Reproductive Behavior in the Bed Bug (Cimex lectularius)

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

By

Scott Atlee Harrison, B.S.

Graduate Program in Evolution, Ecology, and Organismal Biology

The Ohio State University

2016

Master’s Examination Committee:

Dr. Susan N. Gershman, Advisor

Dr. Susan C. Jones

Dr. J. Andrew Roberts

Copyright by

Scott Atlee Harrison

2016

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Abstract

The common bed bug, Cimex lectularius, has resurged in the last 20 years, renewing interest in understanding the biology of this pest. Although much is known about chemical communication in bed bugs, there have been no studies on sexual selection in this species. In this thesis, I have explored reproductive behavior and the possibility of sexual selection in bed bugs and conducted four behavioral experiments to determine I) where bed bugs are most likely to mate, II) how feeding and mating status influence female attraction to harborages, III) the effect of male feeding status on male mating success, and IV) the effects of inbreeding and outbreeding on fitness, and if females exercise mate choice for non-relatives. I found that bed bugs were most likely to mate in a harborage or near the blood feeder than in the open. There was some evidence that feeding status had a positive correlation with harborage attraction. There was a trend for virgin females to be more attracted to harborages than mated females, but it was not statistically significant. Replete (fed) female bugs may be seeking refuge to digest and oviposit while virgin females may be seeking mating opportunities at the harborage.

Replete males were less likely to successfully mate than unfed males due to their large body size and inability to properly mounting a female. I did not find any evidence for inbreeding depression, but did find considerable variation in fitness between two laboratory strains. As I did not find inbreeding depression it stands to reason that bed bugs do not preferentially mate with non-related partners. ii

Acknowledgements

First I would like to thank my advisor, Dr. Susan Gershman for her support and guidance. Susan has fostered an environment of learning through sheer will and a passion for science. She challenged me to become a better researcher, thinker, and writer.

I’d like to thank Dr. Susan Jones for serving on my committee and for giving me free run of the Jones lab. Without her, my project would have been truly impossible. I’d also like to thank Dr. Andrew Roberts for serving on my committee. His thoughtful comments and assistance with experimental design were always appreciated.

Thanks to Steven Nagel, Zac Beres, Kara Baker, Nina Bogart, and Owen Miller for their camaraderie and friendship during stressful times.

Thank you to Kayley Jaquet for assistance with graphical images, and for feeding me.

Finally, I’d like to thank my family and close friends for their encouragement, love, and support.

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Vita

2007……….………………….…….. Gahanna Lincoln High School

2011………………………………….B.S. Entomology, The Ohio State University

2011-2013………………….………..Research Assistant, The Ohio State University

2013-Present…………….……..……Graduate Teaching Associate, Department of

EEOB, The Ohio State University

Publications

Jones, S. C., Bryant, J. L., & Harrison, S. A. (2013). Behavioral responses of the bed bug

to permethrin-impregnated ActiveGuard™ fabric. Insects, 4, 230-240.

Fields of Study

Major Field: Evolution, Ecology, and Organismal Biology

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Table of Contents

Abstract…………………………………………………………………………………....ii

Acknowledgements………………………………………………………………………iii

Vita………………………………………………………………………………………..iv

List of Tables……………………………………………………………...……………..vii

List of Figures……………………………………………………………………...……viii

Chapter 1: Introduction…………...……………………………………………………….1

Chapter 2: Bed Bug Mating Location

Introduction………………………………………………………………………..8

Methods………………………………………………………………………..…10

Results……………………………………………………………………………13

Discussion………………………………………………..………………………15

Chapter 3: Female Condition and Harborage Attraction

Introduction………………………………………………………….……..…….17

Methods…………………………………………………………………………..20

Results……………………………………………………………………………23

Discussion……………………………………………………………………..…27

Chapter 4: Feeding Limits Male Reproductive Success

Introduction………………………………………………………………………31

Methods…………………………………………………………………………..34 v

Results……………………………………………………………………………35

Discussion………………………………………………………………………..38

Chapter 5: Consequences of Inbreeding in the Bed Bug

Introduction………………………………………………………………………40

Methods…………………………………………………………………………..42

Results……………………………………………………………………………45

Discussion…………………………………………………………………...... …49

References………………………………………………………………………………..51

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List of Tables

Table 2.1 Hypothesized and actual probabilities of mating in different locations……....14

Table 2.2 Feeding combinations, mating status, and mean mating duration ± SE……….14

Table 3.1 Treatment, sample size, and mean ± SE for average distance to male zone – average distance to blank zone, duration in male zone – duration in blank zone, and frequency in male zone – duration in blank zone………………………………………..27

Table 4.1 The non-significant effects of male and female pre-feeding weight and blood meal on latency to mate, clutch size, and egg viability…………………………….……37

Table 4.2 Treatment, sample size, and mean ± SE for latency to mate, mating duration, mean clutch size, and egg viability………………………………….………...…………37

Table 5.1 Treatment, sample size, and mean ± SE for latency to mate and mating duration…………………………………………………………………………………..47

Table 5.2 Treatment, sample size, and mean ± SE for clutch size and hatch rate……….49

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List of Figures

Fig 2.1 Top view of the arena………...………………………………………………….12

Fig 2.2 Frequency of mating of a male and female pair in each possible location: on the female harborage (n=13), on the male harborage (n=3), on or near the feeder (n=6), and

in the open (n=17). Mating location was non-random ( =207.00, P<0.001)…….……13

Fig 3.1 Top view of the arena. The arena was filmed from this view…………………...23

Fig 3.2 Side view of the arena. A male harborage (25 bugs and their excrement on a filter paper tent) is on the right and a blank harborage (a clean filter paper tent) is on the left.23

Fig 3.3 Example of Ethovision output for a virgin replete female. The green line is her path………………………………………………………………………..……………...24

Fig 3.4 The effect of female feeding and mating status on mean (±1SE) (a) average distance to the blank harborage - distance from the male harborage, (b) duration in male zone – duration in blank zone, (c) frequency in male zone – frequency in blank zone….26

Fig 4.1 A replete male and female, where a) the male partially mounts the female, struggles to contact the spermalege and b) dismounts and retreats……………………...33

Fig 4.2 The effect of male and female feeding status on mean (±1SE) a) latency to mate and (b) clutch size. For each variable, statistical differences between treatments are represented by letter groups……………………………………………………………...36

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Fig 5.1 The effect of male and female colony on mean (±1SE) (a) latency to mate (b) mating duration. Statistical differences between treatments were found as represented by letter groups……………………………………………………………………………...46

Fig 5.2 The effect of male and female colony on mean (±1SE) (a) clutch size (b) egg viability. Statistical differences between treatments were found as represented by letter groups…………………………………………………………………………………….48

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Chapter 1

Introduction

Sexual selection, differential reproductive success based on variation among individuals in traits that affect competition for mates (Andersson 1994), is found in some species, but not others. Whether or not sexual selection occurs can depend on the inherent behavioral, environmental, and biological properties of a species. One way to assess the intensity of sexual selection in a species is to use the operational sex ratio (OSR), the ratio of fertilizable females to sexually active males at any time (Clutton-Brock and

Parker 1992). When the OSR is skewed to one sex we expect strong intraspecific competition in that sex for mates (Clutton-Brock and Parker 1992). Any factors that affect the ratio of adult males to adult females will affect the intensity of sexual selection, including biased sex ratios at birth or hatching and sex differences in survival or age of sexual maturation (Clutton-Brock and Parker 1992). Mating competition can also be affected by spatiotemporal variance in OSR. For example, in adders (Vipera berus) males compete for females in combat and the larger male usually wins (Madsen et al. 1993).

