Wolbachia in Bedbugs Cimex Lectularius
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Wolbachia in bedbugs Cimex lectularius Louise Lynne Heaton Submitted for the degree of Doctor of Philosophy, September 2013 Department of Animal and Plant Sciences, University of Sheffield To Dora and Elizabeth Lewis who inspired in me a passion to learn and explore and helped set me on the path to where I am today. Abstract The global rise in numbers of bedbug infestations has been facilitated by increased global travel and climate change, and further compounded by widespread pesticide resistance. The resurgence of this blood-sucking pest is costing the hospitality industry millions of pounds each year and non-chemical control strategies are urgently needed. The recent discovery that bedbugs rely on symbiotic Wolbachia bacteria for the B vitamins necessary for normal egg production, growth and development, has highlighted a possible biocontrol strategy through reducing Wolbachia infection loads. The aim of my thesis is to better understand the nature of the Wolbachia-bedbug symbiosis and the mechanisms by which Wolbachia achieve effects in their host in order to determine whether such biocontrol strategies are likely to succeed. I develop protocols to manipulate Wolbachia infection loads using heat or antibiotic treatment to test hypotheses relating to (i) the fitness effects of Wolbachia infection for male hosts; (ii) the fitness effects of Wolbachia infection for female hosts and (iii) the underlying mechanisms through which Wolbachia achieve their effects. My research has revealed that Wolbachia increase the fitness of both male and female hosts via a range of mechanisms. The male’s Wolbachia increase the male host’s fitness through increasing the egg laying rate of the female he has mated with. I found no evidence that Wolbachia are transferred in the male’s ejaculate. Wolbachia may affect egg laying rate through raising the quality of the male’s ejaculate, such as via direct nutritional provisioning or by enabling the male to invest in a higher quality ejaculate. Another symbiont in male bedbugs may also influence ejaculate production. Wolbachia increase the female host’s fitness by increasing her egg hatch and development success (proportion of eggs that hatched or reached adulthood), increasing offspring body size (which will increase her indirect fitness by increasing the fitness of her offspring) and reducing the fitness costs (wounding and infection) associated with traumatic insemination. This latter effect was associated with an enhanced melanisation response. i Despite the widespread fitness benefits that Wolbachia provide to both male and female hosts, biocontrol methods involving a reduction in Wolbachia loads in natural bedbug populations are unlikely to be effective. (i) It is not possible to reduce the Wolbachia load (population size) of all individuals in a bedbug infestation due to the logistical difficulties in administering antibiotic treatment. Heat treatment to reduce Wolbachia loads is logistically feasible but does not present an effective alternative to antibiotics, as the reduction in bedbug fitness is too subtle (even though significant) and short-lived. (ii) A reduction in Wolbachia load does not kill the bedbugs outright, so infestations will remain after biocontrol has taken place. (iii) The ecology of bedbugs precludes the release of sterile males – a biocontrol strategy used for other pest insects –as a biocontrol strategy for this system. ii Acknowledgements First a huge thank you to Mike Siva-Jothy who has been a continual source of encouragement, creativity, blood and a sounding board for ideas – Thank you! I would like to thank past and present members of the E3 lab who critiqued my work and with whom many a lively discussion was shared. Special mention must go to Oliver Otti and Richard Naylor who always had time to offer help and advice. A special thank you to Rich imparting some of his knowledge of all things in the lab, for feeding my bug stocks enabling my experiments to keep on schedule and for being a good friend. I would also like to thank all the students that volunteered their time to carry out mini-projects in the lab over their summer holidays, in particular Eva Lartigau, Dominic Swain, Rebecca Wilson and Conor Waldock. A special thank you to Jennie Garbutt who helped me learn how to carry out qPCR, Toby Fountain who taught me DNA extraction (with an electric toothbrush!), Andy Krupa who helped ensure my qPCRs ran smoothly; Chris Hill who patiently helped with TEM imaging; and to Andrew Beckerman for help with statistics. I must also thank my family, in particular my mother and late father Lynne and David Eaves for their support, encouragement and much, much more. I am grateful also for their understanding in my choice to change career direction and return to university as a mature student to study ecology; this was the best choice I ever made. Last, but by no means least, I must thank my husband James Heaton for his patience, support, love, encouragement ... I couldn’t have done it without you! iii Table of Contents Abstract.......................................................................................................... i Acknowledgements....................................................................................... iii Table of contents........................................................................................... iv List of figures................................................................................................. xiii List of plates.................................................................................................. xvi List of tables.................................................................................................. xvii Abbreviations................................................................................................. xix Terms and definitions................................................................................... xxii Chapter 1: Introduction................................................................................. 1 1.1 Bedbugs................................................................................................ 2 1.2 Insights from the bedbug laboratory system......................................... 3 1.3 Life cycle............................................................................................... 3 1.4 Reproduction......................................................................................... 4 1.5 Immunity................................................................................................ 6 1.5.1 Cellular immunity........................................................................ 6 1.5.2 Humoral immunity...................................................................... 6 1.5.3 Symbiont-mediated immune effects........................................... 7 1.6 Wolbachia.............................................................................................. 7 1.6.1 Symbiosis................................................................................... 7 1.6.1.1 Primary and secondary symbionts............................... 8 1.6.1.2 Reproductive parasites................................................ 9 1.6.2 General biology.......................................................................... 10 1.6.2.1 Characteristics and occurrence.................................... 10 1.6.2.2 Host phenotypes.......................................................... 11 1.6.2.3 Phylogeny.................................................................... 11 1.6.2.4 Application.................................................................... 12 1.7 Symbionts in bedbugs........................................................................... 13 1.7.1 Overview of previous research................................................... 13 1.7.2 Questions arising from the literature.......................................... 16 iv 1.7.2.1 What is the role of Wolbachia in males?...................... 16 1.7.2.2 What are the long-term fitness consequences of Wolbachia?.............................................................................. 16 1.7.2.3 Can Wolbachia operate through non-nutritional mechanisms?........................................................................... 17 1.8 Summary............................................................................................... 17 1.9 Thesis outline and core questions......................................................... 17 Chapter 2: General Methods......................................................................... 20 2.1 Introduction............................................................................................ 20 2.1.1 Aims of chapter.......................................................................... 20 2.2 Insect techniques.................................................................................. 20 2.2.1 Bedbug husbandry..................................................................... 20 2.2.2 Experimental cohorts................................................................. 21 2.2.2.1 Sex determination........................................................ 21 2.2.2.2 Measurement of body size........................................... 22 2.2.2.3 Distinguishing individuals............................................. 22 2.2.2.4 Standardised mating.................................................... 23 2.2.3 Feeding...................................................................................... 23