Inherited Parasites in the Butterfly Hypolimnas bolina (Lepidoptera: Nymphalidae)
by
Emily Ann Dyson
A thesis submitted for the degree of Doctor of Philosophy of the University of London
September 2002
Department of Biology University College London ProQuest Number: 10010111
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 “The first sight of such a thing as the big
Hypolimnas bolina,Linn., black, flashing with violet-blue,
excited an emotion better imagined then described.
At all events, the creatures took me fairly by storm:
collect I must!”
G.B. Longstaff
‘Butterfly hunting in many lands: notes of a field naturalist’, 1912
Inherited parasites in the butterfly Hypolimnas bolina Abstract
Male killing bacteria are known to be widespread in insects, but the factors determining their presence and effects on natural populations are little studied. Studies of the island- inhabiting Hypolimnas bolina were therefore conducted to elucidate the causes and consequences of prevalence variation. Initial investigation of//, bolina in Fiji revealed the presence of a male killing Wolbachia bacterium associated with low egg hatch rates and all-female broods. The prevalence of the male killing Wolbachia is heterogeneous between islands. Sampling in the islands of Independent Samoa indicated the continued presence of highly female-biased populations in this country, associated with the same male killing Wolbachia strain identified from Fiji. The prevalence of the male killer in
Independent Samoa is extreme, and this has severe implications on the host population: the lack of males is associated with increased female virginity and decreased female fertility. The effect of infection on female host survival was examined in Fiji: larvae infected with the male killing Wolbachia bacterium show significantly higher survivorship and are significantly heavier as adults indicating a direct benefit to infection. A prevalence survey was carried out using//, bolina samples from eight different countries. Prevalence is heterogeneous across the butterfly’s range. Another strain of Wolbachia (that does not kill males) is identified from H. bolina populations in both American Samoa and Fiji. The history of the two different Wolbachia infections was investigated through sequence analysis of the mitochondrial COI genes from butterflies deriving from each of the populations. It is concluded that whilst the male killer has undergone a recent selective sweep, the non male killing Wolbachia strain is ancient. The data also indicated that horizontal transmission of the male killing symbiont must be rare.
Inherited parasites in the butterfly Hypolimnas bolina Acknowledgements
One of the most amazing aspects of this Ph.D. study has been working for such long periods of time in so many different countries. Of course this increased the numbers of people - not only who made the work side of things possible - but, equally importantly, made me feel welcome and at home on the other side of the world. Moving around so much has meant adjusting to new places and new ways of life constantly. There are many people to whom Pm indebted for making this last three years an unforgettable experience, here are a few of them:
In Fiji: the Ministry of Forestry provided a laboratory, equipment, friends and assistance. Everyone made me feel welcome and included. Particular thanks go to Eminoni, Eliki, Peni, Willi and Rashmi for their carpentry, butterfly-catching, caterpillar^feeding, sweet potato growing and grog-drinking skill’s. Wilco Liebregts and Madhu Kamath made it possible for me to work at Forestry and provided invaluable advice throughout my 7. months with them. Lashings of gratitude to Suzie, Joe and Joshua without whom I might have had a place to stay/but I wouldn’t have had a home and family. Thanks to you guys and the regular. . faces at the Colonial Lodge: Amelia, Emosi, Tina, Paul, Sue and Yolanda...and, of course, to Angelina. To my bodyguards Craig, Glen and Rob and my friends Tanja, Lynette, Diana and Louisa. Wayalailai Island will always be my epitome of paradise - huge thanks to Glenbo for introducing us, and for all the entertaimnent. To the islanders, especially Si and Big Jerry for putting up with all the butterflies and ferrying me between islands to collect. Thanks to all at the Nadi Bay for always finding room for me and the bugs, especially to Elanoa and Mariah. To Mai and Vani in Taveuni for all their help with collection and to Andrew, Angela and all at Matareva, Kadavu for teaching me to dive, breaking my foot and saving my life.
In Independent Samoa: the biggest basket of thank yous must go to Paul Davies and Raema Von Reiche - my landlords, advisors, drinking partners and friends. Also to their children: Chris, Blake, Jordon and especially Luana ‘demon-butterfly-catcher’ Milroy. Finally thanks to my friends Oscar, Christine and Helen.
In American Samoa: thanks to Barry and Charles for all the advice, transport around the islands and rescuing me from the killer dogs.
In Australia: thanks to Darrell Kemp for his collaboration, help and interest and also to Sarah and Brad Love for a fantastic week’s break and silliness.
Menno Shilthuizen and Sabah University, Borneo: for arranging my work permit, collecting butterflies, driving us around and attempting to drown us with the aid of a landrover and deceptively large puddle...thanks Menno!
To Walter and Lalith in Sri Lanka, and of course the countless children throughout the countries I visited who helped out with the collecting side of things...not always the correct species, but catching butterflies using a fishing net is never easy!!
Thanks to Niklas Wahlberg and El in Claridge for providing butterflies from areas that I didn’t make it to.
Inherited parasites in the butterfly Hypolimnas bolina In the UK: Firstly to the BBSRC, without whom none of this would have been possible. To Gwyneth and Bernard Johnston for their help and fascinating stories about carrying out the original survey oiH. bolina with Sir Cyril Clarke.
Mike Majerus and Frank Jiggins in Cambridge provided advice to a novice butterfly hunter, thanks for all the help and information on all aspects of my research.
A huge thank you to all at UCL, particularly Claire, Imogen, Michelle, Mel, and Jim Mallet, my second supervisor. To the past and present chosen elite of room 417, Wolfson House: infinite thank yous to Mary Webberly because she’s fantastic...for her unstinting kindness, generosity, support, advice and friendship. Also to Jo Bentley for putting up with me and my various crises, and being lovely.
Everyone has down times doing a Ph.D., but it’s hard when you’re thousands of miles from home, so a big thank you to all my friends for their support, via e-mail and back at home: To Miki, Kevin, Adam, Dan, Tim, Nick, Olivia, Vicky, Rachel, Marc, Erica, Charlie, JP, Robj the UCL girls and of course the Seth Effrikans: Ben, Paul, Taryn, Shannon, Marc, Jules, Craig, Heidi and Ma and Pa Travis.
To my housemates Adam, Tam and Andrew for putting up with me and all my papers scattered around the place in various states of disarray.
Massive thank you hugs to my mum and dad for being so supportive, wonderful and keeping calm when their daughter kept disappearing to the other side of the world chasing insects, and to Tom who is, and always will be, the fattest brother in the world.
Throughout all the island-hopping, round-the-world travelling and long frustrating spells in the lab in London, one person constantly provided support and direction, had cheeky ‘holidays’ in the sun, showed endless patience in teaching not the most natural molecular biologist the world will ever see, and made this last 3 years enjoyable and successful: my supervisor, Greg Hurst. As the first of (I’m sure) many Ph.D. students, a huge thank you to Greg.
This thesis is dedicated to Adrian Travis for more reasons than you can shake a stick at: his excellence at butterfly catching (whilst obtaining a perfectly even tan), advice, constant support, putting up with my tantrums, always making me laugh and of course the best chicken impressions this side of the Watford Gap. Kuo la na kana, my Adrian: this one’s for you...
Inherited parasites in the butterfly Hypolimnas bolina Declaration
This dissertation represents, except
where specifically mentioned in the text, the results of my own research.
The dissertation is not substantially the same as any
that I have submitted for a degree or other qualification this or any other University.
No part of my dissertation has already been or is being currently submitted
for any such degree or other qualification.
Emily Dyson Dr. Gregory Hurst
Candidate Supervisor
Inherited parasites in the butterfly Hypolimnas bolina Contents
List of abbreviations ...... 16 Glossary...... 17 Chapter 1: Introduction 1.1. Symbiotic Interactions ...... 21 1.1.1. Endosymbionts ...... 22 1.1.2. Hereditary symbioses ...... 22 1.2. Reproductive Parasitism ...... 25 1.2.1. Cytoplasmic incompatibility (Cl) ...... 25 1.2.2. Féminisation ...... 28 1.2.3. Parthenogenesis induction (PI) ...... 29 1.2.4. Male killing ...... 30 1.3. Embryonic Male Killing ...... 32 1.3.1. Characteristics and incidence of male killers ...... 32 1.3.2. Why kill males? ...... i... 34 1.3.3. Direct (physiological) effects of infection with male killers 35 1.3.4. Prevalence variation in natural populations ...... 36 1.4. Male Killers: Questions That Need To Be Addressed ...... 37 1.4.1. What is the mechanism of male killing? ...... 38 1.4.2. Can male killers drive the evolution o f sex determination ? ...... 38 1.4.3. What causes prevalence and incidence variation? ...... 39 1.4.4. What effects do male killers have on the host population ?...... 40 1.4.5. Phylogeographic inferences from mitochondrial DNA ...... 42 1.5. Study System: Hypolimnas bolina (Lepidoptera: Nymphalidae) ...... 43 1.5.1. Historical evidence of all-female broods in H. bolina ...... 44 1.5.2. Island nature ...... ,...... 45 1.5.3. Indication of prevalence variation ...... /.... 45 1.6. Questions To Be Addressed Using The Yio^X Hypolimnas bolina ...... 46 1.6.1. Is H. bolina infected with a male killing symbiont? ...... 46 1.6.2. Does infection prevalence vary across populations? ...... 46 1.6.3. Inferences from mitochondrial D NA ...... 48 1.6.4. Consequences on host population of variations in prevalence 48 Chapter 2: Association of All-Female Broods in Hypolimnas bolina with Wolbachia infection Summary...... 53 2.1. Summary Of Previous Work On Hypolimnas bolina In F iji ...... 54 2.2. Aims...... 55 2.3. General Methods For Fieldwork In Fiji ...... 55 2.3.1. Sample collection...... 55 2.3.2. Rearing specimens ...... 56 2.4. Do All-Female Broods Still Occur In The Fiji Islands? ...... 59 2.4.1. Egg hatch rates ...... 59 2.4.2. Adult sex ratio ...... 62 2.5. Are Hypolimnas bolina Larvae Cannibalistic? ...... 64 2.5.1. Method ...... 64 2.5.2. Results...... 65 2.5.3. Conclusion...... 67
Inherited parasites in the butterfly Hypolimnas bolina 2.6. Are The All-Female Broods In Hypolimnas bolina Caused By A Bacterium?...... 67 2.6.1. Method ...... 67 2.6.2. Results ...... 69 2.6.3. Conclusion...... 71 2.7. Are Microorganisms Present In Female-Biased Lines? ...... 72 2.7.1. Method...... 72 2.7.2. Results...... 72 2.7.3. Conclusion...... 73 2.8. Is The Infecting Microorganism A Eukaryote? ...... 73 2.8.1. Method: protocol of specimen preparation...... 73 2.8.2. Results...... 75 2.8.3. Conclusion...... 76 2.9. Molecular Analysis Of Laboratory-Bred Specimens ...... 77 2.9.1. Molecular analysis of original wild-collected female H. bolina 79 2.9.2. Molecular analysis offemale progeny from antibiotic-treated lines...... »... 80 2.10. Phylogenetic Position Via Sequence Analysis ...... 81 2.10.1. Method ...... 81 2.10.2. Results and phylogenetic position ...... 82 2.10.3. Conclusion...... 85 2.11. Prevalence Of The Male Killing Wolbachia Across Fiji ...... 85 2.11.1. Historical evidence of prevalence variation ...... 85 2.11.2. Method...... 87 2.11.3. Results...... 88 2.11.4. Conclusion...... ;..... 88 2.12. Discussion ...... 89 Chapter 3: Extraordinary Sex Ratios In Independent Samoa Summary...... 94 3.1. Why 8\\x6y Hypolimnas bolina In Independent Samoa and American Samoa? ...... 95 3.1.1. Historical evidence of female-bias in H. bolina in the Samoas 95 5.7.2. Implications of historical evidence ...... 96 3.2. Aims Of Fieldwork In The Samoas ...... 97 3.3. Introduction To Independent Samoa And American Samoa ...... 97 3.4. General Methods For Fieldwork In Independent Samoa ...... 99 3.4.1. Rearing specimens ...... 100 3.5. Observed Population Sex Ratios In Fiji, American Samoa and Independent Samoa ...... 103 3.5.1. Method ...... 103 3.5.2. Results ...... 103 3.5.3. Conclusion...... 104 3.6. Do All-Female Broods Occur \n Hypolimnas bolina In Independent Samoa? ...... 105 3.6.1. Egg hatch rates ...... 105 3.6.2. Adult sex ratio ...... 107 3.7. What Is The Causative Agent Of All-Female Broods In Independent Samoa? ...... 108 3.7.1. PCR analysis for Wolbachia ...... 109 3.7.2. Sequence analysis ...... 109
Inherited parasites in the butterfly Hypolimnas bolina 3.8. Prevalence Of The Male Killing Wolbachia In Independent Samoa And American Samoa ...... 110 3.8.1. Method...... 110 3.8.2. Results...... 110 3.8.3. Conclusion...... I l l 3.9. Comparison Of Female Virginity In Fiji, American Samoa And Independent Samoa ...... 112 3.9.1. Method...... 112 3.9.2. Results...... 112 3.9.3. Conclusion...... 113 3.10. Comparison Of Female Fertility In Fiji And Independent Samoa ...... 114 3.10.1. Method...... 114 3.10.2. Results...... 115 3.10.3. Conclusion...... ;...... 117 3.11. Comparison Of Spermatophore Sizes ...... 117 3.11.1. Method...... 117 3.11.2. Results...... 118 3.11.3. Conclusion ..^...... 120 3.12. Does The Presence Of A High Prevalence Male Killer Alter Behaviour . Oi Hypolimnas bolina In Independent Samoa? ...... 120 3.12.1. General methods for site fidelity experiments ...... 121 3.12.2. Site fidelity experiment one: Taveuni Island, Fiji ...... 123 3.12.3. Site fidelity experiment two: Wayalailai Island, Fiji ...... 125 3.12.4. Site fidelity experiment three: Upolu Island, Independent Samoa. 127 3.12.5. Site fidelity experiment four: Upolu Island, Independent Samoa... 129 3.12.6. Conclusions from site fidelity experiments ...... 131 3.13. Discussion ...... 133 3.13.1. Why has resistance to the Wolbachia male killer not evolved in the Independent Samoan population ofH. bolina ? ...... !.>...... 135 3.13.2. Population genetic consequences of a high prevalence male killing symbiont ...... 136 3.13.3. Why hasH. bolina in Independent Samoa not been driven to extinction ?...... 139 3.13.4. Migration from other islands: specimen SAM155 ...... 139 Chapter 4: Looking For Direct Effects Of Wolbachia Infection On Hypolimnas bolina Survival And Fecundity Summary...... 143 4.1. Introduction ...... 144 4.1.1. Direct effects associated with cytoplasmically-inherited reproductive parasites (excluding male killers) ...... 145 4.1.2. Direct effects associated with male killing ...... 150 4.2. Hypolimnas bolina - Wolbachia Symbiosis As A Study System For Examination Of Direct Effects Of Male Killing ...... 153 4.2.1. What indirect benefits might be expected? ...... 153 4.2.2. What direct benefits might be expected? ...... 155 4.3. Experimental Design ...... 156 4.4. A im ...... 157
Inherited parasites in the butterfly Hypolimnas bolina 10
4.5. Method ...... 157 4.5.1. PCR assay for Wolbachia ...... 157 4.5.2. Experiment one: method ...... 158 4.5.3. Experiment two: method ...... 159 4.5.4. Experiment three: method...... 161 4.6. Results...... 162 4.6.1. Experiment one: group-reared Fijian larvae ...... 162 4.6.2. Experiment two: singly-reared Fijian larvae ...... 163 4.6.3. Experiment three: singly-reared Independent Samoan larvae 170 4.7. Conclusions And Discussion ...... 175 Chapter Five: Prevalence And History OfWolbachia Infection In Hypolimnas bolina Across The Butterfly’s Range Summary...... 182 5.1. Introduction ...... 183 5.1.1. Why does male killer prevalence vary within populations of certain host species ...... 183 5.1.2. H. bolina/Wolbachia symbiosis as a case study for prevalence vdriation ...... 186 5.2. Aims...... 187 5.3. Prevalence Survey Oi Hypolimnas bolina ...... 187 5.3.1. Molecular analysis ...... 188 5.3.2. Prevalence o f the B-group (male killing) Wolbachia ...... 189 5.3.3. Prevalence oftheA-group Wolbachia ...... 190 5.3.4. Comparison o f wild-collected female matedness rates ...... 193 5.3.5. Comparison of wild-collected female spermatophore sizes ...... 194 5.3.6. Conclusions from prevalence surveys ...... 198 5.3. 7. Sequence analysis and phylogeny of the A-group Wolbachia .... 200 5.4. Introduction To The Use of Mitochondrial DNA In Evolutionary Studies.... 203 5.4.1. Can mtDNA divergence be employed to resolve the phylogenetic history of a species?...... 203 5.4.2. Effect o f spread o f a selfish genetic element on mtDNA diversity.... 205 5.4.3. Interpretation o f schematic diagram ...... 208 5.5. Previous Mitochondrial DNA Diversity Studies In Male Killer Infected Hosts ...... 212 5.5.1. Host: Adalia bipunctata ...... 212 5.5.2. Host'. Acraea encedon ...... 212 5.6. A im ...... 213 5.7. Analysis Oi Hypolimnas bolina Infection History Using Mitochondrial DNA...... 214 5.7.1. Method...... 214 5.7.2. Results...... 215 5.7.3. Population genetic analysis ...... 221 5.7.4. Interpretation o f results and conclusion ...... 223 5.8. Discussion ...... 225 5.8.1. Wolbachia prevalence in H. bolina ...... 225 5.8.2. Phylogenetics o f Yiy^oXimndiS...... 226 5.8.3. The paradox o f transmission efficiency ...... 227
Inherited parasites in the butterfly Hypolimnas bolina 11
Chapter 6: General Discussion 6.1. Summary Of Results...... 230 6.1.1. Identification of the causal agent and prevalence survey: Fiji Islands ...... 231 6.1.2. Questions addressed using the H.bolina/Wolbachia symbiosis 231 6.1.3. High prevalence infection: Independent Samoa ...... 232 6.1.4. Evidence for a direct benefit to infection ...... 233 6.1.5. Prevalence o f the male killer across the butterfly’s range ...... 234 6.2. Future Work ...... 235 6.2.1. Prevalence variation ...... 235 6.2.2. Applications o f prevalence variation in the study of mating systems...... 238 6.2.3. Effects on genetic diversity ...... 241 6.2.4. The last word ...... 242
R e fe re n c e s ...... 245
A p p e n d ix I A.I. Protocol For DNA Preparation ...... 264 A.2. Primer Sequences ...... 265 A.3. PCR Protocols ...... ! 266 A.3.1. Assay for Wolbachia via amplification o/wsp gene...... 266 A.3.2. Amplification of CO I gene ...... 267 A.3.3. Assay for Wolbachia A-groi//? using amplification of 16s subunit.. 267 A.3.4. Assay for Wolbachia using amplification of the ftsZ gene...... 268 A.4. Assessing PCR Results ...... 268 A.5. Sequence Analysis ...... 269 A. 5.1. Preparation o f purified PCR product for sequence analysis 269 A. 5.2. Calculation of DNA concentration ...... 269 A.5.3. Preparation of purified PCR product for sequence analysis 269 A.6. Restriction Digest ...... 270
A p p e n d ix II: Photographs ...... 271
Inherited parasites in the butterfly Hypolimnas bolina 12
List Of Tables Chapter 1: Introduction 1.1. Viability of crosses between host strains of different infection status in a population infected with a single Cl strain ...... 26 1.2. Viability of crosses between host strains of different infection status in a population infected with two different Cl strains ...... 27 1.3. Biodiversity of early male killers with examples of a host species from each of the orders infected by a male killing symbiont in each clade...... 33 1.4. Prevalence of male killing bacteria in natural populations of their insect hosts ...... 37 Chapter 2: Association of All-Female Broods in Hypolimnas bolina with Wolbachia infection 2.1. Egg hatch rates of all Fijian females grouped into low and high hatch 61 rates...... 2.2. Sex ratios produced by wild-collected (parental) female H. bolina from Viti Le vu Island, and in the subsequent FI generation ...... v.... . 63 2.3. Results of cannibalism experiment.» ...... 66 2.4. The seven different types of antibiotic treatment regime received by FI fourth instar larvae ...... 69 2.5. Total amounts of antibiotic consumed by each larva in each matriline, following one of the seven treatment regimes ...... 70 2.6. Egg hatch rates and adult sex ratio in the F2 generation from FI individuals that were treated with antibiotics ...... 71 2.7. Numbers of all-female and normal sex ratio matrilines previously reported by Simmonds and Clarke on five different Fijian Islands ...... 85 2.8. Percentage prevalence of the Wolbachia male killing bacteria in female H. bolina from three different island populations across Fiji ...... 88 Chapter 3: Extraordinary Sex Ratios In Independent Samoa 3.1 Summary of FI adult sex ratios from Independent Samoan H. bolina 108 3.2. Percentage prevalence of the Wolbachia male killer in H. bolina females across different islands of Independent and American Samoa ...... I l l 3.3. Summary of results of site fidelity experiment one ...... 124 3.4. Summary of results of site fidelity experiment two ...... 126 3.5. Summary of results of site fidelity experiment three ...... 128 3.6. Summary of results of site fidelity experiment four ...... 130 Chapter 4: Looking For Direct Effects Of Wolbachia Infection On Hypolimnas bolina Survival And Fecundity 4.1. Summary of studies of host fitness effects associated with cytoplasmically-inherited parasites (excluding male killers) ...... 144 4.2. Studies of host fitness effects resulting from male killer infection ...... 151 4.3 Putative direct physiological benefits to females infected with a cytoplasmically-inherited symbiont relative to uninfected controls ...... 153 4.4. Summary of individual survivorship data, sex ratios and egg hatch rates from each of the nine matrilines within each of the three replicates of experiment two ...... 165 4.5. Development rate of infected and uninfected female larvae in each of the three replicates of experiment two ...... 167
Inherited parasites in the butterfly Hypolimnas bolina 13
4.6. Summary of individual survivorship data, sex ratios and egg hatch rates from each of the five matrilines in experiment three ...... 171 4.7. Development rate of infected and uninfected female larvae in experiment three ...... 173 Chapter Five: Prevalence And History OfWolbachia Infection In Hypolimnas bolina Across The Butterfly’s Range 5.1. Summary of Clarke et al ’s 1975 results on the numbers of female-biased broods recorded from different populations of H. bolina indicating variation in prevalence of the male killing trait ...... 186 5.2. Prevalence of Wolbachia male killer across different populations of 77. bolina ...... 190 5.3. Prevalence of the A-group Wolbachia among the different study populations of 77. W m a...... 192 5.4. Proportion of mated females from the different populations as evidenced by the presence of a spermatophore in the bursa copulatrix ...... 194 5.5. Median diameter and length of spermatophores dissected from mated77. _ bolina females from the different study populations ...... 195 5.6. Results of Kruskal-Wallis test of spermatophore length across all study populations ...... 196 5.7. Results of Kruskal-Wallis test of spermatophore diameter across all study populations except Independent Samoa ...... 197 5.8. Results of Kniskal-Wallis test of spermatophore diameter across all study populations...... 197 5.9. The eight different mitochondrial haplotypes identified among the COI sequences of thirty-four 77. bolina samples together with infection status and geography of the host...... 217 5.10. Position of variable sites within the eight different 77. bolina haplotypes identified from sequence analysis of part of the mitochondrial COI gene 218 5.11. Haplotype frequencies and Nei’s diversity by infection status ...... 222 5.12. Fst values inferred from pairwise comparison of populations of differing infection status ...... 222 5.13. AMO VA analysis of differentiation between samples of different infection status ...... 223
Inherited parasites in the butterfly Hypolimnas bolina 14
List Of Figures Chapter 2: Association of All-Female Broods in Hypolimnas bolina with Wolbachia infection 2.1. Map of Viti Levu Island ...... 56 2.2. Transmission electron micrograph of ovarian tissue taken from an F2 female H. bolina from an all-female matriline showing a prokaryotic cell enclosed in a vacuole ...... 76 2.3. Flow chart showing the sequence in which PCR analyses were performed and how results were interpreted ...... 78 2.4. Photograph of agarose gel showing results of PCR for Wolbachia wsp 80 gene ...... 2.5. Maximum likelihood tree of the Wolbachia ftsZ gene ...... 83 2.6. Maximum likelihood tree of the Wolbachia wsp gene ...... 84 2.7. Map of the Fiji Islands ...... 87 Chapter 3: Extraordinary Sex Ratios In Independent Samoa 3.1.a. Map of Independent Samoa ...... 98 3.1.b. Map of American Samoa ...... 99 3.2. Diagram showing the construction of the laying cages ...... 101 3.3. Proportion of female H. bolina observed whilst collecting in Independent Samoa, American Samoa and Fiji ...... 104 3.4. Percentage of fertilised eggs that hatch from Independent Samoan female H. bolina collected from Savaii and Upolu islands, and in the subsequent 106 generation ...... 3.5. Proportion of wild-caught H. bolina females from different island populations that had mated once, twice and three times ...... 113 3.6. Median proportion of fertile eggs laid by wild-collected females from different islands, and virgin Independent Samoan females crossed with American Samoan or Fijian males ...... 116 3.7. Median size of spermatophore (a) diameter, (b) length ...... 119 3.8. Layout of study area, experiment one ...... 123 3.9. Layout of study area, experiment two ...... 125 3.10. Layout of study area, experiment three ...... 127 3.11. Layout of study area, experiment four ...... 129 3.12. Proportion of adults recaptured during consecutive days in all four site fidelity experiments ...... 132 Chapter 4: Looking For Direct Effects Of Wolbachia Infection On Hypolimnas bolina Survival And Fecundity 4.1. Measurement of right fore- and hind- wings of 77. bolina females...... 161 4.2. Median proportion of larvae, reared in groups of ten, surviving to adulthood in infected and uninfected matrilines in experiment one ...... 163 4.3. Median proportion of infected and uninfected individually-reared larvae that survived to adulthood in each of the three replicates of experiment two 166 4.4. Summary of development rates of infected and uninfected female larvae in experiment tw o ...... 168 4.5. Median dry weight of infected and uninfected adult females in each of the three replicates of experiment two ...... 169 4.6. Summary of mean wing measurements from experiment two, replicate one ...... 170
Inherited parasites in the butterfly Hypolimnas bolina 15
4.7. Proportion of infected and uninfected individually-reared larvae that survived to adulthood in experiment three ...... 177 4.8. Median dry weight of infected and uninfected adult females in experiment three ...... 174 4.9. Summary of mean wing measurements from experiment three ...... 175 Chapter Five: Prevalence And History OfWolbachia Infection In Hypolimnas bolina Across The Butterfly’s Range 5.1. Phylogeny of Wolbachia based on wsp gene sequences showing the phylogenetic positions of the 77. bolina A- and B- group infections ...... 201 5.2. Schematic illustration of the effect of horizontal transfer on mitotype diversity...... 211 5.3. mtDNA phylogeny of77. bolina ...... 220 5.4. Schematic illustration of results of mitotype analysis of 77. bolina from different populations associated with differing infection statuses ...... 221
Appendix I A.I. Example results gel from Dral cut PCR products from a wsp PCR...... 270
Appendix II: Photographs A2.1. Laboratory workbench in Colo-I-Suva, Fiji Islands ...... 272 A2.2. Specially built mating cage, Colo-I-Suva, Fiji Islands ...... 271 A2.3. ‘Laboratory’ in Independent Samoa...... 271 A2.4.77. bolina mating pair ...... 273 A2.5. Mosquito net mating cage ...... 273 A2.6. Oviposition cages, Independent Samoa ...... 273
Inherited parasites in the butterfly Hypolimnas bolina 16
List of abbreviations
COI Mitochondrial cytochrome oxidase subunit I gene
Cl Cytoplasmic incompatibility
D API 4 ’ ,6 ’ -Diamidino-2-pheny lindole fisZ Filamentation temperature sensitivity gene
PCR Polymerase chain reaction
PI Parthenogenesis induction
TEM Transmission electron microscope wsp Wolbachia surface protein gene
Inherited parasites in the butterfly Hypolimnas bolina 17
Glossary
Arrhenotoky A form of reproduction in which unfertilised eggs develop
parthenogenetically into haploid males, and fertilised eggs
sexually into diploid females.
Direct benefit An increase in host fitness-related traits deriving directly
from the symbiont.
Fitness compensation The death of one individual releasing resources for
another, i.e. death of A is ‘compensated’ by increasing the
fitness of B.
Haplotype A non-recombining sequence of DNA (single copy type).
Horizontal transmission Transmission of a symbiont to individuals other than the
progeny of the infected individual (infectious
transmission).
Host The larger of two organisms in a symbiosis.
Incidence The distribution of a parasite across species.
Indirect benefit An increase in host fitness-related traits deriving from
effects of the infection within other hosts (usually
brothers).
Mitotype Haplotype of a mitochondrial gene or genes.
Modifier A gene that alters the expression or action of another gene
within the genome.
Mutualism A symbiosis where both organisms have increased fitness
as a result of the relationship.
Parasitism A symbiosis where one member derives an increase in
fitness at a cost to the other.
Inherited parasites in the butterfly Hypolimnas bolina 18
Parthenogenesis Development of an unfertilised gamete into a new
individual (commonly an egg cell).
Phylogeography The study of the evolutionary history of populations within
a species, associated with geographical location.
Prevalence The frequency of a parasite within a population (or unit) of
a species.
Symbiont The smaller of two organisms in a symbiosis.
Symbiosis An association, involving physical contact, between
members of two different species throughout a significant
proportion of their respective life histories.
Selfish genetic element A genetic element that spreads, despite causing damage (in
inclusive fitness terms) to individuals that bear it.
Thelytoky Completely parthenogenetic reproduction.
Vertical transmission The proportion of progeny of an infected individual that efficiency inherit the infection.
Vertical transmission Transmission of a symbiont to the progeny of the infected
individual.
Inherited parasites in the butterfly Hypolimnas bolina 19
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 20
Chapter 1: Introduction
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 21
Chapter 1: Introduction
1.1. Symbiotic Interactions
‘Symbiosis’ is derived from the Greek words ‘Sym’ and ‘Bios’, literally meaning life as one, and in its original sense refers to the intimate ‘living together’ of dissimilar organisms (de Bary, 1879). It is a word that has taken on a multitude of different uses and applications today: a ‘Web of Science’ search for symbiosis reveals documents as diverse as South African politics (Krabill, 2001), paint finishes (Rothbarth, 2001)and computing systems (Dhem & Feyt, 2001), as well as a host of biological literature. In the biological kingdom, symbiotic interactions are widespread and diverse, involving organisms from many different phyla. Symbioses generally involve two distantly related species. The smaller species is known as the symbiont, the larger is the host. The symbiont is generally a microorganism and its host a multicellular eukaryote. Such interactions often enable host species’ to exploit environments they would otherwise be unable to inhabit. For example, virtually all reef-building corals can only inhabit illuminated, nutrient poor waters because of the presence of intracellular photosynthetic algal symbionts that provide their host with essential photosynthate. Many biological fields benefit from research into symbiont/host interactions: aquatic ecology, biotechnology, population biology (Smith, 2001) and evolutionary biology (Watson &
Pollack, 1999) to name but a few. The latter represents the focus of this thesis, in particular reference to the evolutionary biology of interactions between endosymbiotic bacteria and their invertebrate hosts. This section discusses the different types of host/symbiont interaction, before focusing on the area of the symbiotic field with which this thesis is particularly concerned. It is important to note that throughout this work, the
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 22 word ‘symbiont’ refers to an organism that lives within the host’s body, especially within host cells, regardless of the effect of the symbiont on the host i.e. symbionts can be parasitic.
1.1.1. Endosymbionts
Microorganisms, particularly prokaryotes, have a high affinity for eukaryotic cells from amoebae through to mammals. This leads to a large variety of possible symbiont/host interactions, which have traditionally been classified as mutualistic (beneficial to host), commensal (neutral) or parasitic (harmful to host) (Hentschel & Steinert, 2001).
Removal of a bacterial symbiont by treating the host with antibiotics is a technique often employed to establish and classify the effect of the symbiont on the host (Stouthamer et al, 1990). However, it is much harder to determine how the association, particularly in supposed mutualisms, affects the symbiont itself. What is reasonably clear is that reciprocal manipulation by both parties plays a strong part in such interactions.
The method by which symbionts are transmitted to new hosts has implications on the biology of the symbiosis (Ewald, 1987) (Lipsitch er a l, 1995). Completely horizontal, or
‘infectious’ transmission occurs when a symbiont is transmitted to individuals other than the progeny of the infected host, for example malaria in humans that is transmitted between unrelated hosts via a mosquito vector (Rosenbaum & Sepkowitz, 2002). In completely vertical or ‘heritable’ transmission, the symbiont is only transmitted from parent to offspring. The majority of inherited symbionts employ this mode of transmission and such interactions are termed ‘hereditary symbioses’.
1.1.2. Hereditary symbioses
In interactions where the symbiont is vertically transmitted from host to host, the reproductive success of the host and symbiont are tightly linked, with both the survival and reproduction of the symbiont being totally dependent on that of their host(Werren &
O'Neill, 1997). Such inherited symbionts represent the most extreme form of symbiosis
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 23 and are widespread throughout the animal kingdom. Typically, symbionts are intracellular and transmitted in the cytoplasm of eggs, although other transmission mechanisms are also known (Buchner, 1965). Since the endosymbionts are maternally inherited, it would be expected that selection on these symbionts should directly select for beneficial effects towards the host sex that is responsible for transmission i.e. the female. These effects have been documented in all phenotypes of hereditary symbiosis.
1.1.2.1. Mutualistic heritable symbioses
Here both the host and the vertically transmitted parasite gain a direct benefit from the association. One of the most well-studied interactions involving an invertebrate host and prokaryotic symbiont is that seen between aphids (Homoptera) and bacteria in the genus
Buchneva (Baumann et a l, 1995). Aphids feed on phloem, a diet rich in carbohydrates but deficient in nitrogenous compounds. The bacterium Buchnera aphidicola is believed to synthesise a number of essential amino acids which the insects are unable to either metabolise or receive from their diet (Sabater et a l, 2001). The bacteria are maternally transmitted from one generation to the next (Wemegreen & Moran, 2001). The association between the host and symbiont is so strong that neither partner is able to survive in the absence of the other (Baumann et a l, 1995; Tamas et al, 2001). In fact the host is so dependent on the symbiont that aphids have evolved a specific structure called the mycetocyte whose sole function is to ‘store’ the symbiont (Moran & Telang, 1998).
It is intuitively sensible that in systems such as this in which symbiont transmission is totally dependent on that of the host, selection will favour symbionts that afford the host a direct benefit of some sort, for example the provisioning of essential nutrients as seen in aphids. Hence it is widely believed that hereditary symbiosis will inevitably evolve to a mutualistic interaction due to the fact that the host and symbiont have such a close association (O'Neill & Werren, 1997). However this is not necessarily the case, and there
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 24 is an ever-growing list of hereditary symbioses in which the symbiont has a negative effect on host phenotype (Bandi et a l, 2000).
1.1.2.2. Parasitic heritable symbioses
Heritable symbioses in which the symbiont has a negative effect on its host are often termed ‘reproductive parasitism’. The frequency of the symbiont increases within the host population despite the fact that it may be detrimental to the host’s overall fitness, with the symbiont gaining increased transmission by manipulating host reproduction to its own advantage. At first glance this seems counterintuitive, often raising questions such as: ‘surely because the symbiont transmission is completely dependent on its host, if the symbiont does not provide a direct benefit, the interaction will never evolve?’
However there are a number of ways in which vertically transmitted symbionts can be maintained in the host population despite the fact that they are not directly beneficial to their host: they can bias the sex ratio produced by infected hosts (usually females) towards the production of daughters, or decrease the fitness of uninfected (female) hosts through their action in males.