Female adders do not reproduce every year and due to variation in spring weather, female reproductive frequency, survival rate, and age at maturity the OSR can vary from year to year (Madsen et al. 1993). When relatively few females were available, large males were able to monopolize the fertile females, and there was directional selection on

1 male body size. In contrast, when more females were receptive to mating, both large and small males were able to mate (Madsen et al. (1993).

When sexes vary in their potential rate of reproduction, the sex with the faster rate of reproduction will have to compete for mates. Females typically have a lower rate of reproduction as they must provision and secure adequate conditions for maturing the eggs; the male’s obligation can end at copulation that frees him to pursue other matings

(Williams 1966). Some behavioral and morphological differences between the sexes can be explained by anisogamy, where females produce large nutritious eggs and males produce small, mobile sperm. A male can make many more gametes than a female and males will compete to mate with as many females as possible. In contrast, females invest more in fewer gametes and are therefore predicted to be the choosier sex. However, differences in other forms of reproductive investment, like allocation of resources towards provisioning offspring, can allow females to reproduce at a higher rate than males. For example, in the cabbage butterfly (Pieris rapae) males provide females with a protein-rich spermatophore that increase female reproductive output (Tigreros et al.

2014). Producing a nuptial gift lowers the male’s reproductive rate and males were found to select for pteridine-based wing coloration in females (Tigreros et al. 2014).

Competition between members of one sex for the opportunity to reproduce with the opposite sex can sometimes lead to conflict between the evolutionary interests of males and females, which is called sexual conflict (Arnqvist and Rowe 2005). Conflict can arise from any differences in optimal trait values between males and females, including mating rate, fertilization efficiency, relative parental effort, remating behavior,

2 and female reproductive rate (Arnqvist and Rowe 2005). Males and females may differ in reproductive effort and therefore can maximize their fitness in different ways. This means that traits favored in one sex are often not those favored in the other sex, leading to sexually antagonistic selection on those traits, called intralocus sexual conflict (Arnqvist and Rowe 2005). For example, male broad-horned flour beetles (Gnatocerus cornutus) use large mandibles in male-male combat, and individuals with larger mandibles are better fighters (Harano et al. 2010). Females from populations selected for larger male mandibles were less fit than females from small mandible population, even though females never develop exaggerated mandibles. This is because changes in mandible development correlated with other traits that were not sex-limited, and caused intralocus sexual conflict over mandible size. In contrast, interlocus sexual conflict occurs when the optimal outcome is different for the average male and female (Arnqvist and Rowe 2005).

In many mating systems, females have a lower optimal mating rate than males and will acquire adaptations to resist mating. In response, males acquire adaptations to overcome female mating resistance and an evolutionary arms race occurs. For example, in the water strider genus Gerris, mating increases predation risk and male harassment is frequent (Arnqvist and Rowe 2002). Males have evolved grasping structures to hold the female in place during copulation and female have evolved antigrasping structures that enable females to slip away from males. Sexual conflict is partly responsible for morphological and behavioral differences between the sexes in many organisms.

Another example of interlocus sexual conflict affecting morphology is traumatic, or hypodermic, insemination. Traumatic insemination is when males bypass the female

3 genitalia, pierce the female through the skin and ejaculate into the body of the female.

This extreme practice is suggested to have evolved as a method of overcoming female mating resistance and is rather uncommon, but taxonomically widespread in invertebrates. It occurs in some flatworms (Michiels 1998), rotifers (Aloia and Moretti

1973), flies (Kamimura 2007), bean weevils (Crudginton and Siva-Jothy 2000), plant bugs (Tatarnic et al. 2006), and several hemipterans related to the bed bug (Usinger

1966). Male genitalia in these animals often resemble sharpened needle-like structures and in response females have evolved internal and external paragenital adaptations collectively termed ‘the spermalege’ that mitigate some of the costs of traumatic insemination (Morrow and Arnqvist 2003). For example, in the plant bug, Coridromius zetteli, males have a sharp corkscrew intromittent organ that is inserted into a copulatory tube on the first abdominal sternite of the female (Tatarnic et al. 2005).

Traumatic insemination has been studied the most in bed bugs and is widespread in the family Cimicidae. There are several costs associated with this method of reproduction including repair of the wound, increased risk of pathogen transmission, and immune defense against sperm or accessory gland fluids that are introduced to the hemolymph (Arnqvist and Rowe 2005). Not surprisingly, female bed bugs have evolved a spermalege that mitigates some of these costs. The spermalege is made of two parts, the ectospermalege and the mesospermalege. The ectospermalege is a groove between the fifth and sixth abdominal segment. Directly under the groove is the mesospermalege, which is a hemocyte-containing membrane bound sac. In Cimex lectularius, the male mounts the female, reaches under her abdomen with his hypodermic intromittent organ,

4 pierces the ectospermalege and ejaculates into the mesospermalege. Sperm travel from the mesospermalege into the hemolymph and make their way to specialized sperm storage structures, and then to the ovaries. Reinhardt et al. (2003) found that the spermalege reduces the cost of mating-associated infections in females. However, traumatic insemination still lowers female fitness under the natural mating rate (Stutt and

Siva-Jothy 2001), and it may be advantageous for females to avoid males if they have already mated.

Interest in bed bug behavioral ecology has intensified as bed bugs have experienced a resurgence. A survey of U.S. pest control companies conducted in 2011 reported that before the year 2000, only 11% recalled treating or being asked to treat for bed bugs (Potter et al. 2011). A decade later, this percentage had grown to 99% (Potter et al. 2011). A survey conducted in England covering the years 2006-2007 found that only

10% of 358 people recognized a live bed bug (Reinhardt et al. 2008), so lack of public awareness has likely contributed to their rapid spread. A bed bug extension fact sheet had

1,312 Google hits in 1998, more than 350,000 hits in 2005, and was projected to have more than 1,000,000 hits in 2006 (Potter 2006). Bed bug research was lacking during the latter half of the 20th century, and we must now play catch-up to fully understand this pest.

There are no previous studies that have attempted to determine whether there is currently sexual selection in bed bugs. I expect the OSR to be male biased because unfed females cannot produce many eggs and under the best conditions bed bugs feed on average once a week, limiting the availability of fertile females. Males, on the other hand,

5 can mate successfully while unfed. Also, females have a lower optimal mating rate than males (Stutt and Siva-Jothy 2001), suggesting that females should be the choosier sex.

Males provide no nuptial gift and females are responsible for providing the nutrients to produce eggs. All of these factors could potentially contribute to male competition for females.

Female bed bugs are typically passive during and prior to mating and show no overt resistance (SH personal observation). Rarely a female may vigorously shake her abdomen and dislodge a male, but this behavior is more common in other members of the family Cimicidae (Usinger 1966). Bed bugs lack ritualized courtship behaviors, and the practice of traumatic insemination suggest that females are not choosing males based on male courtship, but there are other ways in which females may be exerting mate choice.

As a pre-copulatory mechanism, females may simply avoid aggregations of bed bugs to limit their chance of coming into contact with a male. For example, in the solitary bee,

Anthophlora plumipes, males harass females and reduce their rate of visiting flowers

(Stone 1995). Females respond with evasive flight and a general change in foraging behavior. Females abandoned flowers on the outer parts of plants and instead foraged on the less nutritious flowers on the inner parts of plants (Stone 1995). Post-copulatory choice can be present in the form of sperm competition, which is competition within a female between the sperm of two of more males (Parker 1970). For example, in the decorated cricket (Gryllodes sigillatus) a male’s spermatophore remains attached to the female genitalia after mating, and females can exhibit post-copulatory choice by removing the spermatophore before sperm transfer has completed (Gershman and

6

Sakaluk 2010). After mating with inbred males, females removed the spermatophore of familiar mates more quickly than that of novel mates (Gershman and Sakaluk 2010).

