 2, 187-200.

Pereira, R.M., Koehler, P.G., Pfiester, M. and Walker, W. (2009) Lethal effects of heat and use of localized heat treatment for control of bed bug infestations. J Econ Entomol 102, 1182-1188.

Pereira, R., Taylor, A., Lehnert, M. and Koehler, P. (2013) Potential population growth and harmful effects on humans from bed bug populations exposed to different feeding regimes. Med Vet Entomol 27, 148-155.

Polanco, A.M., Brewster, C.C. and Miller, D.M. (2011) Population growth potential of the bed bug, Cimex lectularius L.: A life table analysis. Insects 2, 173-185.

Posada, D. (2008) jModelTest: Phylogenetic model averaging. Mol Biol Evol 25, 1253- 1256.

Potter, M.F. (2011) The history of bed bug management. Am Entomol 57, 14-25.

Potter, M., Haynes, K., Gordon, J., Hardebeck, E. and Wickemeyer, W. (2012) Dual- action bed bug killers. Pest Control Technol 40, 75-76.

Potter, M., Haynes, K., Romero, A., Hardebeck, E. and Wickemeyer, W. (2008) Is there a new bed bug answer? Pest Control Technol 36, 116-118.

128

Pritchard, M.J. and Hwang, S.W. (2009) Cases: Severe anemia from bedbugs. CMAJ 181, 287-288.

Raupach, M.J., Hendrich, L., Küchler, S.M., Deister, F., Morinière, J. and Gossner, M.M. (2014) Building-up of a DNA barcode library for true bugs (Insecta: Hemiptera: Heteroptera) of Germany reveals taxonomic uncertainties and surprises. PLoS One, 9, e106940.

Redfern, R., Kelly, T., Bořkovec, A. and Hayes, D. (1982) Ecdysteroid titers and molting aberrations in last-stage oncopeltus nymphs treated with insect growth regulators. Pestic Biochem Physiol 18, 351-356.

Reid, B., Appel, A., Demark, J. and Bennett, G. (1992) Oral toxicity, formulation effects, and field performance of flufenoxuron against the German cockroach (Dictyoptera: Blattellidae). J Econ Entomol 85, 1194-1200.

Reinhardt, K., Kempke, D., Naylor, R. and Siva-jothy, M. (2009) Sensitivity to bites by the bedbug, Cimex lectularius. Med Vet Entomol 23, 163-166.

Reinhardt, K. and Siva-Jothy, M.T. (2007) Biology of the bed bugs (Cimicidae). Annu Rev Entomol 52, 351-374.

Rivnay, E. (1932) Studies in tropisms of the bed bug Cimex lectularius L. Parasitology 24, 121-136.

Romero, A. and Anderson, T.D. (2016) High levels of resistance in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), to neonicotinoid insecticides. J Med Entomol 53, 727-731.

Romero, A., Potter, M.F. and Haynes, K.F. (2010) Evaluation of chlorfenapyr for control of the bed bug, Cimex lectularius L. Pest Manag Sci 66, 1243-1248.

Romero, A., Potter, M.F., Potter, D.A. and Haynes, K.F. (2007) Insecticide resistance in the bed bug: A factor in the pest’s sudden resurgence? J Med Entomol 44, 175-178.

Ronquist, F. and Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572-1574.

Ryne, C. (2009) Homosexual interactions in bed bugs: alarm pheromones as male recognition signals. Animal Behaviour 78, 1471-1475.

Sabou, M., Imperiale, D.G., Andres, E., Abou-Bacar, A., Foegle, J., Lavigne, T., Kaltenbach, G. and Candolfi, E. (2013) Bed bugs reproductive life cycle in the clothes of a patient suffering from alzheimer's disease results in iron deficiency anemia. Parasite, 20, 16.

129

Saenz, V.L., Booth, W., Schal, C. and Vargo, E.L. (2012) Genetic analysis of bed bug populations reveals small propagule size within individual infestations but high genetic diversity across infestations from the eastern united states. J Med Entomol 49, 865-875.

Sáenz-de-Cabezón, F., Pérez-Moreno, I., Zalom, F.G. and Marco, V. (2006) Effects of lufenuron on Lobesia botrana (Lepidoptera: Tortricidae) egg, larval, and adult stages. J Econ Entomol 99, 427-431.

Salazar, R., Castillo-Neyra, R., Tustin, A.W., Borrini-Mayorí, K., Náquira, C. and Levy, M.Z. (2015) Bed bugs (Cimex lectularius) as vectors of Trypanosoma cruzi. The Am J Trop Med Hyg 92, 331-335.

Seong, K.M., Lee, D., Yoon, K.S., Kwon, D.H., Kim, H.C., Klein, T.A., Clark, J.M. and Lee, S.H. (2014) Establishment of quantitative sequencing and filter contact vial bioassay for monitoring pyrethroid resistance in the common bed bug, Cimex lectularius. J Med Entomol 47, 592-599.

Seri, M., Siti, N. A., and Ab Majid, A.H. (2017) Genetic diversity and phylogenetic relationships of cytochrome C oxidase subunit I in Cimex hemipterus (Hemiptera: Cimicidae) populations in Malaysia. J Med Entomol 54, 974-979.

Shaarawi, F., Radwan, W. and Soliman, S. (1982) Effects of juvenoids on some biological aspects of the bed bug Cimex lectularius L. II. effect on egg viability and embryogenesis. Bull Faculty Sci 6, 35-47.

Sierras, A. and Schal, C. (2017) Comparison of ingestion and topical application of insecticides against the common bed bug, Cimex lectularius (Hemiptera: Cimicidae). Pest Manag Sci 73, 521-527.

