Bed Bug (Cimex lectularius) Defecation Behavior Following a Blood Meal THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Courtney Lynn Darrington Graduate Program in Entomology The Ohio State University 2015 Master's Examination Committee: Susan C. Jones, Advisor Pete Piermarini Diana Ortiz Copyright by Courtney Lynn Darrington 2015 Abstract Bed bugs are reported to harbor >40 human pathogens, however, their potential for disease transmission remains poorly understood. Basic feeding behaviors have been documented, but few studies record defecation, and none report whether a bed bug defecates while feeding. We tested the hypothesis that bed bug defecation behavior could facilitate pathogen transmission, and we used the stercorarial system of pathogen transmission documented in triatomines as a model for comparison. We describe post- feeding defecation behaviors of bed bugs, including a long-term laboratory strain, Harlan, and two field collected populations, EPM and Shalamar. In initial trials with Harlan fifth instars and adults on an artificial feeder, the majority defecated less than a minute after withdrawing their mouthparts and within 25 mm of the feeding site. However, there were qualitative differences depending on the stage, with adult feces dropping onto the substrate whereas nymph feces remained on the anus. The defecation index (DI) of Harlan fifth instars was 1.71; Harlan adults had a DI of 2.31, with females having a much higher DI (2.72) than males (1.90); similar trends for the sexes have been documented in triatomines. Adult bed bugs were the subjects in all subsequent trials, and we observed that they moved a relatively short distance from their feeding site to the site of their first defecation (mean 1.0-2.5 cm), regardless of whether they fed on an artificial feeder or a live host (naked rat). Adult females took the largest blood ii meals and defecated closer to the feeding site than adult males. In studies with live rats, nearly one-third of all bed bugs defecated on the host after their meal was completed, and defecation occurred within 5 min or less. Replete bed bugs were almost ten times more likely to defecate than non-replete bed bugs. Our studies indicate that bed bug post-feeding defecation behavior could facilitate potential disease transmission. Furthermore, based on a stercorarial system of pathogen transmission, female bed bugs would be most epidemiologically important stage. A significant barrier to successfully rearing bed bugs in the laboratory is their typical unwillingness to feed from an artificial system, which perhaps stems from the absence of host cues. Carbon dioxide (CO2) is a major host signal detected by bed bugs, but there are no studies documenting its effect on feeding behavior. We compared bed bug feeding success on an artificial feeding system when exposed to a high flow rate of CO2 (2700 mL/min) or no CO2 (control). We tested bed bugs from four populations: Harlan; Shalamar, a recently collected (2014) field population; and Cuyahoga and Marcia, both collected in 2010. Our study documented an increased feeding response of bed bugs from four populations when CO2 was present at a high flow rate rather than absent. Within populations, Cuyahoga and Marcia had the greatest feeding response, with Cuyahoga significantly so. Exposing feeding bed bugs to a high flow rate of CO2 may be a useful solution for rearing bed bug populations that feed poorly from an artificial system. iii This document is dedicated to my family. iv Acknowledgments I would like to thank my advisor, Dr. Susan C. Jones, for getting the best work possible out of me, even when we both wanted to pull our hair out. I also thank my committee for their support and feedback on my research project. I am profoundly grateful to Frances Sivakoff for assistance with statistical analyses. I thank my past and present lab mates Josh Bryant, Scott Harrison, Kara Baker, Alex Tyrpak, Nina Bogart, Olimpia Ferguson, and Tyler Eaton, who helped me with presentations, statistics, observation set-up and cleanup, feeding bugs, refining methodology or just being a sounding board, and so many other things. Thanks to Dave Shetlar for being a referee and for always coming up with sensible wording. I also thank my colleagues and friends for their encouragement and support. Very special thanks to my family for their unending prayers and love, and to my Heavenly Father for instilling in me a desire to learn all I can about His creations, and my Savior Jesus Christ for helping me to be more than I could be on my own. My heart goes to Matt Wojnar whose patience is without measure and whose love knows no bounds. v Vita June 1993 .......................................................Bellbrook High School April 2000 ......................................................B.S. Animal Science, Brigham Young ........................................................................University 2000 to 2010 ..................................................Certified Veterinary Technician, ........................................................................Timpanogos Animal Hospital 2012................................................................MPH-VPH, Veterinary Public Health, The ........................................................................Ohio State University 2012 to present ..............................................Graduate Teaching Associate, Center for Life Sciences Education, The Ohio State University Publications Tiao N., C. Darrington, B. Molla, W.J.A. Saville, G. Tilahun, O.C.H. Kwok O.C.H., W. A. Gebreyes, M. R. Lappin, J. L. Jones, J. P. Dubey. 2012. An investigation into the seroprevalence of Toxoplasma gondii, Bartonella spp., feline immunodeficiency virus (FIV), and feline leukaemia virus (FeLV) in cats in Addis Ababa, Ethiopia. Epidemiol. Infect. 141: 1—5. Dubey J. P., C. Darrington, N. Tiao, L. Ferreira, S. Choudhary, B. Molla, W.J.A. Saville, G. Tilahun, O.C.H. Kwok, W. A. Gebreyes. 2013. Isolation of viable Toxoplasma gondii from tissues and feces of cats from Addis Ababa, Ethiopia. J. Parasitol. 99: 56—58. vi Dubey, J. P., G. Tilahun, J. P. Boyle, G. Schares, S. K. Verma, L. R. Ferreira, S. Oliveira, N. Tiao, C. Darrington, W. A. Gebreyes. 2013. Molecular and biological characterization of first isolates of Hammondia hammondi from cats from Ethiopia. J. Parasitol. 99: 614—618. Fields of Study Major Field: Entomology vii Table of Contents Abstract ............................................................................................................................. ii Dedication ........................................................................................................................ iv Acknowledgements ........................................................................................................... v Vita ................................................................................................................................... vi List of tables ..................................................................................................................... xi List of figures .................................................................................................................. xii Chapter 1: A review of previous bed bug research ........................................................... 1 Taxonomoy and biology ................................................................................................ 1 Resurgence .................................................................................................................... 3 Resistance to pesticide .................................................................................................. 4 Social and economic implications ................................................................................ 5 Public health implications ............................................................................................. 7 Disease transmission ..................................................................................................... 9 Vector status................................................................................................................ 14 References ................................................................................................................... 16 Chapter 2: Bed bug (Cimex lectularius) defecation behavior following a blood meal .. 24 Introduction ................................................................................................................. 26 viii Materials and methods ................................................................................................ 29 Bed bugs .................................................................................................................. 29 Food sources and feeding procedures ...................................................................... 30 Artificial feeder ..................................................................................................... 30 Live host................................................................................................................ 31 Response variables ................................................................................................... 32 Trials ........................................................................................................................ 33 Initial trial.............................................................................................................