In this thesis I focused on reproductive biology and behavior of the bed bug. In chapter 2, I explore where mating is most likely to take place. In chapter 3, I examine the effects of feeding and mating status on harborage attraction in females. I examine how female motivation to mate affects female tendency to visit or avoid bed bug aggregations.

In chapter 4, I examine how feeding status affects males’ ability to successfully mate.

There is anecdotal evidence that replete males were unable to mate successfully, which would temporarily skew the OSR affecting the strength of sexual selection, and I tested this assumption. In chapter 5, I use two inbred lines to determine if bed bugs avoid inbreeding depression by preferentially mating with inbred versus outbred lines. A study by Fountain et al. (2014) found very low genetic differentiation within infestations and very high genetic differentiation between infestations ( =0.59). Inbreeding reduces fitness in many animals, but its effects on the bed bug are not clearly understood.

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Chapter 2

Bed Bug Mating Location

Introduction

Bed bug mating practices has been studied in many contexts (Stutt and Siva-Jothy

2001, Siva-Jothy and Stutt 2003, Ryne 2009), but it is still not known where mating takes place. It seems likely that matings occur in a harborage, as that is where bed bugs spend the majority of their time. Bed bugs are attracted to harborages via volatile and contact aggregation pheromones (Gries et al. 2015) and may reliably find mates there as all stages of life can occur in a harborage. However, male bed bugs preferentially mate with replete females (Stutt & Siva-Jothy 2001), and males may encounter replete females on or near the blood source. It is also possible that bed bugs mate somewhere between the harborage and blood source. Bed bugs are more active during the scotophase (Romero et al. 2010), and more specifically the early part of the scotophase (Weeks et al. 2011).

Since bed bug individuals are active at the same time of day, it is possible that they meet and mate somewhere other than the harborage and host.

Bed bugs are attracted to carbon dioxide and heat (Wang et al. 2009) and typically feed during the scotophase. In an experimental arena bed bugs were stimulated to leave the harborage at the beginning of the scotophase with a heated artificial feeder (Reis and

Miller 2011). Those that fed returned to the harborage within 30 minutes while unfed

8 bugs continued to search the arena. However, all bugs returned to shelters beginning two hours prior to the photophase.

Bed bug harborages have a patchy distribution, leaving unoccupied space near the host and containing anywhere from just a few to a hundred bugs (Naylor 2012). Their patchy distribution may help bed bugs avoid detection. Small infestations are usually located very close to the host and in 6 of 7 apartments with fewer than 100 bed bugs, all bugs were confined to the bed (Naylor 2012). Large infestations tend to have more peripheral harborages suggesting that the harborage population is limited by environmental constraints (Naylor 2012). It is possible that females choose harborages with fewer males in order to avoid excessive traumatic insemination (see Stutt & Siva-

Jothy 2001). Bed bugs defecate in their harborages and may avoid particularly unsanitary harborages as traumatic insemination introduces environmental microbes into the body cavity of the female (Reinhardt & Siva-Jothy 2007).

In this study, I set up an arena to evaluate where matings were more likely to take place, or if bed bugs mated in random locations. This information was useful for designing my other experiments. My hypothesis was that:

1) Bed bugs would be more likely to mate in a harborage or near the blood source rather than in the open because conspecifics can reliably be found in a harborage and the blood source.

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Methods

For this experiment and others except when noted, I used the bed bug strain EPM, which was collected in Columbus, OH on August 25, 2010. Bed bugs were fed with defibrinated rabbit blood approximately 2-4 times a month using the Hemotek® membrane feeding system. Bugs were held in an environmental chamber at 30±0.5 ºC and 50±1% humidity on a 12:12 light-dark cycle.

I used a rectangular arena (See Fig 2.1) consisting of a plastic bin (22 x 28 cm) with a piece of paper taped to the floor. Bed bugs were unable to walk up the side of the bin and the piece of paper provided bed bugs a traversable substrate. The paper was replaced each trial to control for bed bug scent accumulation in the arena. A hole in the side of the arena was covered with organza fabric and connected to a blood-feeder so that bugs could feed while in the arena. I counted bugs as fed when they had pierced the parafilm (plastic paraffin film) on the blood-feeder and had visibly enlarged their abdomen by taking a blood meal. I wanted to provide bugs with a blood source to evaluate if bugs were more likely to mate when fed or unfed.

Virgin adults were used for this experiment. I obtained virgins by removing fifth instar bugs from colony jars and holding them individually in a 24-well plate until they molted to adults. I used these virgin unfed bugs in experiments 2-3 days post-eclosion. A female and a male were housed for two days in separate harborages consisting of filter paper (3.5 x 4 cm) folded into a tent. The female and male harborages (each containing a single bug) then were placed in the middle of the arena on opposite ends.

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In an effort to make the arena similar to what a bed bug may experience in a natural infestation, I observed the bugs during the first 3 hours of the scotophase when bed bugs are most active (Weeks et al., 2011). I also observed bugs under red light because there is evidence that bed bugs cannot perceive the color red (Singh et al. 2015), and bed bugs are active at night.

I observed if and where mating took place, using four categories: I) on the blood feeder (area=25 cm²), II) in the open (area=602.6 cm²), III) on/in the male harborage

(area= 14 cm²), IV) and on/in female harborage (area= 14 cm²). I also recorded the feeding status of bugs (unfed, replete) and copulation duration. When bugs mated I ended the trial, or trials were stopped at one hour and bugs that had not mated by that time were counted as non-maters. While most matings were obvious, I observed that replete (fed) males were unable to successfully mate with unfed females due to the difference in body size, although they still mounted females and in some cases remained in contact for several minutes. Because the purpose of this experiment was to see where mating occurred and not if mating was successful, I considered mating to be any mating attempt that lasted longer than one minute. Out of 58 trials, 39 mated in the arena.

The assumptions of the one-way ANOVA were tested for mating duration; all samples were independent random samples, variance was homoscedastic, and outliers were checked for with the Mahalanobis distance method. Mating duration did not have a normal distribution and was log transformed to achieve normality. Pearson’s chi-squared test was used to evaluate if bed bugs preferentially mated in certain areas at a significance level of α=0.05. The null hypothesis for the test is that bed bugs mate in random locations

11 based on the area of each possible mating location in the arena, 0.02135 for the male harborage (14 cm²/655.6 cm²), 0.02135 for the female harborage (25 cm²/655.6 cm²),

0.03813 for the blood feeder (25 cm²/655.6 cm²), and 0.91916 for the open (602.6 cm²/655.6 cm²). I used JMP 11 (SAS Institute Inc.) to analyze all data.

Feeder

5 cm

♀ Harborage ♂ Harborage

4 cm 21.6 cm

3.5 cm

27.9 cm

Fig 2.1 Top view of the arena.

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Results

Bed bugs did not mate in random locations (Fig 1, =207.00, P<0.001). I tested the effect of male and female feeding combinations (unfed female x unfed male, unfed female x replete male, replete female x unfed male, replete female x replete male) on the likelihood of mating and found no effect ( =1.21, P=0.315). I tested the effect of male and female feeding combinations (unfed female x unfed male, unfed female x replete male, replete female x unfed male, replete female x replete male) on log(mating duration) and found no effect ( =0.96, P=0.424).

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16

14

12

10

8

6

4

2

0 ♀ Harborage ♂ Harborage Feeder Open

Fig 2.2 Frequency of mating of a male and female pair in each possible location: on the female harborage (n=13), on the male harborage (n=3), on or near the feeder (n=6), and

in the open (n=17). Mating location was non-random ( =207.00, P<0.001).

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Mating location Hypothesized Probability Actual Probability

♀ Harborage 0.02135 0.33333

♂ Harborage 0.02135 0.07692

Blood Feeder 0.03813 0.15385

Open 0.91916 0.43592

Table 2.1 Hypothesized and actual probabilities of mating in different locations.