Reproductive parasites are known to occur throughout the arthropod groups (insects, mites and crustaceans) and have been assigned to different prokaryotic lineages and to the eukaryotic lineage of the microsporidia (O’Neill et al, 1997). Four main phenotypes of reproductive parasitism have been identified in arthropods. Their mechanism of action is different but all share the same underlying principles.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 25
1.2. Reproductive Parasitism
As a consequence of living inside cells, reproductive parasites are all vertically transmitted through maternal hosts. It follows that a male host is an ‘evolutionary dead end’ to the symbiont as a male is unable to transmit it. The symbiont lineage will disappear with the death of its male host, unless it is able to ‘escape’ to another host via horizontal transmission. One parasitic phenotype, cytoplasmic incompatibility (Cl) affects the viability of progeny depending on the infection status of the parents, without distorting the host sex ratio (Hoffmann & Turelli, 1997). Any effect of the microorganism’s metabolism that induces biases in the sex ratio towards the transmitting
(usually female) sex will also be selectively advantageous to the symbiont, provided the host’s ability to reproduce is not adversely damaged. There are three main phenotypes of reproductive parasitism resulting in sex ratio distortion: parthenogenesis induction, féminisation and male killing (Rigaud, 1997; Stouthamer, 1997; Hurst et a l, 1997a). The most widespread reproductive parasite is a diverse group of related alpha-Proteobacteria, collectively known as Wolbachia pipientis (referred to as Wolbachia throughout this work). Wolbachia is known to manipulate arthropod reproduction by employing all of the different parasitic phenotypes in different hosts (Stouthamer et a l, 1999); an explanation of these different phenotypes is given below.
1.2.1. Cytoplasmic incompatibility (Cl)
Parasites causing Cl are found only in the rickettsial genus Wolbachia. In Cl-infected populations, crosses between certain host strains are inviable, producing few, if any, adult progeny (Table 1.1.).
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 26
Table 1.1. Viability of crosses between host strains of different infection status in a population infected with a single Cl strain (V - viable cross; X - inviable cross)
Male Host
Infected Uninfected
Female Infected V V Host Uninfected X V
Mechanistically, the symbiont causes condensation of paternal chromatin, resulting in death of progeny in diploid hosts and conversion of daughters into sons in haplodiploid hosts (Hoffmann & Turelli, 1997; Turelli, 1994). A recent study has shown that there is a delay in breakdown of the paternal nuclear envelope prior to this chromosome condensation (Tram & Sullivan, 2002). Cl has been reported in many arthropod species, having been found in numerous insects as well as mites and isopod crustaceans
(Breeuwer, 1997; Cordaux et a l, 200.1; Breeuwer & Jacobs, 1996; Hoffmann & Turelli,
1997; Werren, 1997).
A particular host species can be infected with many different strains of Cl-causing
Wolbachia, and a single host individual may also exhibit more than one Cl infection
(Bordenstein & Werren, 1998; Rousset & Solignac, 1995). In this situation, crosses between hosts of differing infection strain may also be inviable (Table 1.2.).
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 27
Table 1.2. Viability of crosses between host strains of different infection status in a population infected with a two different Cl strains: A and B (V - viable cross; X - inviable cross)
Male Host
Infection Infection Uninfected A B
Infection A V X V
Female
Host Infection B X V V -
Uninfected X X V
In all cases, Cl-inducing Wolbachia exploit infected male hosts in order to reduce the fitness of uninfected female hosts and hence indirectly induce the spread of infection; as discussed earlier, males cannot normally transmit the bacterium.
Cl is probably the most widespread of the different parasitic phenotypes and as such has been extensively studied and its invasion dynamics modelled (for example, Charlat et a l ,
2001). Field studies oi Drosophila simulans infected with Cl-inducing Wolbachia show how selfish genetic elements can spread throughout the host population with extraordinary rapidity (Hoffmann cr a l, 1990; Turelli & Hoffmann, 1995). It is true however, that once a Cl-inducing Wolbachia strain has spread to a high prevalence within the population, there will be little death of hosts as the numbers of uninfected females will be extremely low. For this reason. Cl can be considered the most benign of the reproductive parasitic phenotypes.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 28
1.2.2. Féminisation
As the name suggests, feminising microorganisms convert nuclear genotypic males into functional, phenotypic females. Several species of crustacean hosts are reported to contain parasitic feminisers, which may be either bacterial or protozoan (Ginsburger-
Vogel, 1991; Smith & Dunn, 1991; Terry et a l, 1999). Féminisation renders the previously male host capable of vertical transmission of a cytoplasmic element, preventing the death of the feminising parasite in a male host that cannot transmit it.
Thus the number of productive hosts is increased, as is the probability of the symbiont reaching the next generation. As the frequency of infection increases, the population becomes (at least phenotypically if not genotypically) highly female-biased (Dunn et a l,
1995; Hurst, 1993).
The crustacean host, Gammarus duebeni has been found to be infected by at least four different microsporidian species all causing sex reversal (Bulnheim & Vavra, 1968;
Dunn et a l, 1993; Terry et a l, 1999; Dunn & Rigaud, 1998). Around half of all terrestrial isopod species (woodlice) are infected by feminising bacteria of the genUs
Wolbachia (Rousset et a l, 1992) with each species carrying a single symbiont lineage
(Bouchon et al, 1998). In some species or populations, for examplt Armidillidium vulgare (the common woodlouse) all females are infected, and the feminising reproductive parasite is at fixation (Marcade et al, 1999; Rigaud et a l, 1999). This situation illustrates one of the main conflicts faced in host/reproductive parasite systems in which the symbiont distorts host sex ratio. Sex ratio distorting parasites that spread to a high prevalence within the host population have implications on both host ecology and the evolution of host sex ratios. The increasingly female-biased population sex ratio will lead to increasing conflict between the host and parasite over host sex ratio. Selection on the parasite will favour a female-biased sex ratio as previously explained, but selection acting on the host genes will generally favour a 1:1 primary sex ratio (Fisher, 1930). In
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 29 the case of féminisation, high prevalence can lead to the emergence of novel sex determining mechanisms in the host (Bull, 1983; Taylor, 1990; Hatcher & Dunn, 1995;
Werren & Beukeboom, 1998). Eventually a situation is reached in which all female hosts are infected despite the fact that they are all genetically male (Hatcher & Dunn, 1995).
This conflict selects for host resistance to the action of the symbiont, as has been demonstrated in A. vulgare infected by the feminising parasite Wolbachia where sex determination has become based on nuclear genes that control the Wolbachia phenotype
(Rigaud & Juchault, 1993; Juchalilt et a l, 1993).
Féminisation has previously been reported only from the Crustacea. However, two cases of féminisation have recently been reported in insects: Osirinia furnaCalis, the Asian com borer (Lepidoptera: Crambidae) dind Eurema hecabe (Lepidoptera: Pieridae) are both infected with a different feminising strains of Wolbachia that appear to have independent evolutionary origins to the crustacean feminisers (Hiroki et a l, 2002;
Kageyama er al, 2002). The Wolbachia feminisers in the two orders show distinct characteristics that are likely to be relevant to differences in the sex determination mechanism of the two orders. In insects, the method of sex determination has only been well studied in D. melanogaster (Schutt & Nothiger, 2000) but it is believed that the mechanism of sex determination in Crustacea allows reproductive parasites to cause féminisation (Rigaud, 1997).
1.2.3. Parthenogenesis induction (PI)
Induction of parthenogenesis is mainly confined to the insect order Hymenoptera. Most uninfected hymenopteran species exhibit arrhenotoky, a form of reproduction in which unfertilised eggs develop parthenogenetically into haploid males, and fertilised eggs sexually into diploid females. However, in some members of the Hymenoptera, for example in certain parasitoid wasps in the genus Trichogramma, thelytoky i.e. complete parthenogenesis, is observed in females that are infected by a parthenogenesis inducing
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 3 0
Strain of Wolbachia (Stouthamer, 1997). The reproductive parasite enhances its cytoplasmic transmission by converting unfertilised eggs (which would normally develop into sons) into diploid daughters. Mechanistically, this is achieved by doubling the chromosome content of unfertilised eggs, thus restoring diploidy and producing females (Stouthamer & Kazmer, 1994). Consequently infected females produce many more daughters (all infected) than uninfected females, and the infection spreads - often to fixation. Eventually the ability to reproduce sexually is lost in the population (Zchori-
Fein et al, 1992). Treatment of infected lines with antibiotics, or exposure to high temperatures (Stouthamer et a l, 1990) both cause a reversion to normal arrhenotoky due to the suppressed function of the reproductive parasite. Parthenogenesis induction also occurs in the Encarsia genus of parasitoid wasps, however in this case PI is not always caused by Wolbachia but appears to be associated with a different bacterial clade named the 'Encarsia CFB bacterium’ (Zchori-Fein et al, 2001).
Outside of the Hymenoptera, PI has been reported in the Thysanoptera (predatory thrips)
(Arakaki et a l, 2001) and the mite Bryobiapraetiosa (Weeks & Breeuwer, 2001). In both cases, hosts are infected with Wolbachia endosymbionts.
1.2.4. Male killing
Male killing represents the most highly parasitic and diverse of all symbiotic phenotypes.
Here the symbiont kills the host in which its fitness is zero: the male. Inherited microorganisms that selectively kill males are found in a wide variety of hosts. The mechanism by which symbionts ‘know’ that they are in males is uncertain. Two categories of male killers are recognised, named ‘early’ and ‘late’ male killers distinguished by their time of action, the former killing males at embryogenesis or first larval instar, the latter acting on males in the fourth larval instar (Hurst et a l, 1997a).
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 3 \
1.2A.1. Late male killing
To date, microsporidia are the only class of symbiont known to cause late male killing.
Prior to last year, this phenomenon had only been recorded from mosquito hosts, being
reported from species within the Genera Amblyospora and Thelohania (Hurst, 1991;
Kellen et a l, 1965). The symbiont can cause lethal infections in fourth instar male
larvae. Late male killing agents are relatively benign in female hosts, which tend to
survive and pass on the infection to their progeny. Mechanistically, death is caused by
massive parasite replication in the fat bodies of late instar male larvae (Kellen et a l ,
1965). Death of the aquatic mosquito larva releases spores into the environment where they can be horizontally transferred to other hosts, either directly or via a copepod
intermediary (Hurst et a l, 1997a). This method of late killing permits the symbiont to maximise its replication potential, and infect more hosts via infectious transmission from males (Hurst, 1991). Microsporidial infections are believed to occur in practically all mosquito populations. However, they do not always cause sex ratio distortion. In some instances, both males and females are killed by the parasite, and in others no host death is seen (Hurst, 1993; Kellen et a l, 1965).
Although it was believed that late male killing was confined to the mosquito group, a recent study has revealed a similar phenomenon in the oriental tea tortrix, Homona magnanima (Lepidoptera: Tortricidae) (Morimoto et a l, 2001). Here, male larvae die at the third larval instar similar to mosquitoes. As yet, the causal element of this sex ratio distortion is unknown, but as it is incurable with antibiotics it is thought not to be of bacterial origin. It seems likely that late male killing may be more widespread than previously believed.
1.2.4.2. Early male killing
All early male killing inherited symbionts identified to date are bacteria (Hurst et al,
1997a). Males are killed during or shortly after embryogenesis. In contrast to late male
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 3 2 killers, early male killing symbionts, for the most part, rely on purely vertical transmission. Horizontal transmission of the symbiont has only been recorded from the parasitic wasp ATaso/zia vitripennis (Huger et ai, 1985; Skinner, 1985). This parasitic phenotype represents the central subject of this thesis, and as such is discussed and reviewed in greater depth in the following section. Throughout this work, the phrase
‘male killing’ should be taken as referring to early male killers only, unless otherwise specified.
1.3 Embryonic Male Killing
1.3.1. Characteristics and incidence of male killers
Embryonic male killing symbionts come from a diverse range of bacterial lineages separated by at least 2, 000 million years (Hurst et a i, 1997a). There are no examples of bacterial clades that contain solely male killing lineages. In all cases, male killing is believed to be a derived state, evolving in bacteria that already show maternal inheritance (Schulenburg et al., 2000). Male killers are known to infect an ever-growing multitude of arthropod hosts (Table 1.3.). In most of the cases illustrated in the table, the systematic affiliations derive from 16S rDNA sequence data from the bacteria associated with the trait. Subsequent polymerase chain reaction (PGR) of infected and uninfected specimens confirms the association of trait and bacterium. Because males are killed at embryogenesis, a low (approximately half) host egg hatch rate is a characteristic of infection, in addition to the female-biased sex ratio. Like all reproductive parasites, male killers are maternally inherited, so breeding subsequent generations of infected hosts reveals characteristic all-female broods throughout the matriline. There are many more examples of insect hosts that are believed to harbour male killers, based on breeding data
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 33
where adult sex ratios have been obtained. However, these cases all require molecular
systematic analysis of the agent causing the putative male killing (Hurst & Majerus,
1993).
Table 1.3. Biodiversity of early male killers (all bacteria) with examples of a host
species from each of the orders infected by a male killing symbiont in each clade
Bacterial Clade Host Reference
Wolbachia Coleoptera Adalia bipunctata Hurst et al., 1999a Tribolium madens Fialho & Stevens, 2000 Lepidoptera Acraea encedon Jiggins et al., 1998 Acraea encedana Jiggins et a i, 2000a Diptera Drosophila bifasciata Hurst et al., 2000
Rickettsia Coleoptera Adalia bipunctata Hurst et al., 1994 Adalia decempunctata Schulenburg et a i, 2001
Brachys tessellatus Lawson et a l, 2001
Unnamed Flavobacteria Coleoptera Coleomegilla maculata Hurst e/û/., 1997b Adonia variegata Hurst et a l, 1999b
Arsenophonus nasoniae Hvmenoptera Nasonia vitripennis Werren et a l, 1986
Spiroplasma Coleoptera Adalia bipunctata Hurst et a l, 1999c Harmonia axyridis Majerus et al, 1999 Lepidoptera Danaus chrysippus Jiggins et al, 2000b Diptera Drosophila willistoni Williamson & Poulson, 1979
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 34
Clearly some male killers are present in many different host groups. What is unclear, as yet, is whether some strains are restricted to particular host taxa. Restrictions might arise following specialisation on a particular host sex determination system: Wolbachia and spiroplasmas maybe the only male killing clades that can easily cross this boundary.
1.3.2. Why kill males?
Sex ratio distorting parasites that cause either féminisation or parthenogenesis induction increase the absolute number of infected female hosts. Male killing appears at first to be more paradoxical because it does not. Whilst males cannot transmit the infection, male ' death is only adaptive if there is a resultant increase in female host survival. It4s straightforward to visualise this, due to the cytoplasmic inheritance of these pathogeiis.
Several factors are believed to affect the spread of a male killer within a host population, by conferring indirect benefits on female hosts; these benefits include:
1. Reduced sibling competition
2. Sibling egg cannibalism
3. Prevention of inbreeding
1.3.2.1. Sibling competition and sibling cannibalism
Either antagonistic actions between siblings, or cannibalism of unhatched eggs (or both) are features of the majority of insects that harbour male killing bacteria, and it is in groups that show these patterns of host ecology that male killer incidence is at its highest
(Hurst & Majerus, 1993). Most male killer hosts lay their eggs in clutches and the larvae are gregarious (although there are exceptions such as Danaus chryssipus that is infected with a male killer at 40% prevalence, despite the fact that females lay single eggs
(Jiggins et al, 2000b)). For example, m Adalia bipunctata, the two-spot ladybird, eggs are laid in clutches, the females hatch out and immediately consume the eggs containing the unhatched embryos of their dead brothers (Hurst & Majerus, 1993), thus giving them a survivorship advantage by way of an immediate ‘ready meal’ that they do not have to
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 35 expend energy in foraging for (Osawa, 1992). Significant mortality of neonates that fail to obtain their first meal has been demonstrated in this species (Hurst et a l, 1997a).
Intuitively, the intensity of sibling competition in gregarious species is also reduced in infected lines because of the death of approximately half of the clutch.
13.2.2. Inbreeding avoidance
For male killing to give the infected host an advantage through inbreeding avoidance the host must both naturally inbreed and exhibit some level of inbreeding depression
(Werren, 1987). The sole example of an investigation of inbreeding in a male killer infected host comes from A. bipunctata (Hurst et a l, 1996a). In this study, despite the fact that inbreeding depression in the laboratory was found to be severe, the authors found very little evidence suggesting that inbreeding was occurring in the natural population. They conclude that due to its rarity, inbreeding is unlikely to be important in the dynamics of male killer invasion in the two-spot ladybird. However, it may be true for other host species that inbreeding avoidance plays a large part in symbiont spread.
1.3.3. Direct (physiological) effects of infection with male killing bacteria
The previous section describes indirect effects of infection with a male killer on female host survival. There may also be direct effects of infection on female host fitness. These effects could be negative: the host suffering a physiological cost to harbouring the bacteria; or positive, such as that seen in the A^hiàtBuchnera symbiosis in which the host gains essential nutrients through its obligate association with the symbiont
(Baumann et al, 1995). To date, all studies looking for direct effects resulting from male killer infection have documented physiological costs to hosts. However, positive effects of infection have been reported in host species infected with other phenotypes of reproductive parasite (for example: Dedeine et a l, 2001; Dobson et a l, 2002).
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 3 6
1.3.4. Prevalence variation in natural populations
Huge variation in the prevalence of early male killers has been documented to date ranging from around 1% Drosophila willistoni females infected with Spiroplasma poulsonii (Williamson & Poulson, 1979), to around 90% Acraea encedana females infected with Wolbachia (Table 1.4.) (Jiggins et a l, 2000a). Whilst an obvious study bias towards highly infected species exists, the facts that many species have early male killers and that they are found in the intensely studied Drosophila, suggests them to be widespread at low prevalence among the insect groups.
Prevalence of an early male killer within a group is affected by differences in host behaviour, ecology, and interaction with host physiology. Low vertical transmission efficiency and physiological costs to hosts will lead to reduced prevalence of a male killer. However, if the female hosts benefit from the loss of males, as discussed above, the male killer might be expected to reach high prevalence in the population as is seen in some species.
Prevalence can also vary between populations of the same host. For example, different symbiont prevalences are documented in populations that are only kilometres apart, in the butterfly Acraea encedon (Hurst & Jiggins, 2000). Explaining these variations is problematic, as the proposed indirect benefits to harbouring the parasite are surely the same in these populations, yet the prevalence is different. This fact illustrates the difficulties in interpreting variation in symbiont occurrence and is perhaps the main reason why there is little quantitative empirical evidence to date concerning the factors determining male killer prevalence.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 37
Table 1.4. Prevalence of male killing bacteria in natural populations of their insect hosts.
Prevalence is expressed as the percentage of females infected, n>30 in all cases.
Host Prevalence (%) Reference
Adalia bipunctata Spiroplasma: 0-22 Hurst et a l, 1999c Wolbachia : 0- 5 Hurst et a l, 1999a Rickettsia : 5-1 Hurst et a l, 1999c; Hurst et a l, 1993
Harmonia axyridis 0-49 Majerus et a l, 1998
Danaus chrysippus 40 Jiggins et a l, 2000b
Drosophila willistoni 0-3 Williamson & Poulson, 19^79
Acraea encedon 61-95 Jiggins cr a/., 1998= - -
Acraea encedana 95 Jiggins et a l, 2000a
Coleomegilla maculata 23 Hurst et a l, 1996b
Brachys tessellatus 50 Lawson et a l, 2001
Nasonia vitripennis 4 Skinner, 1985
Drosophila bifasciata 0-7 Ikeda, 1970
Gastrolina depresa 0-50 Chang era/., 1991
1.4. Male Killers: Questions That Need To Be Addressed.
Although huge advances in the study of selfish genetic elements have been made in the last ten years, due in no small part to improved technology and molecular analysis techniques, there are still many unanswered questions within this field, particularly within the arena of male killers.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 3 §
1.4.1. What is the mechanism of male killing?
Male killing is an example of selective parasitism, with the symbiont being pathogenetic only to the male sex. Little is known about either how host sex is detected or how the host is killed. The only functional study has been conducted in Drosophila willistoni infected with the symbiont Spiroplasma poulsonii. In this symbiosis, death of male embryos has been shown to occur at two stages of embryogenesis (Counce & Poulson,
1962). Transinfection of this symbiont into Drosophila melanogaster and study of the interaction with Tra mutations has shown that the pathogenetic interaction is not associated with somatic sex (Sakaguchi & Poulson, 1963).
Related male killers (within both the Wolbachia bacteria and spiroplasmas) have been found to act in host taxa as diverse as Lepidoptera and coccinellids, which have widely different sex determination mechanisms: coccinellids being male heterogametic and
Lepidoptera being female heterogametic. This observation suggests that the mechanism of male killing is focussed on either somatic or germ-line sex determination, and not dosage compensation (Hurst & Jiggins, 2000).
1.4.2. Can male killers drive the evolution of sex determination?
Sex determining mechanisms are incredibly diverse in plants and animals. Selfish genetic elements that distort the sex ratio of their hosts are thought to be one of the reasons why such diversity exists (Werren & Beukeboom, 1998). Genetic conflict exists between the non-Mendelian reproductive parasite and the Mendelian host nuclear genes.
Nuclear genes are selected to produce a Fisherian 1:1 sex ratio (Fisher, 1930); male killers on the other hand are acting to bias the sex ratio towards females. Hence, selective pressures are acting in opposite directions, resulting in genetic conflict that can shape the system of host sex determination. As discussed earlier (section 1.2.2.) this has been shown in the woodlouse, A. vulgare infected with a sex ratio distorting symbiont that causes féminisation (Rigaud, 1997). In some cases, as the feminising symbiont increases
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 3 9 in prevalence within the population, the host ‘fights back’: a dominant masculinising factor has evolved, that overrides the action of the reproductive parasite. In effect, an outcome of male heterogamety can be reached, in a population that originally (prior to invasion by the feminising symbiont) was female heterogametic (Rigaud & Juchault,
1993).
Such conflicts exist, and are likely to occur in male killer/host interactions due to the extreme nature of the parasitism. No examples have yet been discovered, and it is not known how often such reproductive parasites have a role in the evolution of sex determination.
1.4.3. What causes prevalence and incidence variation in male killers?
The population dynamics of male killer invasion and maintenance have been extensively modelled (Hurst, 1991; Hurst et a l, 1997a; Randerson & Hurst, 1999; Randerson et a l,
2000). The basic model contains the following five parameters that have previously been discussed: a, the vertical transmission rate; s, the direct effect on infected female hosts
(survivorship and fecundity); F, the rate of inbreeding; (1 - 1) the subsequent inbreeding depression; and b, a multiple of the increased fitness of female progeny, following death of their brothers (i.e. 6 is a function of a).
If the vertical transmission rate (a) and direct effect on infected females ( 5 ) vary, then the frequency of the male killing trait in the subsequent generation (p’) as a function of its present frequency ip) can be shown as:
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 4 0
abp{l + s) œ
Where: 0) = /?[a(l + 5 ) + (l - a)] +1 - and invasion is possible if: 1 b, > —7 r > 1 , a (l + 5 ) leading to a stable equilibrium frequency at:
P* = (Equation 1.1.) h(l + as) - 1
It follows that variation in vertical transmission efficiency, direct effect on infected females and/or rate of inbreeding all combine as above to determine the prevalence and incidence of the male killing symbiont. For example, in a species where there is perfect transmission of the male killing element that has no direct effect on its host (either positive or negative) then the agent may spread by drift. However, in a situation where the host gains a direct benefit from the selfish genetic element (s is greater than 0 and
(1 + s)a > 1), the male killer will spread deterministically in the population. The extent of this spread is dependent on all of the above factors, higher prevalences being seen in species in which the infected females gain a large indirect advantage over their uninfected counterparts, in terms of reduced competition and consumption of sibling eggs as discussed previously (for example the A. encedana!Wolbachia symbiosis: Jiggins et a l, 2000a). So prevalence and incidence of male killers vary based on the ecology, transmission efficiency and rate of inbreeding of their host species.
1.4.4. What effects do male killers have on the host population?
In reference to equation 1.1. it is clear that in a species that shows near perfect transmission efficiency of a male killing element, which has an associated benefit to
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 41 female survival and reproductive success, (i.e. a = 1,5 = 1) prevalence of the symbiont will be extremely high. Such a situation has implications on the host population:
1.4.4.1. Behavioural changes
As previously discussed, in populations harbouring a high prevalence male killer the female host gains an indirect survival benefit resulting from the action of the element.
What is interesting to note in such populations is the effect on the host population as a whole of the widespread prevalence of the inherited symbiont. One can imagine that a male killer at extremely high prevalence could have serious consequences on its host population. The African butterfly A. encedana is an example of such a species. Female
Acraea butterflies form swarms of virgins, or leks on hilltops (Jiggins et a/., 2000a). This is thought to be due to the lack of males resulting from the spread of a male killing
Wolbachia. However, d\\ Acraea butterflies exhibit this ‘hill-topping’ behaviour, so it is impossible to tell whether this is an evolved state in response to the presence of the male killer.
1.4.4.2. Population dynamic effects
A high prevalence male killer has potential implications on its host population arising from lack of males. Such effects have been little reported and hence little studied, although extensively modelled. In 1967, Hamilton modelled invasion dynamics of selfish genetic elements, showing that extremely high prevalence of infection may drive the host population to extinction (Hamilton, 1967). This has been demonstrated in a laboratory study oi Drosophila melanogaster in cage populations infected with an artificially introduced selfish genetic element, a Y chromosome meiotic driver (Lyttle, 1977).
Hamilton’s paper concludes that in such a system, evolution of genes resistant to the action of the selfish genetic element is inevitable. However, if resistance does not evolve
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 4 2 and the population existence is threatened we might expect to see a change in allocation of host sexual resources, as well as alterations in host mating behaviour.
1.4.4.3. Resistance evolution
As a male killer spreads throughout a population, the host genome would be expected to
‘fight back’ in terms of evolving resistance to the selfish genetic element. Although resistance has been documented to other sex ratio distorting symbionts, such as the woodlouse/feminising Wolbachia symbioses (Juchault et a l, 1993), there is currently scant empirical evidence regarding evolution of host resistance to male killers. This seems surprising, as we would expect to see some evidence of resistance evolution especially in populations harbouring a male killer at high prevalence. Perhaps the fact that there are few definitive studies indicates that resistance is difficult to evolve.
1.4.5. Phylogeographic inferences from mitochondrial DNA.
In both evolutionary and ecological studies, variation in mitochondrial DNA is used for phylogenetic resolution over a variety of timescales (for example: Lundrigan et al, 2002;
Roehrdanz et al, 2002; Streelman et a l, 2002). Mitochondria are passed from host to host through the female line, hence maternally transmitted parasites such as Wolbachia are expected to be in linkage disequilibrium with mitochondrial genes, with a particular reproductive symbiont becoming associated with a particular mitotype (Johnstone &
Hurst, 1996). As the selfish genetic element increases in prevalence in the host population, the frequency of the particular mitotype with which it is associated increases at the same rate. Several studies have revealed how selfish genetic elements can significantly reduce the level of mitochondrial diversity, confounding interpretations of host evolutionary history (Ballard et a l, 1996; Jiggins, 2002; Johnstone & Hurst, 1996).
In a situation in which populations of the same species are infected with different selfish genetic elements, the linkage disequilibrium of mitochondria and reproductive parasites can be positively misleading to phylogeographers, although informative about the
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 43 infection history of the host population. Sequencing mitochondrial genes from individuals from geographically distinct populations that bear different, or no, infections, enables the history and pattern of infection of the reproductive symbiont to be traced.
1.5. Study System:Hypolimnas bolina (Lepidoptera: Nymphalidae)
It is clear from the previous sections that different hereditary symbioses show a large amount of variability both within the specific host/parasite interaction and between different systems. The majority of differences can be classified in terms of: -
1. Phylogenetic affiliation of host and symbiont
2. Reproductive parasitic phenotype
3. Geographical location of host population(s)
4. Prevalence of symbiont within each host population
5. Approximate age of symbiosis
6. Effect of infection on host fitness
The majority of unanswered questions within the reproductive parasitism (and particularly male killer) field of study, concern why and how these variations occur.
To address these questions, a single host-parasite system is required in which all these effects can be ‘isolated’ and studied. The ideal system would consist of a single host species infected with a sex ratio distorting selfish genetic element that exhibits variation in prevalence across different host populations. These host populations should be geographically separated with a low level of mixing through migration. One can imagine that the most successful host species would be an aquatic one, with different populations occurring in separate pools, such as John Endler used for his experimental evolutionary studies on the trade off between mating system and predation pressure in Trinidadian
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 4 4
Guppies (Reznick et al, 1994). Ponds are contained controlled environments with minimal mixing between populations, which allow evolutionary responses to be isolated and monitored successfully without a high degree of genetic ‘noise’ resulting from invasion by immigrants. Unfortunately, the majority of selfish genetic elements occur in non-aquatic insects, so a controlled ‘pool’ study is not an option. However, the terrestrial approximation for an aquatic study such as John Endler’s is to use naturally occurring islands.
Based on the above requirements, the butterfly Hypolimnas bolina was chosen, as a suitable study system with which to address unanswered questions concerning reproductive parasitism. Hypolimnas bolina occurs throughout a wide geographical range, with many populations being established on islands. Historically, this butterfly species is suspected to harbour a sex ratio distorting selfish genetic element, and variation in prevalence of infection has been recorded across different populations
1.5.1. Historical evidence of all-female broods in H. bolina
Since the times of the earliest amateur naturalists, there has been an emphasis on collection and breeding of Lepidoptera, particularly butterflies, due to their aesthetically pleasing appearance. In the Fiji Islands in the 1920’s, H.W. Simmonds demonstrated, using breeding experiments, the occurrence of all-female broods in the butterfly H. bolina (Poulton, 1923; Poulton, 1927; Poulton, 1928; Simmonds, 1926). Sir Cyril Clarke also recorded this phenomenon (Clarke et a l, 1975) and resurveyed77. bolina in the Fiji
Islands in 1983, finding the all-female broods persisting some sixty years later (Clarke et a l, 1983). Clarke noted that the phenomenon could not be caused by parthenogenesis due to characters carried by the male, and not the female, being present in the offspring.
Both Simmonds and Clarke reported that the observed sex ratio distortion was maternally inherited, and that the egg hatch rates from all-female matrilines were never
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 45 significantly different from 50% (Poulton, 1928). Clarke’s results from Fiji are presented in more detail in the next chapter.
1.5.2. Island nature
Throughout much of its vast distribution, H. bolina is an island species, being found on many of the South Pacific archipelagos as well as islands in the Indian Ocean.
Carrying out fieldwork on island species allows study of replicated evolution in different environments. Islands are small, discrete and isolated from larger landmasses. In such a setting, diversifying evolution can occur reqiarkably rapidly. Studying evolution in the relative simplicity of the island environment, allows inferences about an organism’s evolutionary history to be made (Grant, 1998). Many of the oceanic islands on which
H. bolina populations occur are isolated and difficult to reach, enabling a comparative approach within a species and making this organism a useful tool for evolutionary studies.
1.5.3. Indication of prevalence variation
Data collected by Clarke et al. (Clarke et al, 1975; Clarke et al, 1983) suggests that there may be huge variation in prevalence of the all-female trait across the.butterfly’s range. For example they found female-biased sex ratio distortion to be at high prevalence in Borneo, but no evidence for the all-female trait in Australia. These findings imply that studies of H. bolina across the whole of its range may provide insights into how and possibly why male killer prevalence varies. The fact that variability is seen between different populations of the same species allow us to factor out effects of host/parasite differences, allowing more easily controlled examination of possible causes and consequences of prevalence variation.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 46
1.6. Questions To Be Addressed Using The Host Hypolimnas bolina
The island nature of H. bolina at certain parts of its range means that certain populations
are ‘discrete’, making it a highly useful system in which to study both evolution and
maintenance of a selfish genetic element. The following questions will be addressed in
this thesis using the H. bolina (supposed) hereditary symbiosis.
1.6.1. Is H. bolina infected with a male killing symbiont?
Species that are infected with male killers show a characteristic half hatch rate and all
female sex ratio on emergence. Breeding/f. bolina females from one of the areas in
which Clarke and Simmonds found evidence of the all-female trait should reveal whether
or not the trait still persists, and whether or not it is antibiotic curable (i.e. the trait is
bacterially-mediated). If the all-female trait is present, molecular analysis will enable
identification of the symbiont causing the sex ratio distortion, and its phylogenetic
position to be established.
1.6.2. Does prevalence of infection vary across differentH, bolina populations?
Clarke et a l ’s (1975) data is highly suggestive of prevalence variation throughout the
range of//, bolina. Hypolimnas bolina is found on many of the South Pacific
archipelagos: French Polynesia, Tonga, Fiji, American Samoa and Independent Samoa to
name but a few. If, as Clarke’s study suggests, prevalence of the all-female trait does vary, looking for ecological correlates associated with this variation may provide an
explanation for why the prevalence of selfish genetic elements varies between host populations.
One factor that could putatively cause variation in male killer prevalence is the existence of direct and indirect on the fitness of infected (female) hosts conferred by the bacterium.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 4 7
1.6.2.1. Indirect effects
Indirect effects, or ‘fitness compensation’, such as sibling egg cannibalism, clutch size
and inbreeding avoidance can be examined in different ‘isolated’ H. bolina populations
that show different levels of infection. Such a study may enable deductions as to whether
the level of fitness compensation is correlated with infection prevalence, and if so, which
indirect effects are most important in determining the spread of a selfish genetic element.
Suggestive evidence that indirect effects may be, in part, responsible for variation in the
occurrence of all-female broods, comes from H. bolina in Australia. In Queensland,
Australia, H. bolina reportedly lays eggs singly (Kemp, 1998), and not in clutches of 10-
12 as has been documented from the South Pacific Islands: Fiji and Independent Samoa
(Clarke et al, 1975; Mathew, 1888). As explained earlier, clutch size and the resulting
sibling competition is believed to play an important role assisting the spread of a male
killing symbiont to high prevalence within the host population. According to Clarke’s
data, the ‘all-female’ trait is not found in Australia, but it occurs in Fiji. Perhaps the
explanation for this difference lies in the different oviposition behaviour of female H.
bolina from the different populations.
1.6.2.2. Direct effects
No evidence for true direct physiological benefits to host fitness resulting from infection
has been reported from male killer symbioses, and very little from other
host/reproductive parasite interactions. However, it can be postulated that if a selfish
genetic element, such as a male killing symbiont, is present at a high prevalence within a population, and if fitness compensation (indirect effects) are minimal, a direct benefit to
the host resulting from infection might be observed. The postulated prevalence variation
and island nature of H. bolina provide a useful system in which to look for direct effects
such as increased fecundity, longevity and survivorship. One of the difficulties that researchers have faced when examining hosts for evidence of direct effects resulting
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 4 8 from infection is that in the majority of cases, the uninfected ‘control’ population is derived from originally infected lines that have been antibiotic cured (for example,
Dobson et a l , 2002). It becomes more difficult to interpret results if, for example, increased fecundity is seen among infected individuals relative to controls it could in fact be that the uninfected control fecundity is reduced due to the action of antibiotics. Clarke et a l ’s (1983) data suggests that, at least in the Fiji islands, a large proportion of the female population is not infected with the all-female trait. Unless this lack of infection is due to resistance evolution, experiments looking for direct effects in this host should be able to be carried out with both naturally-occurring and previously infected antibiotic- cured uninfected controls. Of course, it is also the case that, as in a number of other male killer symbioses (Ebbert, 1991; Hurst et a l, 1994), any direct effects on host fitness may well be negative.
1.6.3. Inferences from mitochondrial DNA (mtDNA)
If prevalence variation in the all-female trait does exist in H. bolina, or if the host species is found to be infected with more than one selfish genetic element, then due to the isolation of populations, this species is an ideal candidate for host mtDNA studies. Such a study should enable conclusions to be drawn about the evolution and age of the symbiosis as well as answering questions about the trait, such as the infection history of uninfected individuals or populations.
