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WOLBACHIA DISTRIBUTION AND REPRODUCTIVE EFFECTS IN POPULATIONS OF A NEOTROPICAL BEETLE, CHELYMORPHA ALTERNANS BOH. (CHRYSOMELIDAE) by GWEN P. KELLER (Under the Direction of KENNETH G. ROSS) ABSTRACT Wolbachia is a group of maternally-inherited endocellular bacteria that manipulate arthropod reproduction to favor infected females. While strains of these bacteria have been found in all insect orders where at least 17% of all insect species infected, few studies have investigated the distribution and effects of Wolbachia in natural populations. Here I present a review of Wolbachia research in the context of host biology (chapter 2) and I describe original research (chapter 3) that details the distribution and effects of Wolbachia in a Neotropical beetle, Chelymorpha alternans, that is infected with two Wolbachia strains, wCalt1 and wCalt2. In chapter 3 I describe the two strains based on three Wolbachia genes, I track their distribution across space and time in host populations throughout the Panamanian isthmus, I determine strain effects on host reproduction, and I show a correlation between environmental effects and the ongoing loss of one strain. This is the first study to show the loss of a Wolbachia strain from natural populations and I discuss the possible factors responsible for the loss. INDEX WORDS: Wolbachia, Chelymorpha, cytoplasmic incompatibility, strain loss WOLBACHIA DISTRIBUTION AND REPRODUCTIVE EFFECTS IN POPULATIONS OF A NEOTROPICAL BEETLE, CHELYMORPHA ALTERNANS by GWEN P. KELLER University of Georgia, 2005 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2005 © 2005 GWEN P. KELLER All Rights Reserved WOLBACHIA DISTRIBUTION AND REPRODUCTIVE EFFECTS IN POPULATIONS OF A NEOTROPICAL BEETLE, CHELYMORPHA ALTERNANS by GWEN P. KELLER Major Professor: Kenneth G. Ross Committee: Patty A. Gowaty Joe V. McHugh Donald Champagne Wyatt W. Anderson Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2005 DEDICATION With great affection I dedicate this work to Lois Edgell and Joaquin Recuero, two loyal friends whose cheerfulness and humor buoyed me through the trials of pursuing this degree. Your faith in me, and constant friendship has made all the difference in my life. Muchisimas gracias mis queridos amigos!! iv ACKNOWLEDGEMENTS I wish to thank my advisors, Ken Ross and Patty Gowaty for always being available, encouraging and marvelous! Cheers to Jack Werren for his willingness to share his knowledge of all things Wolbachia, and for his amazing grant writing abilities that enabled much of this research. Special thanks to Don Windsor for introducing me to the world of leaf beetles, especially Chelymorpha, and to Jenniffer Saucedo whose extraordinary beetle husbandry contributed greatly to this work. I also fondly thank the kind folks at the Smithsonian Institution in Washington, D.C. and the Smithsonian Tropical Research Institute in Panama for logistical and financial support. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ........................................................................................................v LIST OF TABLES....................................................................................................................vii LIST OF FIGURES .................................................................................................................viii CHAPTER 1 INTRODUCTION TO THE DISSERTATION..........................................................1 2 INTRODUCTION AND LITERATURE REVIEW OF THE BIOLOGY OF WOLBACHIA IN ARTHROPODS ........................................................................4 INTRODUCTION.................................................................................................5 EARLY HISTORY ...............................................................................................6 WOLBACHIA MANIPULATION OF HOST REPRODUCTION..........................8 REFERENCES ...................................................................................................22 3 REPRODUCTIVE EFFECTS AND GEOGRAPHIC DISTRIBUTIONS OF TWO WOLBACHIA STRAINS INFECTING THE NEOTROPICAL BEETLE, CHELYMORPHA ALTERNANS BOH. (CHRYSOMELIDAE)............................28 INTRODUCTION...............................................................................................29 MATERIALS AND METHODS.........................................................................31 RESULTS...........................................................................................................39 vi CHAPTER 3 DISCUSSION.....................................................................................................46 REFERENCES ...................................................................................................56 4 CONCLUSIONS.....................................................................................................71 vii LIST OF TABLES Page Table 1: Collection and site information....................................................................................60 Table 2: Egg hatch rates from crosses .......................................................................................62 Table 3: Mitochondrial CO1 haplotype and nucleotide diversity estimates................................63 Table 4: Parameters for invasion model ....................................................................................64 viii LIST OF FIGURES Page Figure 1: Chelymorpha alternans populations sampled in Panama ............................................65 Figure 2: Wolbachia infection status and C. alternans haplotype distributions in Panama populations ...............................................................................................................66 Figure 3: Frequency of the wCalt2 strain in populations with varying length of dry season in 19 populations ...............................................................................................................67 Figure 4: Egg hatch rates for females ........................................................................................68 Figure 5: The effects of double infected males from Remedios (Rem), Gamboa (Gam) and Cana (Can) on egg hatch rates............................................................................................69 Figure 6: Statistical parsimony network of C. alternans haplotypes...........................................70 ix CHAPTER 1 - INTRODUCTION 1 INTRODUCTION Increasing interest in reproductive parasites of arthropods over the past 10 years has led to the discovery of several species of maternally-inherited endosymbiotic bacteria that manipulate host sex ratios to favor infected female hosts. Among these bacteria, Wolbachia is the most common and shows a broad range of host effects including parthenogenesis, male feminization, male-killing, and cytoplasmic incompatibility (O’Neill et al. 1997; Werren 1997; Stouthamer et al. 1999). Standard techniques of bacterial identification rely on morphology and nutritional require- ments in culture. However, endocellular bacteria, such as Wolbachia, have eluded culture because of their fastidious requirements. Wolbachia survive only within host cells, thus culturing Wolbachia requires culturing insect cells, which is difficult to do. Thus, culturing techniques for identification of Wolbachia are impractical considering that Wolbachia infect at least 17% of species in the class Insecta, a group that is estimated to contain 106 - 306 species. The recent and rapidly evolving development of molecular genetic techniques such as clon- ing, polymerase chain reaction (PCR), and automated DNA sequencing has significantly advanced the study of fastidious microbes. These techniques allow researchers to quickly isolate and identify DNA of bacterial strains and thus avoid the time-consuming tasks involved in culturing fastidious bacteria. Using eubacterial DNA primers, strings of oligonucleotides that are conserved across eubacterial species, researchers isolated Wolbachia genes from insect tissues and created Wolbachia-specific primers for those genes. These primers specifically amplify Wolbachia products. Because of their specificity, researchers are able to quickly screen host tissues for the presence of Wolbachia strains. By cloning Wolbachia products from PCR reactions and then sequencing them, it is possible to determine the number of strains present in a single host by determining the number of variable sequences present in the sample taken from 2 that host. To verify the identity of strains, additional genes should be sequenced because cryptic strains produced by recombination could be present but not obvious if they share the same DNA sequence at one locus. Likewise, to determine whether the Wolbachia genes represent true strains rather than DNA sequences that have been transferred to the host genome, it is best to sequence several Wolbachia genes as it is unlikely that the entire Wolbachia genome would be transferred to the host. The development of molecular techniques for identifying Wolbachia strains has created a landslide of studies investigating the distribution, variation, and evolution of Wolbachia in insects. However, there is a paucity of studies investigating the effects of Wolbachia in natural insect populations. My research aims to shed light on the effects of Wolbachia in natural populations of Neotropical insects. In the following

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