Feeding combination N Mated Mean mating duration

(s) ± SE

Unfed ♀ X Unfed ♂ 33 23 316±77

Unfed ♀ X Replete ♂ 9 4 701±330

Replete ♀ X Unfed ♂ 12 10 273±58

Replete ♀ X Replete ♂ 3 2 272±148

Table 2.2 Feeding combinations, mating status, and mean mating duration ± SE.

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Discussion

These results indicate that bed bugs are more likely to mate in a harborage or near the blood feeder than in the open. Bed bugs can likely reliably find mates on the harborage and simultaneously be shielded from host detection. Mating on or near the blood source is more dangerous in terms of host detection but bed bugs were likely to encounter each other at the blood source.

Interestingly, more matings occurred on the female harborage than the male harborage. Males may have been attracted to the female harborage, or alternatively were more active than the female and encountered her there. Previous studies have shown that males walk significantly faster, have a higher proportion of activity, and a greater turning rate while walking over odor pots (harborages separated from physical contact with bed bugs) compared to no-odor pots (Weeks et al. 2013). Levinson (1971) showed that males are attracted to both female and male harborages, so it is possible that the male was attracted to the female harborage.

Although bed bugs were more likely to mate on the harborages or blood source based on area, there was still a fairly high chance of mating in the open. This seems odd because mating in the open exposes the bugs to predators or to the host. However, bed bugs may have not felt threatened because observations were conducted under red light, which bed bugs do not avoid (Singh et al. 2015). Males usually attempted to mount females the first time the male and female encountered each other in each trial, which in many cases was in the open (SH personal observation). Bed bugs may have randomly encountered each other while searching for a host, as bed bugs were not fed prior to the

15 trial and unfed bed bugs typically begin to search for hosts at the beginning of the scotophase (Reis and Miller 2011). Indeed, while males showed interest in feeding, if they encountered a female at the feeding site they attempted to copulate instead of feeding.

In conclusion, my data supports the hypothesis that mating location is non- random. Mate choice may be based on scramble competition because bed bugs often mated the first time they encountered each other. It is surprising that feeding status had no effect on the likelihood of mating as many sources have reported that replete females are more likely to mate (Johnson 1941, Usinger 1966, Stutt & Siva-Jothy 2001). It is possible that the arena was too big and replete females were simply not encountered by males.

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Chapter 3

Female Condition and Harborage Attraction

Introduction

In many systems, a female visits male territories and assesses resources or male displays to choose a mate. For example, in lek systems males aggregate and these aggregations function as a center of attraction for sexually reproducing members of that species. For example, in the bullfrog, Rana catesbaiana, males aggregate and defend territory within the aggregation using threats and physical combat (Emlen 1976). Females then visit the aggregation and spend many hours moving from territory to territory assessing males.

In some systems, females may receive direct material benefits from mating that increase reproductive output (Kokko et al. 2003). Females can gain indirect genetic benefits by mating with males that have superior genes, or genes that combine well with the female’s own genes. Females can also gain indirect genetic benefits by choosing novel or unrelated partners to increase the genetic diversity of their offspring (Garant et al. 2008). Outbred salmon had increased freshwater juvenile survival compared to inbred salmon (Garant et al. 2008).

Despite many benefits of sexual reproduction, there are also costs, particularly for females. For example, females increase their disease/pathogen risk, waste energy and time, and may be harmed during or as a result of mating (Parker 2006). In Drosophila 17 melanogaster females with elevated mating rates died earlier than controls (Chapman et al. 1995). Seminal fluid products from the male accessory gland were responsible for this female mating cost.

The bed bug mates by traumatic insemination; the male bed bug pierces the female abdominal wall with his intromittent organ and ejaculates into her body cavity

(Usinger 1966). The male pierces a structure called the spermalege which helps defend against pathogens introduced during traumatic insemination (Reinhardt et al. 2003). Stutt and Siva-Jothy (2001) showed that traumatic insemination results in last male sperm precedence, suboptimal re-mating frequencies for female fertility, and reduced longevity and reproductive success in females. Females did not receive indirect benefits from multiple matings and under natural conditions received 20 times more traumatic inseminations than was required to maintain fertility. A single mating was shown to provide females with enough sperm to maintain maximum fertility for at least four weeks, so females have a direct cost for superfluous matings.

Bed bugs aggregate in harborages, which are typically in cracks and crevices and are scented with volatile aggregation pheromones (Gries et al. 2015). All life stages congregate in these harborages. Although we know that both male and female bed bugs are attracted to the odor of harborages (Levinson & Bar Ilan 1971; Weeks et al. 2011,

2013), it is not known whether bed bugs associate harborages with mating opportunities.

Weeks et al. (2011) found that bed bugs are attracted to harborages that had been exposed to 100 bed bugs for one month. She places pots with harborages under an arena and measured attraction to the harborage. She termed a pot with a harborage in it an odor pot

18 and a pot with clean filter paper a non-odor pot. In a choice between an odor pot and a non-odor pot bed bugs uniformly preferred the odor pot. Bed bugs moved faster and spent more time near scented areas (Weeks et al. 2013). Feeding status did not significantly affect visits to pots or time spent above them, although engorged females made more visits to the odor pot than engorged males and starved adults of either sex.

Mated females made more visits to the odor pot than males, virgin females, and nymphs.

This may be because mated females are seeking a known safe place to oviposit and an established harborage indicates that bugs are able to obtain blood meals nearby.

Engorged males may make fewer visits to the odor pot because other males may try to mount them and because engorged males have difficulties mating successfully (SH personal observation). Weeks et al. (2013) did not test how the combination of feeding status and mating status affects harborage attraction.

In my study, I tested how female feeding and mating status affect the strength of female attraction to olfactory cues from an all-male harborage versus a blank harborage. I used all-male odor pots to assay female attraction to males because I was interested in the possibility of females exercising habitat choice that would increase or decrease the possibly of meeting a male. Since reproduction is necessary, but superfluous mating decreases female fitness, I hypothesized that:

1. Virgin replete females should be the most attracted to the odor pot because they

have the nutritional resources to produce eggs and can find a male to fertilize their

eggs and a safe place to digest at a harborage.

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2. Mated replete females should be the second most attracted to the odor pot because

they already have the resources to produce viable eggs and can rely on a

harborage as a safe place to digest and oviposit.

3. Virgin unfed females should be more attracted to the odor pot than mated unfed

females because virgin females need to mate.

Methods

I designed a circular arena using an acrylic sheet as a base. Two holes (dia 2.5 cm) were cut out of the acrylic sheet 13.1 cm apart (Fig 3.1). Muslin fabric was then placed on the acrylic sheet, and a section of PVC pipe (dia 18.1 cm) was placed on top of the muslin fabric. The 2.5 cm holes were placed on opposite sides of the 18.1 cm circle because bed bugs are positively thigmotactic and seek contact with other objects (Aboul-

Nasr & Erakey 1969). They are more likely to encounter the odor zones if they are on the edge of the arena. Harborages of bed bugs could then be placed under the 2.5 cm holes

(Fig 3.2) and a bed bug could walk on top of them without seeing or making physical contact with the harborages. This is an approximation of what a female may experience in natural environments as harborages are not visually obvious.

An all-male harborage was used to account for possible differences between male and female aggregation pheromones as suggested in the study by Siljander et al. (2007), and I was interested in the possibility of females exercising choice by selecting a harborage. I chose to use 25 males per harborage as Levinson and Bar Ilan (1971) showed that paper discs scented with 25 male bed bugs caused 92% of female bed bugs

20 to aggregate on a disc. Harborages of males were made by isolating 25 males from the colony jar, feeding them to ensure they would defecate, and then placing them on a 3.5 x

4 cm filter paper tent in a plastic container for a week. Blank harborages consisted of a clean 3.5 x 4 cm filter paper tent held under the same conditions. I refer to the area above each harborage as the male odor zone (male harborage underneath) and the non-odor zone (blank harborage underneath).