1.6.4. Consequences on host population of variations in infection prevalence
1.6.4.1. Behavioural changes
If prevalence of the all-female trait is variable across different populations oiH. bolina, we might expect to find correlates in host behaviour reflecting this variation. Studies of the African butterfly Acmca encedon (Jiggins et al, 2000c) that is infected with a male killing Wolbachia at high prevalence, reveal ‘sex role reversal’ in which females are observed to form leks of virgins. Looking for variation in host behaviour across different
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 49
H. bolina populations may reveal whether or not such sex role reversal occurs in this species and if it does, whether or not behaviour is correlated with the presence and prevalence of the all-female trait. Due to the isolation oiH. bolina populations and indicated prevalence variation in the all-female trait, this butterfly provides the ideal system in which to search for such behavioural changes, and to look for any other population dynamic effects associated with prevalence variability.
I.6.4.2. Resistance evolution
Carrying out a phylogeographic study oiH. bolina (previous section 1.6.2.) should reveal the approximate age of the causative agent of all-female broods in this species. If the symbiont has been present in the host population for a considerable time, we might expect to find evidence for resistance to the reproductive parasite to have evolved in certain populations. Resistance evolution has not been documented in symbioses in which male killing is the reproductive phenotype. We would expect to see evolution of resistance in populations in which a reproductive symbiont has achieved a very high prevalence. Perhaps H. bolina can help address questions concerning resistance evolution, in populations where prevalence of the all-female trait is high.
Hypolimnas bolina is a useful study system in which to look at both evolution and maintenance of selfish genetic elements. The fact that we already have historical ‘pilot’ data indicating the existence of a male killer that exhibits prevalence variation across host populations seem to make this butterfly an ideal subject of research.
In this thesis, the above points will be addressed. In Chapter 2, the existence of a male killing symbiont is established in H. bolina populations from the Fiji Islands. The causative agent of the all-female trait is identified and the prevalence variation of the male killer calculated across different populations within the Fijian archipelago. In
Chapter 3, H. bolina populations from the Island countries of Independent and American
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1: Introduction 50
Samoa are investigated for the presence of the male killing symbiont and the population dynamics of variation in male killer prevalence is examined across the different countries. In Chapter 4, evidence for direct physiological effects deriving from infection with a selfish genetic element are investigated. In Chapter 5, variation in male killer prevalence across the range oiH. bolina is examined together with the phylogeography of the male killing trait. In Chapter 6 the implications of this work are discussed together with ideas for the direction of future work on this system based on the results presented in this thesis.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 1 : Introduction 5 \
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 52
Chapter 2: Association Of All-Female
Broods In Hypolimnas bolina With
Wolbachia Infection
This chapter is adapted from: Dyson, E.A. et al. 2002. Wolbachia infection associated with all-female broods in Hypolimnas bolina (Lepidoptera: Nymphalidae): evidence for
horizontal transmission of a butterfly male-killer. Heredity 88:166-171.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 5 3
Chapter 2: Association Of All-Female Broods In
Hypolimnas bolina With Wolbachia Infection
Summary
This chapter presents results from the initial field study of Hypolimnas bolina. Based on historical evidence from Clarke and Poulton (Clarke et al, 1983; Poulton, 1923; Poulton,
1927; Poulton, 1928), the Fiji Island populations were chosen for study. All-female broods were identified and egg hatch rates obtained from both infected and uninfected lines. Experiments using larvae revealed no evidence for egg cannibalism in this species.
The all-female trait was inherited and not due to parthenogenesis. Investigation of this system revealed the causal agent of sex ratio distortion in H. bolina to be a male killing
Wolbachia bacterium. This Wolbachia strain is identical in wsp and ftsZ gene sequences to a Wolbachia male killer in the Tanzanian butterfly Acmca encedon in Tanzania, suggesting it has moved between host species yet retained its phenotype. The prevalence of the male killing Wolbachia was calculated for populations of H. bolina collected from three different island groups of Fiji, and found to vary significantly across the archipelago. Antibiotics failed to cure either the male killing trait or the Wolbachia infection. The implications of these results are discussed.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 54
2.1. Summary Of Previous Work OnHypolimnas bolina In Fiji
In the early 1920’s, H. W. Simmonds bred//, bolina in the Fiji Islands, recording the occurrence of all-female broods in this species (Poulton, 1923; Poulton, 1927; Poulton,
1928; Simmonds, 1926). He reported that the “all-female trait” was passed from mother to daughter, and was not due to parthenogenesis. Some fifty years later, Clarke and
Sheppard (Clarke & Sheppard, 1975; Clarke & Sheppard, 1977) studied the genetics of the female wing polymorphism in this species, collecting and breeding//, bolina from all over the Indo-Pacific. During their course of study, they recorded all-female broods in matrilines from Borneo, Sri Lanka and Hong Kong, reinforcing Simmonds’ findings that the trait was passed through the female line (Clarke et a l, 1975). Cytological sexing of larvae and embryos indicated that the deficiency of males was due to their very high mortality in the pre-adult stage (Clarke et a l, 1975). Despite obtaining negative results when testing infected females for viral particles and spirochaetes, they suggested a cytoplasmic factor to be the cause of the observed sex ratio distortion. Clarke et al carried out a re-survey of//, bolina in Fiji in 1983. They demonstrated, through breeding experiments, the persistence of the phenomenon some 150 generations after it was originally recorded (Clarke et a l, 1983) concluding that, “no entirely satisfactory explanation has yet been given for the persistence of all-female broods”. Based on this historical data, H. bolina populations in Fiji were re-examined.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 55
2.2. Aims
The aims of this first field season were threefold:
1. To establish that all-female broods still occur in the Fiji Islands.
2. To discover the causal agent of the sex ratio distortion.
3. To establish the variation in prevalence of the male killer across different populations
within the Fiji island group.
2.3. General Methods for Fieldwork in Fiji
All fieldwork in this section was carried out in the Fiji Islands between May and
November 1999. Laboratory work whilst in the field was carried out at the Ministry of
Forestry, Colo-I-Suva, Suva, Fiji (see map. Figure 2.1.).
2.3.1. Sample collection
Although specimens were collected from several different islands, all of the breeding work was carried out using butterflies collected on the main island of Viti Le vu, Fiji. Viti
Levu is the largest and most developed island in the Fiji group. The two main collection sites on this island were situated at Suva and Nadi, the country’s two most prominent towns, situated at opposite ends of the island (Figure 2.1. shows a map of Viti Levu)
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 56
50km
Figure 2.1. Map of Viti Levu Island adapted from Microsoft Encarta interactive world
atlas (2001) Experimental H. bolina females were collected from Suva and Nadi
Hypolimnas bolina in Fiji tends to occur in areas of secondary scrubland, and around weed patches in built up areas (pers. obsn.). One of the preferred nectaring plants in Fiji,
Stachytarpheta urticifolia (the blue rat’s tail) is found on roadside verges and newly cleared areas, typically found in semi-urban environments (Whistler, 1995). Butterflies were collected using a 40cm diameter four-fold butterfly net (Watkins and Doncaster,
E674) with a 70-180 cm aluminium extension pole (Watkins and Doncaster E6763).
Once collected, specimens were stored live in stamp envelopes and taken back to the laboratory.
2.3.2. Rearing specimens
For the dual purpose of larval feeding and oviposition, Ipomoea batatas (sweet potato) plants were cultivated in a garden outside the laboratory, and in greenhouses belonging to the Ministry of Forestry (Vane-Wright et al, 1977; Clarke and Sheppard, 1975).
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 5 7
2.3.2.1. Maintaining adults and obtaining eggs
All butterflies collected were coded alphabetically according to area of collection: ‘S’ for
Suva and ‘N’ for Nadi, and numerically according to the number collected, i.e. the first butterfly collected in Suva was coded ‘S I’, the second ‘S2’ etc. Live specimens were kept in the laboratory in cages of one of two types, either metal mesh cube cages measuring 50cm in length (provided by the entomology section of the Ministry of
Forestry), or cylindrical 60cm diameter hanging cages made of black fabric mesh
(Watkins and Doncaster E60911) (Figure A2.1., Appendix II). Adults were provided with “false flowers” containing 0.4M sugar solution on which to feed. Wild-caught and
FI mated females were caged singly and provided with a potted sweet potato plant (J. batatas) for oviposition (Clarke & Sheppard, 1975; Mathew, 1888; Vane-Wright et al,
1977). Males and virgin females were kept in single sex cages. Only healthy plants showing signs of new growth were used, as H. bolina females prefer new growth for oviposition (Kemp, 1998). Egg clusters were removed from plants daily, each clutch laid by the same female on the same day stored in a separate 9 cm Petri dish, and the following recordings made:
1. Number of eggs laid
2. Date laid
3. Date hatched
4. Number of eggs that hatch
5. Number of unhatched grey eggs (dead embryo visible through egg chorion)
6. Number of unhatched yellow eggs (no embryo visible through egg chorion)
Each egg clutch was coded according to matriline and numbered depending on the number of clutches previously laid by the particular female. For example, the first clutch laid by the first female collected in Suva (female ‘S I’) was coded IS l, the second 2S1 etc.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 5 8
23.2.2. Rearing larvae
An FI generation was reared, larvae being fed an excess oil. batatas leaves (Vane-
Wright et a l, 1977), fresh leaves being provided twice daily. Containers were lined with tissue paper and cleaned out once a day. Caterpillars were initially reared in 9 cm transparent Petri dishes, then transferred after the third larval moult to large plastic yoghurt pots (450g size) covered with fine mesh mosquito netting secured with an elastic band. Ten larvae were reared in each container, where numbers permitted, with never more than ten caterpillars in a single container. Pupae were removed from containers the day after forming, to prevent disturbance by other larvae. Duration of larval and pupal stages was recorded. The times of any larval deaths within each matriliiie during the course of rearing were also noted. On emergence, sex ratio and wing-type of adults were recorded following the classification of female wing polymorphisms in 77. bolina females outlined in Clarke & Sheppard, 1975.
2.3.2.3. Mating laboratory-reared adults
These adults were then mated in an eight cubic meter outdoor flying cage. FI mated females were coded alphabetically according to matriline (based on the code of their mother), i.e. the first mated FI female from the first clutch of the first field-collected female from Suva (‘ST) would be coded ‘ISIA ’ etc. The flying cage was specially constructed from metal mosquito mesh and timber, and situated in the grounds of the laboratory in an area that received a lot of sunshine (Figure A2.2., Appendix II). Large- leaved plants were placed on the cage top if shade was required. Prior to use, the cage was checked to ensure there were no predatory spiders. Virgin females and males were flown in the cage for periods of up to six hours. The average duration of mating in this species is approximately 45 minutes (pers. obsn.), so the cage was checked every half an hour for signs of mating. Mating pairs were removed to a separate small cage for identification purposes. Outbreeding was ensured by only allowing interaction between
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 59 unrelated FI males and females. The F2 generation was reared from each matriline following the methods previously outlined.
2.4. Do All-Female Broods Still Occur In The Fiji Islands?
2.4.1. Egg hatch rates
Freshly oviposited//, bolina eggs are bright blue or yellow in colour (Mathew, 1888).
Fertilised eggs turn grey several hours prior to hatching due to the development of the tanned head capsule of the embryo. This is also seen in coccinellids (Hurst et âl, 1992) and other butterflies (Jiggins et al, 1998). If the clutch is infected by a male killer, eggs that contain male embryos also turn grey prior to hatching, but do not hatch out due to death of the male (Jiggins et al, 1998). It is therefore straightforward to tell whether an unhatched egg is infertile (blue/ yellow), or probably male killed (grey), due to this convenient ‘colour-coding’. Throughout this thesis, unfertilised//. bolina eggs are referred to as ‘yellow eggs’.
2.4.1.1. Method
For the first ten days that a captured or lab-mated female laid eggs in the laboratory, hatch rates of all egg clutches were recorded as number of eggs that hatch, and numbers of unhatched grey and yellow eggs.
2.4.1.2. Results
Egg clutches from seven out of the twelve wild-collected H. bolina females show the low egg hatch rates indicative of the presence of a cytoplasmic sex ratio distorter (Table 2.1.).
The hatch rates of broods from these seven matrilines are homogeneous (%^ 6.419; df =
6; ns) as are those of the other five matrilines that show high egg hatch rates (x^l5.167; df =4; ns) however, there is a significant difference between the two data sets (Mann-
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 60
Whitney U test: Ni=5; N2=7; U=0;p< 0.001). The egg hatch rates produced by FI females demonstrates that this trait is inherited (Infected: x^=3.8775; df = 1; ns.
Uninfected: %^=3.6324; df = 1; ns), consistent with the findings of both Simmonds and
Clarke (Clarke et a l, 1975; Clarke et a l, 1983; Poulton, 1923; Poulton, 1928;
Simmonds, 1926). Eggs that did not hatch in a normal brood were yellow and very rarely grey whereas in an all-female brood, the number of grey eggs and the number of hatched eggs were approximately equal.
Once egg hatch rates had been obtained, not all larvae were reared to adult. In most cases only ten or twenty larvae were reared in order to obtain the adult sex ratio.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 61
Table 2.1. Egg hatch rates of all Fijian females grouped into low and high hatch rates.
(Wild-collected females in bold, FI generation in normal type).
Matriline N®. Hatched N®. Eggs that Don’t Hatch Proportion Eggs Hatch N®.Grey N®. Yellow
Hiah hatch rate S4 279 1 2 0.99 S23 64 2 6 0.89 FS23 56 0 0 1.00 S43 104 0 7 0.94 AS43 36 0 0 “ 1.00 BS43 6 0 0 1.00 N3 128 2 1 0.98 N41 12 0 0 1.00 Low Hatch rate S9 28 40 3 0.39 S45 53 48 3 0.51 AS45 4 3 0 0.57 BS45 15 12 0 0.55 S47 16 16 1 0.48 S48 42 35 2 0.53 BS48 3 3 0 0.50 CS48 166 170 4 0.49 S49 61 85 2 0.41 AS49 26 20 0 0.57 CS49 15 18 0 0.45 N1 5 7 1 0.38 N7 23 31 4 0.40 AN7 39 51 3 0.42 BN7 15 14 0 0.52
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 62
2.4.13. Conclusion
These findings strongly implicate the presence of a male killer in some matrilines.
2.4.2. Adult sex ratio
2.4.2.I. Method
Adult sex ratio of all reared broods was recorded on emergence from pupae.
2.4.22. Results
The adult sex ratio of broods produced by parental female H. bolina and in the subsequent FI generation is given in Table 2.2. Out of the twelve parental matrilines, seven show all-female, or heavily female-biased broods. The other five broods.show an approximately 1:1 adult sex ratio, which is also seen in the F2 generation (%^ = 4.737; df
= 2; ns [Test for homogeneity between lines: yf- = 0.79; df = 10; ns]) . Males only occurred in the FI generation of a single all-female matriline, in one brood, but even here there was a strong female-bias. The seven female-biased matrilines are the same seven that gave lower egg hatch rates (Table 2.1.). Consistent with the findings of both
Simmonds and Clarke (Clarke et a l, 1975; Clarke et a l, 1983; Poulton, 1923; Poulton,
1928; Simmonds, 1926), the adult sex ratio and low egg hatch rates in broods produced by FI females demonstrate that all tested females inherited the all-female trait.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 63
Table 2.2. Sex ratios produced by wild-collected (parental) female//, bolina from Viti
Levu Island, Fiji, and in the subsequent FI generation. Matrilines are coded according to area of collection of wild female, S = Suva, N = Nadi.
N.B. An F2 generation was not obtained from all matrilines due to time and weather constraints.
Parental females F I females
Matriline Male Female Male Female number Progeny Progeny Progeny Progeny a) All-female broods
S9 0 16 S45 0 37 0 12 S47 0 15 S48 5 33 0 2 0 38 S49 0 54 0 19 0 12 N1 0 5 N7 0 40 0 24 0 12 b') Normal sex ratio broods
S4 13 11 S23 54 53 9 9 S43 38 33 8 7 2 1 N41 3 5 N3 18 20
2.4.2.3. Conclusion
All-Female broods still exist in Fiji some eighty years after they were first recorded. The fact that all-female broods occur in matrilines that have a low egg hatch rate, and that
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 6 4
both these traits are inherited, implicate the causal agent of sex ratio distortion in H.
bolina to be a cytoplasmic selfish genetic element.
2.5. Are Hypolimnas bolina Larvae Cannibalistic?
The above breeding and rearing data are suggestive of the presence of a male killing
symbiont in this species. It also appears that the prevalence of this suspected selfish genetic element is fairly high in Fiji, at least on Viti Levu Island. In species with high " prevalence male killers, for example the African butterfly Acraea encedana (Jiggins et a l , 2000a) larvae are laid in clutches and indulge in sibling egg cannibalism. This cannibalistic habit is thought to provide a large benefit to infected hosts in terms of fitness compensation (Hurst & Majerus, 1993; Chapter 1: section 1.3.2.), and is thought to explain the high prevalence of the infection. Hypolimnas bolina in Fiji lays eggs in clutches of ten or twelve (Mathew, 1888) but it is unknown whether or not other larvae will eat these eggs. The following experiment was carried out to ascertain whether or not
H. bolina larvae cannibalise eggs.
2.5.1. Method
All larvae used in these experiments were taken from the F2 generation. Caterpillars from four different matrilines were used, two from suspected infected lines (low hatch rate, all-female adult sex ratio) and two from supposed uninfected lines. Two replicates of each were completed.
Immediately after larvae had hatched and dispersed from the egg cluster, and egg hatch rates recorded (see above) all but five larvae were removed from each Petri dish. The removed larvae were reared and studied as normal. The remaining five experimental larvae were left with the egg cluster that contained discarded shells from hatched siblings
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 65 and unhatched (grey or yellow) eggs. The egg clutches were still attached to the leaves on which they had been laid, but as it takes four days from oviposition to egg hatching, these leaves were desiccated and inedible. No food was provided to any of the larvae during the course of the experiment. The experimental lines were monitored daily for evidence of egg or sibling cannibalism, eggs being checked for perforations using a lOx magnification hand lens. The day of hatching of the experimental larvae was counted as
‘day one’. All deaths of larvae were recorded daily.
A second set of similar experiments was performed in which larvae were provided with
■ five freshly laid unrelated eggs. These experiments were carried out using two-replicates of five larvae from each of two matrilines (one suspected infected, one suspected uninfected). Again, larvae were provided with no other food and any evidence of cannibalism and larval death recorded. This second manipulation was carried out to control for kin recognition; it has been postulated that recognition of kin occurs in certain coccinellid species (coccinellids are highly cannibalistic of conspecific eggs and larvae)
(Agarwala & Dixon, 1993).
2.5.2. Results
Table 2.3. summarises the results of these experiments.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 66
Table 2.3. Results of cannibalism experiment, in each replicate five freshly-hatched F2 larvae from all-female or normal sex ratio matrilines, presented with either sibling or non-sibling eggs. Evidence of cannibalism and larval deaths recorded daily
Matriline Replicate Female- Sibling Evidence of Cannibalism? N®. biased? eggs? (N°. Larvae dead) Day 1 Day 2 Day 3
S23 1 No No No No - (1 dead) (5 dead)
S23 2 No No No No No (4 dead) (5 dead)
S43 1 No No No No - (2 dead) (5 dead)
S43 2 No No No No - (1 dead) (5 dead)
S48 1 Yes No No No - (1 dead) (5 dead
S48 2 Yes No No No - (5 dead)
S49 1 Yes No No No - (2 dead) (5 dead)
S49 2 Yes No No No No (4 dead) (5 dead)
S43 1 No Yes No No No (4 dead) (5 dead)
S43 2 No Yes No No No (1 dead) (4 dead) (5 dead)
N7 1 Yes Yes No No - (1 dead) (5 dead)
N7 2 Yes Yes No No No (4 dead) (5 dead)
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 67
2.5.3. Conclusion
These experiments show no evidence of egg cannibalism by larvae in//, bolina. Larvae are unable, or unwilling to consume either sibling or non-sibling eggs. Perhaps they do not have the physiological ability to eat eggs, or perhaps the eggs are protected as seen in many coccinellid species (Agarwala & Dixon, 1992). It is unlikely that the larvae in this experiment were dying from any cause other than starvation. Siblings of all experimental larvae were reared to adult and did not show high first instar larval morbidity.
2.6. Are The All-Female Broods In Hypolimnas bolina Caused By A
Bacterium? r
There are many examples in the literature of antibiotics being used to cure arthropods of sex ratio distorting selfish genetic elements (Stouthamer et a l, 1990). The same is true with male killing symbionts, infected coccinellids fed with golden syrup containing tetracycline revert to producing normal sex ratio broods (Hurst et a l, 1992). Jiggins
(1998) observed that fourth instar larvae of the African butterfly Acraea encedon infected with a male killing Wolbachia symbiont that were fed leaves treated with tetracycline produced broods with an approximate 1:1 sex ratio as adults. Subsequent
PCR analysis revealed the antibiotics had cured the butterflies of the male killing symbiont. In the following experiment, H. bolina larvae were fed with antibiotics in order to test if this would cure the suspected bacterially-mediated trait.
2.6.1. Method
A measured amount of antibiotic (tetracycline or rifampicin) was dissolved in water and the solution painted onto fresh I. batatas leaves such that the amount of antibiotic per leaf was known. Both antibiotics have been previously demonstrated to cure insects
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 68 infected with cytoplasmic bacteria (Stouthamer et al, 1990). Treated leaves were presented to different groups of FI fourth instar larvae following one of seven treatment regimes, detailed in Table 2.4. The treatment regimes vary according to the concentration and frequency of presentation as well as the type of antibiotic presented. Larvae were kept in containers according to matriline, all caterpillars in the same container receiving the same antibiotic treatment. Larvae from five suspected infected and three suspected uninfected matrilines were tested, with between two and ten replicates per treatment.
Treatment was commenced when larvae reached the fourth larval instar, and continued until the first caterpillar in the container pupated (approximately 8-10 days). To establish the amount of antibiotic presented to larvae under each of the different treatment regimes, the leaf area consumed per day for each treatment regime was recorded. This data, together with the concentration of antibiotic supplied and the number of larvae in the experiment, was used to calculate the mean amount of antibiotic consumed by each larva. Control groups from the same matrilines were fed on untreated plants.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 69
Table 2.4. The seven different types of antibiotic treatment regime received by FI fourth
instar larvae
Treatment Code Antibiotic Antibiotic Frequency Concentration Of Treatment
T1 Tetracycline 1% Once only
T2 Tetracycline 1 % Every other day
T3 Tetracycline 1% Every day
R1 Rifampicin 0 .1% Once only
R2 Rifampicin 0 .1% Every other day
R3 Rifampicin 1% Once only
R4 Rifampicin 1% Every third day
2 .6.2 . Results
The average amount of antibiotic consumed by each larva was calculated based on the
amount of antibiotic presented, and the number of larvae within the particular
experimental manipulation (Table 2.5.). In spite of the fact that some larvae consumed a
large amount of antibiotic (far greater in some cases than has previously been
demonstrated to cure the trait in other species, for example: Jiggins et al, 1998), it is
evident from breeding data that neither the sex ratio bias nor the half-hatch rate observed
in suspected infected matrilines were affected by treatment with antibiotics (Table 2.6.).
R4 treatment (1% rifampicin daily) caused host sterility, so it seems unlikely to be the
case that the antibiotics presented were not strong enough to kill a bacterial infection. No other effect resulting from antibiotic treatment was observed in either all-female or control lines.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 70
Table 2.5. Total amounts of antibiotic consumed by each larva in each matriline, following one of the seven treatment regimes outlined in Table 2.4, Numbers in brackets indicate the number of larvae subjected to the particular treatment. R = rifampicin, T = tetracycline.
Total Amount of Antibiotic received per larva (mg)
Suspected Uninfected Suspected Infected Matrilines Antibiotic Matrilines Treatment Regime S23 S43 N41 S45 S47 S48 S49 NB7
R1 0.04 0.04 0.04 0.09 0.05 ■ 0.06 0.08 (10)(10) (4) (2) (3) (4) (3)
R2 0.19 0.19 0.29 0.34 0.30 0.13 0.17 (10) (10) (6) (3) (3) (4) (3)
R3 0.45 0.50 0.48 0.85 0.75 0.63 0.83 (10)(10) (4) (2 ) (3) (4) (3)
R4 1.43 1.45 2.14 2.85 2.17 1.88 0.83 (10)(10) (6) (3) (3) (4) (3)
T1 0.45 0.63 0.83 0.83 1.00 (8) (8) (3) (4) (3)
T2 3.20 1.88 2.25 2.50 2.08 2.67 (6)(8) (4) (3) (4) (3)
T3 8.43 3.33 4.81 (6) (4) (4)
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 71
Table 2.6. Egg hatch rates and adult sex ratio in the F2 generation from FI individuals that were treated with antibiotics according to the treatment regimes detailed in Table
2.4.
Matriline and N Hatched N®. Eggs that Don’t Hatch Proportion Adult Antibiotic Eggs Hatch Sex Ratio Treatment (M:F) N®.Grey N®. Yellow
Suspected Uninfected Matrilines
AS23R3 33 0 0 1.00 - 10:7
DS23T3 77 1 1 0.97 11:7,
GS23 T3 79 1 1 0.98 8:7
Suspected Infected Matrilines
CS45 R2 169 166 5 0.50 0:53
DS45 R2 98 78 3 0.55 0:35
DS49 T3 4 4 1 0.44 0:4
ES49 T3 43 34 1 0.55 0:26
FS49 T1 9 9 0 0.50 0:9
DN7 T1 25 24 0 0.51 0:17
2.6.3. Conclusion
Based on these results, it appears that the cause of sex ratio distortion in H, bolina is not curable with antibiotics, which suggests that it is not a bacterium. This was also indicated by Clarke et al (1975) who looked for, but found no evidence of bacteria in 77. bolina ovarian tissue.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 72
2.7. Are Microorganisms Present In Female-Biased Lines?
In order to ascertain whether or not there were any microorganisms present in the cell cytoplasm of suspected infected females, specimens were DAPI stained and examined for presence of microorganisms within the oocyte cytoplasm under a fluorescence microscope.
2.7.1. Method
A number of live F2 specimens from all-female matrilines were dissected and their ovaries removed. Ovaries were fixed on a grease free slide with a drop of 4% v/v formaldehyde in balanced salt solution (lOmM Tris pH 7.5, 55mM NaCl, 40mM KCl, 7 mM MgC12, 5mM CaC12, 20mM glucose and 50 mM sucrose) (Ashbumer, 1989). After five minutes, excess fixative was removed using tissue paper. The fixed material was then stained for ten minutes with a drop of 1% 4’,6’-diamidino-2-phenylindole (DAPI) in vectashield (Vector Laboratories). A cover slip was applied and the tissue examined under ultra-violet light at high magnification.
DAPI fluoresces blue under ultra-violet light when bound to DNA. This fluorescence is only visible by eye if the DNA is present in large amounts: the nucleus and any microbial elements will fluoresce, but host cytoplasmic DNA will not be visible as the genome of animal mitochondria is too small to produce observable fluorescence.
2.7.2. Results
All suspected infected specimens showed a common trait, having numerous fairly large structures containing DNA around the outside of the developing egg cells.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 73
2.7.3. Conclusion
It appears that there are microorganisms present in the ovarian cells of female-biased lines. Based on the negative results from the antibiotic treatments, these structures were believed to be eukaryotic. Investigation at higher magnification was warranted.
2.8. Is The Infecting Microorganism A Eukaryote?
The failure of antibiotics to cure the all-female lines with low hatch rate but the confirmed presence of microorganisms in infected host tissue, indicate that a non- bacterial microorganism is the cause of the all-female trait in H. bolina. This would be an unusual situation as all currently known male killing symbionts are bacterial (Hurst et al, 1997) and all breeding work points towards the presence of a male killer ini/, bolina in Fiji. However, other instances of sex ratio distortion in arthropods, for example féminisation in isopods (Rigaud, 1997) and late male killing in mosquitoes (reviewed by
Hurst, 1991) are caused by eukaryotic microorganisms, namely microsporidia. Possibly the microorganism causing sex ratio distortion in H. bolina is also a member of this eukaryotic lineage. This was investigated by examining//, bolina ovarian tissue using transmission electron microscopy (TEM).
2.8.1. Method: Protocol of specimen preparation
Two F2 female H. bolina from all-female matrilines were killed and their ovaries removed immediately. Following primary fixation in a 2% ^uteraldehyde based fixative, the infected ovarian tissue was embedded in Araldite resin according to the following protocol:
1. Tissue washed in 0.1 M sodium cacodylate buffer, with two changes each of 10
minutes duration.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 7 4
2. Tissue placed in a solution of 1% osmium tetraoxide made up in O.IM sodium
cacodylate buffer and left in the fridge for 1 hour for peptide crosslinking
(optimum rate of osmium tetraoxide penetration into tissue occurs at 4°C).
3. Excess osmium tetraoxide washed off in O.IM sodium cacodylate buffer for five
minutes.
4. En-bloc stained in 2% uranyl acetate at 4°C.
5. Tissue washed in distilled water for 5 minutes.
Dehydration: '
6. 25% ethanol in water for 5 minutes » _
50% ethanol in water for 5 minutes
70% ethanol in water for 5 minutes
90% ethanol in water for 10 minutes
Absolute alcohol, four changes for 10 minutes each
7. After dehydration, specimen soaked in propylene oxide at room temperature for
four changes of 10 minutes each. Care was taken to ensure enough propylene
oxide remained in each vial when changing solution to avoid solvent evaporation
from the tissue.
8. Tissue placed in a 50:50 mixture of Araldite resin (composition when mixed and
heated: lOg dodecenyl succinic anhydrite; lOg Araldite; O.Sg dibutyl pthalate;
0.4ml benzyldimethylamine) and propylene oxide, and left at room temperature
for 45 minutes.
9. Propylene oxide mixture removed, and the specimen soaked in fresh Araldite
resin for 24 hours at room temperature on a rotator.
10. Specimen polymerised in the oven at 60°C overnight.
11. Specimen sectioned using an ultramicrotome, cutting 70 - 90nm sections on a
diamond knife.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 75
12. Sections were collected on 200 mesh copper grids.
13. Sections counterstained with lead citrate for 6 minutes.
14. Tissue sections examined in a Jeol 1010 transmission electron microscope at
80kV.
2.8.2. Results
On analysis of the infected tissue, no eukaryotic cells were observed within the//, bolina oocytes. However, bacteria contained in vacuoles were seen in the cytoplasm of egg cells. These bacteria were found to be particularly numerous in tiie youngest developing eggs. (Figure 2.2.).
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 76
i «C m
a 4m
Figure 2.2. Transmission electron micrograph of ovarian tissue taken from an F2 female
H. bolina from an all-female matriline showing a prokaryotic cell enclosed in a vacuole.
2.8.3. Conclusion
It is possible, despite the fact that antibiotics failed to cure the trait, that the causal element of sex ratio distortion in H. bolina is a bacterium.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 7 7
2.9. Molecular Analysis Of Laboratory-Bred Specimens
The microorganisms (Figure 2.2.) present in ovarian tissue of all-female matrilines are double-membraned prokaryotes contained in the host vacuole. The appearance of these bacteria is similar to that of the sex ratio distorting rickettsial bacterium Wolbachia
(O'Neill & Werren, 1997). Wolbachia are known to cause all phenotypes of sex ratio distortion in a variety of host organisms (Stouthamer et a l, 1999). In most cases,
Wolbachia has been identified as the causal agent of sex ratio distortion, using polymerase chain reaction (PCR) (Werren, 1997). A variety of different PCR assays for
Wolbachia have been developed. Four different clades of Wolbachia bacteria are known: . the A, B, C, and D clades (Werren et a l, 1995). The A-strain and the B-strain have been shown to cause sex ratio distortion in arthropod groups and members of both clades are known to cause male killing (Hurst et al, 1999; Hurst & Jiggins, 2000; Werren et al,
1995). The C and D clades are only associated with nematode host species (Casiraghi et a l, 2001). Assays were therefore carried out using both B-specific and A-specific primers. Primers that amplify the B-group specific wsp (Wolbachia surface protein) and the A-group specific 16S subunit were used (full details of primer sequences in
Appendix I, section A.2). If a particular sample did not amplify with these primers, a
PCR assay was carried out using primers that amplify part of the mitochondrial COI block (Brunton & Hurst, 1998) in order to check that amplifiable DNA was present in the sample. If amplifiable DNA was absent, the sample was reprepared and the process repeated. A summary of the PCR assays performed on each DNA sample is given by the flow chart below (Figure 2.3.)
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 78
Tissue from single Hypolimnas bolina specimen (usually female)
DNA prepared following protocol A.l, Appendix I
wsp B PCR
Negative Positive
16S A PCR 16S A PCR
Positive Negative Negative Positive
Butterfly is Butterfly is Butterfly is - infected with COI PCR infected with infected with Wolbachia A Wolbachia B Wolbachia A and B
Positive Negative
wsp PCR
Positive Negative
Butterfly is not infected with Wolbachia
Figure 2.3. Flow chart showing the sequence in which PCR analyses were performed and how results were interpreted
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 79
2.9.1.1.Method
For these first twelve original female samples, DNA was extracted from the ovaries and prepared for PCR analysis using the Qiagen DNA preparation kit, following the manufacturer’s protocol. All specimens were assayed for the presence of both A-strain and B-strain Wolbachia (details of all primer sequences and PCR protocols are given in
Appendix I sections A.2. and A.3. respectively. Gels were mixed and visualised as detailed by the protocol A.4., Appendix I).
2.9.1.2. Results
Seven out of the twelve DNA samples derived from the original wild-collected H. bolina females gave positive results for wsp B-strain PCR assays indicating the presence of a B- group Wolbachia bacterium in these specimens (Figure 2.4.). These seven samples are the same females that gave female-biased adult sex ratios. The remaining five matrilines all gave high egg hatch rates and normal sex ratio broods, and showed no evidence for
Wolbachia presence. All twelve samples produced a positive result when assayed with
CO I primers, showing that amplifiable DNA was present in all positive and negative samples. All twelve samples gave a negative result when tested using the Wolbachia A- group specific PCR.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 80
Figure 2.4. Photograph of agarose gel showing results of PCR assay for B-group
Wolbachia wsp gene. Matrilines reading from left to right; Uninfected (no band except positive): S4, S23, S43, Positive control (Adalia bipunctata infected with B-strain
Wolbachia), N3,N41; Infected (all show bands except negative): S9, S45, S47, S48,S49,
Nl, N7, Negative (no DNA present).
2.9.13. Conclusion
There is an association between the existence of all-female broods and the presence of B group Wolbachia in the original 12 ‘founder’ female 77. bolina. This result was something of a surprise, considering that antibiotics had failed to cure the male killing trait.
2.9.2. Molecular analysis of female progeny from antibiotic-treated matrilines
2.9.2.1. Method
DNA was prepared from all F2 females whose mothers had been antibiotic-treated, following the Chelex preparation protocol A.I., Appendix I. All samples were assayed for the presence of Wolbachia as detailed in section 2.9.1.1.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 81
2.9.2.2. Results
All F2 progeny from antibiotic-treated infected matrilines gave a positive result when
assayed for the presence of B-group Wolbachia. All control F2 progeny from antibiotic-
treated uninfected matrilines gave a negative result when assayed.
2.9.2.3. Conclusion
As indicated by the adult sex ratios obtained during fieldwork, tetracycline and
rifampicin antibiotics failed to cure H. bolina of the infecting symbiont. Given the
association of B-group Wolbachia with the all-female trait and the incurability of both the trait and the bacterium, Wolbachia is the likely causative agent of male killing in H.
bolina. Sequence analysis of the bacterium would reveal more about this male killing element.
2.10. Phylogenetic Position Via Sequence Analysis
To date, a large number of Wolbachia phylogenies have been published. Many of these phytogenies are based on sequencing of two Wolbachia genes, wsp and ftsZ. In order to establish the position of the H. bolina strain of Wolbachia in the phylogeny of
Wolbachia, these genes were both sequenced, to control against any effects of recombination between Wolbachia strain (Jiggins et a l, 2001a).
2.10.1. Method
DNA from three female specimens from different infected matrilines was selected and
PCR amplification carried out following protocols A.3.1. {wsp) and A.3.4. (ftsZ),
Appendix I. The amplified DNA was prepared for sequence analysis and sent for sequencing (protocol A.5., Appendix I). Th& ftsZ and wsp sequences were manually
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 8 2 aligned to previously determined Wolbachia sequences of these genes (Cabot, 1997), and the phylogenetic affiliation of the H. bolina Wolbachia recorded.