Siljander, E., Gries, R., Khaskin, G. and Gries, G. (2008) Identification of the airborne aggregation pheromone of the common bed bug, Cimex lectularius. J Chem Ecol 34, 708-718.

Slama, K. and Williams, C.M. (1965) Juvenile hormone activity for the bug Pyrrhocoris apterus. Proc Natl Acad Sci 54, 411-414.

Smith, R.D., Paul, A.J., Kitron, U.D., Philip, J.R., Barnett, S., Piel, M.J., Ness, R.W. and Evilsizer, M. (1996) Impact of an orally administered insect growth regulator (lufenuron) on flea infestations of dogs in a controlled simulated home environment. Am J Vet Res 57, 502-504.

Stephens, M., Smith, N.J. and Donnelly, P. (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68, 978-989.

Stutt, A.D. and Siva-Jothy, M.T. (2001) Traumatic insemination and sexual conflict in the bed bug Cimex lectularius. Proc Natl Acad Sci 98, 5683-5687.

130

Su, N. (1994) The termite bait age dawns. Pest Control 62, 36-38.

Su, N. and Scheffrahn, R.H. (1993) Laboratory evaluation of two chitin synthesis inhibitors, hexaflumuron and diflubenzuron, as bait toxicants against formosan and eastern subterranean termites (Isoptera: Rhinotermitidae). J Econ Entomol 86, 1453- 1457.

Su, N. and Scheffrahn, R.H. (1990) Comparison of eleven soil termiticides against the formosan subterranean termite and eastern subterranean termite (Isoptera: Rhinotermitidae). J Econ Entomol 83, 1918-1924.

Su, N., Thoms, E.M., Ban, P.M. and Scheffrahn, R.H. (1995) Monitoring/baiting station to detect and eliminate foraging populations of subterranean termites (Isoptera: Rhinotermitidae) near structures. J Econ Entomol 88, 932-936.

Suchy, J.T. and Lewis, V.R. (2011) Host-seeking behavior in the bed bug, Cimex lectularius. Insects 2, 22-35.

Susser, S.R., Perron, S., Fournier, M., Jacques, L., Denis, G., Tessier, F. and Roberge, P. (2012) Mental health effects from urban bed bug infestation (Cimex lectularius L.): A cross-sectional study. BMJ Open, 2, 10.1136/bmjopen-2012-000838. Print 2012.

Szalanski, A.L., Austin, J.W., McKern, J.A., Steelman, C.D. and Gold, R.E. (2008) Mitochondrial and ribosomal internal transcribed spacer 1 diversity of Cimex lectularius (Hemiptera: Cimicidae). J Med Entomol 45, 229-236.

Tawatsin, A., Lorlertthum, K., Phumee, A., Thavara, U., Boon-Long, J., Boonserm, R. and Siriyasatien, P. (2013) Discrimination between tropical bed bug Cimex hemipterus and common bed bug Cimex lectularius (Hemiptera: Cimicidae) by PCR-RFLP. The Thai J Vet Med 43, 421-427.

Tawatsin, A., Thavara, U., Chompoosri, J., Phusup, Y., Jonjang, N., Khumsawads, C., Bhakdeenuan, P., Sawanpanyalert, P., Asavadachanukorn, P. and Mulla, M.S. (2011) Insecticide resistance in bedbugs in Thailand and laboratory evaluation of insecticides for the control of Cimex hemipterus and Cimex lectularius (Hemiptera: Cimicidae). J Med Entomol 48, 1023-1030.

Tiwari, S., Clayson, P.J., Kuhns, E.H. and Stelinski, L.L. (2012) Effects of buprofezin and diflubenzuron on various developmental stages of Asian citrus psyllid, Diaphorina citri. Pest Manag Sci 68, 1405-1412.

Todd, R.G. (2006) Efficacy of bed bug control products in lab bioassays: do they make it past the starting gate? Am Entomol 52, 113-116.

Usinger, R.L. (1966) Monograph of Cimicidae (Hemiptera, Heteroptera). Entomological Society of America. College Park, MD.

131

Walpole, D.E. and Newberry, K. (1988) A field study of mating between two species of bedbug in northern KwaZulu, South Africa. Med Vet Entomol 2, 293-296.

Wang, C., Gibb, T., Bennett, G.W. and McKnight, S. (2009) Bed bug (Heteroptera: Cimicidae) attraction to pitfall traps baited with carbon dioxide, heat, and chemical lure. J Econ Entomol 102, 1580-1585.

Weeks, E.N., Birkett, M.A., Cameron, M.M., Pickett, J.A. and Logan, J.G. (2011) Semiochemicals of the common bed bug, Cimex lectularius L.(Hemiptera: Cimicidae), and their potential for use in monitoring and control. Pest Manag Sci 67, 10-20.

Wigglesworth, V. (1940) Local and general factors in the development of "pattern" in Rhodnius prolixus (Hemiptera). J Exp Biol 17, 180-201.

Yoon, K.S., Kwon, D.H., Strycharz, J.P., Hollingsworth, C.S., Lee, S.H. and Clark, J.M. (2008) Biochemical and molecular analysis of deltamethrin resistance in the common bed bug (Hemiptera: Cimicidae). J Med Entomol 45, 1092-1101.

Yu, S. (2008) The Toxicology and Biochemistry of Insecticides, Boca Raton, FL, Taylor & Francis Group.

132

BIOGRAPHICAL SKETCH

Brittany Elise Campbell was born in Rome, Georgia and received her undergraduate degree in biology at a small, private college in her hometown. She earned her Master of Science degree from Virginia Tech in 2013 with a major in

Entomology. Upon completion of her PhD, she plans on continuing pursuit of a career that allows advancement of science and education in the pest management industry.

133