A factorial design was used to evaluate how female mating status and feeding status affected female attraction to harborages. There were four female condition treatments: I) virgin unfed, II) virgin replete, III) mated unfed, and IV) mated replete. To obtain mated females, I paired virgin females with unfed virgin males until copulation occurred for one minute, ensuring that females had received a relatively equal amount of ejaculate as ejaculate volume and copulation duration have a linear relationship (Siva-

Jothy and Stutt 2003). Females were brought into the lab approximately one hour before testing and each was tested one at a time for 15 minutes in the arena. To control for preferences for certain sides of the arena I rotated it 90º every three trials.

I filmed bed bugs under red light and used Ethovision XT 8.5 (Noldus

Information Technology) to analyze bed bug behavior based on x-y coordinates (Fig 3.3).

Distance from the center of each zone (cm), duration in each zone (s), and frequency entering the zones were used as proxies for choice. For each variable, the difference between the male zone and the blank zone was calculated, with a positive value indicating a larger mean for the male zone than the blank zone.

21

The assumptions of the one-way ANOVA were tested for the variables of distance, frequency, and duration; all samples were independent random samples, responses followed a normal distribution, and no outliers were found with the

Mahalanobis distance method. I found homoscedasticity of variance for distance, but not for duration and frequency. I also tested the assumptions of the one-way ANOVA for average speed and found that homoscedasticity of variance was violated. Therefore, one- way ANOVA was used to make comparisons between female condition treatments for distance, and the Welch ANOVA (which controls for heteroscedasticity of variance) was used to make comparisons between treatments for frequency, distance, and speed. The null hypothesis for the ANOVA and Welch’s ANOVA tests was that there is no statistical difference between female condition treatments. A Wilcoxon signed rank test was used to test if bed bugs preferred the male zone using the three proxies of choice. The null hypothesis for these tests was that the difference between the male zone and blank zone was not different from zero. I used significance level of α=0.05 for all statistical methods.

I used JMP 11 (SAS Institute Inc.) to analyze all data.

22

18.1 cm

2.5 cm

Fig 3.1 Top view of the arena. The arena was filmed from this view.

Fig 3.2 Side view of the arena. A male harborage (25 bugs and their excrement on a filter paper tent) is on the right and a blank harborage (a clean filter paper tent) is on the left.

23

Blank ♂ Zone Zone

Fig 3.3 Example of Ethovision output for a virgin replete female. The green line is her

path.

24

Results

I tested the effect of female feeding status, mating status, and their interaction on female distance to each zone. There was no effect of female condition on female distance to each zone (Fig 3.4a, =0.79, P=0.501). I tested the effect of female feeding status, mating status, and their interaction term on duration in each zone. The effect of female condition on female duration in each zone approached significance (Fig 3.4b,

=1.49, P=0.054). I then tested the effect of female feeding status on female duration in each zone and found that replete females spent more time in the male zone than unfed females ( =4.59, P=0.034). I tested the effect of female feeding status, mating status, and their interaction term on frequency in each zone. There was no effect of female condition on female frequency in each zone (Fig 3.4c, =1.49, P=0.223).

No interaction term between feeding status and mating status was significant (T=-1.05,

P=0.294 for distance; T=0.15, P=0.883 for duration; and T=0.00, P=0.997 for frequency).

I pooled females from all feeding and mating combinations and tested if females spent more time, made more visits, and were on average closer to the male zone or the non-odor zone. Females spent more time in the male odor zone than the non-odor zone

( =4.37, P<0.001) and made more visits to the odor zone than the non-odor zone

( =4.29, P<0.001). Females on average were not statistically significantly closer to the male zone or the blank zone ( =1.67, P=0.076).

25

a) 1

0.5 A A Distance to 0 male zone - A distance to blank zone -0.5 A

-1

-1.5

14 b) A 12 10 A Duration in 8 A male zone- duration in 6 A blank zone 4 (seconds) 2 0 -2

7 A 6 A 5 A Frequency in 4 male zone - 3 A frequency in blank zone 2 1 0 -1 Mated Replete Mated Unfed Virgin Replete Virgin Unfed Treatment

Figure 3.4 The effect of female feeding and mating status on mean (±1SE) (a) distance from the male zone - distance from the blank zone, (b) duration in male zone – duration in blank zone, (c) frequency in male zone – frequency in blank zone. Positive mean values indicate a greater attraction to the male zone than the blank zone. For (a), (b) and (c) no statistical difference was found between the four female condition treatments.

26

N Mean Average Mean Duration (s) Mean Frequency Distance (cm) ± SE ± SE ± SE Mated Replete 34 -0.81±0.46 6.57±2.17 3.12±1.27 Mated Unfed 38 0.15±0.44 1.21±2.05 1.16±1.20 Virgin Replete 34 -0.46±0.46 9.23±2.17 4.51±1.27 Virgin Unfed 32 -0.45±0.48 4.60±2.33 2.56±1.31

Table 3.1 Treatment, sample size, and mean ± SE for average distance to male zone – average distance to blank zone, duration in male zone – duration in blank zone, and frequency in male zone – duration in blank zone.

Discussion

Replete females spent more time in the male zone than unfed females. This observation supports the idea that replete bugs may be seeking refuge from predators and a safe place to digest their blood meal in the harborage. Overall, females were more attracted to the male zone than the blank zone in terms of time spent therein and number of visits. Distance from the odor zone showed a non-significant trend of females remaining in closer distance to the male zone. This agrees with previous studies that bed bugs are attracted to harborages via volatile pheromones (Levinson & Bar Ilan 1971;

Weeks et al. 2011, 2013; Gries et al. 2015) and demonstrates that the arena was effective at measuring attraction to harborages. Video showed that bed bugs often circled near the male zone for a period of time before exploring another part of the arena. Females may have given up on searching the area after they found no harborage or other bed bugs. This 27 observation agrees with the findings of Gries et al. (2015) that bed bugs are attracted to volatile aggregations pheromones, but arrestment is only caused by contact with non- volatile histamine, which is found in bed bug exuviae. Females also repeatedly circled the edge of the arena in most trials. Bed bugs are positively thigmotactic, that is, they are attracted to any object that provides a mechanical stimulus (Aboul-Nasr & Erakey 1969) and could simultaneously touch two surfaces at once by remaining on the edge of the arena.

Female condition did not have a statistically significant effect on frequency of visits and average distance from each zone, although replete females spent more time in the odor zone than the blank zone. Differences between the virgin and mated treatments may have been greater if females were multiply mated. My hypothesis that virgin unfed bugs would be more attracted to the odor pot than mated unfed bugs was not statistically supported, but virgin unfed bugs did spend more time and make more visits to the odor pot than mated unfed bugs. My hypothesis that virgin replete bugs would be more attracted to the odor pot than mated replete bugs was not statistically supported, but virgin replete bugs did spend more time and made more visits to the odor pot than mated replete bugs. Virgin replete bugs may be seeking a mate and mated replete bugs may be seeking a safe place to oviposit as an established harborage is proof that bed bugs are able to obtain a blood meal there. Replete bugs made more visits and spent more time above the odor pot than unfed bugs. My study agrees with Weeks et al.’s (2013) findings that fed females make more visits to the odor zone than unfed females. However, I observed

28 that virgin females made more visits to the male zone than mated females, whereas

Weeks et al. (2013) found the opposite using a mixed sex odor pot.