2.10.2. Results and phylogenetic position
Sequence analysis of the wsp MidftsZ genes of the H. bolina Wolbachia revealed them to be identical in sequence to the male killing Wolbachia strain from the butterfly A. encedon in Tanzania (Jiggins et al, 2000d) (Accession numbers for the//, bolina
Wolbachia: ftsZ, AJ307075; wsp, AJ307076 ). Thus the phylogenetic position of the male killing Wolbachia in H. bolina is the same as that of the Tanzanian A. encedon.
Phylogenies based on the ftsZ and wsp gene sequences showing how this male killing
Wolbachia relates to other Wolbachia is given by (Jiggins et al, 2001b), and reproduced here (Figure 2.5. ftsZ phylogeny and Figure 2.6. wsp phylogeny).
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 83
A aJthoffi A peneieos t C.pipiens L australis N. vitripennis Ad bipunctata Z 99 ' Ad. bipunctata Y ■ A equatoria T S. oryzae £ formosa A tesselatus A encedon U A encedana Ae. albopictus A macarista A acerata 13 A pentapoiis Ar. vulgare 67 99j— T. deion 1 T deion TX 65 T. confusum N^ J A alcinoe 59 A pharsalus \ j A acerata 11 A encedon T/H. bolina ^ C. cauteila 86“ A eponina Protocalliphora sp. BE N. giraulti ■ N. longicornis 100 G. pennsylvanicus G. integer G. pennsylvanicus r D. melanogaster CS T. drosophiiidae 0.1
Figure 2.5. Maximum likelihood tree with 1000 bootstrap replicates of the Wolbachia ftsZ gene labelled by host species. The A-groupWolbachia of T. drosophilae and D.
melanogaster are set as outgroups.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 8 4
H. bolina 83 A encedonT Aacerata 4 C. pipiens D. mauritiana A alcinoe A pharsalus A acerata 3 83 A peneieos A macaristâj A pentapoiis 2 A althoffi Ae. altx)pictus ^ i Ar. vulgare Portugal Ar. \/u/gare Wageningen O. asellus P. scaber 98jH A encedon U 80 A encedana) 1 2 2 j ~ ” T. deioni 63 65 T. deionSW436 91 ' L striatellus T. bedeguaris [ ~ A /. bipunctataY f 9990 Ad. bipunctata! T. confusum 100 0 0 ^ E stauferri L australis E formosa 99 C. cauteila 83 Di. rosae ^ T. orizicolus A eponina Ap. diversicornis — A equatoria D. Melanogaster CantonS D.sechellia 0.1
Figure 2.6. Maximum likelihood tree with 1000 bootstrap replicates of the Wolbachia wsp gene labelled by host species. The A-group Wolbachia of D. sechellia and D. melanogaster are set as outgroups.
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 85
2.10.3. Conclusion
The fact that the Wolbachia male killers infecting both /f. bolina and Tanzanian A.
encedon (Jiggins et a l, 2000d) are identical in the sequence of the wsp zndftsZ genes,
despite the fact that the two host species are both phylogenetically and geographically
distinct, indicates the possible horizontal transmission of the male killing Wolbachia.
2.11. Prevalence Of The Male Killing Wolbachia Across Fiji
2.11.1. Historical evidence of prevalence variation
Both Simmonds and Clarke (Clarke et a l, 1983) bred specimens from different islands of Fiji. Table 2.7. provides a summary of their results, based on the adult sex ratios obtained from different lines.
Table 2.7. Numbers of all-female and normal sex ratio matrilines previously reported by
Simmonds (1926) and Clarke (1983) on five different Fijian Islands, for details of island locations, see Figure 2.6. (below).
Simmonds (1926) Clarke (1983) Fijian Island All-Female Normal Sex All-Female Normal Sex Ratio Ratio
Viti Levu 14 0 1 1
Vanua Levu 1 0 3 3
Ovalau 0 1 1 4
Taveuni 1 1 0 2
Kadavu 1 1 0 1
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 8 6
The data are fairly sparse, but the sex ratio on emergence from the different specimens
indicates the male killing trait to have been present in all studied Fijian islands at some
time in the last 80 years.
Modem molecular analysis techniques such as PCR allow larger sample sizes to be
easily checked for presence of a symbiont of known sequence. These give increased
power when examining different populations for evidence of heterogeneity in prevalence
of the infection. A survey was carried out in 1999 using larger sample sizes, in order to
see if the same patterns were obtained. Variation in prevalence across islands would
allow examination of the causes and consequences of different levels of male killing
infection. During this survey, PCR assay was carried out using Wolbachia B-group
specific primers. General wsp primers that amplify the wsp gene of all Wolbachia were
used to amplify specimens for sequence analysis (all primer sequences section A.2.
Appendix I).
Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 87
0 1------,---- s1 o mi 543 km ra/wcr Yas4Twa ^ Croup NèbùuWài BJtih" W Uvio K o m I S e a iV/ievu Nako! ausori uvm »vva Oïotjtp Vütuftt* 1 Moaioep I Kcd^fiavti . * fofoyo*^ Kabafo% , W ayalailai tC(X««NU Island MutttikKt Pcieî/ïc Ocean Figure 2.7. Map of the Fiji Islands. H. bolina were collected from Suva and Nadi (Viti Levu Island), Taveuni Island, and Wayalailai Island (inset). Map adapted from http//:www.nationalgeographic.com 2.11.2.Method Female H. bolina were collected from three island locations within the Fijian archipelago: Viti Levu, Taveuni and Wayalailai (Figure 2.7.). Kadavu and Ovalau islands were also visited, but unfortunately no specimens were obtained due to poor weather conditions. The specimens were preserved in 95% ethanol immediately following death. DNA was extracted from each specimen (protocol A.I., Appendix I) and the supernatant used directly in PCR assay using the Wolbachia B-group specific wsp primers (protocol A.3.1., Appendix I). As per section 2.10, samples were checked for DNA presence (see Figure 2.4.). Single stranded sequence of the wsp gene was obtained from a single infected individual from each island location to ensure the infection identical at this level (protocol A.5., Appendix I). Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 8 8 2.11.3.Results Prevalence of the male killer was calculated in the three different areas of Fiji, as summarised in Table 2.8. Chi-squared analysis rejects the null hypothesis of homogeneity between islands = 7.778, df = 2; p = 0.02) indicating variation in prevalence of the male killing trait across the Fiji Islands. Table 2.8. Percentage prevalence of the Wolbachia male killing bacteria in H. bolina females from three different island populations across Fiji. (*N.B. 26 of these females were collected in 20 0 1 ) Area of Fiji Prevalence n (%) Viti Levu Island 58.8 34 Wayalailai Island 53.9 76* Taveuni Island 25.0 24 2.11.4. Conclusion In agreement with the earlier work of both Simmonds and Clarke et al. (1983), the male killing trait is found throughout the Fiji islands. Variation in prevalence is observed, with the prevalence of the male killing Wolbachia in the Taveuni population being approximately half that of the other two areas surveyed. Reasons for this are investigated and discussed in Chapter five. Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 89 It is interesting that prevalence variation seems to have altered since Simmonds’ survey (Poulton, 1923; Simmonds, 1926). He reported a very high prevalence (all fourteen females tested) of the all-female trait from Suva (Viti Levu) in 1924. Perhaps the reason for this decrease is due to evolution of resistance to the male killer trait, although we found no evidence suggesting that this was the case. It could also be that Simmonds unwittingly bred from a single matriline, or reported all-female broods with bias. 2.12. Discussion Correlation between the presence of B-group Wolbachia and the all-female trait across different matrilines, as revealed by PCR assay, indicates that Wolbachia is the cause of male killing in the butterfly H. bolina in Fiji. Wolbachia bacteria represent one of the most widespread causal agents of embryonic male killing, being responsible for one third of reported cases to date. Knowing this, and given the high frequency of occurrence of Wolbachia in insects and other arthropods (thought to be 17-42% across all insect species) (Stouthamer et a l, 1999) it is perhaps unsurprising to find that we are dealing with another Wolbachia male killer. These findings support the view that there will be many unreported cases of Wolbachia male-killing in the wild, and that male-killing will be a common phenotype within the clade Wolbachia (Jiggins et a l, 2001c). The result that a male killing Wolbachia is responsible for all-female broods in H. bolina is further reinforced by the fact that the sequences of the wsp dûctdiftsZ genes of this Wolbachia are identical to those in a known causal agent of male killing. The two host species H. bolina and A. encedon are only distantly related (they are in different tribes within the family Nymphalidae) but share an identical male killing Wolbachia, strongly implicating horizontal transmission of the male killing element. This suggests that Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 9 0 certain Wolbachia strains specialise on certain host sex determination systems. In butterflies, in contrast to the majority of other insects, the male is the homogametic sex. Perhaps only particular strains of male killer are able to act on host systans of this type. More Lepidopteran male killers need to be examined in order to verify this. One notable result from this study is the failure of antibiotics to cure the all-female trait. This does not rule out Wolbachia as the cause of male killing in H. bolina, as antibiotics did not cure the Wolbachia infection either. There are many examples of cases in which just a single treatment with antibiotic will cure infected arthropods of sex-ratio distorting bacteria (Stouthamer et a l, 1990). Why this should not be the case in//, bolinorxtmdXns a mystery for further investigation. Potential explanations of the cause of failure of the two different antibiotics to cure both the Wolbachia infection and the all-female trait in H. bolina are that either the Wolbachia are antibiotic-resistant, or that the host did not sequester the antibiotics. Antibiotic-resistant Wolbachia would be a very useful tool for manipulation of this bacterium, and thus the investigation of the lack of response of the trait and bacterium to antibiotics should be carried further. However, there are huge implications on other WolbachiajhosX systems if these bacteria can develop antibiotic resistance: Wolbachia is thought to play an essential non- pathogenetic role in the biology and metabolism of filarial worms that cause diseases such as river blindness in humans (Frankish, 2002; Saint Andre et a l, 2002). Elimination of Wolbachia using tetracycline will also kill the adult nematodes (Langworthy et al, 2000). The ability of Wolbachia to evolve antibiotic resistance, as suggested in this chapter, may lower the long-term utility of antibiotics in drug treatment of filiariasis. Wolbachia prevalence is well understood in theory, but not in practice. Data from Clarke et. al (1975) indicates a high degree of variation in prevalence of the male-killer in H. bolina across different populations. In this study, prevalence is seen to vary across the different islands of Fiji. The cause of this variation will be discussed further in Chapter Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 91 five. It should be possible to examine genetic and ecological characteristics in different populations to see if there is any evidence for the presence of resistance genes on certain islands, or if any correlates with life history traits and male-killer prevalence exist in this species. Notably, Æ bolina lays eggs in clutches of 1012 eggs in the Fiji Islands (pers.obsn) whereas in Australia (where Clarke found no evidence for the presence of a male killer) the species lays eggs singly or in pairs (Kemp,1998). Given the importance of sibling competition in the spread of male-killers (Hurst et a l, 1993), the aims of this thesis are to carry out further investigation into this phenomenon across a range of populations that may give an insight into possible causes and maintenance of a male killing cytoplasmic factor within a host population. Inherited parasites in the butterfly Hypolimnas bolina Chapter 2: Association of all-female broods with Wolbachia infection 9 2 Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 93 Chapter 3: Extraordinary Sex Ratios In Independent Samoa Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 9 4 Chapter 3: Extraordinary Sex Ratios In Independent Samoa Summary This chapter presents the results of studies of Hypolimnas bolina in Independent Samoa. The same male killing Wolbachia identified from the Fiji Islands is present in all study populations in this country. The prevalence of the trait is exceedingly high, in fact the highest recorded prevalence of a male killer in a natural population. The effects of this high prevalence infection on the reproductive biology of the host are investigated and compared with the islands of Fiji (see previous chapter) and neighbouring American Samoa (where H. bolina populations are uninfected with male killing Wolbachia). The dearth of males on the islands of Independent Samoa increases female virginity and decreases female fertility, demonstrating for the first time the dramatic effects a highly prevalent selfish genetic element can have on its host population. The size of spermatophores produced by males from Independent Samoa is smaller than those produced by Fijian and American Samoan males, and this is independent of male mating history. This data is consistent with the evolution of ejaculate partitioning in Independent Samoa. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 95 3.1. Why StudyHypolimnas bolina In Independent Samoa And American Samoa? Independent and American Samoa are neighbouring South Pacific island nations in which//, bolina is common (section 3.3. describes the countries in detail). Historical evidence suggests that H. bolina populations on these islands may help to address previously unanswered questions concerning male killing symbioses:- 3.1.1. Historical evidence of female-bias in if. in the Samoas No record was found of breeding data of//, bolina from either Independent or American Samoa. 3.1.1.1. Independent Samoa Although there is no actual data indicating the presence of the male killer in Independent Samoa, G.H.E. Hopkins in his book ‘Insects of Samoa’ (1927), wrote: “The female (//. bolina) is very common everywhere on all the islands of Western (Independent) Samoa, but the male is extremely rare, much less than 1 percent.” Hopkins also records that Rechinger in 1905 failed to observe a single, male//. bolina in Independent Samoa. Hopkins speculates that H. bolina females in Independent Samoa must be parthenogenetic, although he provides no evidence for this deduction, other than the observed population sex ratio and the fact that all six females dissected were unmated (Hopkins, 1927). 3.1.1.2. American Samoa Hopkins (1927) reports that in Tutuila (American Samoa) in contrast to Independent Samoa, males are seen to outnumber the females. This does not necessarily indicate that the male killing parasite is absent from this country as it is often the case (as in Fiji) that Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 96 despite the presence of a male killer at a fairly high prevalence, males are more commonly sighted. 3.1.2. Implications of historical evidence Hopkins’ suggestive reports indicate the presence (at least in 1927) of a male killing symbiont at a very high prevalence in the Independent Samoan populations oîH. bolina. These populations could help address a number of previously unanswered questions concerning male killer symbioses. Initially, it is important to establish (on both American and Independent Samoa) if there is a male killer infection and if there is, whether dr not it is the same Wolbachia male killer identified from Fiji. Secondly, the prevalence of infection needs to be examined in order to assess how the situation on these islands has altered (or not) since 1927. This may have occurred in one of three ways: 3.1.2.1. Independent Samoan population unchanged since 1927 If the populations oiH. bolina on Independent Samoa appear as described by Hopkins, with the numbers of females far exceeding the numbers of males, then resistance to the male killing symbiont has not evolved despite intense selection. If fieldwork and molecular analysis of 77. bolina show that infection prevalence is very high, the implications of high prevalence infection on the reproductive biology and behaviour of the host can be examined. 3.1.2.2. Independent Samoan population appears ^normal* If the populations of H. bolina on Independent Samoa exhibit an observed sex ratio closer to 1:1 than indicated by Hopkins and Rechinger, then it would appear that the populations have evolved resistance to the male killing symbiont (Hopkins, 1927). 3.1.2.3. H. bolina absent from Independent Samoa If no H. bolina specimens can be found on Independent Samoa, the population has clearly been driven to extinction due to the spread of the male killer. Such an outcome Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 9 7 would demonstrate the effect that a highly prevalent selfish genetic element can have on its host population, showing that resistance evolution is not always inevitable. Assessment of the American Samoa populations of H. bolina will provide information as to whether the situation in these populations has altered since Hopkins’ 1927 observations, and whether or not the male killing symbiont is present in American Samoan H. bolina populations. If prevalence of the male killing symbiont is different from that observed in Independent Samoa, the populations from the different countries can be compared and contrasted to//, bolina populations from the Fiji Islands,-to attempt to explain the causes and consequences of any observable variation in prevalence of infection. 3.2. Aims of Fieldwork In The Samoas 1. To establish whether the population on Independent Samoa has remained in its female-biased state. 2. To test whether the female-biased population is the product of high prevalence of the same male killing Wolbachia identified from Fiji. 3. To compare the reproductive biology of the//, bolina populations from Independent Samoa with those of the Fiji Islands and American Samoa. 3.3. Introduction To Independent Samoa And American Samoa. The Samoas are a South Pacific archipelago situated in Micronesia. Independent Samoa consists of two large islands: Savaii and Upolu (where all field work was carried out). Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 98 and three small islands. To the East, American Samoa consists of four islands: butterflies were collected from Tutuila and Olosega islands. Forty kilometres of ocean separate the Eastern tip of Upolu (Independent Samoa) and the Western tip of Tutuila (American Samoa) (see Figures 3.1. a and b, below). 0 (9 mi ------1 ,— I Ù id km ' Pacific Ocean »t|W lo (vvala«u S a ls'iiu ttxio\^>gA lei Apoiint^ Strait Afiofhna * L eu (u m < » g Solojoio fftidaUt Tt'ave* Sd/dra U*S £ Ja^ S aU n l Figure 3.1.a. Map of Independent Samoa taken from www.nationalgeographic.com (Western tip, Latitude: 172°45’W, Longitude: 13°30’S; Eastern tip. Latitude: 17r25’W, Longitude: 14°00’S) Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 99 0 lOmi I J 1 ' I 0 10 km ; :# I Marraa islanet ] ^ ) ^^Ofosêga AMERICAN SAMOA Pago Pag Aumta ' I tctij Tt^tuffa Pacific Ocf CM Figure 3.1.b. Map of American Samoa taken from www.nationalgeographic.com (Western tip, L>atitude: 170°50’W, Longitude; 14°20’S; Eastern tip, Latitude: 169°20’W, Longitude: 14”14’S) 3.4. General Methods For Fieldwork In Independent Samoa All laboratory work whilst in the field was carried out in Apia (Figure 3.1.a), the capital of Independent Samoa, in two separate three month field seasons: July through to October 2000 and June through to September 2001. For breeding and rearing purposes, all female butterflies were collected from Independent Samoa: from Apia (Upolu Island), and Fagamalo (Savaii Island) unless otherwise specified (see map. Figure 3.La) For the purposes of crossing experiments, male H. bolina were collected from the same areas of Independent Samoa, and also from Pago Pago (Tutuila Island) American Samoa, and Wayalailai Island, Fiji (Figure 3.1.b-American Samoa; Map of Fiji Islands - Figure 2.7., Chapter 2). Apia and Pago Pago are semi-urban areas, Fagamalo and Wayalailai more rural. All butterflies were collected following the collection method detailed in Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 100 Chapter 2, section 2.3.1. These butterflies were strictly maintained in captivity to prevent contamination of the population. 3.4.1. Rearing specimens Sida rhombifolia, a favoured host plant of H. bolina for both oviposition and larval feeding, was used for all fieldwork in Independent Samoa (Vane-Wright et a l, 1977). Sida rhombifolia is a perennial shrub that is common in disturbed areas throughout Independent Samoa (Whistler, 1995). Some plants were cultivated in pots in the 'laboratory’, but the majority of food plant was collected daily from the field. 3.4.1.1. Maintaining adults and obtaining eggs All female butterflies collected in Independent Samoa, were coded ‘SAM’ for Independent Samoa. Butterflies collected on Upolu Island were coded numerically according to the order of collection, i.e. the first butterfly collected in Apia was coded ‘SAM 1’, the second ‘SAM2’ etc. Butterflies collected on Savaii Island were coded alphabetically in order of collection, i.e. the first butterfly collected in Fagamalo was coded ‘SAM A ’, the second ‘SAM B’ etc. Live specimens were kept in the laboratory in cylindrical 60cm diameter hanging cages made of black fabric mesh (Watkins and Doncaster E60911) (Figure A2.3., Appendix II). Adults were provided with “false flowers” containing 0.4M-sugar solution on which to feed. All butterflies were labelled using a small Tipp-Ex blob on the wing tip, with their specific code written on in permanent pen (Figure A2.4., Appendix II). No more than fifteen adults were kept in a single cage. For ovipostition purposes, small laying cages were designed (Figure 3.2. and photograph A2.6., Appendix II,). A fresh sprig of 5. rhombifolia was cut and placed in the water bottle, and a single female placed on the plant and caged in using mosquito netting secured by the elastic band. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 101 Wire Frame. Mosquito netting placed Hypolimnas over this and bolina wild secured with an caught elastic band female placed in Freshly cut here sprig of Sida rhombifolia Plastic 1 litre bottle, filled with water. Hole cut in lid to hold plant. Figure 3.2. Diagram showing the construction of the laying cages, also see photograph (Figure A2.6., Appendix II) The cages containing females were placed in direct sunlight for thirty minutes, and after this in dappled shade to encourage oviposition. Only healthy plants showing signs of new growth were used, as H. bolina females prefer new growth for oviposition (Kemp, 1998). At the end of the day, the butterfly was removed back to the hanging cage, and the plant sprig checked for presence of eggs. Leaves containing egg clusters were removed and each clutch laid by the same female on the same day stored in a separate 9 cm Petri dish. The following recordings were made: 1. Number of eggs laid. 2. Date laid 3. Date hatched 4. Number of eggs that hatch Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 1 0 2 5. Number of unhatched grey eggs. 6. Number of unhatched yellow eggs. Each egg clutch was coded according to matriline and numbered depending on the number of clutches previously laid by the particular female. For example, the first clutch laid by the first female collected in Apia (female ‘SAMI’) was coded ISAMl, the second 2 SAM1 etc. 3.4.L2. Rearing larvae An FI generation was reared, larvae being fed an excess of S. rhombifolia leaves (Vane- Wright et a l, 1977), fresh leaves being provided twice daily. As in the Fijian fieldwork, containers were lined with tissue paper and cleaned out once a day. Caterpillars were initially reared in 9 cm transparent Petri dishes, then transferred after the third larval moult to large plastic yoghurt pots (450g size) covered with fine mesh mosquito netting secured with an elastic band. No more than ten caterpillars were reared per container. Pupae were removed from containers the day after forming, to prevent disturbance by other larvae. Duration of larval and pupal stages was recorded. The times of any larval deaths within each matriline during the course of rearing were also noted. On emergence, sex ratio and wing-type of adults were recorded following the classification of female wing polymorphisms in 77. bolina females outlined in Clarke & Sheppard (1975). 3.4.1.3, Mating laboratory-reared adults Adults from the FI generation and wild-collected virgin females were mated in a double bed sized mosquito net erected outside in a sunny position, usually with a small amount of dappled shade (Figure A25., Appendix II). The cage was checked every half an hour for mating pairs. These were removed to a separate small cage, for identification purposes. As in the Fijian fieldwork, FI mated females were coded alphabetically according to matriline (based on the code of their mother), i.e. the first mated FI female Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 103 from the first clutch of the first field-collected female from Apia (‘SAMI’) would be coded ‘ISAM IA’. Outbreeding was ensured by only allowing interaction between unrelated FI males and females. Egg hatch rates were obtained for the F2 generation, but larvae were not reared to adult in most cases due to time constraints. 3.5. Observed Population Sex Ratios In Fiji, American Samoa And Independent Samoa In direct contrast to Fiji, where territorial male//, bolina are much more commonly sighted than females, in Independent Samoa only 5 males were seen over the total period of study, whereas females were observed daily flying in numbers. In order to quantify this and compare the Independent Samoan population with that of Fiji and American Samoa, a basic survey of the observed sex ratios in each population was carried out. 3.5.1. Method The numbers of male and female H. bolina observed were recorded on a walking census of the different island populations of Fiji, American Samoa and Independent Samoa. The observed proportion of females was calculated for each population. Many, but not all, of these butterflies were collected. 3.5.2. Results The numbers of male and female//, bolina observed in the three populations from Fiji and the two populations from American Samoa are very similar, all showing an observed proportion of females of around 0.3 (%^=2.401, df = 4, ns.). However, the populations in Independent Samoa show a completely different pattern. On both Upolu and Savaii Islands, the observed proportion of females is much higher, 0.99 and 0.98 on either island respectively. The two populations from Independent Samoa are homogeneous with Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 104 respect to observed proportion of females (x^=0.785, df = 1, ns.). However there is a significant difference between the two Independent Samoan populations and the five populations from American Samoa and Fiji (%^363.00, df = 1, p<0.0001) (Figure 3.3.). n = 327 n = 41 n = 28 n — 63 n = 43 n = 88 n = 26 Upolu Savaii Olosega Tutuiia Viti Levu Taveuni Wayalailai Independent Samoa American Samoa Fiji Figure 3.3. Proportion of female H. bolina observed whilst collecting in Independent Samoa, American Samoa and Fiji. 3.5.3. Conclusion As in Hopkins’ (1927) survey, the population sex ratio in Independent Samoa is drastically skewed towards females in comparison to both the American Samoa and Fijian populations. The Independent Samoan population was investigated more closely to discover why this should be the case. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 105 3.6. Do All-Female Broods Occur In Hypolimnas bolina In Independent Samoa? 3.6.1. Egg hatch rates 3.6.1.1. Method Hatch rates of all egg clutches laid in the laboratory were recorded as number of eggs that hatch, and numbers of unhatched grey and yellow eggs. 3.6.1.2. Results Only butterflies that had mated (spermatophore present in the bursa copulatrix) gave rise to fertile eggs. Figure 3.4. provides a summary of egg hatch rates obtained from fertile females collected in the field from Upolu and Savaii Islands, and in the subsequent FI generation. These hatch rates are calculated based on the number of eggs that hatch and the number of grey (male killed) eggs that did not hatch, and do not include the number of infertile (yellow) eggs that did not hatch. The percentage egg hatch rate in each case is calculated as shown: ______N° Eggs Hatch ______(N° Eggs Hatch + N° Grey Unhatched Eggs) Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 106 30 ^ Upolu 25 (I) □ Savaii 0 20 g ■ FI Ü ‘S 15 n0 I 10 I 35-40 41-45 46-50 51-55 56-60 61-65 66-70 71-75 76-80 81-85 86-90 91-95 95-100 Percentage of Fertilised Eggs that Hatch Figure 3.4. Percentage of fertilised eggs that hatch from Independent Samoan female//. bolina collected from Savaii and Upolu Islands, and in the subsequent generation. (N.B. due to time, weather constraints and lack of male//, bolina, only a few FI females were mated.) In total, 113 out of 114 wild-collected Independent Samoan females gave rise to fertile eggs that showed the embryonic mortality consistent with the presence of a male killing symbiont. The mean proportion of fertile eggs that hatch is 0.533 and egg hatch rate is homogeneous across these 113 matrilines (%^=35.36, df = 82, ns.). Sixty-four of these broods, including the single high hatch rate brood, were reared to adulthood. The egg hatch rate data obtained from captive mated FI generation females show that the trait is inherited, as previously shown in the Fiji Islands. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 107 3.6.1.3 Conclusion These data suggest the strongly female-biased population sex ratio is associated with the presence of a male killing symbiont at a high prevalence throughout the Independent Samoan H. bolina populations. 3.6.2. Adult sex ratio 3.6.2.1. Method Sex ratio of all reared broods was recorded on emergence from pupae. Due to time constraints, only the FI generation was reared to adulthood. 3.6.2.2. Results Of the 64 matrilines scored for sex ratio produced, 61 gave all-female broods on emergence (Table 3.1). Of these, 34 were all-female with brood size ^6 (statistically female biased on a binomial test) and 27 were all female with brood size < 6. Of the remaining three matrilines, two broods were highly female-biased (2 males: 12 females and 2 males: 14 females) with the few sons dying soon after emergence as adults. Only one normal sex ratio brood from Independent Samoa was recorded during this study, this being the same matriline that gave a high egg hatch rate (adult sex ratio, 22 males: 22 females). Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 108 Table 3.1. Summary of FI adult sex ratios from Independent Samoan i/. bolina, grouped according to the number of individuals in a brood. (Female-biased broods are those that gave a sex ratio significantly different from 1:1). Independent Normal Sex Ratio Female-Biased Female-Biased Samoan Broods Broods (n s: 6) Broods (n < 6) Population Upolu 1 30 26 Savaii 0 6 1 3.6.2.3. Conclusion All-female broods do occur in Independent Samoa. Both the egg hatch rate data, and the adult sex ratio indicate the presence of a high prevalence male killing symbiont in Independent Samoa. We investigated further, to identify the causal agent of the sex ratio distortion and establish if it was the same male killer as that found in the Fijian H. bolina. 3.7. What Is The Causative Agent Of All-Female Broods In Independent Samoa? Based on the results of the field study, we decided to test the samples for presence of Wolbachia, and to obtain the sequence of the Wolbachia if it was present, in order to establish if the same male killer identified from the Fijian population was causing sex ratio distortion in H. bolina in Independent Samoa. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 109 3.7.1. PCR analysis forWolbachia 3.7.1.1. Method Five of the original wild-collected Independent Samoan H. bolina females were randomly selected (three from the Upolu population and two from the Savaii population) together with the single Upolu female that gave a normal sex ratio brood (the other five females had given low egg hatch rates and all-female broods). The specimens were prepared for molecular analysis following the DNA preparation protocol A.I., Appendix I. The specimens were assayed for the presence of the hdiCienumWolbachidiwsp gene) using the B-group specific PCR (protocol A.3., Appendix I; primers w sp^li and wsp69\i were used, see A.2., Appendix I for sequences). 3.7.1.2. Results PCR assay revealed an association between the presence of Wolbachia and the presence of the all-female trait, as observed in Fiji: the five female-biased matrilines tested positive for the presence of Wolbachia, the single normal sex ratio matriline was found to be uninfected with Wolbachia. Analysis of the wsp sequence of the infecting strain would reveal the relatedness of the Independent Samoan Wolbachia strain to the Wolbachia strain previously identified as the causal agent of male killing in//, bolina in the Fiji Islands. 3.7.2. Sequence analysis 3.7.2.1. Method DNA from the five infected matrilines was prepared for PCR following protocol A.3.I., Appendix I. The amplified DNA was prepared for sequence analysis and sequenced following protocol A.5, Appendix I. The wsp sequence was manually aligned to the previously determined Wolbachia sequence from Fijian//, bolina (Cabot, 1997). Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 110 3.7.2.2. Results The causal agent of sex ratio distortion in Independent Samoa is a B-group Wolbachia bacterium, identical in wsp sequence to the male killer previously identified from Fiji. 3.7.2.3. Conclusion The production of all-female broods in Independent Samoa is associated with the same strain of Wolbachia previously identified from H. bolina in the Fiji Islands. 3.8. Prevalence Of The Male Killing Wolbachia In Independent Samoa And American Samoa 3.8.1. Method Further wild-collected female H. bolina from Upolu and Savaii Islands, Independent Samoa were subsequently assayed for the presence of the male killing Wolbachia using PCR. Female H. bolina were also collected from Tutuiia and Olosega Islands, American Samoa and assayed for Wolbachia presence. No field data was obtained from American Samoa, specimens were simply collected and killed. All specimens were preserved in 95% ethanol immediately following death. DNA was extracted from each specimen (A.I., Appendix I) and the supernatant used directly in PCR assay using the Wolbachia B-group specific wsp primers (sequences A.2, protocol A.3.I.; Appendix I). Specimens that gave negative results were checked for the presence of amplifiable DNA template (protocol A.3.2., Appendix I). 3.8.2. Results As expected from the breeding data, the male killing B-group Wolbachia was very common on Independent Samoa; the average prevalence of the Wolbachia male killer among//, bolina females across both islands being 99.6% (n=292) (Table 3.2). This is in Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 111 sharp contrast to the prevalence of 59% (n=35) recorded on Viti Levu, Fiji (Chapter 2, section 2.11). No evidence was found for the presence of the male killing Wolbachia from the American Samoan samples. On Upolu Island, one of the two uninfected individuals is the same female that gave rise to the normal sex ratio brood; the other female was unmated. Table 3.2. Percentage prevalence of Wolbachia male killer in H. bolina females across different islands of Independent and American Samoa Country Island Prevalence (%) N Independent Upolu 99.2 257 Samoa Savaii 100 35 American Olosega 0 23 Samoa Tutuiia 0 6 3.8.3. Conclusion Prevalence of the male killing Wolbachia among female H. bolina in Independent Samoa is the highest ever recorded for a male killing element. This has huge implications on the Independent Samoan host population. The male killing symbiont does not appear to be present in the American Samoan population. This seems remarkable as, at their closest point, the two countries are separated by a mere 40 kilometres of ocean. The Independent Samoan populations were examined in more detail and compared to H. bolina populations in both Fiji and American Samoa. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 112 3.9. Comparison Of Female Virginity In Fiji, American Samoa And Independent Samoa The observations detailed in the previous section indicate that the H. bolina populations in Independent Samoa are very different from those in the other two countries. With the sex ratio so skewed towards females we would expect to see evidence of population-level effects in the Independent Samoan populations. Initially, the matedness rate in the different wild populations was examined to analyse effects of prevalence. 3.9.1. Method All wild-caught females from each of the populations were dissected under a Leica MZ6 stereomicroscope. The abdomen was split and the presence or absence of a spermatophore recorded. The number of spermatophores present in the bursa copulatrix was also noted. A single spermatophore is transferred from the male to the female during copulation, so the presence of a single spermatophore indicates that the butterfly has mated once. If no spermatophore is present in the bursa copulatrix then the female is a virgin. It is important to note that the spermatophore is still present in the bursa copulatrix of the female regardless of whether or not the contents of the spermatophore have been ‘used up’. 3.9.2. Results The proportion of mated females from each of the populations sampled was calculated. Within each population, the proportion of H. bolina females that had mated once (one spermatophore present in the bursa copulatrix), twice (two spermatophores) and three times (three spermatophores) was recorded (Figure 3.5.). The results from American Samoa are presented together: most of the females were collected from Olosega (23 specimens) the other six specimens were from Tutuiia and were all mated. There was no Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 113 significant difference between the proportion of mated H. bolina females from American Samoa and Fiji (10,000 Monte Carlo Simulations, p<0.03, ns.). Compared with American Samoa and Fiji, Independent Samoan females exhibited a significantly higher level of unmatedness (x^=82.77, df = 1, p<0.0001). 1 ■ 3 Spermatophores 0.9 □ 2 Spermatophores 0.8 m 1 Spermatophore w Î 0.7 i Ü- 0 .6 I 0.5 • | 0.4 I 0.3 CL 0.2 0.1 0 Upolu Savaii A. Samoa Viti Levu Taveuni Wayalailai (n=174) (n=40) (n=29) (n=28) (n=27) (n=75) Independent Samoa American Fiji Samoa Figure 3.5. Proportion of wild-caught females from different island populations that had mated once, twice and three times, as evidenced by the presence of spermatophores in the bursa copulatrix. Error bars represent binomial 95% confidence intervals on the proportion of females that had mated. 3.9.3. Conclusion The virginity rate among wild-caught female H. bolina from Independent Samoa is significantly higher than that among wild-caught H. bolina from either Fiji or American Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 1 1 4 Samoa. This is undoubtedly due to the sex ratio being dramatically skewed towards females, with males being the limiting resource in Independent Samoa. It is also interesting that there were no examples of females mating more than once in the Independent Samoan populations. Multiple-mating was observed in each of the other populations, however the paucity of female H. bolina with more than a single spermatophore in the bursa copulatrix from any of the study populations indicate that multiple-mating is clearly not characteristic of the species. 3.10. Comparison of Female Fertility in Fiji and Independent Samoa Female-biased sex ratios may affect the fertility of mated females in addition to their opportunity to mate. In Lepidoptera, all fertilized eggs turn grey prior to hatching due to the development of the tanned head capsule of the embryo. If these eggs are infected with a male killer, approximately half of these developed eggs hatch (these being daughters). Unfertilised H. bolina eggs remain yellow. In order to examine differences in fertility between H. bolina populations from Fiji and Independent Samoa, the proportion of infertile eggs laid by females from each population was compared. 3.10.1. Method For each Fijian and Independent Samoan wild-collected female that laid eggs in the laboratory, the numbers of fertile and infertile (yellow) eggs laid within the first week after capture were recorded. As soon as larvae had dispersed from the egg cluster after hatching, the egg cluster was removed and the numbers of infertile (yellow), grey (fertilised, male killed) and hatched (fertilised) eggs noted. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 115 3.10.1.1. Effect of cross-population mating In order to determine if the fertility of Independent Samoan females was a function of the females themselves, or the male to which they had mated, virgin Independent Samoan females were mated to males from a population in which males were not a limiting resource. Virgin Independent Samoan females were mated to males from either Fiji (14 matings) or American Samoa (7 matings) and the same recordings of egg fertility made. 3.10.1.2. Does male fatigue effect female fertility? It is possible that the female-biased population sex ratio in Independent Samoa leads to the few males that are present mating so many times that they become exhausted, such that matings later on in their lives produce fewer fertile offspring. In order to control for male fatigue, a virgin Independent Samoan male (obtained from a laboratory cross) was mated to a virgin Independent Samoan female, and the subsequent fertility of the female recorded. No replicates of this cross were performed due to the shortage of Independent Samoan males, and the unwillingness of these males to mate. 3.10.2. Results Field-mated Independent Samoan 77. bolina females laid a significantly higher proportion of unfertilised eggs than Fijian females, indicating a much lower rate of fertilization in Independent Samoa iyf = 40.79, df = 1, p<0,0001) (Figure 3.6.). 3.10.2.1. Effect of cross-population mating The 21 virgin Independent Samoan females crossed with males from either Fiji or American Samoa showed equivalently high fertilization rates to wild collected Fijian females, with 99% of eggs being fertilized (yf = 1.54, df = 1, ns) (Figure 3.8.). This manipulation demonstrates that reduced fertility of Independent Samoan females is not due to the Wolbachia infection of the females but to the lack of sperm from Independent Samoan males. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 116 1.0 1 O)CO o 0.8 0) ■ ■ C CD c 0.6 o o Q. 2 0.4 Q. C CO TD 0.2 CD 0.0 CO CO Æ GO 3 LO jO C\J o > T— i II CO ë II c E CD CO CO II CO ÎA I f C If S 's . CO E c < Independent Fiji American Crosses: Ind. Samoa Samoa Samoa female x Figure 3.6. Median proportion of fertile eggs laid by wild-collected (i.e. locally-mated) females from different Islands, and virgin Independent Samoan females crossed with American Samoan or Fijian males. Error bars represent interquartile range. 3.10.2.2. Does male fatigue effect female fertility? The single virgin Independent Samoan female crossed with a laboratory-reared virgin local male showed an egg fertilization rate of 0.68, similar to that observed from wild- collected Independent Samoa females (median fertilized 0.72). The fact that fertility is low, whether or not the Independent Samoan male is a virgin, suggests that the effect of lowered fertility observed in Independent Samoa is not due to male fatigue, although the data is rather limited. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 117 3.10.3. Conclusion The combined data on virginity rates and the fertility levels of mated females suggest a high degree of sperm limitation in the Independent Samoan population, which is apparently not due to male fatigue. The fact that there is little variance among samples from each population also indicates that male fatigue does not effect female fertility: if females mated to older (more ‘tired’) males showed reduced fertility compared with those mated with ‘fresher’ males, we would expect to obtain a larger variation in spermatophores collected from wild-caught females due to random sampling. 3.11. Comparison Of Spermatophore Sizes The fact that fertility of Independent Samoan females is low, and that this is a male effect not associated with males tiring, indicates that males are somehow limiting the amount of sperm per ejaculate. In order to examine whether this was a possible explanation for the observed fertility decrease, the size of spermatophores of males from the different populations was measured. 3.11.1. Method Spermatophores were dissected from the bursa copulatrix of wild-collected females from American Samoa (Olosega Island), Fiji (Viti Levu Island) and Independent Samoa (Upolu Island). Spermatophores were also dissected from the Independent Samoan females that had been cross-mated with either American Samoan or Fijian males (see section 3.9.1.1.). All spermatophores were stored separately in 95% ethanol. Spermatophores were taken individually and placed on a slide on a video microscope. They were visualised on screen using NIH image (version 1.55, National Institute of Health, USA). Drawing tools and a graticule were used to calibrate the software allowing Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 118 measurement of the spermatophore image to the nearest 0.005 mm. The length and diameter of each spermatophore was measured and recorded. 3.11.2. Results Spermatophores from Independent Samoan males are significantly smaller than those from either American Samoan or Fijian males (Length: nl=29, n2=42; U=0; p<0.0001. Diameter: nl=29, n2=42; U=0; p<0.0001) (Figure 3.7. a and b). This difference persists regardless of the source of the female, with Fijian and American Samoan males mated to Independent Samoan females producing large spermatophores. That this difference is not due to male fatigue is indicated by the size of the spermatophore from a virgin Independent Samoan male mated to a local female, which is not significantly different from those found in wild collected Independent Samoa females (diameter = 1.090mm, length = 0.950mm compared to wild female: median diameter = 1.165mm, median length = 0.960). Again, variance is low among the different populations reinforcing that the observed differences are not due to male fatigue. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 119 (a) £ 1.5 c Q) £ sz0 Q. 1 - § 1 Q) Q. V) .1 0.5 Independent American Samoa Fiji (n = 11 ) I.Sam female x I. Sam female x Samoa (n = 28) (n = 10) Fiji male (n = 14) A. Sam male (n = 7) 2.5 (b) I ^ IE ••§ 1 .5 0£ JO. CL 10) ' c .55 m 0.5 Independent American Fiji (n = 11) I.Sam female x I. Sam female x Samoa (n = 28) Samoa (n = 10) Fiji male (n = 14) A. Sam male (n = 7) Figure 3.7. Median spermatophore (a) diameter (b) length removed from the bursa copulatrix of wild-collected females from different islands, and virgin Independent Samoan females crossed with American Samoan, or Fijian males. Error bars represent interquartile range. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 120 3.11.3. Conclusion Males from Independent Samoa produce significantly smaller spermatophores than males from either Fiji or American Samoa. This effect is independent of the origin of the female mating partner, and there is no evidence it is associated with fatigue in males that re-mate frequently. This effect appears to be limited to the Independent Samoan population, where the Wolbachia male killer is at an excessively high prevalence. 3.12. Does The Presence Of A High Prevalence Male Killer Alter Behaviour Of Hypolimnas bolina In Independent Samoa? In the African butterfly A. encedon that is infected with a male killing Wolbachia at high prevalence, Frank Jiggins has reported sex role reversal, with virgin females forming leks, and apparently competing for males (Jiggins et a l, 2000c). However, no populations of A. encedon are known that do not show this behaviour, so it is impossible to conclude whether this apparent ‘role-reversaF is due to the presence of a male killer at high prevalence in this host population, or a case of behavioural plasticity. Hypolimnas bolina presents an ideal opportunity to examine whether or not mating system is affected by the presence of a high prevalence male killer within a population. Contrasting populations from Independent Samoa with Fijian populations enables the effect of the male killer on the population to be examined. Territoriality of if. bolina males has been well documented, for example Darrell Kemp’s studies of male perch site selection and contest behaviour in Queensland, Australia (where no evidence has been found for male killer presence) (Kemp, 2000; Kemp, 2001; Kemp, 2002). Observations of butterfly behaviour in Fiji and American Samoa reinforce these findings. In these countries, males are observed flying in numbers, are very active and territorial and Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 121 constantly searching for mates (pers.obsns). In Fiji, even in areas around Suva where the male killer is at fairly high prevalence, females were rarely seen. Females tended to be flying fast and high, hence difficult to catch. Conversely males, were easy to find, patrolling, nectaring and resting on S. urticifolia (Blue rat’s tail) plants. In contrast, the few males caught in Independent Samoa were very inactive and reluctant to mate, whereas the females were more active and prone to chasing each other. Reporting from the South Pacific Islands in 1888, Mathew describes female//, bolina as showing high site fidelity (Mathew, 1888): “Females seem to have regular beats, and appear to stick to the same spot for days, probably for the whole period of their existence”. My own observations in the field seemed to contradict this idea in Fiji, but reinforce it in Independent Samoa. In an attempt to examine territorial behaviour of//, bolina in Fiji and Independent Samoa, small-scale mark release recapture experiments were carried out. 3.12.1. General methods for site fidelity experiments Four mark release recapture experiments were carried out at the following locations: 1. Todranisiga, Taveuni Island, Fiji; November 1999 2. Moto’o’tua, Apia, Upolu Island, Independent Samoa; September 2000 3. Wayalailai Island, Yasawa Island group, Fiji: May 2001 4. Moto’o’tua, Apia, Upolu Island, Independent Samoa; June 2001 Details of the specific sites are provided in the following sections. In all cases, for ease of recording, the area was arbitrarily divided up into patches based on distribution of Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 1 2 2 nectaring plant. These patches were recorded on an outline plan of the collection area that was used to record the positions of the butterflies on a daily basis. In both countries, the most apparent sex was used for the experiment, i.e. the Fijian experiments were carried out using males, and the Independent Samoan experiments using females. Ideally, the same sex would have been used in both countries, but due to the rarity of males in Independent Samoa, and the apparent scarcity of females in Fiji, this was not possible. The following protocol was followed for all four experiments: 1. On the first day of the experiment (designated day one), as many//, bolina specimens as possible (up to a maximum of 20) were captured between 10 a.m. and 11 a.m. 2. All insects were uniquely marked using Tipp-Ex and coloured marker pens. 3. The position at which each butterfly specimen had been collected was marked on the outline plan. 4. At the end of the collection hour, all specimens were released (to save time, caught specimens were placed in specific cages depending on area of collection, and labelled at the end of the hour). 5. Every day for the next five days, the area was revisited between 10 a.m. and 11 a.m and points 6 to 10 carried out: 6. All butterflies sighted within the collection area were caught. 7. If the caught specimen was one of those previously marked, its position of collection was recorded on the outline plan. 8. A note was made of the number of unmarked specimens of the study sex (males in Fiji, females in Independent Samoa) that were caught. 9. All specimens of the study sex were released at the end of the hour Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 123 10. A note was made of the number of specimens of the non-study sex that were caught. In Independent Samoa these specimens were not released as they were required for other aspects of fieldwork (males were so scarce in Independent Samoa that they had to be collected for breeding work if they were sighted). In the Taveuni study, five females were also marked and released on day one. The following sections provide details of the layout of each of the experimental areas, and the results obtained from each of these areas in turn. N.B. The schematic diagrams show approximate measurements only and are not to scale. 3.12.2. Site fidelity experiment one: Taveuni Island, Fiji 3.12.2.1. Description of experimental area This experiment was carried out at Todranisiga, Taveuni Island, Fiji, in November 1999. The area chosen was an open patch of scrubland that was arbitrarily divided up into four patches based on the distribution of the nectaring plant, S. urticifolia. These patches were arbitrarily designated B 60m A, B, C, and D. In this experiment, twenty males were captured and marked. Five females were also marked. D Recordings were taken for 6 days, and the weather noted. 50m Figure 3.8. shows the schematic outline plan of the Figure 3.8. Layout of study area. experimental area. 3.12.2.2. Results Results are summarised below (Table 3.3.). None of the five marked females were recaptured during the course of the experiment. Marked males were consistently found in the same area. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 124 Table 3.3. Summary of results of side fidelity experiment one. Males marked on day one are numbered, and their position of recapture on each of the subsequent days is recorded (Figure 3.8. details the layout of the experimental area). Numbers of unmarked males and females captured on each day are also recorded as are daily weather conditions. Marked Day Day Day Day Day Day Male 1 2 3 4 5 6 1 A BB D B 2 A C A -- D 3 B BD A - D 4 B - C -- “ A 5 B - BB - - 6 B - ■ B B - A 7 B B C C D B 8 B - D D - C 9 B - A A - A 10 B - B B -- 11 B -- - - B 12 C AD D - B 13 C - -- -- 14 CD - A - - 15 B B AD - D 16 D D B - - - 17 D D D C - B 18 D - --- - 19 D - B -- C 20 D B DB - C Weather Sunny Dull Sunny Dull Raining Sunny Conditions No. males 10 6 11 7 1 7 (Unmarked) No. females 0 1 4 2 0 5 (Unmarked) No. Marked 20/30 10/16 16/37 13/20 1 /2 14/21 males/Total No. males Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 125 3.12.3. Site fidelity experiment two: Wayalailai Island, Fÿi 3.12.3.1. Description of experimental area This experiment was carried out above on a small, uninhabited island next to Wayalailai Island, Fiji, in May 2001. The area chosen was a partly wooded / partly open patch of scrubland just above a beach. The study area was arbitrarily divided up as before, this time into three areas labelled A, B, and C 20m (approximate measurements only). Areas ‘A’ and ‘B’ consisting of 5. urticifolia (nectaring plant) in B 50m open ground, approximately 2.5m x 2m and Im x 4m respectively. Area ‘C’ is the shady wooded area, containing some flowering weeds and nectaring Figure 3.9. Layout of plants (Figure 3.9.). In this experiment, sixteen study area males were captured and marked. 3.12.3.2. Results Results are summarised below (Table 3.4.). Again, marked males were consistently found in the same area. N.B. In this experiment, unmarked females were collected for other fieldwork, so consequently could not be recaptured. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 126 Table 3.4. Summary of results of side fidelity experiment two. Males marked on day one are numbered, and their position of recapture on each of the subsequent days is recorded (Figure 3.9. details the layout of the experimental area). Numbers of unmarked males and females captured on each day are also recorded as are daily weather conditions. Marked Day Day Day Day Day Day Male 1 2 3 4 5 6 1 A 2 A A A - B A 3 A B A B - 4 A - C - C C 5 AA B - B - 6 B - B BA - 7 B C - C - B 8 B - A A - A 9 B - ---- 10 B A B BA B 11 B C C C - B 12 BA B - B B 13 CC - -- - 14 C B - C - C 15 C A -- - B 16 CC B - C - Weather Sunny Sunny Sunny Sunny Sunny Sunny Conditions No. males 0 5 6 10 9 9 (Unmarked) No. females 0 2 1 3 2 1 (Unmarked) No. Marked 16/16 11/16 10/16 7 /1 7 7 /1 6 9 /1 8 males/Total No. males Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 127 3.12.4. Site fidelity experiment three: Upolu Island, Independent Samoa 3.12.4.1. Description of Experimental Area This experiment was carried out in a large cultivated garden in the Moto’o’tua suburb of Apia. The study area was divided up into three sections (Figure 3.10.). Area ‘A ’ consists of one large nectaring plant in open ground that covers a ground area of approximately 5m^. Area ‘B’ is a tree- shaded cultivated flowerbed measuring approximately 40m B 6m X Im and containing several plants at which H. bolina had been observed to nectar. Area ‘C’ is an open ‘scrubby’ area approximately 4m x 2m in size 25m- containing some flowering weeds and several low Figure 3.10. Layout growing specimens of the H. bolina host plant, S. of study area rhombifolia. Seventeen females were captured and marked. 3.12.4.2. Results Results are summarised below (Table 3.5.). No males were observed or collected during the course of the experiment. Marked females were consistently found in the same area. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 128 Table 3.5. Summary of results of side fidelity experiment three. Females marked on day one are numbered, and their position of recapture on each of the subsequent days is recorded (Figure 3.10. details the layout of the experimental area). Numbers of unmarked males and females captured on each day are also recorded, as are daily weather conditions (N.B. Temperature and humidity not recorded, it was constantly both hot and humid). Marked Day Day Day Day Day Day Female 1 2 3 4 5 6 1 A B A AA B 2 AB - A - B 3 A CC - - 4 A A - - - A 5 A - --- - 6 A A - CC - 7 A - BB - - 8 A A C - A A 9 AB - A - - 10 A A B - A - 11 B - B B -- 12 B B CB - C 13 B A - --- 14 B - A B -- 15 C --- -- 16 C B - B - B 17 C C A C C - Weather Sunny Sunny Sunny Sunny Overcast/ Sunny Conditions showery No. males 0 0 0 0 0 0 (Unmarked) No. females 0 4 6 6 1 8 (Unmarked) No. Marked 17/17 12/16 9 /1 5 10/16 4 /5 7 /1 5 females/Total No. females Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 129 3.12.5. Site fidelity experiment four: Upolu Island, Independent Samoa 3.12.5.1. Description of experimental area This experiment was carried out along a rough pedestrian track bordering the edge of a horse paddock in the Moto’o’tua suburb of Apia. The area of track chosen is approximately 30m in length, and was B 30m arbitrarily divided into three 10m long areas as shown (Figure 3.11.). The centre ‘road’ of the area consisted of scrubby rough grass and a few low growing specimens 2m of the H. bolina host plant, S. rhombifolia. The edges of Figure 3.11. Layout the track were very overgrown containing various trees, of study area nectaring plants and a lot of rotting fruit. 3.12.5.2. Results Results are summarised below (Table 3.6.). No males were observed or collected during the course of the experiment. Marked females were consistently found in the same area. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 130 Table 3.6. Summary of results of side fidelity experiment three. Females marked on day one are numbered, and their position of recapture on each of the subsequent days is recorded (Figure 3.11. details the layout of the experimental area). Numbers of unmarked males and females captured on each day are also recorded, as are daily weather conditions (N.B. Temperature and humidity not recorded, it was constantly both hot and humid). M arked Day Day D ay D ay Day Day Fem ale 1 2 3 4 5 6 1 A C 2 A --- AA 3 AA - B - A 4 A - - A -- 5 B B --- B 6 B B ---- 7 B C -- C B 8 B -- - - - 9 C ---- - 10 C B ---- 11 C C - C B C 12 CC - -- - 13 CC - - B c 14 C A ---- 15 C A - A AA Weather Sunny Sunny Severe Overcast/ Sunny Sunny Conditions Rain showery storm No. males 0 0 0 0 0 0 (Unmarked) No. females 0 5 0 1 3 11 (Unmarked) No. Marked 1 5 /1 5 11/16 0 /0 4 / 5 5 / 8 7/18 females/Total No. females Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 131 3.12.6. Conclusions from site fidelity experiments Figure 3.12. provides a summary of all four experiments for ease of comparison. Unsurprisingly, the weather plays a large part in the numbers of butterflies observed. This appears to be particularly important in experiment four (Table 3.7.), following a severe rain storm on day 3, the numbers of butterflies flying decreases considerably for the next couple of days before rising again, probably due to emergence of new adilts. However, despite the effect of the weather, results strongly suggest that in Fiji male//. bolina are territorial whereas in Independent Samoa, the females are territorial, being found consistently in the same areas at the same times on consecutive days. No marked Fijian females were recaptured indicating that female//, bolina behave differently in the different populations. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 132 m Experiment 1 □ Experiment 2 ■ Experiment 3 0.8 □ Experiment 4 ■a § i-0.6 0s CC c o V o 0.4 OQ. 0.2 Day 2 Day 3 Day 4 Day 5 Day 6 Figure 3.12. Proportion of adults recaptured during consecutive days in all four site fidelity experiments: Experiments 1 and 2 carried out in the Fiji Islands, using male//. bolina (n=20 and n= 16 respectively); experiments 3 and 4 carried out in Independent Samoa using female H. bolina (n=15 and n=17 respectively). The male killing Wolbachia at high prevalence seems to have an effect on the behaviour of adult H. bolina. At high prevalence of the male killer, females start to behave in the way that males do in uninfected or low prevalence male killer host systems. We might expect to witness female competition for mates, and although certain ‘altercations’ between different females in the field were recorded, so few males were seen in Independent Samoa, and these were always collected so it was impossible to quantify the way the females behaved towards these males. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 133 3.13. Discussion The H. 6o/mfl/male killing Wolbachia symbiosis in Independent Samoa provides an example of sex ratio distortion in the extreme. It seems incredible that this situation has persisted for at least 80 years (i.e. at least 300 generations) and in all probability, considerably longer (Hopkins, 1927). The data presented in this chapter represent the highest prevalence of a male killing bacteria ever recorded in a natural host population. This has huge implications on the host population. It can be postulated that such a situation will result in one of three different outcomes for the host population: extinction, evolution of resistance or population damage. In his 1967 paper, ‘Extraordinary Sex Ratios’, W. D. Hamilton modelled invasion dynamics of selfish genetic elements (Hamilton, 1967). He suggested that if selfish genetic elements were to reach an exceedingly high level of prevalence, the host population would be damaged and could eventually be driven to extinction due to lack of one sex. Lyttle’s (1977) laboratory study demonstrated Hamilton’s earlier model in artificial caged host populations. His experiments used Drosophila melanogaster as the host of an artificially introduced selfish genetic element, a meiotically driving Y chromosome. Host extinction was recorded from a number of replicates as the level of infection increased in the artificial laboratory populations. Such effects have not been reported in natural populations but, for obvious reasons, it is difficult to record extinction in nature. If extinction does not occur, as it clearly has not in the Independent Samoan77. bolina populations, then we might expect to see evidence of host resistance to the selfish genetic element, as has been previously reported in natural populations of woodlice infected with feminising Wolbachia (Rigaud & Juchault, 1993). Resistance evolution seems a distinct Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 134 possibility for the Independent Samoan populations, given the rarity with which infected females produce sons and the scarcity of uninfected females in the population. Surprisingly, neither extinction nor resistance evolution are seen in if. bolina in Independent Samoa. That is not to say that the Wolbachia male killer ‘monopoly’ in these populations does not have dramatic ‘knock on’ effects on the host species. The resultant lack of male H. bolina in Independent Samoa increases female virginity and decreases female fertility in the study populations. The net result is a decrease in female reproductive success, even when females are mated, as demonstrated by the crossing experiments: Independent Samoan females mated to males from populations in which the male killing Wolbachia is at a much lower, or nonexistent prevalence, showing ‘normal’ levels of fertility. Based on the significant differences in sizes of spermatophores between males from Independent Samoa and those from other populations, it is tempting to suggest that Independent Samoan males have evolved the ability to partition their ejaculate, reducing the number of sperm allocated per mating. Even virgin Independent Samoan males produce significantly smaller spermatophores than males from populations with much lower prevalences of the Wolbachia male killer. This apparent difference in ejaculate allocation requires more stringent analysis. Butterflies produce two different types of sperm: eupyrene or fertilising sperm, and apyrene non-fertile sperm (Wedell, 2001). It has been demonstrated in Pieris rapae that the proportion of eupyrene and apyrene sperm allocated per mating vary with the degree of sperm competition in the population (Wedell & Cook, 1999). An investigation of H. bolina males from Independent Samoa, Fiji and American Samoa could be carried out, analysing the average proportion of eupyrene and apyrene sperm allocated per mating in each of these host populations. Based on the results presented in this chapter, one could hypothesise that Independent Samoan//. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 135 bolina males would allocate less sperm per ejaculate than males from the other host populations, although we would expect to see a higher ratio of fertilising (eupyrene) sperm reflecting the lack of sperm competition in the Independent Samoan population. 3.13.1. Why has resistance to theWolbachia male killer not evolved in the Independent Samoan population ofH. bolina’! Two reasons for the lack of host resistance to the action of the male killer ini/, bolina in the Independent Samoan population can be postulated. Firstly, a mutation that would bring about suppressive modification of sex ratio distortion may be difficult to evolve. Perhaps two separate mutations are required to achieve such an eventuality - a process that could take a very long time in evolutionary terms. Alternatively, the mutation rate and standing genetic diversity in //. bolina is low, due to restrictions in population size and genetic variability in this population. The probability of a host species evolving resistance to a selfish genetic element is lowered in an isolated island population due to the small population size. For example, if the probability of a mutation arising that confers resistance on its bearer is 10 ^ for every individual in the population, if the host population size is 10^ we would expect evolution of resistance within a single generation. However, an island population size is small, therefore it follows that there is less probability of resistance evolving. In an island population in which a male killer is at very high prevalence, for example H. bolina in Independent Samoa, the probability of evolving resistance is even lower. 3.13.1.1. Is reduction in spermatophore size a form of resistance? While resistance in the form of a suppressive modifier of the sex ratio trait has not been found, the significantly smaller spermatophore size of//, bolina males in Independent Samoa could be said to be a form of resistance evolution, albeit one that does not affect male killer prevalence. The data suggest that persistence of the host population may, in Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 136 part, be associated with the evolution of increased capacity of males to fertilise more females on these islands. If the production of small ejaculates by males is associated with the production of more ejaculates then selection for ejaculate partitioning in males has increased mean female fertility. This increase in male fertility is a modifier that decreases the probability of extinction of the host population. Perhaps if the spermatophores of these males were of an equivalent size to those of Fijian or American Samoan males then the Independent Samoan population would have been driven to extinction by the extraordinarily high male killer prevalence. Of course, it could be the case that spermatophore size is correlated with body size of males. However, whilst it is~true that Independent Samoan//, bolina are smaller than their Fijian, or American Samoan counterparts, the size difference in females is approximately 10%, and this difference is less pronounced, although not measured for males (pers. obsn). There is a much greater difference in spermatophore volume (arbitrarily calculated based on length and diameter measurements) than in overall size difference of the butterflies between the Independent Samoan population and those from American Samoa and Fiji: Independent Samoan spermatophores being, on average 50% smaller. Crossing experiments also reveal that the size of female H. bolina from Independent Samoa does not restrict the size of spermatophore that can be stored in the bursa copulatrix. 3.13.2. Population genetic consequences of a high prevalence male killing symbiont The ‘pool’ of genetic diversity within a population is a function of the effective population size, and is a key element of the evolutionary biology of the population. It dictates how selection acts in response to environmental change, the importance of drift in producing fixed differences, and the strength of selection required for the deterministic spread of a mutation. The effective population size (NJ can be expressed as: Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 137 Equation 3.1. = 4N„N/N„+Nf. (Crow & Kimura, 1988) Where and Nf are proportion of males and females in the population respectively. In a 1:1 sex ratio population N, is equal to N, the number of individuals in the population i.e. in a population composed of 50 individuals, = N = 50. Equation 3.11 can be applied to the Independent Samoan population oiH. bolina. Based on the PGR results from Upolu Island, the sex ratio in this population is approximately ImaleilOO females; i.e. N = 1:100, = N/lOO, Nf = 99N/100. a) Assuming full female fertility, is approximately 3.96% of that in a similarly sized population with a 1:1 sex ratio. b) Of course, in Independent Samoa, the females show increased virginity and decreased fertility. A rough estimate of decline in female fertility of 50% would give Ng s 1.98% of that which would be observed if the population sex ratio was 1:1. Such a low effective population size allows massive fluctuation in gene frequency by drift and loss of genetic diversity over each subsequent generation. As genetic variability is lost from the population, the capacity for adaptation is also reduced. Suggestive evidence for a decrease in variation of host genetic background in Independent Samoa is provided by Mathew (1888). In both American Samoa and Fiji, H. bolina females are highly polymorphic for wing-pattem (pers.obsn.; Clarke and Sheppard, 1975). During this course of study at least eight different female wing types were identified from these populations (classified as in Clarke & Sheppard, 1975). In contrast, in Independent Samoa, females showed much less variability in wing-pattem. Only two different morphs were identified throughout the course of study. Hopkins (1927) reports a similar lack of variation, but Mathew’s reports indicate that Æ bolina Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 138 females in Independent Samoa in 1888 showed a great deal of wing-pattem variation, similar to that observed in both Fiji and American Samoa. If this is indeed the case, then it appears that the genetic variation in the host species has decreased over time perhaps making it more difficult for mutations to arise and hence resistance to evolve. There is an alternative explanation for this reduction in female wing-pattem polymorphism in Independent Samoa:- Female wing-pattem polymorphism is believed to evolve in non-mimicking species such as H. bolina as a mechanism for escaping predation. Having a number of morphs that look completely different prevents formation of a search image by predatory birds, with scarcer morphs having an advantage over common morphs (Krebs & Davies, 1993a). Males however are monomorphic. This is thought to be due to sexual selection. In the standard Fisherian scenario, females are ‘choosy’ and will only mate with males that ‘look right’ (Krebs & Davies, 1993b). However, in Independent Samoan populations ofH. bolina there is suggestive evidence of sex role-reversal, with females apparently being territorial, like their male counterparts in Fiji. Females were also observed to perch for extended periods of time and chase other butterflies, as Fijian males also do. The operational sex ratio in Independent Samoa is highly skewed towards females, perhaps females have to compete for males, and as such have evolved to be less polymorphic. Perhaps a male will ‘choose’ not to mate with a female that does not look like all the others and, in Independent Samoa, this has caused a reduction in the number of different types of female wing-pattems. Whilst the loss of wing pattern variation is consistent with effects of the biased sex ratio on effective population size, it is not necessarily caused by it. It will be necessary to compare genetic diversity at an array of neutral markers (e.g. microsatellites) between islands with different infection prevalence to fully assess the importance of Wolbachia in determining this aspect of the evolutionary biology oiH. bolina. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 139 3.13.3. Why hasH. bolina in Independent Samoa not been driven to extinction? The suggested modification of increase in male ability associated with the high prevalence male killer in H. bolina may, in part explain why the butterfly population persists under such extreme conditions. It is also true that the viability of host populations in the face of extreme lack of males is dependent on the rate of natural increase (R^) that the host population can achieve in the absence of the sex ratio distorting parasite. Most insect species exhibit values of between 1.5 and 15 (Hassell et al, 1976). Chris Thomas’ work on population dynamics of Lepidopteran species suggests that species of butterfly that colonize, that is to say invade and persist in new environments, have much higher than average rates of natural increase (Thomas et al, 1998; Thomas & Hanski, 1997). Hypolimnas bolina is a colonizing species and as such a single colonising female will be expected to leave, on average, three daughters (i.e. Rq = 3). So, relating this to the Independent Samoa population, despite the observed 50% loss of fertility among females in this host population, Rq is still high enough (1.5) to render the population viable. Were H bolina not a colonizing species, i.e. if Rq was much lower, then the population could not persist and we would expect host extinction to occur. 3.13.4. Migration from other islands: specimen SAM155 This study presented one//, bolina specimen, butterfly Saml55, that appears to be an anomaly. This female was collected in Upolu, but in appearance is vastly different to all other Independent Samoan females sighted throughout the course of study. This female was considerably larger, and of a wing pattern (pallescens) not previously seen in Upolu (Clarke and Sheppard, 1975). The initial impression, especially given the battered near transparent state of her wings, was that she must have migrated from a different population, for example that of American Samoa. It is also highly coincidental, that she was one of only 2 uninfected individuals collected. She was unmated, and was not mated Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 1 4 0 when flown with local males. The presence of migrants, with new genes, and their compatibility with indigenous individuals, may be an important aspect of the evolution of this population. In particular, if uninfected individuals arrive each generation, this may explain the persistence of the population at extreme prevalence, without fixation of the bacterium and death of the population from lack of males. Inherited parasites in the butterfly Hypolimnas bolina Chapter 3: Extraordinary sex ratios in Independent Samoa 141 Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 142 Chapter 4: Looking For Direct Effects Of Wolbachia Infection On Hypolimnas bolina Survival And Fecundity Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 143 Chapter 4: Looking For Direct Effects Of Wolbachia Infection On Hypolimnas bolina Survival And Fecundity Summary In this chapter the Hypolimnas bolina/Wolbachia male killer symbiosis is examined to ascertain whether or not infection has a direct physiological effect on host fitness. Positive effects have been recorded in arthropods infected with Cl-causing microbes, but a direct ‘physiological’ advantage to a male killer infection has never been reported. Past studies have suggested that there are costs to infection. Repeated experiments examining the effects of infection with the male killing Wolbachia on host fitness of Fijian//. bolina reveal that infection confers an advantage in terms of increased survivorship to adult even when host density is controlled. Infected adult females are slightly heavier and larger than uninfected controls, indicating that increased survivorship does not trade off with decreased size or fecundity. That the effect persists in randomly mated FI individuals demonstrates that the observed increase in survivorship is not due to fitness effects deriving from inbreeding avoidance. Results obtained from Independent Samoa show a similar pattern but are inconclusive owing to small sample sizes. The implications of these findings are discussed. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 1 4 4 4.1. Introduction As discussed in Chapter 1 (section 1.1.2.), selection will be expected to act on both mutualistic and parasitic symbionts to increase their host’s (and hence their own) survival and reproduction (Ewald, 1987; Fine, 1975; Lipsitch et a l, 1995). In some mutualistic symbioses, such as those of aphids and the syrxAiionX Buchneva, the relationship between host and parasite is obligate, and treatment with antibiotics results in host death (Baumann et a l, 1995; S abater et a l, 2001). Reproductive parasites indirectly increase fitness of maternal hosts at the expense of male hosts (Stouthamer et a/., 2000) and, as they show cytoplasmic transmission, selection acts only on host females and not on males. Just as in mutualistic interactions, we might still expect to see a resultant increase in host (in this case female host only) survival and reproduction in such systems. As previously discussed, (Chapter 1, section 1.3.2.) indirect benefits are sufficient to explain the incidence and prevalence of male killing symbionts, however this does not exclude the possibility of direct benefits resulting from infection. Direct effects could have a positive or negative effect on host fitness, the former case would facilitate spread of infection through exploitation of males (indirect effect) and directly benefiting infected females. This chapter concerns an investigation into any direct effects (positive or negative) gained by female 77. bolina as a result of infection with the previously identified Wolbachia male killing symbiont. Wolbachia is the most widespread reproductive parasite, exhibiting various parasitic phenotypes: cytoplasmic incompatibility, parthenogenesis induction, féminisation and male killing in a diverse range of arthropod hosts (Stouthamer et al, 1999; Werren, 1997). Many models describing populations infected with a cytoplasmically-inherited parasite such as Wolbachia, assume that there must be some degree of benefit to infection, otherwise the symbiont would not spread Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 145 (O'Neill & Werren, 1997). In response to these models, many host-parasite systems have been investigated in an attempt to identify and isolate evidence of direct effects on host fitness resulting from infection. However, remarkably few examples of direct effects, beneficial or otherwise, have been reported. 4.1.1. Direct effects associated with cytoplasmically-inherited reproductive parasites (excluding male killers) Studies of direct effects on host fitness resulting from infection with non-male killing symbionts are summarised in Table 4.1. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 146 Table 4.1. Summary of studies of host fitness effects associated with cytoplasmically- inherited reproductive parasites (excluding male killers) Host Causal Parasitic Fitness Comments Reference Element Pheno - Effect type Nematodes Brugia Wolbachia None Positive: (Bandi et pahangi Enhances al, 1999) reproduc Litomosides Wolbachia None tion and (McCall et sigmidontis develop a /, 1999) ment Onchocera Wolbachia None Positive: ochengi obligate symbiosis Diptera Drosophila Wolbachia Cl Positive: Temporary (Poinsot & simulans increased effect Mercot, offspring 1997) production Wolbachia None Neutral Field study (Hoffmann et al, 1996) Wolbachia Cl Negative: (Hoffmann decreased et a l, 1990) fecundity Drosophila Wolbachia Cl Neutral (Hoffmann melanogaster et a/., 1994) Drosophila Wolbachia Cl Neutral (Giordano et mauritiana a l, 1995) Drosophila Wolbachia Cl Neutral (Bourtzis et ananassae al, 1996) Drosophila Wolbachia Cl Neutral sechellia Sphyracephala Wolbachia Positive: No effect of (Hariri et becarii increased Incompati a l, 1998) male bility fertility Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 147 Table 4.1. (continued) Host Causal Parasitic Fitness Comments Reference Element Pheno Effect type Hymenoptera Nasonia Wolbachia Cl Postitive: Only found (Stolk & vitripennis increased in double- Stouthamer, offspring and not 1996) production single- infecteds. Trichogramma Wolbachia None Positive: Infected line (Girin & bourarachae increased has double Bouletreau, offspring the number 1995), production of offspring ■ ;= Trichogramma Wolbachia PI Negative: (Stouthamer deion and reduced & Luck, T. pretiosum offspring 1993) production Asobara tabida Wolbachia PI Positive: (Dedeine et necessary for a l, 2001) oogenesis Encarsia Encarsia , PI Positive: (Zchori- pergandiella bacterium alters Fein et al, oviposition 2001) behaviour Mosquitoes Aedes Wolbachia Cl Positive: (Dobson et albopictus increased a l, 2002) fecundity Acari Dermacentor Rickettsia Inefficient Abnormalities (Burgdorfer andersoni rickettsii Trans - in oviposition & Brinton, mission and egg 1975) development Coleoptera Tribolium Wolbachia Cl Positive: Infected (Wade & confusum increased male sperm Chang, male fertility precedence 1995) Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 148 4.1.1.1. Direct effects associated with Cl (cytoplasmic incompatibility) Most examples of studies in which direct effects have been measured concern selfish genetic elements that cause cytoplasmic-incompatiblity (Cl), for example Dobson et. al’s investigation of fecundity in the mosquito A albopictus (2002). One of the main reasons for this is that Cl represents the most widely-studied of all reproductive parasitic phenotypes. It is also true in Cl that because the selfish genetic element does not distort the host’s sex ratio, the symbiont is more likely to spread to fixation. The very nature of Cl, with crosses between infected host males and uninfected females resulting in decreased offspring viability indirectly increases the relative fitness of infected hosts (Chapter 1, section 1.2.1.). Once at, or near, fixation, selection pressure for a beneficial or mutualistic effect on host fitness to evolve is strong. 