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 i 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. iii 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 iv 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 vi 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 vii 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 viii 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 ix 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