Average distance to each zone may not be as reliable a proxy for female choice as frequency and duration in each zone which necessitate that bed bugs actually visit the odor zone. Distance may be confounded with arena exploration and edge seeking behavior, and may not reflect attraction to the male odor zone.

While my study generally agreed with other studies that found bed bugs are attracted to harborages (Levinson & Bar Ilan 1971; Weeks et al. 2011, 2013), there were some notable differences. One drawback to Levinson and Bar Ilan’s (1971) study is that

10 bugs were released at a time, and bed bugs are known to aggregate in response to physical contact with each other (Usinger 1966). Weeks et al. (2011) controlled for this effect and tested one bug at a time. Weeks et al. (2011) used odor pots which contained a filter paper tent that had previously harbored bed bugs. These harborages did not contain live bed bugs, which may affect attraction to the odor pot.

The costs of superfluous matings can be high for females, and includes suboptimal re-mating frequencies, reduced longevity, and reduced reproductive success

(Stutt & Siva-Jothy 2001). However, the benefits of resting in a harborage may outweigh any fitness consequences from unwanted matings. Bed bugs that aggregate together have greater resistance to desiccation (Benoit et al. 2007). The harborage may also be a sign of a safe location to digest a blood meal and/or be used to avoid humans and predators.

In conclusion, my experimental evidence demonstrates that replete bugs have increased attraction to harborages compared to unfed bugs, and that females in general

29 are attracted to a male harborage. Interestingly, feeding status had a stronger effect on harborage attraction than mating status. While I did not find statistical support for my hypotheses, it is apparent that body condition affects the degree to which bed bugs are attracted to harborages. In future studies, the odor stimulus and the mating rate for mated females may be increased to obtain stronger results.

30

Chapter 4

Feeding Limits Male Reproductive Success

Introduction

Increased body size is typically thought of as improving fitness but it can also have viability costs in adults and juveniles due to increased predation, parasitism, or starvation (reviewed in Blanckenhorn 2000). In some species, there is sexual selection favoring small males (Andersson 1994). Large males can also have decreased mating success due to decreased agility or physiologically incompatibility with females

(Blanckenhorn 2000). For example, in midges small males have higher mating success than large males due to being more aerobatic (Neems et al. 1990).

Feeding and reproduction are tightly linked in the bed bug; males will preferentially mate with replete females rather than unfed females (Reinhardt et al. 2008).

Replete females have nutrients to produce eggs and may also be less able to resist male mating attempts due to their bulbous and vulnerable post-feeding state. Since optimal female and male mating rates are at odds, males may attempt to copulate during situations when females are unable to resist. Reinhardt et al. (2008) found that males have increased mating success when mating with replete females rather than unfed females and prefer to mate with replete and experimentally enlarged females. While there have been anecdotal reports of replete males having difficulty mating I found no studies on this subject. Females increase their body volume by >300% when they feed (Titschak 1930). 31

Males have smaller blood meals than females but still increase their body volume by at least 150% (Titschak 1930). Replete males appear to struggle reaching the female spermalege with their intromittent organ due to their large body size. In previous behavioral experiments, I have observed replete males attempt to mount a female, struggle for several minutes, and then dismount (SH personal observation).

If feeding status affects male and female mating ability, then as the opportunity for feeding changes, the ratio of reproductively viable males to females will change. In my study, I examined how feeding status of males and females affected their mating success. My hypotheses were that

1. Unfed males with replete females should have the highest mating success due to

being the most morphologically compatible and having female mating resistance

lowered by her blood meal.

2. Replete males with replete females should have the next highest mating success as

they are near the same size and are usually morphologically compatible.

3. Unfed males with unfed females will be the third most successful. They are

morphologically compatible but females have no nutritional resources to produce

eggs.

4. Replete males with unfed females will be the least successful because they are

morphologically incompatible.

32

a)

b)

Fig 4.1 A replete male and female, where a) the male partially mounts the female, struggles to contact the spermalege and b) dismounts and retreats.

33

Methods

I used a factorial design to evaluate how male and female feeding statuses affect mating success. Four treatments were I) replete male x replete female (RM x RF), II) replete male x unfed female (RM x UF), III) unfed male x replete female (UM x RF), and

IV) unfed male x unfed female (UM x UF). Females and males used in this study were virgin, and were fed one hour before the trial. Because bed bugs are more likely to feed if they see or make contact with a replete bed bug (SH personal observation), I fed them communally. To keep track of female identity, I marked females with paint markers on the dorsal side of their abdomen as minimally as possible. I weighed both sexes pre- and post-feeding to calculate their blood meal size.

To determine the fecundity of the four types of mating pairs, I paired a single female and male in close quarters in a 24-well plate. Any bugs that did not mate in 20 minutes were considered non-matings and latency to mate was recorded as 20 minutes. I also recorded mating duration, clutch size, and egg viability. Clutch size and egg viability were measured by examining female wells with a dissection scope five to eight days after oviposition. Viable eggs can be readily distinguished from non-viable eggs by red eye spots on the developing embryo. Additionally, non-viable eggs can be diminished in size and may be brown instead of the usual white color (SH personal observation).

I tested assumptions of the two-way ANCOVA for latency to mate, mating duration, clutch size, and egg viability. All samples were independent random samples, variance was homoscedastic, and outliers were checked for with the Mahalanobis distance method. However, all dependent variables had non-normal distributions. Mating

34 duration achieved a normal distribution by log transformation but the other variables did not. For the non-normally distributed variables I used the Kruskal-Wallace H test to examine the effect of male pre-feeding weight, female pre-feeding weight, and male and female blood meal size on latency to mate, clutch size, and egg viability. I used a significance level of α=0.05 and JMP 11 (SAS Institute Inc.) to analyze all data.

Results

I tested the effects of male pre-feeding weight, female pre-feeding weight, and male and female blood meal size on the log of mating duration and found non-significant results ( =0.73, P=0.729). Replete males had a longer latency to mate than unfed males (Fig 4.2a, H=29.37, P<0.001). I also tested the effects of male and female pre- feeding weight, mating duration, male and female blood meal size, and feeding status on whether or not females laid eggs. After dropping non-significant parameters (male and female pre-feeding weight, female blood meal size) I saw a significant effect of female feeding status, male feeding status, and male blood meal on whether or not females laid eggs ( =5.54, P =0.002). Replete females laid more eggs than unfed females (Fig

4.1b, H=13.77, P<0.001). Refer to table 4.1 for statistics on the effect of non-significant variables on latency to mate, clutch size, and egg viability.

35

1200 a) A 1000 A 800

Latency to B 600 B mate 400

200

0 R♂ x R♀ R♂ x U♀ U♂ x R♀ U♂ x U♀ Treatment

8 b) 7 6 5 B A 4 Clutch 3 Size 2 C C 1 0 -1 -2 R♂ x R♀ R♂ x U♀ U♂ x R♀ U♂ x U♀ Treatment

Fig 4.2 The effect of male and female feeding status on mean (±1SE) a) latency to mate and (b) clutch size. For each variable, statistical differences between treatments are represented by letter groups.

36

Male Pre-feeding Male Blood Meal Female Pre- Female Blood Weight feeding Weight Meal Test P- Test P- Test P- Test P- Statistic Value Statistic Value Statistic Value Statistic Value Latency to H=98.84 0.373 H=47.94 0.475 H=87.06 0.681 H=46.28 0.584 Mate Clutch Size H=30.56 0.437 H=7.00 0.423 H=32.00 0.467 H=25.00 0.462 Egg Viability H=32.96 0.371 H=8.00 0.433 H=33.00 0.467 H=25.00 0.462

Table 4.1 The non-significant effects of male and female pre-feeding weight and blood meal size on latency to mate, clutch size, and egg viability.

N Mean latency Mean mating Mean clutch Mean egg

to mate ± SE duration ± SE size ± SE viability ± SE

Replete ♂ x 24 858.20±88.32 148.90±28.13 3.14±0.76 0.91±0.06

Replete ♀

Replete ♂ x 25 1056.50±86.60 137.50±36.32 0.08±0.70 0.00±0.17

Unfed ♀

Unfed ♂ x 27 406.75±83.45 107.70±18.55 6.36±0.66 0.92±0.04

Replete ♀

Unfed ♂ x 29 442.83±80.62 137.96±18.55 0.53±64 1±0.06

Unfed ♀

Table 4.2 Treatment, sample size, and mean ± SE for latency to mate, mating duration, mean clutch size, and egg viability.

37

Discussion

As predicted, replete males had lower mating success than unfed males. Replete males also had a longer latency to mate than unfed males, representing their difficulty in properly mating. While replete males could mate successfully with replete females, replete males and unfed females were completely incompatible. I often observed replete males mount a female, struggle to contact the spermalege with their paramere, and then dismount. I also observed replete males sitting on top of replete and unfed females for extended periods of time without any mating attempt. This may be a form of mate guarding wherein males have prolonged contact with females post-insemination in order to reduce mating attempts and sperm competition from other males (Alcock 1994). Half of the blood meal is lost as excreted fluid in the first 5 hours after feeding (Omori 1941), so if a male can prevent other males from mating with a guarded female he will be able to successfully mount her in a reasonable amount of time. The size of the male blood meal was negatively correlated with clutch size, indicating that there may be selection for males to have smaller blood meals in order to have a better chance of mating successfully.

It is not surprising that female feeding status positively correlated with mating success. Females need a blood meal to lay a full clutch of eggs (Usinger 1966), although unfed females may have some nutritional reserves from their last molt to lay a few eggs.

If males have difficulty mating when replete, and females cannot produce eggs unless they’ve recently had a blood meal, then the operational sex ratio can vary week to week based on host availability. Lab-reared populations of bed bugs typically have a sex

38 ratio of 1:1 (Johnson 1941), although there is potential for populations to become male- biased due to male harassment and excessive traumatic insemination (Usinger 1966). It is uncertain if these factors affecting the OSR are strong enough to influence sexual selection and more studies are needed exploring the OSR in natural populations.

In conclusion, male feeding status has a strong effect on male mating success and mating strategy. When feeding can negatively affect a male’s mating success, it is in his best interest to mate before feeding. Indeed, as mentioned in Chapter 2 of my thesis, unfed males encountered females at the blood source and attempted to mate instead of feed. While these results may predict selection for smaller male blood meals and reduced male feeding rate, bed bugs are relatively long lived insects (up to1 year) (Usinger 1966) and males likely still need nutrients to replenish energy and ejaculate.

39

Chapter 5

Consequences of Inbreeding in the Bed Bug

Introduction

Lack of genetic diversity decreases fitness. A meta-analysis found a significant positive correlation between measures of genetic diversity and measures of population fitness in invertebrates, plants, and vertebrates (Reed and Frankham 2003). Deleterious mutations can accumulate due to drift in small populations and homozygosity increases expression of recessive alleles resulting in inbreeding depression (Keller and Waller

2002). Heterosis, or improved fitness via outbreeding is hypothesized to be the result of decreased expression of recessive deleterious mutations (Charlesworth and Willis 2009).

One way that females can increase the genetic diversity of their offspring is to mate with unrelated males, or novel males. Females can gain direct or indirect benefits from mating with unrelated or novel males. Direct benefits of mating may be in the form of increased fertility, nutritional resources, or parental care. Indirect benefits of mating include genes that increase the survival or reproductive success of offspring. For an inbred population, indirect benefits may also include increased genetic diversity. There are several ways that animals can avoid inbreeding: dispersal, extra-pair copulations, cryptic sperm choice, recognition and avoidance of kin, and delaying maturation or reproductive senescence (Pusey and Wolf 1996). For example, in the orb-web spider,

Argiope lobata, females preferentially store sperm from non-siblings rather than siblings

(Welke and Schneider 2009). Inbreeding in another spider, Oedothorax apicatus, led to

40 major declines in fecundity and hatching rates of eggs (Bilde et al. 2007). When inbreeding depression has high costs, females that mate with non-relatives stand to gain indirect benefits.

The vast majority of bed bug populations are inbred (Fountain et al. 2014, Saenz et al. 2012). Bed bugs infestations can be started by as few as one mated female, which result in extreme population bottlenecks. Fountain et al. (2014) found very low genetic differentiation within infestations and high genetic differentiation between infestations

( =0.59). Saenz et al. (2012) also found high genetic differentiation between infestations ( =0.68) and no geographic pattern of genetic structure was found.

Populations that were close in proximity were nearly as genetically differentiated as those separated by hundreds of kilometers (Saenz et al. 2012). These results suggest that infestations are usually started by a single source population, founded by a few individuals and that there is a low amount of migration between infestations. Since bed bug populations are naturally inbred, it stands to reason that bed bugs have either adapted to inbreeding or have strong mechanisms to avoid inbreeding.

In this study I examined how inbreeding and outbreeding in two inbred lines of bed bugs affected their clutch size, hatching rate, and mating interactions. My hypotheses were that:

Part 1: Female choice for non-related males

1. Outbred pairs will have a shorter latency to mate and shorter mating durations due

to behavioral adaptations to choose non-related mates.

41

Part 2: Effects of inbreeding on clutch size and egg viability

2. Outbred pairs will lay more eggs than inbred pairs and the hatch rate will be

higher in outbred pairs than inbred pairs due to inbreeding depression.

Methods

I used EPM and Jersey colonies for both parts of this experiment. EPM was collected in Columbus, OH on August 25, 2010 and has had approximately 28 generations in the lab. Jersey was collected in Jersey City, New Jersey on June 7, 2012 and has had approximately 18 generations in the lab. Considering that bed bugs were fed approximately 2-4 times a month, and not all bugs fed every time, I estimated that a bed bug would develop from egg to adult in approximately 2 months, when a new generation would start.

Part 1: Female choice for non-related males

I used a factorial design was used to see how colony identity affected mating interactions. Mating interactions were between strains EPM and Jersey and the four treatments were I) EPM males x Jersey females (Me x Fj), II) EPM males x EPM females

(Me x Fe), III) Jersey males x EPM females (Mj x Fe), and IV) Jersey males x Jersey females (Mj x Fj). Females and males used in this study were virgin, and females were fed one hour before the trial. Because bed bugs are more likely to feed if they see or make contact with a replete bed bug (SH personal observation), I fed them communally.

To keep track of female identity, I marked females with paint markers on the dorsal side of their abdomen as minimally as possible. I weighed both sexes before mating and weighed females post-feeding to obtain their blood meal size.

42

To observe matings, I placed bed bugs in a plastic circular arena with filter paper on the bottom and replaced the paper each trial to control for bed bug scent accumulation in the arena. I filmed under red light during their scotophase; bugs were shown to prefer red and black harborages over other colors (Singh et al. 2015). I gave bed bugs 20 minutes to mate and if they did not mate in 20 minutes their latency to mate was recorded as 20 minutes.

I tested assumptions of the one-way ANOVA. For latency to mate and mating duration; all samples were independent random samples, variance was homoscedastic, and outliers were checked for with the Mahalanobis distance method. However, latency to mate and mating duration had non-normal distributions, so these variables were log transformed which made the distributions normal. The null hypothesis for the one-way

ANOVA tests was that there is no statistical difference between treatments. I used a significance level of α=0.05. JMP 11 (SAS Institute Inc.) was used to analyze all data.

Part 2: Effects of inbreeding on clutch size and egg viability

I used a factorial design to evaluate how colony identity and sex affected fitness.

The four treatments were the same as in part 1. I only used virgin bugs for this experiment. I marked females with paint markers on the dorsal side of their abdomen as minimally as possible. This enabled me to feed them communally and keep track of their identity as I noticed that bed bugs are more likely to feed if they see or make contact with a replete bed bug. If females fed, I paired them with an unfed male in close quarters until they mated. I verified mating was taking place by observing the paramere making contact with the ectospermalege and started the clock once any female shaking had subsided. I allowed bugs to mate for one minute so that females received a relatively equal amount

43 of ejaculate as ejaculate volume and copulation duration have a linear relationship (Siva-

Jothy and Stutt 2003). I then separated them with gentle forceps and females were held in

24-well plates. I weighed both sexes before mating and weighed females post-feeding to obtain their blood meal size. I inspected the females daily and kept a tally of their eggs and if they were viable, first by checking for red eye spots and then verifying that they eclosed. Eye spots typically appear when eggs are five days old.

The assumptions of the one-way ANOVA were tested. For clutch size and egg viability, all samples were independent random samples, variance was homoscedastic, and outliers were checked for with the Mahalanobis distance method. Clutch size followed a normal distribution, but egg viability did not. Therefore, one-way ANOVA was used to make comparisons between treatments for clutch size, and the Kruskal-

Wallace test used to make comparisons between treatments for egg viability. I used two- sample t-tests to make comparisons between populations for variables female weight pre- feeding, female blood meal size, and male weight. The assumptions of the two-sample t- tests were verified for each variable: populations followed a normal distribution, standard deviations were equal, and samples were independent. The null hypothesis for all tests was that there is no statistical difference between treatments. A significance level of

α=0.05 was used for all statistical methods. JMP 11 (SAS Institute Inc.) was used to analyze all data.

44

Results

Part 1: Female choice for non-related males

I examined the effect of female colony, male colony, the interaction between female and male colony, blood meal size, and male and female unfed weight on log(latency to mate) and found no significant effect. I dropped highly insignificant parameters (male colony, male and female blood meal size, male unfed weight), and found a statistically significant effect of female unfed weight and female colony on log(latency to mate) (See Fig 5.2a, =3.51, P=0.033). I examined the effect of female colony, male colony, the interaction between female and male colony, blood meal size, and male and female unfed weight on log(mating duration) and found no effect.

After dropping highly insignificant paremeters (male identity, the interaction between male and female colony, and blood meal size), I found that female colony and male weight had a statistically significant effect on log(mating duration) (See Fig 5.2b,

=6.43, P=0.002).

45

300 A A a) A 250 A

200

Latency to 150 Mate (s) 100

50

0 Jer♀ x Jer♂ EPM♀ x Jer♂ Jer♀ x EPM♂ EPM♀ x EPM♂ Treatment

250 b) A A A 200 A

150 Mating Duration (s) 100

50

0 Jer♀ x Jer♂ EPM♀ x Jer♂ Jer♀ x EPM♂ EPM♀ x EPM♂ Treatment

Fig 5.1 The effect of male and female colony on mean (±1SE) (a) latency to mate (b) mating duration. Statistical differences between treatments were found as represented by letter groups.

46

Treatment N Mean Latency to Mate ± SE Mean Mating Duration ±

SE

Jersey ♀ X Jersey ♂ 29 221±57 175±42

EPM ♀ X Jersey ♂ 30 179±56 134±42

Jersey ♀ X EPM ♂ 30 205±56 151±42

EPM ♀ X EPM ♂ 29 210±57 173±42

Table 5.1 Treatment, sample size, and mean ± SE for latency to mate and mating duration.

Part 2: Effects of inbreeding on clutch size and egg viability

I tested the effects of female identity, female weight pre-feeding, female blood meal size, male identity, male weight, and if it was an inbred or outbred pairing on clutch size and found the model to be statistically significant (See Fig 5.1a, =10.37,

P<0.001), with female weight pre-feeding (P=0.002), female blood meal size (P<0.001), and male identity (P=0.012) significantly contributing to clutch size. I also looked at the effect of female identity, female weight pre-feeding, female blood meal size, male identity, male weight, eggs laid, and if it was an inbred or outbred pairing on hatch rate and found that no parameter estimates had a statistically significant on hatch rate (See Fig

5.1b). There were no significant differences in female weight pre-feeding, female blood meal size, or male weight in between colonies. Hatch rate was not affected by any parameter, although there was a slight trend for lower clutch sizes to have lower hatch rates, except for one female who laid many infertile eggs.

47

14 A a) 12 AB 10 B B 8 Clutch Size 6

4

2

0 Jer♀ x Jer♂ EPM♀ x Jer♂ Jer♀ x EPM♂ EPM♀ x EPM♂ Treatment

1 b) 0.98 A A 0.96 A

0.94 Hatch A 0.92 Rate 0.9

0.88

0.86

0.84 Jer♀ x Jer♂ EPM♀ x Jer♂ Jer♀ x EPM♂ EPM♀ x EPM♂ Treatment

Fig 5.2 The effect of male and female colony on mean (±1SE) (a) clutch size (b) egg viability. Statistical differences between treatments were found as represented by letter groups

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Treatment N Clutch Size ± SE Hatch Rate ± SE

Jersey ♀ X Jersey ♂ 28 11.38±0.81 0.97±0.03

EPM ♀ X Jersey ♂ 27 9.61±0.83 0.92±0.03

Jersey ♀ X EPM ♂ 28 8.07±0.81 0.94±0.03

EPM ♀ X EPM ♂ 29 7.17±0.81 0.93±0.03

Table 5.2 Treatment, sample size, and mean ± SE for clutch size and hatch rate.

Discussion

Inbreeding did not have a negative effect on fitness and bed bugs did not preferentially mate with non-related partners. Several meta-analyses have found a negative correlation between inbreeding and fitness across a wide diversity of organisms

(Reed and Frankham 2003, Crnokrak and Roff 1999, Keller and Waller 2002). For example, in the butterfly, Melitaea cinxia, larval survival, adult longevity, and egg- hatching rate were all negatively affected by inbreeding. In that system inbred individuals increased their fitness by mating with unrelated individuals from another population

(Saccheri et al. 1998).

In part 1, I found that female colony and male weight had an effect on mating duration with Jersey females mating longer than EPM females. Female unfed weight had an effect on latency to mate, and female identity, while not significant, still had a trend where Jersey females had a longer latency to mate than EPM females. Male weight negatively correlated with mating duration, agreeing with my findings in Chapter 4. Since

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I did not find inbreeding depression in bed bugs, it stands to reason that there is no mate choice for non-related mates.

Male colony affected clutch size with Jersey males fathering more eggs than EPM males. This is likely due to heritable properties of each line because there was no difference in male or female weight between the sources. This finding gives the impression that the identity of the father is more important than the mother in determining egg clutch size, but the mechanism for this phenomenon is not clear. It is not surprising that female pre-feeding weight and blood meal size have an effect on egg number as each can be considered a measure of nutrition available for producing eggs. I found no evidence supporting my hypothesis that outbred pairs lay more eggs and have higher egg viability than inbred pairs.

My results suggest that inbreeding in bed bugs may not have a negative effect on reproductive success as has been found in many species. One theory predicts that inbreeding can positively affect the inclusive fitness of parents because an individual that mates with a relative will help that relative to spread identical genes (Kokko and Ots

2006). If mating success is not diminished by inbreeding, then there could a great benefit via inclusive fitness to reproduction among close relatives. Additionally, if a population has local adaptations it may be more beneficial to mate with a colony member than an individual from another population. Bed bugs live in a stable environment compared to other animals. In the bed bug’s environment temperature and humidity are relatively constant, and a food source is readily available. Heterozygosity and genetic variance may not confer an advantage in such a stable environment.

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