4.1.1.2. Direct effects associated with PI (parthenogenesis induction) Three studies have been carried out concerning direct effects resulting from infection with a parthenogenesis inducing reproductive symbiont. Infection with PI Wolbachia is found to decrease lifetime fecundity in terms of numbers of eggs laid relative to antibiotic cured controls in two Trichogramma species (Stouthamer & Luck, 1993). In the parasitic wasp, Asobara tabida, treatment of hosts infected with PI Wolbachia results in oogenesis cessation (Dedeine et a l, 2001). Encarsia pergandiella is infected with a PI bacterium, named the ‘Encarsia’ bacterium. Removal of infection via antibiotic treatment causes an alteration in host selection behaviour (Zchori-Fein et a l, 2001). 4.1.1.3. Direct effects in nematode hosts Symbionts that infect nematode hosts are all Wolbachia and do not exhibit a parasitic phenotype. Wolbachia that infect nematodes fall into two different clades: C and D, both of which are unique to this host genus (Bazzocchi et a l, 2000; Casiraghi et a l, 2001). The phytogeny of the nematodes and their infecting Wolbachia are remarkably congruent, in stark contrast to those of other arthropod/Wb/bac/i/a associations. This Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 149 suggests that the relationship between the host and symbiont is long-standing, and suggestive of an intimate species-specific association. The fact that removal of the symbiont results in host death strengthens the idea of ntmdXodt[Wolbachia co-evolution and co-dependence. 4.1.1.4. Problems with measurement o f direct effects In most of the studies cited in Table 4.1., uninfected controls are obtained by antibiotic treatment of infected lines. It is therefore difficult to conclude from many of these studies whether or not the direct effect recorded is due to the presence of the symbiont, or to the antibiotic treatment. For example, in the case of oogenesis-cessation in Asobara tabida (Dedeine et al. , 2001), although there is no effect of antibiotics on a closely-related species and there are no other apparent changes in host function post-treatment, the idea that the tetracycline treatment is causing the effect cannot be definitively ruled out. Controlling for host genetic background is a factor that is often overlooked, but has been demonstrated to confound the original interpretation of results. InN. vitripennis, a direct benefit on fecundity is recorded initially where the host is doubly infected with two CI- strains of Wolbachia (Stolk and Stouthamer, 1996). However this effect no longer apparent when effects of host genetic background are controlled (Bordenstein & Werren, 2000). Although the majority of investigations into the physiological effect of inherited parasites have concerned selfish genetic elements that cause Cl, there is no reason to expect other parasitic phenotypes to exhibit different patterns of host-parasite co evolution. Over time we would expect to see a reduction in host fitness cost to harbouring a reproductive parasite in all such systems. Looking at a wider variety of reproductive phenotypes should give an indication as to whether in fact fitness costs to the host are reduced, eventually becoming more mutualistic and possibly even evolving Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 150 to a State in which the maternal host gains a physiological benefit to harbouring the cytoplasmic element. This ‘end-point’ in co-evolution, could be expected to be seen more readily in the most extreme reproductive parasite systems of all: the male killing bacteria. 4.1.2. Direct effects associated with male killing Male killing is much less studied than Cl. However, direct effects of infection have been examined in a variety of host species (Table 4.2.). Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 151 Table 4.2. Studies of host fitness effects resulting from male killer infection Host Male Killing Fitness Details Reference Bacteria Effect? Coleoptera Adalia Rickettsia Neutral Larval development (Hurst et al, bipunctata rate and body mass 1994) Negative Decreased fecundity and longevity Harmonia Spiroplasma Negative Increased sterility. (Mâtsiika et axyridis (anecdote) decreased egg al, 1975) production Adonia Ravobacteria Neutral Larval development (Hurst et al, variegata rate and body mass 1999b) Negative Decreased fecundity (very weak effect) Diptera Drosophila Wolbachia Negative 10-15% decrease in (Ikeda, 1970) bifasciata egg production Drosophila Spiroplasma Positive Increased larval (Ebbert, 1991) willistoni development rate (Malogolowkin Negative Increased sterility & Rodriguez- Decreased longevity Pereira, 1975) Lepidoptera Epiphyas postvittana Unknown Negative 50% decrease in (Geier et al, egg production 1978) Spodoptera Unknown Negative 40% decrease in (Brimacombe, littoralis egg production 1980) It is apparent that the majority of direct host effects that have been attributed to male killing bacteria are negative, or at best neutral. InD. willistoni, although there is a positive effect in terms of increased larval development (Ebbert, 1991), there is also a Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 152 physiological cost to infection (Malogolowkin & Rodriguez-Pereira, 1975). There are currently no definitive examples illustrating a male killing infection that gives a direct benefit to its host. 4.1.2.1. Why have no direct benefits to male killing been found? Generally, bacteria that make a beneficial contribution to host physiology are found in hosts with poor diets, for example obligate phloem feeders such as aphids whose symbionts provide them with essential amino acids (Baumarm et al., 1995). The majority of the case studies presented above concern host species whose diet is nutritionally balanced. It follows that, due to the energetic requirements of the reproductive parasite, there is more likely to be a physiological cost to infection in such host species, as there is no necessity for nutrient provision by the symbiont. However, just because it is ‘more difficult’ for a beneficial symbiosis to evolve, does not mean that this will not happen. Intuitively, direct benefits to hosts are most likely to evolve in high prevalence male killer systems, for example the A. encedana! Wolbachia symbiosis (Jiggins et a l, 2000a). Despite the fact that in this system selection on both host and symbiont will be strong, recent investigation has revealed no evidence of a physiological benefit to the host resulting from infection (Jiggins et a l, 2003). Although there is no evidence for a direct host benefit from this association, there are indirect benefits associated with the male killing symbiont, known as ‘fitness compensation’, such as inbreeding avoidance, reduced competition and sibling caimibalism (Chapter 1, section 1.3.2.). What is unclear from other symbioses is how the symbiont can spread without fitness compensation, or by fitness compensation alone if indirect benefits are small. In these populations, in order for the male killing element to spread to such a high level, we would expect to find evidence of a direct benefit to the host. The virulence level observed in male killers may well be a balance between selection for increased rate of Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 153 transmission to the germline and selection for increased host fecundity. This being the case, and suspecting male killing to be widespread amongst the arthropod groups, a direct physiological benefit to male killing does not seem beyond the realms of possibility. With this in mind, H. bolina populations were examined to assess any direct effects, positive or negative, associated with infection with the male killing Wolbachia. 4.2. Hypolimnas bolina ! Wolbachia Symbiosis As A Study System For Examination Of Direct Effects Of Male Killing Certain H. bolina populations are infected with a Wolbachia male killing symbiont at high (Fiji, around 60%: Chapter 2, section 2.11.; Dyson et a l, 2002) to exceedingly high prevalence (Independent Samoa, over 99%: Chapter 3, section 3.8.) with no evidence indicating that resistance has evolved. In such systems, as in previously studied high prevalence male killing symbioses (Jiggins et a l , 2000a) we would expect to find evidence of some sort of benefit to the host species, be it direct or indirect (fitness compensation), to account for the widespread incidence of the bacteria. 4.2.1. What indirect benefits might be expected? 4.2.1.1. Reduced sibling competition The average clutch size of//, bolina in both Fiji and Independent Samoa is 10-12 eggs (Mathew, 1888). Data from egg clutches collected in the field in Independent Samoa indicate that this figure may be a slight over-estimate (mean clutch size: 10.25 ± 0.47; n=28). Compared to A. encedon (clutch size usually exceeding 100 eggs - Jiggins et a l, 1998), clutch size is relatively small, so there is unlikely to be a high degree of sibling competition. Also, unlike A. encedon, H. bolina larvae disperse after hatching, and are Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 154 only gregarious until the second larval instar, if at all (pers.obsn). So sibling interactions are reduced, and although there is probably a degree of sibling competition, this is unlikely to be a major factor aiding the spread of a male killer. 4.2.1.2. Sibling cannibalism Sibling cannibalism has been reported in male killer infected populations of both ladybirds and Lepidoptera (Hurst et a l, 1992; Jiggins et a l, 1998) and is often held as an example of fitness compensation resulting from infection. However, as demonstrated in Chapter 2 (section 2.5.), controlled experiments using Fijian //, bolina reveal that larvae starve to death, rather than consuming sibling eggs or each other. 4.2.1.3. Inbreeding avoidance It is possible that male killer infected H. bolina could be gaining fitness compensation through inbreeding avoidance. Inbreeding avoidance indirectly benefits the host as it reduces homozygosity, thereby decreasing the expression of deleterious mutations. Inbreeding avoidance has been little quantified in male killer systems as it is difficult to access accurately (Hurst et a l, 1993). In other systems it appears to be of low magnitude and is not thought to be a major factor facilitating male killer spread to high prevalence (Hurst & Majerus, 1993). Other than the fact that H. bolina may gain an indirect benefit of inbreeding avoidance, there seems to be no immediately obvious evidence for fitness compensation resulting from male killer infection in this species. Because the Wolbachia male killing symbiont is recorded at such high prevalence, there must be some form of benefit to the host facilitating the spread of the symbiont. Does this benefit come in the form of a direct effect on the host? Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 155 4.2.2. What direct benefits might be expected? Examples of the different direct effects that have been recorded in host/reproductive parasite symbioses are given in Table 4.3. Based on previous studies looking for direct effects, the best methods of testing for these effects are also detailed. Table 4.3. Putative direct physiological benefits to females infected with a cytoplasmically-inherited symbiont relative to uninfected controls, and how these could be measured in 7/. bo/ma Direct Benefit How to Measure (relative to uninfected controls) Accelerated Larval Development Record duration of : 1. Larval stage 2. Pupal stage and timing of larval moults Increased Survivorship Record any larval deaths Increased Adult Mass Measure fresh or dry weight on emergence from pupae Increased Body Size Measure wing sizes Increased Fecundity Either: 1. Post-mating, record number of eggs present in ovaries 2. Record number of eggs laid over natural lifespan Increased Longevity Record duration of adult stage and time of death These facts were used as a springboard for the design of an experiment looking for direct effects of Wolbachia infection in H. bolina. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 156 4.3. Experimental Design The best method of comparing hosts for evidence of direct physiological benefits such as those detailed in Table 4.3. is to carry out experiments involving paired replicates, with each pair consisting of one uninfected, and one infected individual. It is impossible to sex H. bolina larvae without first killing them, so carrying out experiments involving paired replicates would mean that half of the replicates would need to be discarded (comparisons can only be drawn between same-sex, i.e. female, hosts and we would expect approximately half of the offspring of an uninfected female to be male). Therefore, more than two larvae were used in each repHcate, but within a replicate the conditions (i.e. food provision, temperature, age of larvae) were consistent. The main experiments were carried out using if. bolina from Fiji, and naturally- occurring (rather than antibiotic-treated) uninfected hosts were used as a control. Only one experiment involved if. bolina from Independent Samoa due to the fact that progeny from the single uninfected matriline (see Chapter 3) were the sole control. Two main experimental designs were employed. The first, a pilot study, compares larvae reared in fixed size groups and only egg to adult survivorship is measured. Both FI and F2 larvae are used in order to establish whether any effects noted are due to inbreeding, the FI being forced to outbreed. The second design is more robust, with larvae being reared singly to factor out effects of competition. Survivorship, larval development rate, pupal duration and adult dry weight are recorded in the second experiment; it was hoped to measure fecundity and longevity of adults, but this proved impossible due to a disease infecting adult butterflies. These two studies were carried out over different time periods, using butterflies from two Fijian island populations. A third study was carried out following the second experimental design but using butterflies from Independent Samoa. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 157 The next sections details these three separate studies analysing differences between infected male killer lines and uninfected normal sex ratio lines. 4.4. Aim To examine populations oiH. bolina for any evidence of direct physiological effects resulting from infection with the male killing Wolbachia. 4.5. Method Three experiments were carried out using butterflies from three different populations: Experiment One: H. bolina from Viti Levu Island, Fiji. Experiment Two: H. bolina from Wayalailai Island, Fiji. Experiment Three: H. bolina from Upolu Island, Independent Samoa. In terms of design, experiment three is a replicate of the second experiment, the sole difference being the population from which the butterflies were collected. 4.5.1. PCR assay for Wolbachia All field-collected females from the three experiments were assayed post-hoc for the presence of the male killing Wolbachia using PCR. The Wolbachia B-group specific primers 81f and 522r that amplify the bacterium’s wsp gene (sequences section A.2., Appendix I) were used following the protocol A.3.I., Appendix I. These females were all assayed for Wolbachia presence for the prevalence surveys detailed in Chapter 2 (Fiji) and Chapter 3 (Independent Samoa). The results of these assays are not presented again here. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 158 4.5.2. Experiment one: method This experiment was carried out at Colo-I-Suva, Viti Levu Island in 1999. The host plant used throughout this experiment, for both oviposition and larval feeding, was Ipomeoa batatas (sweet potato) (Vane-Wright et a l, 1977). 4.5.2.1. Replicate one: survival of the FI generation Parental adults were field-collected, maintained, and eggs obtained as detailed in Chapter two (section 2.3). Eggs laid in the laboratory were removed from the plant and each clutch stored in a separate Petri dish coded according to matriline, and egg hatch rates were recorded (General methods and egg hatch rate table: Chapter 2, section 2.4.). On hatching, larvae were removed from the egg clutch and groups of ten larvae from the same matriline were placed in separate Petri dishes. Survival of larvae from six infected and five uninfected matrilines were tested. An FI generation was reared, larvae being fed on an excess of/, batatas leaves (Vane-Wright et a l, 1977). The groups of ten caterpillars were transferred to large 450ml yoghurt pots after the third larval moult. All larval deaths after the first larval moult were recorded. Larvae dying before they reached the first larval moult were not included in the experiment as sometimes males from male killing lines hatch as larvae but are killed before they reach first instar (Jiggins et al, 1998). On emergence from pupae, the adult sex ratio was recorded and, within each group of ten larvae, the proportion of//, bolina surviving to adulthood was calculated. 4.5.2.2. Replicate two: survival o f the F2 generation FI adult females from normal sex ratio and all-female matrilines were mated to FI adult males from different matrilines to control for effects of inbreeding (following the method outlined in Chapter 2, section 2.3.). Survival of larvae from eight infected and five uninfected matrilines was tested. An F2 generation was reared following the method detailed in the previous section, and survivorship to adult measured and calculated as before. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 159 4.5.3. Experiment two: method This experiment was carried out at Moto’o’tua, Apia, Upolu Island, Independent Samoa between June and August, 2001. Adult female H. bolina were collected from Wayalailai Island, Fiji and transported to Independent Samoa. The host plant used throughout this experiment, for both oviposition and feeding, was Sida rhombifolia (Clarke et al, 1975). Adults were maintained and eggs obtained as detailed in Chapter 3 (section 3.4.). Eggs laid in the laboratory were removed from the plant and each clutch stored in a separate Petri dish coded according to matriline, and egg hatch rates were recorded (General methods and egg hatch rate table: Chapter 3, section 3.6.). All ‘infected’ and ‘uninfected’ eggs laid on the same day were pooled into one ‘paired’ replicate. Within a single replicate, conditions are identical i.e. larval age, type and amount of food provided and temperature conditions. To avoid confounding effects of food and transmission efficiency, comparisons are only carried out within replicates. Three replicates were carried out in total: replicate one involving 32 individual larvae from 5 matrilines (three infected, two uninfected); experiment two involving 36 individual larvae from four matrilines (two uninfected, two infected) and experiment three involving 54 individual larvae from four matrilines (two uninfected, two infected). 4.53.1. Survivorship to adult On hatching, larvae were confined singly in Petri dishes and numbered. The experiments were carried out ‘blind’ with respect to infection status, i.e. larval identification number was not related to matriline number. For example, in replicate one the thirty-two larvae were arbitrarily numbered 1.1 up to 1.32 irrespective of matriline by a friend who kept the data relating each particular larvae with its matriline until the end of the experiment. Within each replicate, larvae were fed from the same S. rhombifolia bush, and provided with an equal (and excess) amount of food. Larvae were moved to large 450ml yoghurt pots following the third larval moult. Any deaths after first instar were recorded. As in Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 160 experiment one, larvae dying before they reached the first larval moult were excluded from analysis. On emergence from pupae, sex ratio was recorded. The proportion of female H. bolina surviving to adult was calculated for both infected and uninfected matrilines within each of the three replicates. 4.53.2, Larval development rate Development rate of each larva was recorded by noting down the day on which each of the larval moults, pupation and emergence occurred. The day that the larvae hatched from the eggs was counted as day one. 4.5.3.3. Adult dry weight Following emergence as adults, FI females were left in a cage on their own for twenty- four hours without food to allow them to dry completely, and their wings harden. They were then freeze killed, their wings removed and their bodies’ desiccated. Each dried specimen was stored in permeable envelope, and envelopes kept in a sealed airtight Tupperware containing desiccant silica gel. On returning to London, the specimens were further desiccated under a weak vacuum and then weighed on a microbalance. Dry weight of each specimen was recorded to the nearest O.Olmg. 4.5.3.4. Wing measurement Due to unforeseen circumstances, wing measurements were only obtained from females involved in replicate one. The removed right fore- and hind- wings of infected and uninfected female Ff. bolina were measured as indicated in Figure 4.1. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 161 i) Forewing ii) Hindwing Figure 4.1. Measurement of right fore- and hind- wings of H. bolina females. Four measurements were taken: a, b and c on the forewings (i) ; d on the hind-wing (ii) Measurements were taken in centimetres to the nearest 0.05 cm. Markers used for measurement were consistent across all females. 4.5.4. Experiment three: method This experiment analysed the effects of Wolbachia infection in H. bolina collected from Apia, Upolu Island, Independent Samoa. The methods for experiment three are exactly the same as experiment two (previous section) i.e. survival to adult, larval development rate, dry weight on emergence as adults and wing sizes were measured. As detailed in Chapter 3, during the course of fieldwork in Independent Samoa, progeny were reared from only a single uninfected female. The progeny from this single uninfected matriline, together with progeny from four infected matrilines were involved in this experiment. All infected larvae were progeny of wild-collected Independent Samoan females mated to wild-collected Fijian males. These cross-matings control for any effect of reduced fertility or fecundity due to sperm limitation, as is seen in wild mated Independent Samoan females (see Chapter 3, section 3.10.). Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 162 4.6. Results 4.6.1. Experiment one; group-reared Fijian larvae Group-reared larvae from Wolbachia-infcctQd matrilines show a significantly greater survivorship to adult than those from uninfected matrilines in both the FI and F2 generations (Mann-Whitney U test. FI: U = 0, nl = 5, n2 = 6; p ^ 0.025. F2: U = 0, n l = 5, n2 = 8; p ^ 0.025) (Figure 4.2.). No significant difference in survivorship to adult between FI and F2 generations was recorded (Mann-Whitney U test. Normal sex ratio broods: U = 67.5, nl = 5, n2 = 5; ns. All-female broods: U = 19, n l = 6, n2 = 8; ns). The adult sex ratio of these broods has been previously presented in Chapter two, section 2.2. The sex ratio produced by uninfected parental individuals was not heterogeneous = 0.194, d.f = 4; ns) and was not significantly different from 1:1 (%^ = 0.120, d.f = 1; ns). Similarly, the sex ratio produced by uninfected FI individuals was not heterogeneous (x^ = 0.702, d.f = 4; ns) and was not significantly different from 1:1 = 0.053, d.f = 1; ns). Therefore the difference in survivorship to adult is not due to increased mortality of males in normal sex ratio broods. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 163 n=180 n = 1G0 u ■S; Q8 I n=37D fae n=140 & Ë.Q4 a cS a Q2 0 nînÉected(6 RUiinÉected(5 KInfected(8 E2Uiinfected(5 iiBtrilines) nBtrilines) matriliiies) nBtrilines) Figure 4.2. Median proportion of larvae reared in groups of ten surviving to adulthood in infected and uninfected matrilines in experiment one, replicates one (FI generation) and two (F2 generation). Error bars represent interquartile range; n is the total number of larvae involved in each category. 4.6.2. Experiment two: singly-reared Fijian larvae 4.6.2.L Survivorship to adult A summary of the egg hatch rates and survivorship in all of the matrilines used in experiment 2 is given in Table 4.4. Egg to adult survivorship of uninfected larvae was found to be homogeneous across replicates (y^ = 0.607, d.f = 2; ns.) as was egg to adult survivorship of infected larvae (%^ = 0.000, d.f = 2; ns) (Figure 4.3.). Larvae from infected matrilines show a significantly greater survivorship to adult than do uninfected larvae (%^13.062, d.f = 1; p rs 0.0001). All infected matrilines gave all-female broods at adulthood. There is no heterogeneity between replicates one, two and three in the sex Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 164 ratio produced by uninfected females = 0.518, d.f. = 2, ns). The adult sex ratio in these broods was not significantly different from 1:1 = 0.00, d.f = 1; ns). It is notable that, across all replicates, egg to adult survivorship was 100% in all five infected matrilines and less than 82% in all four uninfected matrilines. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct ejfects o/Wolbachia infection in H.bolina 165 Table 4.4. Summary of individual survivorship data, sex ratios and hatch rates from each of the nine matrilines within each of the three replicates of experiment two Matriline Proportion Infection N°. N°. Proportion Hatch (n) Status Larvae Adult Adult Dying As Reared Males Females Larva/Pupa Replicate One A 0.33 (6) Infected 2 0 2 0 C 0.50(8) Infected 4 0 4 0 D 0.53(17) Infected 9 0 9 0 B 1.00 (12) Uninfected 12 5 4 0.25 E 1.00 (5) Uninfected 5 2 1 0.4 Replicate Two A 0.40 (5) Infected 2 1 1 0 F 0.45 (20) Infected 9 0 9 0 G 1.00 (14) Uninfected 14 5 6 0.21 H 1.00 (12) Uninfected 12 5 5 0.17 Replicate Three F 0.56 (18) Infected 10 0 10 0 I 0.46 (26) Infected 12 0 12 0 E 0.95(22) Uninfected 21 8 9 0.19 H 0.91 (11) Uninfected 10 4 4 0.2 Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct ejfects of Wolbachia infection in H.bolina 166 n = 15 n = 11 n = 23 n = 26 ■D n = 31 > 0.8 £ n = 17 Î 3 W 1 § 0.6 ■ ■ C o Q. 2 0.4 Q. c 0) 0.2 Infected Uninfected Infected Uninfected Infected Uninfected (3) (2) (2) (2) (2) 1 (2) Replicate 1 Replicate 2 Replicate 3 Figure 4.3. Median proportion of infected and uninfected individually reared larvae that survived to adulthood within each of the three replicates of experiment two. Error bars represent range; n is the total number of larvae involved in each category. The number of matrilines involved in each manipulation is given in brackets. 4.Ô.2.2. Larval development rate Development rate of larvae was calculated as the median time (in days) to each larval moult (Table 4.5.). Infected and uninfected larvae moult at approximately the same times during development. No significant difference was observed between rate of development from egg to adult, between infected and uninfected lines (Mann-Whitney U tests; Replicate 1: Ni = 10, N 2 = 11, Ucrit = 19, U = 43, ns; Replicate 2: Ni = 15, N 2 = 6, Ucrit = 19 , U = 46.5, ns; Replicate 3: Ni = 22, N 2 = 13, Ucrit = 47 , U = 47.5, ns). A graphical summary of these results across all three replicates, including the day and larval stage of all deaths is given in Figure 4.4. All deaths recorded (after first instar) were from matrilines that are not infected with the male killer. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 167 Table 4.5. Development rate of infected and uninfected female larvae in each of the three replicates expressed as median day on which each larval moult took place, and median day on which adults emerged, interquartile range in brackets. Larval Moult Adults First Second Third Fourth Fifth Emerge Replicate 1 Infected 5 7 10 13 16 31 (n= 15) (4,5) (7,8) (10/10) (13/14) (16/18) (31/32) Uninfected 4.5 7 10 13 16 31 (n= 6) (4,5) (7,7) (10/10) (13/14) (16/18) (31/31.75) Replicate 2 Infected 5 8 10.5 14 18 30.5 (n= 10) (5,5) (8,8.75) (10/11) (14/14) (17.25/18) (30/31) Uninfected 5 8 10 14 18 31 (n= 11) (5,5) (7,8) (10/11) (13/14) (17/18.25) (30.25/31) Replicate 3 Infected 5 8 10 13 17 31 (n= 22) (4.25,5) (8,8) (10/11) (13/14) (17/18) (31/32) Uninfected 5 8 10 13 17 31 (n= 13) (4,5.5) (8,8) (10/11) (13/14) (17/17) (31/31) Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 168 35 -A - - Infected - B - Uninfected 30 • Death in Replicate 1 ♦ Death in Replicate 2 25 + Death in Replicate 3 E15 10 5 First Second Third Fourth Fifth Pupa Butterfly Larval Moult Figure 4.4. Summary of development rates of infected (n=47) and uninfected (n=30) female larvae in experiment two, presented as the mean day (across all three replicates) on which each larval moult took place. Larval deaths that occurred in each of the replicates are recorded; all larvae that died were from uninfected matrilines. 4.6.2.3. Adult dry weight In all three replicates, infected FI adult females were significantly heavier than their uninfected counterparts (Mann-Whitney U tests. Replicate 1: Ni = 15, N] = 6, Ucrit = 10, U = 19, p=0.05; Replicate 2: Ni = 10, N 2 = 11, Ucrit = 14, U = 14, p< 0.05; Replicate 3: Nl = 22, N2 = 13, using z approximation, z = -3.26, U = 47, p< 0.005) (Figure 4.5.). Only adults in the same replicate were compared to control for differences in environment and feeding regime. (Insects within the same experiment were fed on the same food, and exactly the same age). Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 169 140 n=10 n=6 I n=22 120 n=11 T n=13 O) E 100 4 O) I 80 £■ •Ü 60 § % 40 20 0 Infected Uninfected Infected Uninfected Infected Uninfected Replicate 1 Replicate 2 Replicate 3 Figure 4.5. Median dry weight of infected and uninfected adult females in each of the three replicates of experiment two. All females within an experiment were reared at exactly the same time, on the same food. Error bars represent interquartile range; n is the total number of larvae involved in each category. 4.6.2.4. Wing measurement Right fore and hind wings were measured following the four criteria (a, b, c, and d detailed in Figure 4.1.). The size of these characteristics was approximately normally distributed (as determined a Shapiro-Wilk W test). In all cases, forewings of infected females are significantly larger than those of uninfecteds. (Measurement a: t = 4.134, d.f = 18, p < 0.001; Measurement b: t = 2.344, d.f = 18, p < 0.05; Measurement c: t = 3.018, d.f = 18, p < 0.005). The hindwings of infected females were also larger than those of uninfecteds, although this difference was not significant (Measurement d: t = 1.943, d.f= 18, ns) (Figure 4.6.). Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 170 5 4.5 4 3.5 I ' £ 2.5 O) 0 2 - 1.5 1 0.5 0 a b e d Part of Wing Measured Figure 4.6. Summary of mean wing measurements from experiment two, replicate one; right fore- and hind- wings of freshly killed females were measured along four different axes: a, b, c and d (see Figure 4.1. for details). Infected matrilines (n=14) are grey bars, uninfected (n=6) are white bars. Error bars represent 95% confidence interval. 4.6.3. Experiment three: singly-reared Independent Samoan larvae 4.63.1. Survivorship to adult The egg to adult survivorship of infected larvae was found to be homogeneous across experiments (all show 100% survivorship) (Table 4.6., Figure 4.7.). Only one uninfected matriline was tested, as this was the only uninfected line reared from Independent Samoa. Larvae from infected matrilines show a significantly greater survivorship to adult than do uninfected larvae (%^= 12.26, d.f =1; p ^ 0.0005). All infected matrilines gave all female broods at adulthood. The adult sex ratio of the uninfected normal sex ratio brood was not significantly different from 1:1 (x^ =0.142, d.f =1; ns). Therefore, the difference Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct ejfects of Wolbachia infection in H.bolina 171 in survivorship to adult is not due to increased mortality of males in normal sex ratio broods. Table 4.6. Summary of individual survivorship data, sex ratios and hatch rates from each of the five Independent Samoan matrilines in experiment three Matriline Proportion Infection N°. N°. N°. N°. Hatch (n) Status Larvae Adult Adult Larval/Pupal Reared Males Females Deaths SAM ll 0.48 (29) Infected 2 0 2 0 SAM12 0.53 (40) Infected 16 0 16 0 SAM14 0.49 (84) Infected 16 0 16 0 SAM15 0.50 (52) Infected 10 0 10 0 SAM13 1.00 (55) Uninfected 35 15 13 7 Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 172 n = 44 n = 35 ±± 0.8 3 < s 1 0.6 E 3 <0 c o 0.4 ■■o e Q. 2 0“ 0.2 Infected (4 matrilines) Uninfected (1 matriline) Figure 4.7. Proportion of infected and uninfected individually reared larvae that survived to adulthood in experiment three, n is the total number of larvae involved in each category. 4.63.2. Larval development rate Similarly to the Fijian experiments, infected and uninfected larvae moult at approximately the same times during development (Table 4.7.). No significant difference was observed between the median rate of development from egg to adult between infected and uninfected lines (Ni = 44, N 2 = 13, using z approximation, z = 0.335, U = 268.5, ns). All deaths recorded (after first instar) were from matrilines that are not infected with the male killer. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 173 Table 4.7. Development rate of infected and uninfected female larvae expressed as median day on which each larval moult took place, and median day on which adults emerged, interquartile range in brackets Larval Moult Adults First Second Third Fourth Fifth Emerge Infected 4 7 10 13 16 31 (n= 44) (4/4.25) (6/7.25) (9/10.25) (13/14) (15/17) (30/32) Uninfected 4 7 10 14 16 31 (n= 13) (4/4) (7/8) (10/10) (13/14) (15/17) (30/31) 4.6.33. Adult dry weight Although male killer infected females are slightly heavier, there is no significant difference in the dry weight of infected FI adult females and their uninfected counterparts (Mann-Whitney U tests: Ni = 43, N 2 = 13, using z approximation, z = -1.28, U = 346, p< 0.005, ns) (Figure 4.8.). As in experiment two, insects within the same experiment were fed on the same food, and were exactly the same digt.N.B. Dry weights are only recorded for 43 of the 44 infected females in experiment three: tissue was lost from a single specimen, so dry weight could not be measured. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 174 100 n = 43 n = 13 .2) 60 Q 50 Infected (4 matrilines) Uninfected (1 matriline) Figure 4.8. Median dry weight of infected and uninfected adult females in experiment three. All females were reared at exactly the same time, on the same food. Error bars represent interquartile range; n is the total number of larvae involved in each category. 4.63.4. Wing measurement Right fore- and hind- wings were measured following the four criteria (a, b, c, and d detailed in Figure 4.1.). The size of these characteristics was approximately normally distributed (as determined by a Shapiro-Wilk W test). There were no significant differences between wing sizes of infected and uninfected female//, bolina (Measurement a: t = 1.438, d.f = 55, ns; Measurement b: t = 0.985, d.f = 55, ns; Measurement c: t = 0.697, d.f = 55, ns; Measurement d: t = 0.954, d.f = 55, ns)(Figure 4.9.). Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 175 4.0 3.5 H 3.0 I 2.5 - DÎ § 2.0 (0c 0) 1.5 1.0 0.5 0.0 a b e d Part of wing measured Figure 4.9. Summary of mean wing measurements from experiment three; right fore- and hind- wings of freshly-killed females were measured along four different axes: a, b, c and d (see Figure 4.1. for details). Infected matrilines (n=44) are grey bars, uninfected (n=13) are white bars. Error bars represent 95% confidence interval. 4.7. Conclusions And Discussion These experiments provide the first clear evidence of a male killing infection that confers a physiological benefit to its host. Infected females have an increased chance of survival to adult, and hence an increased reproductive success. Although there is no significant difference in development rate between infected and uninfected lines, infected female H. bolina are also significantly heavier and slightly larger than their uninfected counterparts. This confers an advantage in terms of increased fecundity and larger size. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 176 The results from experiment one suggest that the benefit of increased survival exists when larvae are reared in groups of ten. It is possible that perhaps infected females have an increased ‘competitive’ ability. However, we still see an increased survivorship to adult even in experiment two, when larvae are reared individually under optimum conditions (i.e. excess food, no competition, excess space) indicating that competition for food and space are not the causes of this pattern. The results from experiment one are almost certain to be an underestimate as H. bolina in Fiji lays clutches of 10-12 eggs in the field (Clarke et al , 1983). So in a typical infected clutch in the field, only five to six larvae would hatch out of the same brood, possibly giving an even greater survival advantage to these larvae than that recorded in the first experiment, due to reduction in competition. The same pattern of increased survivorship was observed at different times of the year in two different Fijian populations, indicating it to be a common phenomenon in Fiji. Independent Samoan H. bolina reared individually exhibit a similar pattern to that seen in the Fijian samples, with infected females having a survivorship advantage. Although these results are suggestive and indicate that H. bolina females from populations other than Fiji gain a survivorship advantage through infection with the male killing Wolbachia, they are inconclusive due to the scarcity of control data (only a single uninfected line). The repeatability of this data, with the same pattern of results being obtained from Fijian H. bolina in five experimental manipulations across two different Fijian populations indicate that the effect is real. The increased survivorship trait is inherited, as demonstrated by replicate two of the first experiment. At first sight, it could be suggested that increased survivorship of infected females is a trade off with other aspects of life history, such as fecundity. However the fact that infected females are significantly heavier as adults than uninfected comparators indicate Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct ejfects of Wolbachia infection in H.bolina 177 this not to be the case. Although data on fecundity was not obtained, adult dry weight strongly correlates to fecundity in butterflies (Hughes c/ a l, 2000). Butterflies obtain all their resources for egg production as larvae, in the form of protein from leaves. As adults they simply feed on nectar, which is protein depauparate. Therefore adult dry mass is indicative of the resources available for egg production, i.e. the fecundity of the female (Hughes et a l, 2000; Lyons, 1996). One could postulate that the increased larval mortality seen in uninfected broods is in fact due to the increased death of males at later instars. It is possible that differential survival of male and female larvae exists such that the observed decrease in survivorship in normal broods was due to death of males, and in fact female survival is identical in both infected and uninfected matrilines. However, normal broods are homogeneous with respect to adult sex ratio and the adult sex ratio in these broods is not significantly different from 1:1. Nevertheless, it is also important to establish the sex ratio in a brood at hatching, as it could be the case that H. bolina broods are male-biased at hatching, such that subsequent male-biased mortality would not be noticed. Clarke et a l , (1975)used the used presence of a heteropycnotic body in the interphase nuclei of the somatic cells in the heterogametic sex (the female) to sex both eggs and larvae oiH. bolina. They showed an approximately 1:1 sex ratio among the developing ova in both infected and uninfected H. bolina broods, concluding that this ratio “stays constant throughout development” as the sex ratio on emergence in all uninfected broods from the same matrilines was found to be not significantly different from 1:1 (Clarke et al, 1975; Clarke et a l, 1983). Clarke’s results demonstrate that 77. bolina broods are not male- biased at hatching, therefore the increase in larval survivorship in Wolbachia-mieoXeé matrilines shown in this chapter is not due to an increase in male-specific mortality in normal sex ratio matrilines. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects of Wolbachia infection in H.bolina 178 These results show a direct physiological benefit to infection with a male killer for the first time. But, how is the Wolbachia providing this benefit? At this stage, without investigation, the effect of the male killer on host physiology can only be speculated. It is possible, looking at the larval development rate data from experiment two, that the bacterium somehow confers a benefit in terms of disease resistance on its host. The larvae that die in uninfected matrilines show, for the most part, a slowing of development prior to death that is characteristic of disease infection (Royama, 2001; Rothman & Myers, 1996). Perhaps the Wolbachia male killer somehow ‘protects’ its host from diseases such as this, thus increasing the likelihood of an infected larvae reaching adulthood. In this light, it is interesting that antibiotics failed to cure the infected female H. bolina of the male killing Wolbachia (see Chapter 2, section 2.6.). Perhaps Wolbachia confers some sort of antibiotic resistance on infected females. In Independent Samoa, and throughout much of its range, H. bolina larvae feed on S. rhombifolia. This common weed is used by the local people in the Samoas as a traditional remedy to prevent infection of cuts and boils. The leaves are mulched up and applied to wounds to speed the healing process. Traditional Samoans believe S. rhombifolia to have antibiotic properties, it is possible that it does, and is difficult for H. bolina larvae to break down. Further investigation is required to clarify whether or not the/f. bolina Wolbachia has a degree of antibiotic resistance. It is possible that the reproductive parasite plays an anabolic role, such as that seen in the dL^hidjBuchnera symbiosis, providing the larvae with essential, or extra nutrients, and thus increasing their chance of survival (Baumann, 1995). The results of experiment one exclude the possibility of infected H. bolina females gaining fitness compensation due to inbreeding avoidance. The fact that the same pattern of results was obtained in both the FI (from field-collected females) and F2 generations Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct effects o/Wolbachia infection in H.bolina 179 (where outbreeding was ensured) indicates that differences in survivorship of uninfected versus infected larvae is not due to inbreeding avoidance. This Chapter presents the first demonstration of a direct effect on the reproductive success of an arthropod host resulting from male killer infection. Physiological advantages to Wolbachia infections have been reported only in systems in which Wolbachia causes cytoplasmic incompatibility, and not in systems where it acts as a sex ratio distorter. Cl-Wolbachia have been shown to augment fecundity, for example Dobson et al (2002), but no record of an effect on larval survivorship has been made prior to this investigation. Inherited parasites in the butterfly Hypolimnas bolina Chapter 4: Direct ejfects of Wolbachia infection in H.bolina 180 Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 181 Chapter 5: Prevalence And History Of Wolbachia Infection In Hypolimnas bolina Across The Butterfly’s Range Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 182 Chapter 5: Prevalence And History Of Wolbachia Infection In Hypolimnas bolina Across the Butterfly’s Range Summary This chapter presents results of a prevalence survey of the male killing Wolbachia across the range oiHypolimnas bolina. Molecular analysis of specimens from Borneo, Malaysia, Thailand, Australia and Tahiti as well as those previously presented from Fiji, Independent Samoa and American Samoa reveals prevalence variation of the male killer across populations. Reasons for the observed prevalence variation are discussed. A second Wolbachia strain, deriving from the A-clade, is identified from American Samoa and certain islands of Fiji. The mechanism of action of this Wolbachia is unknown, but its presence in adult male H. bolina makes it unlikely to be a male killer. Mitochondrial DNA sequences are obtained using uninfected specimens and specimens of differing infection status and type from all populations sampled, in order to ascertain the history of infection of this species with Wolbachia, and to investigate the infection dynamics of the two different infections. The implications of these results are discussed. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 183 5.1. Introduction Prevalence of male killers in natural populations is seen to vary between host species (see Table 1.4., Chapter 1) from very low {1% in Drosophila willistoni: Williamson & Poulson, 1979) to very high (greater than 90% in the African hnXitïüyAcraea encedana: Jiggins et al., 2000a). Variability of the prevalence of male killer infection between different host species’ permits examination of factors that influence both the prevalence and spread of male killing microorganisms, enabling conclusions to be drawn as to whether male killers can only reach high prevalences in particular host taxa. Although such comparisons are informative, examination of variable prevalence within a single host species factors out confounding inter-host differences permitting a more controlled analysis of the determinants of prevalence variation. Several examples illustrate variation in male killer prevalence between populations within the same host. For instance in the African butterfly, Acraea encedon, prevalence of the infecting male killing Wolbachia shows huge variability across neighbouring populations (Jiggins et a l, 2000c). Between- population prevalence variation has also been demonstrated in populations of Harmonia axyridis and Gastrolina depressa. In both cases, prevalence of the male killing agent is seen to vary from 0-50% in different populations (Chang et a l, 1991; Majerus et al, 1998). 5.1.1. Why does male killer prevalence vary between populations of certain host species? To date there is scant empirical data to explain why such dramatic variations in prevalence of male killing agents exist within certain host populations. However, we can postulate a number of factors that might be responsible for the observed variation: Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 184 5.1.1.1. Variations in indirect benefits resulting from infection Within-species differences in host ecology and behaviour could result in different indirect benefits conferred to the host through infection with the male killing agent. If the indirect benefit resulting from infection is high, then the spread of the infection to high prevalence will be facilitated. For example, clutch size of the host species is believed to be an important factor determining the invasion and spread of male killing parasites, indirect benefits to female hosts being greater in hosts with higher clutch sizes. Here, survival of infected female hosts is increased due to both decreased sibling competition, and in some cases sibling egg cannibalism (Hurst & Majerus, 1993). If within-species variation exists in clutch size, it can be postulated that the prevalence of the male killer infection will vary between different host populations that exhibit different oviposition behaviours. 5.1.1.2. Variations in direct (physiological benefits) If the male killer confers a direct physiological advantage to an infected female in certain populations, but not in others, or if the direct benefit varies according to host ecology and behaviour, we would expect to see an effect on prevalence between different host populations. For example, if infection with the male killing bacterium aids physiological breakdown of toxins, host larvae could exploit this by feeding on host plants that would be toxic to their uninfected counterparts. Such a direct benefit would be dependent on the existence and availability of the novel noxious host plant, and if the distribution of this plant exhibited variation, we might expect to see variation in male killer prevalence reflecting this. 5.1.1.3. Infection with more than one selfish genetic element It is more difficult for a new strain to invade a population in which a single strain is established than it is for a new strain to invade an uninfected population. If host populations are isolated, it is possible that male killer invasion could be inhibited or Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 185 excluded in certain populations due to the prior existence of a cytoplasmic factor (not necessarily another sex ratio distorting microorganism); for example male killer invasion could be prevented in a population infected with a Cl-causing selfish genetic element, as incompatibility would make the male killer infected females effectively sterile. 5.1.1.4. Evolution o f resistance If host populations are widely spaced, with little gene flow between them, it is possible that genes conferring resistance to the male killing bacterium could have evolved in certain host populations, but not in others. Resistance to the action or transmission of a male killing bacterium will reduce its prevalence in females. The extent to which this is reduced depends on the mode of action of resistance (i.e. producing inefficient transmission or inefficient male-killing) and on the cost of resistance to uninfected individuals. This latter feature determines the frequency to which resistance genes rise within the population. If the resistance gene prevents the action of the bacterium, the bacterium will then become apparent in male as well as female hosts. 5.1.1.5. Study system The ideal system in which to look for and examine the underlying reasons for variation in prevalence of male killers would be a single species, known to be host to a male killing microorganism, with different host populations being isolated with little gene flow between them: the H. bolinafWolbachia symbiosis. 5.1.2. H. bolinaJWolbachia symbiosis as a case study for prevalence variation 5.1.2.1. Historical evidence Clarke et al.'s original survey oîH. bolina (1975) revealed tantalising evidence of variation in the prevalence of the male killing trait from different populations of//. bolina. Table 5.1. summarises Clarke’s results from areas surveyed other than Fiji (Fijian results are presented in Chapter 2, section 2.11.1.). The sample sizes are small, and evidence is based on existence of female-biased broods only. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H, bolina 186 Table 5.1. Summary of Clarke et al.’s (1975) results on the numbers of female-biased broods recorded from different populations of H. bolina indicating variation in prevalence of the male killing trait Population Prevalence of Number Sampled Comments Sampled Female-Biased (n) Broods Sarawak, Malaysian 87.5% 8 Two ‘all-female’ Borneo broods produce a small number of males. Papua New Guinea 0% 1 Sample size inconclusive Sri Lanka 33.3% 3 Hong Kong 100% 1 Sample size inconclusive Australia 0% 5 5.1.2.2. Previous evidence of prevalence variation from this thesis Clarke et al. ’s (1975) results suggest that prevalence of the causal agent of all-female broods in H. bolina shows extreme variation across populations. To date in this thesis, populations of H. bolina have been examined from three different South Pacific island countries, with each country’s population showing a different level of prevalence of the male killing Wolbachia, as well as variations across island populations within a single country (Chapter 2 section 2.11.; Chapter 3, section 3.8.). The results from Independent Samoa illustrate that high prevalence of male killer infection can have dramatic effects on the host population. Hypolimnas bolina has a huge range, being found throughout the Indo-Pacific, with many host populations occurring on isolated South Pacific islands. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 187 Such ‘discrete’ populations may be expected to show variations in prevalence of the male killing Wolbachia. Assaying for the presence of the male killing Wolbachia across as many of these populations as possible should provide insights as to how, and possibly explain why, prevalence variation exists within a host population. 5.2. Aims To survey the prevalence of the male killing Wolbachia in H. bolina across as much of the butterfly’s range as possible, and attempt to interpret causes and consequences of any observed variation. 5.3. Prevalence Survey OfHypolimnas bolina Adult H. bolina (females and in some cases, males) were collected from American Samoa, Fiji, and Independent Samoa as previously detailed (Chapter 2, section 2.3.; Chapter 3, section 3.4.). Butterflies were also collected from Sabah province, Malaysian Borneo in November 2000. More H. bolina specimens were obtained (collected by friends or ordered from breeders) from: 1. Kota Kinabalu, Sabah province, Malaysian Borneo (May 2001) 2. Rurutu Island, French Polynesia (August 2001) 3. Townsville, Queensland, Australia (DNA sent by Niklas Wahlberg, University of Stockholm) 4. Peninsular Malaysia (DNA sent by Niklas Wahlberg) 5. Thailand (London Butterfly Co.) Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 188 Adult butterflies were preserved in 95% ethanol immediately following death. If butterflies were collected by persons other than the author, wings were also removed, and kept in separate specifically-labelled envelopes to verify species identification. DNA was prepared for PCR assay following protocol A.I., Appendix I. 5.3.1. Molecular analysis 5.3.1.1. Method All samples were assayed for the presence of Wolbachia using primers that specifically amplify the wsp gene, 81f and 691r (these primers amplify both A- and B- group Wolbachia, see primer sequences, section A.2., Appendix I). PCR was carried out following protocol A.3.I., Appendix I, As previously, PCR for the presence of the mitochondrial COI gene was used as a control to ensure negative results for Wolbachia were true negatives and DNA was present. Sequence of part of the wsp gene from at least two samples from each population was obtained following protocol A.5., Appendix I, using PCR products obtained following PCR assay for the wsp gene using general primers. Sequence analysis was used to verify that the same Wolbachia symbiont was present in infected individuals from different populations. 5.3.1.2. Initial results Sequence analysis revealed certain specimens to be infected with a Wolbachia bacterium that did not have the same sequence as that identified as being the causal agent of male killing in H. bolina in both Independent Samoa and Fiji. A BLAST search revealed that this bacterium derives from the A-group of Wolbachiapipientis (Altshul et al, 1997). Calculation of the prevalence of the A-group Wolbachia among all sample populations might help to reveal details concerning the mechanism of action of this selfish genetic element. Two prevalence surveys were therefore carried out: Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 189 1. Prevalence of B-group (male killing) Wolbachia using B-specific primers. 2. Prevalence of A-group (action unknown) Wolbachia using A-specific primers. These surveys were carried out on all//, bolina samples (both male and female) collected from all study populations. PCR analyses were carried out in the order detailed in the flow chart, Figure 2.3. (Chapter 2). 5.3.2. Prevalence of the B-group (male killing) Wolbachia Table 5.2. details the results of the prevalence survey for the presence of the male killing Wolbachia from different populations of//, bolina. The table includes the previously reported results from Fijian and Samoan populations for ease of comparison. These data show that prevalence of the male killing trait shows variation across different populations of H. bolina (yf- = 285, df = 11, p<0.001). Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 190 Table 5.2. Prevalence of Wolbachia male killer across different populations ofH. bolina. Survey carried out using both males and females. Country Population Male Killer Prevalence In Females (n) In Males (n) Malaysian Borneo Kota Kinabalu, 96% (25) 100% (19) Sabah province Peninsular Unknown 100% (1) - Malaysia Thailand Unknown 100% (3*) - Australia Townsville, 0% (1) - Queensland American Samoa Olosega Island 0% (23) 0% (25) Tutuila Island 0% (6) 0% (10) Fiji Viti Levu Island 58.8% (34) 0% (9) Taveuni Island 25% (24) - Wayalailai Island 53.9% (76) 0% (8) Independent Upolu Island 99.2% (257) 33% (3) Samoa Savaii Island 100% (35) 50% (2) French Polynesia Rurutu Island 50% (2) 0%(7) *These specimens were bought through a breeder, it is probable that they are sisters. 5.3.3. Prevalence of the A-group Wolbachia The action of the A-group Wolbachia is unknown, so investigation of the prevalence of the trait, and analysis of both males and females may help reveal details about the mechanism of action of this selfish genetic element. The A-group Wolbachia was found Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 191 to occur in H. bolina in American Samoa as well as in samples from Taveuni Island, Fiji. Single specimens from Viti Levu, Fiji and Upolu Island, Independent Samoa were also infected with this Wolbachia (Table 5.3.). The A-strain Wolbachia was not found in any of the individuals from either Fiji (Chapter 2) or Independent Samoa (Chapter 3) that were identified through breeding data as bearing the male killing Wolbachia. No incidence of a single individual being infected with both Wolbachia strains was recorded. Prevalence of the A-group Wolbachia shows significant variation across the study populations (x^ = 397, df = 11, p<0.001). The result from the single specimen from Australia is inconclusive as the DNA supplied was from the leg of a female//, bolina. N.B. In certain cases, PCR assay revealed the presence of Wolbachia but did not give a clear indication as to whether a particular specimen was infected with Wolbachia A, or Wolbachia B. In such cases, a restriction digest was carried out following PCR assay, according to the protocol A.6., Appendix I. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 192 Table 5.3. Prevalence of A-group Wolbachia across different populations of//, bolina. Survey carried out using both males and females. Country Population Wolbachia A Prevalence In Females (n) In Males (n) Malaysian Borneo Kota Kinabalu, 0% (25) 0% (19) Sabah province Peninsular Unknown 0% (1) - Malaysia Thailand Unknown 0%(3) - Australia Townsville, 0% (1) - Queensland American Samoa Olosega Island 100% (23) 96% (25) Tutuila Island 66.7% (6) 70% (10) Fiji Viti Levu Island 2.9% (34) 0% (9) Taveuni Island 75.0% (24) - Wayalailai Island 0% (76) 0%(8) Independent Upolu Island 0.4% (257) 0%(3) Samoa Savaii Island 0% (35) 0% (2) French Polynesia Rurutu Island 0%(2) 0% (7) Prevalence of both the A-group and B-group Wolbachia vary significantly across the range of//, bolina. Levels of virginity and spermatophore sizes were measured across the different populations in order to see if these correlated with prevalence of either of these infections. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 193 5.3.4. Comparison of wild-collected female matedness rates 53.4.1. Method Female H. bolina from Borneo and Tahiti were dissected as detailed in Chapter 3 (section 3.9.), and the presence or absence of a spermatophore recorded. If no spermatophore is present in the bursa copulatrix then the female is a virgin. Butterflies from the other study populations were all virgins (from breeders) or received as DNA samples. Data from Australia detailing virginity rates oiH. bolina collected in Queensland are reproduced by permission of Darrell Kemp (Kemp, 2002). 5.3.4.2. Results Where it was possible to dissect specimens, the proportion of mated females collected from each of the sample populations was calculated (Table 5.4.). The Fijian and Samoan data are presented, but not divided into different populations as these results have already been illustrated and discussed (Figure 3.5., Chapter 3): they are summarised here simply for ease of comparison with the other data presented. There is no significant difference between the proportion of mated females from Borneo, Fiji and American Samoa (10,000 Monte Carlo simulations, p=0.068, ns). The two females from Tahiti are both mated, however the sample size is too small for statistical analysis. The Independent Samoan population shows a significantly lower matedness rate, as discussed in Chapter 3. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 194 Table 5.4. Proportion of mated females from the different population as evidenced by the presence of a spermatophore in the bursa copulatrix. Number of spermatophores present indicates the number of times the female has mated. (* - From Kemp, 2002) N® Females Number of Spermatophores Proportion Population Dissected Mated (n) One Two Three Females Borneo 25 24 0.96 Tahiti 2 2 1.00 Australia* 78 70 8 1.00 American 29 27 1 0.97 Samoa Independent 214 116 0.54 Samoa Fiji 130 112 9 1 0.94 5.3.5. Comparison of wild-collected female spermatophore sizes 53.5.1. Method Spermatophores dissected from the abdomens of females from Borneo and Tahiti were measured following the protocol detailed in Chapter 3, section 3.11. Australian data were obtained from Darrell Kemp (2002). 5.3.5.2. Results Table 5.5. presents the median diameter and length of spermatophores dissected from wild collected females from different populations, where these could be obtained. Data from Fiji, and the Samoas has previously been illustrated (Chapter 3 Figure 3.7.) and is presented here simply for comparison. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 195 Table 5.5. Median diameter and length of spermatophores dissected from mated if. bolina females from the different study populations, interquartile range in brackets. Population (size) Median Spermatophore Median Spermatophore Length (mm) Diameter (mm) Borneo (n = 10) 1.725 (1.700-1.790) 2.025 (1.923-2.028) Tahiti (n = 2) 1.345 (1.333-1.358) 1.475 (1.458-1.493) Australia (n = 78) 1.700 (1.500-1.844) 1.775 (1.650-1.900) American Samoa (n = 10) 1.685 (1.603-1.813) 1.940 (1.903-2.265) Independent Samoa (n = 28) 0.960 (0.890-1.025) 1.035(1.165-1.225) Fiji (n = 11) 1.760 (1.640-1.870) 2.030 (1.960-2.145) There is no significant difference in spermatophore length between if. bolina from Borneo, Australia, American Samoa and Fiji (Kruskal-Wallis test H = 6.77, df = 3, ns - adjusted for ties); however, as discussed in Chapter 3, length of spermatophores from Independent Samoan wild-mated females are significantly smaller (Kruskal-Wallis, H = 67.19, df = 4, p=0.00 - adjusted for ties; Table 5.6.). There is no significant difference in spermatophore diameter between ff. bolina from Borneo, American Samoa and Fiji (Kruskal-Wallis test H = 0.07, df = 2, ns - adjusted for ties). However, there is a significant difference between spermatophore diameter from these populations compared with diameter of spermatophores dissected from Australian H. bolina (Kruskal-Wallis, H = 32.61, df = 3, p=0.00 - adjusted for ties; Table 5.7.). Diameter of spermatophores from Independent Samoan wild-mated females are significantly smaller than those from all the other populations (Kruskal-Wallis, H = 85.36, df = 4, p=0.00 - adjusted for ties; Table 5.8.). Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 196 The two specimens from French Polynesia show spermatophores of intermediate size. Unfortunately the sample size is too small for statistical analysis. Table 5.6. Results of Kruskal-Wallis test of spermatophore length across all study populations, revealing significant difference between length of spermatophores from Independent Samoan populations compared with the other study populations Population n Median (mm) Average Rank z-value Independent 28 0.960 15.1 -8.09 Samoa Fiji 11 1.760 95.6 2.14 American 10 1.685 81.4 0.89 Samoa Borneo 13 1.725 92.5 2.06 Australia 78 1.700 81.8 3.69 Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H, bolina 197 Table 5.7. Results of Kruskal-Wallis test of spermatophore diameter across Æ bolina populations from Fiji, American Samoa, Borneo and Australia, revealing significant difference between diameter of spermatophores from Australian populations compared with the other study populations. Population n Median (mm) Average Rank z-value Fiji 11 2.030 84.2 2.98 American 10 1.940 81.3 2.53 Samoa Borneo 13 2.025 83.3 3.17 Australia 78 1.775 44.9 -5.17 Table 5.8. Results of Kruskal-Wallis test of spermatophore diameter across all study populations, revealing significant difference between diameter of spermatophores from Independent Samoan populations compared with the other study populations Population n Median (mm) Average Rank z-value Independent 28 1.165 15.7 -8.00 Samoa 1 Fiji 11 2.030 112.2 3.55 American 10 1.940 109.3 3.14 Samoa Borneo 13 2.025 111.3 0.66 Australia 78 1.775 72.5 3.81 Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 198 5.3.6. Conclusions from prevalence surveys As Clarke et alfs data indicated (1975), prevalence variation in the male killing trait does exist across H. bolina populations. Variation in prevalence of the A-group Wolbachia is also shown. The existence of the A-group Wolbachia seems to explain why the male killing B-group Wolbachia is either at a lower prevalence (in Taveuni) or absent (in American Samoa) from certain populations. Prevalence of the A-group Wolbachia varies between the two different island populations sampled in American Samoa (Fisher exact test, p = 0.037). Why this should be the case is unknown, especially in the light of the fact that the B-group male killing Wolbachia appears to be absent from these populations. It is difficult to explain the cause of this variation when the action of the Wolbachia A symbiont can only be speculated on. 53.6.2. Prevalence of male killing Wolbachia in Borneo The prevalence of the male killing Wolbachia in H. bolina collected in Sabah, Borneo is not significantly different from the exceedingly high prevalences recorded from Independent Samoa (Fisher exact test: p=0.22, ns). Thus, the population effects recorded in Independent Samoa such as increased infertility, reduced spermatophore size and increased virginity might be expected to be observed in Borneo. However, observations of the populations reveal significant differences. Whilst collecting in Borneo, my ‘PCR- blind’ impression was that if the male killer was present in the Kota Kinabalu population it would be at around the same level of prevalence observed in Fiji. The reason for this was due to the number of males observed. As in Fiji, males were observed to be flying in numbers, active and territorial, and females were much more scarce. In Independent Samoa, the reverse was true (see section 3.5., Chapter 3). The virginity rates and spermatophore sizes obtained from the Borneo samples are also equivalent to those obtained from American Samoa and Fiji, further indicating that the population in Borneo is not ‘damaged’ to the level of the Independent Samoan populations. The observation Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 199 that the bacterium is also present in males (all males sampled tested positive for the Wolbachia male killer) suggests that resistance to action of the male killing Wolbachia has evolved in H. bolina in Borneo. Alternatively, it could be the case that the infecting Wolbachia present in H. bolina in Borneo is different to that identified as the male killing bacterium identified from Fiji and Independent Samoa. However, this seems unlikely as the^i^^Z and wsp gene sequences are identical to those of the known male killing Wolbachia, hence an explanation of resistance evolution is the best explanation of the data. It may not be that all females have developed resistaiice to the ihale killing symbiont, but unfortunately little egg hatch rate or rearing data was obtained from Borneo. Egg hatch rate data was obtained from two wild collected females in Borneo, both showing high hatch rates typical of uninfected individuals, with no unhatched ‘grey’ eggs being recorded: Female 1: Proportion hatch - 0.95 (n = 21) Female 2: Proportion hatch - 1.0 (n=12) The existence of resistance in this population therefore is strongly suggested, but only backed up by scant empirical data. It is important to note that these findings are also consistent with the male killing symbiont in Borneo having lost the ability to kill males. 53.6.3. What is the action of the A-group Wolbachia? The A-group Wolbachia is found at high prevalence on Taveuni Island, Fiji and in both Island populations studied in American Samoa. In American Samoa, there is no significant difference between the prevalence of the Wolbachia among males and females (Fisher Exact tests, Olosega Island: p=l,ns; Tutuila Island: p=l,ns). The fact that the Wolbachia is found in males at such high prevalences indicates that it is probably not a symbiont that causes male killing, although it could be the case (as seen with the B- group Wolbachia in Borneo) that the A-group Wolbachia does cause male killing, but the host populations have developed resistance to its action. Unfortunately no male H. bolina Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 0 0 were collected from Taveuni Island, Fiji for verification of the American Samoa data. More stringent sequence analysis of the A-group Wolbachia, and determination of the phylogenetic position of this bacterium might provide insights into the mechanism of action of this selfish genetic element: 5.3.7. Sequence analysis and phylogeny of the A-groupWolbachia 5.3.7.1. Method Sequence of the wsp gene of the A-group Wolbachia was determined from at least two infected specimens from each population. Sequence analysis Was performed following protocol A.5., Appendix I. Sequences were manually aligned (Cabot, 1997). 5.3.7.2. Results All H. bolina specimens that had tested positive for the presence of the A-group Wolbachia were found to be infected with the same Wolbachia strain. A BLAST search for this sequence (Altshul et al, 1997), and subsequent phylogenetic analysis showed the Wolbachia A-group present in some populations of H. bolina is most closely related to a Wolbachia A-group found in another Lepidopteran s^tcits,Ephestia kuhniella. The Wolbachia in E. kuhniella is known to cause cytoplasmic incompatibility (Sasaki & Ishikawa, 1999). The position of the H. bolina A and B group Wolbachia are given in the following phylogeny based on wsp gene sequences adapted from Jiggins et al, 2002 (Figure 5.1.). Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history’ o/Wolbachia infection in H. bolina 201 M. uniraptor D. simulans Ha H.botina . kuehniella E. cautella A D. simulans Ri N. VitripenmsA C. perengainus D. bifasciata A. fuscipennis D. meianogaster As. albopictus A T. deion SW ' \ /V. formosa T. kaykai B r sibericum T. nubilale A rth ro p o d Wolbachia T. deion G ro u p A F. vespiforrnis ' A. bipunctata Z encedon U striatellus A. bipunctata Y S furcifera T. nawai striatellus T. bedeguaris E. formosa A. diversicornis A. eponina E. staufferi T. taiwanemma E. cautella 8 D. rosae S. fuscipes T. orizicolus G. micromorpha Arthropod Wolbachia A. alcinoe Group B H. bolina A. en ced o nT C. pipiens Ae. albopictus B D. simulans No A. equatoria L. sigmodontis A. vulgare A. vulgare Nematode worm symbionts W. bancrofti pahangL malayi 0 . volvulus _ O.ocheni O.gibsoni O Q repens D. immitis Figure 5.1. Phylogeny of Wolbachia based on wsp gene sequences showing the phylogenetic positions of the H. bolina A and B group infections (from Jiggins et ai, 2002). î^iU oritari r in m c if û v itt hiêft£>r-flM W\mr>limnQC Krvlina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 2 0 2 53.7.3. Conclusion The same A-group Wolbachia is found in both males and females from populations in American Samoa, Taveuni and Viti Levu Islands (Fiji), and in a single anomalous specimen from Independent Samoa. InE. kuhniella, the A-group Wolbachia causes cytoplasmic incompatibility (Sasaki & Ishikawa, 1999). Due to the overall similarity of the wsp sequences of the A-group Wolbachia from E. kuhniella and H. bolina and the fact that this strain is present at high and approximately equal prevalences in both sexes in the populations in which it occurs, it is probable that the H. bolina A-group Wolbachia also causes cytoplasmic incompatibility. However, host effects on Wolbachia phenotype make it possible that identical strains have different phenotypes in different host species (Sinkins et a l, 1995). Indeed the observation that matings between A-infected males from American Samoa, and B-infected females from Independent Samoa showed no deviation from normal male killer egg hatch rates suggests that the A-strain Wolbachia may not cause Cl in//, bolina (Chapter 3 section 3.10.), although it is possible that somehow the B-strain ‘rescues’ the effect of incompatibility brought about by the A- strain. The true test as to whether or not the A-Wolbachia causes Cl would be to cross an A-infected male with a female known to have no Wolbachia infection; if the A-strain causes Cl we would expect such a cross to be infertile. Unfortunately, I was unaware of the existence of the A-strain during fieldwork, so this simple cross was not performed. No occurrences of both infections in a single individual were recorded. On the basis of these results, it was decided to carry out mitochondrial DNA sequence analysis of H. bolina in an attempt to establish the infection history of the two Wolbachia strains. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 203 5.4. Introduction To The Use Of Mitochondrial DNA In Evolutionary Studies The selective forces that influence the dynamics of distinct biological processes have been extensively researched for more than a century. Mitochondrial DNA (mtDNA) has been used as a tool for such investigations, particularly intraspecific population genetic studies, for the last forty years. Mitochondria are thought to be non-recombinant, and, together with their maternal mode of inheritance and relatively high rate of nucleotide substitution, often provide multiple haplotypes (multiple alleles) within a population, or between species. Phytogenies composed using mtDNA haplotypes reflect the maternal component of an organism’s history. Many studies have shown specific and closely- related mtDNA variants to be limited to specific geographical regions of the species range, representing limited gene flow with respect to females. Phytogenies constructed using mtDNA variants can therefore be utilised to infer population history in terms of size and spread of the species across the whole of its range (for example see Wahlberg & Zimmermann, 2000). The mtDNA diversity can also provide details of colonising events due to the founder effect: low diversity of mtDNA in a particular region being indicative of a recent colonisation event or other bottleneck (for example see Gillespie, 2002). However, the evolutionary dynamics of mtDNA are not necessarily neutral, and other factors such as maternally inherited symbionts can have far-reaching effects on mtDNA diversity. 5.4.1. Can mtDNA divergence be employed to resolve phylogenetic history of a species? The simple answer to this question is yes, it can: there are many examples in the biological literature of both intra- and inter- species relationships that have been Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 0 4 determined using mtDNA phylogenies (Brunton & Hurst, 1998; Wahlberg & Zimmermann, 2000). However, within-species phylogenies composed using mtDNA haplotypes can be confounded by the spread of a selfish genetic element within the host population (Ballard et a l, 1996). Mitochondria are maternally transmitted in the cytoplasm of egg cells. For this reason they will be in linkage disequilibrium with other cytoplasmic elements such as inherited bacteria and, in female heterogametic species (e.g. Lepidoptera) the W sex chromosome. The linkage disequilibrium is so ‘strong’ that introgression of mtDNA into a closely related species associated with introgression of an inherited bacterium has been reported in both fruit flies (Ballard, 2000) and butterflies (Jiggins, 2002). The association may well also explain the common finding that mtDNA haplotypes and clades within many species are found to be geographically localised and may be an alternative to other interpretations in many cases (Avise et a l, 1987). Maternally inherited selfish genetic elements such as Wolbachia have been shown to influence the evolution of mtDNA in many cases (Behura et a l, 2001; Marcade et al, 1999). For example, different populations of Drosophila simulans are known to be infected with several different strains of Cl-causing Wolbachia; in each case the bacterial strain is associated with a distinct mitochondrial haplotype (Montchamp-moreau et a l, 1991; Rousset & Solignac, 1995). It can easily be extrapolated that compiling a phyfogeny of D. simulans populations based on mtDNA haplotypes, without prior knowledge of the different Wolbachia strains, would produce a confused and incorrect basis from which to derive the history of the species. Recently several studies have compared phylogenies composed using nuclear DNA markers, with those of the same host species composed using mitochondrial haplotypes. In most cases, the two types of phylogeny suggest vastly different relationships (Ballard et a l, 2002; Jiggins, 2002). These studies illustrate that although mtDNA can be used to Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 205 genetically define the phylogenetic history of a species in some cases, the widespread existence of cytoplasmically inherited selfish genetic elements can cause incorrect phylogenetic inferences to be made. 5.4.2. Effect of spread of a selfish genetic element on mtDNA diversity As a selfish genetic element spreads in a population, we would expect to see spread of the particular mitochondrial haplotypes with which the infection was first associated. So the presence of a selfish genetic element can give rise to reduced mtDNA diversity. The following section explains how the diversity of mtDNA would expect to be affected due to invasion and spread of a selfish genetic element. In the following diagrams, numbers represent different mitochondrial haplotypes, haplotypes that are associated with infection with a selfish genetic element are numbered in blue, those in uninfected hosts are numbered in black. A circle represents either the uninfected (black) or the infected (blue) gene pool of mitochondrial haplotypes. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 206 Stage 1. Prior to invasion of selfish genetic element: uninfected population Diverse range of mitochondrial haplotypes or ‘mitotypes’ (1-8), new types emerging due to mutation, with spread and loss by drift only. Stage 2. Invasion of new cytoplasmically inherited infection, e.g. a male killer The new infection is initially found in only a single individual. Due to its maternal inheritance, it will become associated with the haplotype of the originally infected individual, (in this case, type 8). As the infection spreads, this haplotype will preferentially spread in the population, and become more common than the other uninfected haplotypes. At this stage, the haplotype associated with infection will be homogeneous, as it is a Mitotype of first infected recent spread of infection. individual The uninfected population is still variable, as above. I n lio r iio fi ttn rn ^ ito v in h>-nifofft\f T 4-»m r\lim n !ic K<->linî Chapter 5: Prevalence and history (^Wolbachia infection in H bolina 207 Stage 3. Leakage of infected mitotype back into uninfected mitotype pool After many generations, the infected mitotype will leak back into the uninfected population due to inefficient transmission of the bacterium. The diversity of the uninfected population will decrease. Leakage due to inefficient transmission Stage 4. Infected mitotype diversifies due to mutation and drift Eventually, neutral evolutionary forces of mutation and drift will result in diversification of the ‘infected’ mitotype. Leakage due to inefficient transmission of the infection continues (as in stage 3). InUoritort rtnrrfviio Stage 5. ‘Monopoly’ of originally infected mitotype As diversification of the infection-associated haplotype (stage 4) and leakage due to inefficient transmission (stage 3) continue, eventually all infected and uninfected mitotypes in the host population are derived from the mitotype of originally infected individual. 5.4.3. Interpretation of schematic diagram Sequencing of mtDNA from different individuals within a species known to be infected with a selfish genetic element, allows the following conclusions concerning infection history and dynamics to be drawn, based on the diversity of mitotypes observed; 5.4.3.1. Selective sweep and timing of infection invasion The diversity of mitochondrial haplotypes present in a population infected with a cytoplasmically inherited reproductive parasite allows deductions to be made as to how recently a sweep of the infection has occurred. For example, if the symbiont-host interaction has reached stage 2, an analysis of mitochondrial haplotypes of the host n n m v i i a i v im W\7nr\limnac Knlino Chapter 5: Prevalence and history o/Wolbachia infection in H, bolina 2 0 9 population would reveal that the same mitotype is present in all infected individuals. The lack of diversity would indicate a recent sweep of infection has occurred, within the last 300,000 years (Ballard et al, 2002). The rate at which the mitochondrial haplotype originally associated with the infection (i.e. type ‘8’ in the schematic diagram) is found to be in the uninfected population, represents the rate of ‘leakage’ of the mitotype from infected to uninfected. In terms of the infection itself, this rate reflects the rate at which the selfish genetic element is being ‘lost’ from the host population. Infected hosts can ‘escape’ infection in this manner by one of two mechanisms: 5.43.2. Rates of ‘leakage’ of infection via inefficient transmission If transmission efficiency of the infection is perfect, i.e. an infected female always passes the symbiont to all of her progeny, we would expect all infected individuals to have a unique ‘infected’ mitotype. However, if leakage is occurring due to inefficient transmission, certain progeny of an infected female will ‘escape’ infection. Although these progeny will be uninfected, they will carry the infected-type mitotype: hence the infected haplotype has leaked back into the uninfected population (stage 3). The rate at which such leakage occurs is dependent on the degree of inefficient transmission of the selfish genetic element. If transmission efficiency is high, we would expect to see a low level of leakage, and (at least initially) a small amount of uninfected individuals bearing the infected-type mitotype. 5.4.3.3. Loss of disequilibrium via horizontal transfer Horizontal transmission of the cytoplasmic element would cause association of the symbiont with more than one mitotype. Imagine a scenario in which two novel Wolbachia infections arise in a host population (‘Y’ and ‘Z’), each associated with a different mitotype (‘A’ and B ’ respectively). If the heritable symbionts can be passed to Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 2 1 0 each other via horizontal, as well as vertical transmission we would eventually expect to see a situation in which both infections are associated with both mitotypes, and with all previously uninfected mitotypes (Figure 5.2. A and B). In heritable symbioses, horizontal transmission of the selfish genetic element is thought to be a rare occurrence. Recently, however frequent horizontal transmission of Vl-Wolbachia from infected to uninfected Trichogramma pretiosum has been reported in wasps sharing a common food source (Huigens et al, 2000). It is unknown how common such transmission events are, and the mtDNA phylogeny of T. pretiosum has yet to be investigated. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history c/Wolbachia itifection in H. bolina 211 A. Invasion of novel infections: A B C D E E D E C B Infection with Infection with /B A ^ Wolbachia ‘Y ’ strain Wolbachia ‘Z’ strain B B B A A A B B B B AAA B B B A A A BA B. Eventually, as the two infections show horizontal transmission: Horizontal Transfer of A B A Infections Y and Z A B A B BAB < C A A B A A B B O B B B B E D A A B C E D E D E C B B A Figure 5.2. Schematic illustration of the effect of horizontal transfer on mitotype diversity. ‘Z’ and ‘Y’ are novel and different Wolbachia strains. Letters A-E represent mitochondrial haplotypes, ‘Z’ infected haplotypes are shown in red, ‘Y’ infected haplotypes in blue, and uninfected haplotypes in black. rtn m v'ilo v rn huttof'-fly) W \mr\limnac KnIinQ Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 2 1 2 5.5. Previous Mitochondrial DNA Diversity Studies In Male Killer Infected Hosts Two previous studies have investigated the phylogeography of two different species, both known to be infected with male killing bacteria, with similar results: 5.5.1. Host: Adalia bipunctata Schulenberg et al (2002) investigated the mitochondrial history of the two spot ladybird, Adalia bipunctata, that is known to be infected with four different male killing bacteria: one Rickettsia, one Spiroplasma and two distinct strains of Wolbachia. Analysis was carried out based on two variable mitochondrial regions: part of the Cytochrome oxidase subunit one gene region (COI) and part of the NADH dehydrogenase subunit 5 gene region. These gene regions were selected as both have been previously indicated to contain some of the most variable parts of the mitochondrial genome in insects (Ballard et a l, 2002; Beard et al, 1993; Simon et a l, 1994). Host insects assayed were of known infection status and varying geographic origin. Results showed that mitochondrial haplotype was not associated with species geography, but with infection status, again illustrating linkage disequilibrium of the selfish genetic element (in this case male killing bacteria) and the mitochondria. The authors also demonstrate that the diversity of mtDNA allows insights into the pattern and history of infection of the different male killing infections (as illustrated in the schematic diagrams, section 5.4.2.) 5.5.2. Host: Acraea encedon Jiggins’ study (2002) confirms the above results, this time in a butterfly host. Again, different male killers are found to be associated with different mitotypes and the selective sweep of male killer infection reduces the diversity of mitochondrial haplotypes compared with that predicted by neutral theory (Kimura, 1983). No diversity of infected Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 2 1 3 cytoplasm is reported, but there is a high diversity of uninfected cytoplasm that correlates with geography. Maternally inherited microorganisms clearly have the ability to distort the mitochondrial haplotype diversity of their host species and disrupt species phylogenies, due to linkage disequilibrium of the symbiont and mitochondria, and their consequent co-inheritance. It is believed that, as has been shown in several host populations, the mitochondrial variant that is associated with the infection increases in frequency throughout the geographical range of infection resulting in a reduced concordance of mitotype with geography. Island populations of insects, such as H. bolina, present an ideal host system to use to test this hypothesis. Due to the isolation of such populations, it can be postulated that the flow of mitotypes between islands will be greatly reduced. Examination of geographically separated H. bolina mitochondrial haplotypes associated with the two different Wolbachia infections should allow this issue to be addressed. 5.6. Aim To investigate the history of the two different Wolbachia infections ini/, bolina using host mitochondrial diversity. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 2 1 4 5.7. Analysis OfHypolimnas bolina Infection History Using Mitochondrial DNA 5.7.1. Method 5.7.1.1. PCR and sequence analysis Individual H. bolina specimens from different populations and countries were assayed for the presence or absence of the two different Wolbachia infections using PCR (See section 5.3. for methods and results). From each population, several individuals were selected that were infected with the different Wolbachia strains. PCR assay for the mitochondrial gene COI (part of the Cytochrome oxidase subunit one gene region) was carried out on the selected specimens following protocol A.3.2., Appendix I (Brunton & Hurst, 1998), and the PCR product sequenced (protocol A.5., Appendix I). The same method was used to obtain the COI gene sequence of selected specimens from each of the study populations that were not infected with either of the Wolbachia strains. This should enable determination of whether or not uninfected specimens have a history of infection, i.e. if they have previously been infected by one of the two Wolbachia infections, but secondarily ‘escaped’ infection due to, say, inefficient transmission (in which case we should find the same mitochondrial haplotype in infected and some uninfected individuals); or if they are from a separate lineage altogether (no evidence of previous infection). 5.7.1.2. Phylogenetic analysis Sequences were manually aligned (Cabot, 1997). Phylogenetic tree estimation was performed with maximum likelihood as implemented in PAUP*, 4.0b 10 (Swofford, 1993). Likelihood scores were obtained for a variety of substitution models and thereafter compared using the programme ‘MODELTEST’ (Posada & Crandall, 1998). Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 215 The Tamura - Nei model with gamma-distributed rate heterogeneity across sites (TrN + G: Tamura & Nei, 1993) was found to provide the most realistic representation of sequence evolution for the data set. This substitution model was subsequently used for tree estimation and nonparametric bootstrapping. Both procedures were performed with the heuristic search mode using branch swapping by nearest-neighbour interchanges and parameter estimates derived from ‘MODELTEST’ (Posada & Crandall, 1998). The stability of the tree was tested via 1000 bootstrap replications. The results were loaded into ‘Treeview’ (Page, 1996) to produce a graphic tree. Variation within samples of a given infection status and differentiation between samples of different infection status were analysed using standard population genetics measures as implemented on ‘ARLEQUIN’ (Schneider et a l, 1997). In brief, haplotype frequencies were calculated for each infection status. Nei’s diversity index (ji) was then calculated for samples of each infection status (A, B or uninfected) in turn. Differentiation between samples of different infection status (F s t values) were calculated, and the statistical significance of differences by infection status analysed by AMOVA (Excoffier et al, 1992; Weir & Cockerham, 1984). In order to determine approximate sequence relationships, a table of pair wise distances between haplotypes was also produced. Uncorrected distances were used in this analysis because the short evolutionary distances between haplotypes suggested little error from saturation. 5.7.2. Results 5.7.27. Sequence analysis The H. bolina specimens surveyed exhibit mitotype diversity. Eight different mitotypes of H. bolina were identified through sequence analysis of specimens from the different study populations The male killing Wolbachia B-strain is associated with a single genetically homogeneous mitotype, regardless of the geography of the host specimen. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history o/Wolbachia infection in H. bolina 2 1 6 The A-strain Wolbachia is associated with four mitotypes: two being found only in Fiji, one in both American Samoa and Fiji, and one being found only in the single anomalous individual collected in Independent Samoa (Chapter 3, section 3.13.4.). One of the A- strain associated mitotypes is also identified from uninfected Fijian specimens. A further two haplotypes are only identified from uninfected Fijian and the single uninfected French Polynesian//, bolina (Table 5.9.). Table 5.10. gives details of regions of variable DNA positions within the COI gene of//, bolina specimens of different infection statuses from the different study populations. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 1 7 Table 5.9. The eight different mitochondrial haplotypes identified among the COI sequences of 34 H. bolina samples together with infection status and geography of the host. Mitotype N°. Specimens Population Location Infection 2 Fiji - Viti Levu Wolbachia B 1 Fiji - Taveuni Wolbachia B 2 Fiji - Wayalailai Wolbachia B 1 2 I. Samoa - Upolu Wolbachia B 2 I. Samoa - Savaii Wolbachia B 1 French Polynesia Wolbachia B 2 Thailand Wolbachia B 2 Borneo - Sabah Wolbachia B 1 Malaysia Wolbachia B 1 I. Samoa - Upolu Uninfected 2 1 I. Samoa - Upolu Wolbachia A 3 1 French Polynesia Uninfected 2 Fiji - Wayalailai Uninfected 4 1 Australia - Uninfected* Queensland 5 1 Fiji - Wayalailai Uninfected 6 2 Fiji - Viti Levu Uninfected 1 Fiji - Wayalailai Uninfected 1 Fiji - Taveuni Wolbachia A 7 2 A. Samoa - Olesega, Wolbachia A 2 A. Samoa - Tutuila Wolbachia A 2 Fiji - Taveuni Wolbachia A 8 2 Fiji - Taveuni Wolbachia A Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 1 8 <<000000 o<<<<<<< <<<<< E-HUUUUHH < o<<<<<<< E-UE-E-E-HE-E- T3 * » 0) ed UUUUUUE-E- oo<<<<<< a Cm Cm (L> 0 <<000000 O JO o<<<<<<< i uuuuuuuo CL 1 I oo<<<<<< 5 3 CL <<00000 cd § § UUUUUUE-E- <000000 £ £ E-'E—OE-'E-'OE^E-' 1 <000000 E - E - < < < < < < < < < < < o o <<000000 o < < < < < < oo<<<<<< o < < < < < < *5 <0000000 o < < < < o o I o o o < o o o o c < < < < < o o < o o o o o o < G o < < < < < < <<000000 Iu & o < < < < < < 1 O UE-E-'E-E-E-E-E- u E- U U U U E- E- I < T-HCNcn'd->nvor~oo Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 1 9 5.7.2.2. Phylogenetic Analysis The relationship between the different haplotypes, and three othQi Hypolimnas species (all received as DNA samples from Niklas Wahlberg), H. deios, H. alimena and H. missipus (outgroup) is given in the following phylogeny (Figure 5.3.). Figure 5.4. provides a schematic illustration of the diversity and infection association of the different H. bolina mitotypes. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence atid history o/Wolbachia infection in H. bolina 220 American Samoa (4) Fiji (2) 99 Fiji (2) Fiji (2) French Polynesia (1) 95 Fiji (I) Fiji (3) Australia (1) F iji(l) Hypolimnas deios ( 1 ) 72 Independent Samoa (1) Independent Samoa (1) Independent Samoa (4) Fiji (5) Thailand (2) Borneo (2) Malaysia (1) French Polynesia ( I ) Hypolimnas alimena (1) Hypolimnas missipus (1) lOutgroup] Figure 5.3. Fifty percent majority rule mtDNA phylogeny of Hypolimnas bolina showing the relationships between different mitotypes. The geographical location of all H. bolina specimens is given, and the infection status is represented by colour: black- uninfected, h\uQ-Wolbachia A infected, red- Wolbachia B infected. Three other Hypolimnas species are included, H. missipus being an outgroup. Numbers represent bootstrap support for particular nodes. Irthorifort nnt-nvhov in tho hnttovflyt t-T^m rtlimnoc Kr»lino Chapter 5: Prevalence and history Wolbachia infection in H. bolina 221 Uninfected Wolbachia A infected Wolbachia B infected Figure 5.4. Schematic illustration of results of mitotype analysis of H. bolina from different populations associated with differing infection statuses. Numbers 1-7 represent the seven different mitochondrial haplotypes identified by differing COI sequences. Colour of box represents infection status of the specimen (red: specimen infected with Wolbachia A; blue: specimen infected with male killing Wolbachia B; black: uninfected). Colours of strain numbers represent the originating country of each specimen: Fiji, , Australia, Independent Samoa American Samoa, Borneo, Malaysia * •■;':! . The frequencies of each strain type represent the number of individuals of that type sequenced. 5.7.3. Population genetic analysis As shown in Table 5.10, there was no haplotype variation within the sample of B (male killer) infected individuals (ti = 0), despite these deriving from geographically disparate populations (from Malaysia to French Polynesia). Genetic diversity was observed in both A-infected {n = 0.644) and uninfected samples (tc = 0.833) (Table 5.11.). When differentiation between samples was investigated, it was clear that the samples infected with the B (male killer) strain were distinct from both A-infected and uninfected samples, as indicated by the high Fst value (Table 5.12.). There is some weak r%nrrtcifov i»> iUi.> T-ï^mr\limnoc Kolino Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 2 2 differentiation between A-infected and uninfected samples, which may be associated with geographical sampling rather than infection status per se. Analysis of differentiation by AMO VA (Schneider et al, 1997) confirmed there is significant variance in haplotype frequencies between samples of different infection status (From 1023 Monte-Carlo permutations: p<0.001; Table 5.13.). 55.8% of haplotypic variation in the total sample of 34 individuals was explained by differentiation associated with infection status. Table 5.11. Haplotype frequencies and Nei’s diversity (jt) by infection status Infection ji Number Haplotypes Haplotype Code (diversity) sampled found 1 2 3 45678 Wolbachia 0.644 10 4 0 1 0 0 0 1 6 2 A ±0.152 Wolbachia 0±0 15 1 15 0 0 0 0 0 0 0 B Uninfected 0.833 9 5 1 0 3 1 1 3 0 0 ±0.098 Table 5.12. Fst values inferred from pair wise comparison of populations of differing infection status Wolbachia A Wolbachia B Uninfected Wolbachia A 0 Wolbachia B 0.732 0 Uninfected 0.237 0.619 0 Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 2 3 Table 5.13. AMOVA analysis of differentiation between samples of different infection status Source of d.f. SSQ Variance Percentage of variation Variation Within 31 6.233 0.201 44.2 infection status Between 2 6.002 0.254 55.8 infection status Total 33 12.235 0.455 5.7.4. Interpretation of results and conclusion The male killing B-group Wolbachia is associated with its own uniform mitotype. The fact that uninfected individuals exhibit diversity of mitochondrial haplotypes, when there is no diversity within the male killer infection-associated lineage indicates that the male killing symbiont has either entered H. bolina relatively recently, or there has been a recent selective sweep of the male killing Wolbachia. The dynamics of this infection indicate it to be at stage 2 of the schematic diagram (section 5.5.2.). Conservatively, this selective sweep of infection with the male killing B-Wolbachia has probably occurred within the last 300,000 years (we expect on average 1 diverged locus per 125000 years in 400 bases of sequence (Brower, 1994). In contrast, the A-strain Wolbachia is found to be associated with four different mitotypes. This diversity indicates that the last selective sweep of the A-strain far predates that of the B-strain. A paucity of uninfected individuals were surveyed, but mitotype diversity was recorded among them, indicating that the A infection is at stage 4 of the schematic diagram. In terms of differentiation, it is clear that there is very little leakage of the B-infection associated haplotype into the uninfected population: only one of 10 uninfected Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 2 4 individuals bore the standard B haplotype (haplotype 1). This specimen was from Independent Samoa, where nearly all individuals are B infected. It is surprising that uninfected females from Fiji, where the B infection was also prevalent, did not show the B haplotype. This suggests that transmission efficiency of this infection in the field is very high. In contrast, the fact that the mitotypes of certain uninfected individuals from Fiji and French Polynesia were identical in sequence to an A-associated mitotype indicates that these strains were previously infected with the A-infection but the infection has been lost due to ‘leakage’, probably via inefficient transmission of the Wolbachia. The only specimen from Independent Samoa not to show the B-strain haplotype, is the anomalous Saml55. The mitotype of this Wolbachia A-infected Æ bolina is most closely related to the B-strain haplotype, although this is not a close relative in distance terms (distance to B-clade: 4.66%; distance to rest of A-clade: 5.7%). It is unfortunate that the true origin of this butterfly is unknown. However, it tells us that the Wolbachia A-strain is very old, and the B-strain originally infected a branch of the diversified A- associated mitotypes, and since this infection event, original mitotypes have diversified further. Alternatively, this relationship could represent an old horizontal transmission event of the A-infection into the B-infection associated mitotype, with subsequent haplotype diversification. It is also apparent that the B-infection has not been, or at least is not often, horizontally transmitted. If horizontal transmission of the B-group male killing Wolbachia was occurring, we would expect to see the male killing infection associated with both uninfected and A-group haplotypes, however there is not a single example in this data indicating such horizontal transmission has occurred. Finally, the mtDNA phylogeny suggests that, based on mtDNA sequence,//, bolina is paraphyletic. This may have occurred through mtDNA introgiession following Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 2 5 hybridisation. For example, if a H. deios female, infected with the male killing B-group Wolbachia hybridised with a H. bolina uninfected male to produce fertile offspring, the hybrid progeny would contain the cytoplasmic elements from the mother, i.e. mtDNA and Wolbachia. If these progeny persisted, over many generations of mating with non hybrid H. bolina the hybrid nuclear genome would die out, but possibly the mtDNA and male killer infection would persist and spread due to the drive associated with male killing, providing an mtDNA phylogeny similar to that shown in Figure 5.4. This possible explanation is beguiling, but it is true that in Lepidoptera, where the female is the heterogametic sex, hybridisation is more unlikely than in other insect groups as hybrid females tend to become sterile before males (Haldane, 1922). More data is required to resolve this. 5.8. Discussion 5.8.1. Wolbachia prevalence in H. bolina Prevalence of the male killing Wolbachia is seen to vary between different host populations. The existence of the A-group Wolbachia in American Samoa and on Taveuni island, Fiji, provides an explanation as to why male killer prevalence is low in these populations. However, this does not explain why prevalence is variable across other populations from which the A infection is apparently absent. As previously discussed, prevalence in Borneo is equivalent to that recorded in Independent Samoa (section 5.3.6.2.) but due to the presence of the male killing Wolbachia in males collected from Borneo, it appears that resistance has evolved in this population. This population requires further investigation to analyse how (and if) the host species has evolved such resistance. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 2 6 It is more difficult to explain variations in prevalence between the other areas: the male killer is present in populations from French Polynesia, Thailand, Malaysia, Borneo, Independent Samoa and Fiji. In the former three cases, more samples are required in order to correctly assess the level of prevalence within the population. The result from the single specimen from Australia as being uninfected with either of the Wolbachia strains is inconclusive as the DNA supplied was from the leg of a female//, bolina. However, based on personal communication from Darrell Kemp who has spent 3 years studying //, ho/ma in Australia and has found no evidence for either low hatch rates or all female broods despite extensive breeding work, it appears that Clarke’s original findings, that the male killing Wolbachia B symbiont is not present in Australia, are likely to be correct (Kemp, 2000). 5.8.2. Phylogenetic status ofHypolimnas bolina The two specimens from Australia and Peninsular Malaysia were kindly sent (as DNA samples) by Niklas Wahlberg (Institute of Zoology, University of Stockholm, Sweden). Dr. Wahlberg is in the process of composing the mitochondrial DNA phylogeny of butterflies in the family Nymphalidae, using the COI mitochondrial marker. Prior to our communication, and based on their vastly divergent COI sequences. Dr. Wahlberg had suggested that his two H. bolina specimens from Australia and Malaysia were in fact different species, being paraphyletic. This example illustrates the difficulties of composing phytogenies based on mtDNA sequence, due to their inheritance in conjunction with cytoplasmic selfish genetic elements. Of course, it is true that specimens from Australia and Malaysia have not (to the best of my knowledge) been cross-mated, and it may be that they are in fact different species. However, during the course of this thesis, female butterflies from Independent Samoa (infected with the B- group Wolbachia, and demonstrated to have the B-type mitotype) were cross-mated with males from American Samoa (infected with the A-group Wolbachia) and produced Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 2 7 fertile offspring. COI analysis has revealed the haplotype of the American Samoan specimen clades with that of the Australian sample sent by Dr Wahlberg (A-infected and uninfected respectively), and the haplotype of the Independent Samoan individual clades with that of the Malaysian sample (both B-infected). The two butterflies are almost definitely from the same species, despite the misleading story that the mitochondrial DNA phylogeny suggests. The fact that maternally inherited symbionts are so widespread (Wolbachia is thought to be found in 17-22% of all arthropod species) makes one wonder how many mtDNA phytogenies are similarly misleading. 5.8.3. The paradox of transmission efficiency Models of the dynamics of male killing agents predict that if transmission efficiency is perfect (i.e. a = 1, referring to equation 1.1., Chapter 1) then the symbiont can invade and will spread to fixation within the population. However, as the results presented here show, prevalence can be much lower than predicted with apparently perfect transmission efficiency. In the Fijian population of77. bolina from Viti Levu for example, the mtDNA diversity provides no evidence for imperfect transmission of the male killing element (i.e. there are no instances of the B-associated mitotype being found in uninfected individuals). However prevalence of the male killing element on Viti Levu, Fiji is found to be 55%, and not the predicted 100%. This phenomenon is also seen in A. encedon, where transmission efficiency has been observed to be perfect through breeding data as well as through mtDNA analysis (Jiggins, 2002). We are apparently missing some selective factor that is not understood, keeping infection from fixation, and this factor is frequency dependent. Further investigation is clearly required. Inherited parasites in the butterfly Hypolimnas bolina Chapter 5: Prevalence and history of Wolbachia infection in H. bolina 2 2 8 Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 2 9 Chapter 6: General Discussion Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 3 0 Chapter 6: General Discussion 6.1. Summary Of Results Historical evidence dating back to the late nineteenth century pointed towards the existence of a sex ratio distorting agent in the butt&rüy Hypolimnas bolina (Clarke et al, 1975; Clarke et al, 1983; Hopkins, 1927; Poulton, 1923; Poulton, 1927; Simmonds, 1926). However, although the existence of sex ratio distortion in a host species implicates a cytoplasmic factor there are many examples of host sex ratio distorting factors that have nothing to do with cytoplasmic elements, for example meiotic drivers or sex-linked lethal genes. In Drosophila simulans, for example, the expression of X-linked genes in the father may kill Y-bearing sperm resulting in a female-bias to the sex ratio (Atlan et a l, 1997; Cazemajor et al, 1997; Montchamp-moreau & Joly, 1997). It follows that although all-female broods and a female-biased adult sex ratio, may be consistent with the existence of a cytoplasmic causal agent it is not necessarily the case that the trait is caused by a cytoplasmic factor. In77. bolina, existence of all-female broods in populations from various countries, as documented by several researchers (Clarke et a l, 1975; Clarke et a l, 1983; Hopkins, 1927; Poulton, 1923; Poulton, 1927; Simmonds, 1926) strongly suggested the presence of a cytoplasmically inherited male killing agent. However Clarke et al, (1975) found no evidence suggesting the presence of microorganisms in infected lines. More stringent analysis of the host species was required in order to draw definitive conclusions: the initial aim of this thesis therefore was to determine the nature of the causative agent of sex ratio distortion in 77. bolina. Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 231 6.1.1. Identification of the causal agent and prevalence survey: Fiji Islands An initial field study in the Fiji Islands revealed the continued existence of sex ratio distortion inH. bolina. Laboratory analysis of infected specimens showed a male killing bacterium to be responsible for the observed all-female trait. It is unsurprising that Wolbachia is the causative agent of male killing in H. bolina as bacteria in the genus Wolbachia are the most widespread agents of sex ratio distortion, causing féminisation, male killing and parthenogenesis induction in a wide range of arthropod hosts ( Stouthamer et a l, 1999;Bourtzis & Braig, 2000). What was more surprising was the finding that both the ftsZ and wsp genes of the H. bolina male killing Wolbachia are identical in sequence to a Wolbachia male killing agent found in another butterfly species: Acraea encedon (Tanzania) (Jiggins et al, 1998). This result suggests that horizontal transfer of this Wolbachia has occurred at some point in the evolutionary history of these two host species. Antibiotics failed to cure either the male killing trait, or the presence of the Wolbachia bacterium in infected lines. Female H. bolina were collected from three different Fijian Islands. PCR assays of these specimens revealed the existence of significant prevalence variation in infection, with the Taveuni island population showing a prevalence of the male killing Wolbachia of approximately half of that recorded on the other two islands. Having established the existence of a cytoplasmically inherited male killing Wolbachia bacteria in the Fijian population oiH, bolina in the first year, the remainder of this work involved investigating the system in detail in an attempt to address previously unanswered questions concerning the effects of male killers in natural host populations. 6.1.2. Questions addressed using Ff. bolinalWolbachia symbiosis Many ‘facts’ concerning the spread and action of male killing symbionts have been derived from a highly diverse range of interactions; in many cases there are few reliable observations of closely related uninfected populations. Theoretical studies of the Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 3 2 population biology of male killers predicts that the prevalence of the male killing symbiont within the host population is dependent on both the indirect and direct advantages to the host resulting from infection. Although there are numerous examples of different species showing different levels of male killer prevalence, the underlying reasons for such variation are largely untested, as is the affect of a high prevalence male killer infection on the host population. Hypolimnas bolina presented the ideal system in which to examine such questions due to its island nature, and to the coexistence of uninfected and infected individuals in some populations. Clarke et al, suggested that the ‘all-female trait’ showed prevalence variation across different island populations. The initial fieldwork in Fiji revealed this to hold true across islands within one country. Populations of H. bolina were then examined in other island countries in order to initially ascertain if male killer prevalence does vary, and further to attempt to establish the underlying factors responsible for any observed prevalence variation. 6.1.3. High prevalence infection: Independent Samoa The populations of H. bolina on the islands of Independent Samoa provide the most extreme example of male killer prevalence recorded to date in a natural population. Over 99% of the females sampled were found to be infected with the same male killing Wolbachia identified from Fijian specimens. Incredibly, historical evidence suggests that this has been the situation for at least three hundred generations but the population still persists despite a paucity of males. PCR assays of//, bolina specimens collected from neighbouring American Samoa revealed no evidence of the male killing symbiont. The lack of males in Independent Samoa has led to a significant increase in the numbers of virgin females compared to that observed in the Fijian and American Samoan populations. No evidence was found suggesting the evolution of resistance to male killer action or transmission. However, Independent Samoan male H bolina were found to produce significantly smaller spermatophores, and females showed increased infertility Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 233 in comparison with the Fijian populations. Cross-mating of butterflies from the different countries revealed these findings to be an effect of Independent Samoan males, suggesting that the reduction in spermatophore size may have evolved through reduced competition due to the incredible female-bias in the natural populations. Throughout this project. Independent Samoa was the only study area in which severe population damage of this kind was recorded. 6.1.4. Evidence for a direct benefit to infection Experiments carried out in the Fiji islands demonstrated that H. bolina larvae do not cannibalise sibling or non-sibling eggs, or each other. Sibling egg cannibalism is held to be one of the indirect advantages to male killer infection, and is widely held to be an important factor in both spread and level of prevalence of a male killing symbiont within a host population (Hurst and Majerus, 1993). Another well-reported example of fitness compensation resulting from male killer infection is that of reduced competition. The butterfly A. encedon, for example, lays clutches of over one hundred eggs, and larvae are gregarious, one can easily see how the death of half the clutch would benefit the remaining infected larvae (Jiggins et al, 1998). In//, bolina, reduction of competition through male killing is theoretically less significant: clutch size is much smaller and larvae are not gregarious. This, together with the fact that//, bolina larvae do not cannibalise but the male killer can spread to high prevalence within certain populations, indicated that some other advantage to infection was an important factor in this system. Experiments to investigate this were carried out, primarily using larvae from the Fiji island populations (using Independent Samoan specimens was problematic due to the paucity of uninfected control lines). Both group-reared and more controlled paired single larvae rearing data revealed the same pattern: a significant increase in survivorship through to the adult stage in infected lines. Larvae within each experiment were reared under identical conditions, and experiments were carried out in two separate field Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 3 4 seasons in two separate countries with remarkably consistent results. As well as an increase in survivorship, infected H. bolina females from the paired experiments showed significantly increased dry weight on emergence compared to uninfected controls, indicating that the observed increase in survivorship does not trade off with reduced fecundity. The adult sex ratio of uninfected lines was approximately 1:1 in all cases showing that the difference in survivorship was not due to death of males in normal sex ratio broods. A similar pattern of results was obtained from the outbred F2 generation in the group-reared experiments showing that the survivorship difference was not due to the indirect benefit of reduced inbreeding depression. 6.1.5. Prevalence of male killer across the butterfly’s range Molecular analysis of H. bolina specimens from various different countries, revealed the presence or absence of the male killing Wolbachia and its prevalence within the particular host population. Certain populations were found to be infected with an A- group Wolbachia, although no butterflies were reared from these populations. The A- strain symbiont was found to be present in both males and females at fairly high prevalence and is closely related to a Cl-causing Wolbachia found in E. kuhniella (Sasaki & Ishikawa, 1999). No incidences of doubly-infected hosts were recorded. Genes from the mitochondria of individuals, both infected (with A or B Wolbachia) and uninfected, from each population were sequenced and mitotype frequencies compared. The A-infection was found to far predate the B-infection, and the lack of mitotype diversity associated with the male killing Wolbachia B-strain indicated a fairly recent selective sweep of this infection. These results reconfirm the perils of using mitochondrial DNA for phylogenetic analysis, as selfish genetic elements can confound interpretation of results (Johnstone & Hurst, 1996; Ballard, 2002). Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 235 6.2. Future Work This thesis represents a starting point in the study of the H. bolina/Wolbachia symbiosis. The original aim, to find the cause of the all-female broods documented in the early part of this century, was achieved in the first year of study. Since that time, study of this system has provided a wealth of information that will hopefully eventually enable certain fundamental questions about male killers to be addressed. This work has involved carrying out a preliminary investigation of this symbiosis. Using this thesis as a springboard, there are a number of directions which future research could take. The primary reasons whyH. bolina is such a useful tool for this and future work is its island nature, the co-existence of infected and uninfected individuals and the existence of the two different types of Wolbachia at different prevalences in different discreet populations. 6.2.1. Prevalence variation 6.2.1.1. Existence of prevalence variation Currently the prevalence of the male killing Wolbachia has been calculated from sixteen populations from eight different countries. Prevalence has been found to vary from 0% (American Samoa and Australia) to over 99% in Independent Samoa. However, in order to draw robust conclusions from these results, more samples need to be analysed from certain populations. For example, the figure of zero prevalence in Australia is based on PCR analysis of a single specimen. Despite the fact that Clarke et a/. (1975) found no evidence suggesting the presence of the male killing symbiont inH. bolina in Australia, more samples are required in order to conclude that the male killer is definitely absent from this population. More samples from French Polynesia, Thailand and Malaysia would also be useful. The populations that were sampled for the purposes of this thesis Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 3 6 represent only a small area of the vast range ofH. bolina. Surveying more populations from as many countries as possible, to which the butterfly is native, would provide more data points in a comparative analysis. Carrying out more thorough surveys of this kind would permit the researcher to address whether or not certain events are unique or general, for example: are male killer infected males only found in H. bolina in Borneo? Is extreme prevalence of the male killer only found in H. bolina from Independent Samoa? Is oviposition behaviour associated with prevalence? (In Australia, 0% prevalence associated with single-egg laying by females). 6.2.1.2. Explanation o f prevalence variation Male killer prevalence in H. bolina may vary because of variations between populations in one of four factors: oviposition behaviour, resistance evolution, alternative infections and benefits to infection. The suggestion that each is occurring in the H. bolinalWolbachia symbiosis has been presented in this thesis. However, in order for appropriate conclusions to be drawn, more stringent analysis is required: 1. Oviposition behaviour: H. bolina in Australia, where the male killer is apparently absent, lays eggs singly, or in pairs (Kemp, 1998). In Fiji and Independent Samoa, where the male killer is present, eggs are laid in clutches of around 10 (pers. obsns). As more different populations are sampled, oviposition behaviour could be recorded from each population to reveal whether or not a correlation between male killer prevalence and clutch size exists in this species. 2. Resistance: PCR analysis of different populations will suggest whether or not each host population has evolved resistance to the male killing element. If the existence of resistance is suggested, i.e. prevalence of the male killing Wolbachia is equal in both male and female hosts (as demonstrated in the Borneo population) then specimens could be cross-mated with butterflies from populations in which the host males are not infected with the male killing Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 3 7 symbiont. For example, crosses could be performed using Borneo males and females mated with Fijian males and females. If the Borneo population is resistant to the Wolbachia male killer, we would expect resistance to appear in infected Fijian females into which the Borneo nuclear genes have been introgressed; we would expect resistance to disappear in infected Borneo females multiply cross-mated to uninfected Fijian males. It would also be possible to transinfect the strain in the laboratory. However, the multiple cross-mating method would be less time consuming, and has the benefit Of providing information on the genetic architecture of resistance: the degree of dominance and the number and action of genes involved. 3. Alternative Infections: Results from American Samoa and Taveuni Island, Fiji, suggest that the presence of the A-group Wolbachia somehow prevents large- scale invasion of the male killing B-group Wolbachia in H. bolina. In order to work out whether or not this is the case, and also to establish whether, as suspected, the A-strain causes cytoplasmic incompatibility, a range of crosses could be carried out. Of the areas studied, Taveuni Island would probably be the best population to use for this study, as both the A- and B- strain Wolbachia bacteria were found within this population. Crosses involving all possible permutations of A-strain, B-strain and uninfected males and females from the Taveuni population should be performed. Examination of the egg hatch rate will reveal whether or not incompatibility is occurring and, if it is, in which direction i.e. whether or not the incompatibility is preventing invasion of the male killing parasite, keeping it at low prevalence within predominantly A-infected populations. 4. Benefits to infection: In this thesis, thorough investigation of two//, bolina populations on the Fiji islands revealed the existence of a direct benefit to Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 3 8 infected hosts through increased survivorship. Such a benefit was also suggested in the Independent Samoan population, although the sample size was small. The existence of a direct benefit to infection has huge implications on the invasion, spread and maintenance of a male killing parasite in a host population i.e. its prevalence. It is important to replicate these experiments in other populations of differing male killer prevalence, in order to see if the magnitude of this beneficial effect is variable. If variation is found, the genetic basis behind this could be examined via crossing, for example infecting Australian (supposedly 0% male killer prevalence) H. bolina with the male killing Wolbachia, then testing to see whether or not the benefit is the same as that observed in Fiji. This might be particularly interesting to look at in Borneo where infection is high, but the bacterium does not kill males. 6.2.2. Applications of prevalence variation in the study of mating systems The Wolbachia male killer is found at a variety of different prevalences within different H. bolina populations. This variation will generate variability in the population sex ratio, as has been demonstrated in this thesis. For example, in Independent Samoa where the population sex ratio is heavily female-biased and male killer prevalence is extremely high compared with the Fiji islands, where sex ratio distortion is much less significant, and male killer prevalence much lower. The existence of variation in sex ratio brought about by variable male killer prevalence makes ff. bolina an ideal model system in which to investigate the evolutionary biology of reproductive systems. One can imagine three scenarios, each of which is probably occurring in certain//. bolina populations: 1. Variable sperm competition: all females mated, some females multiply mated, e.g. Australia, and some Fijian specimens. Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 3 9 2. No competition but full matedness: most females have a single spermatophore, e.g. Fiji. 3. No competition but high virginity: many females unmated (no spermatophore in bursa copulatrix) e.g. Independent Samoa. Various male and female traits would be predicted to covary with these different degrees of sperm competition. 6.2.2.1. Spermatophore size, sperm number and sperm type This thesis has demonstrated variations in spermatophore size and, indirectly, sperm number, apparently reflecting reduced sperm competition 'mH. bolina in Independent Samoa due to the high prevalence of the male killing Wolbachia. However a more stringent analysis of this occurrence is required with more cross-matings between butterflies of different populations, as well as direct calculation of the average number of sperm present in a spermatophore from each of the study populations. We might also expect to see a change in allocation of sperm type, reflecting the level of sperm competition in the local population, as is seen in Pieris rapae (Wedell & Cook, 1999). The ratio of eupyrene (fertilising) sperm to apyrene (non-fertilising) sperm could be calculated for males from each population, both locally mated, and cross-mated to females from different populations. The prediction would be that males from populations with reduced levels of sperm competition i.e. those with a high prevalence of the male killing element, such as Independent Samoa, would have a lower percentage of non fertilising sperm. 6.2.2.2. Sex role-reversal Preliminary experiments carried out in Independent Samoa and the Fiji islands, indicated that the behaviour oîH. bolina adults varied according to the level of prevalence of the male killer in the host population. In Fiji, Australia (Kemp, 2000; Kemp, 2001; Kemp & Rutowski, 2001), American Samoa and Borneo (pers. obsn.) male Æ bolina are very Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 4 0 active and territorial, constantly searching for mates. However, the situation seems to be reversed in Independent Samoa: prevalence of the male killer is so high that hardly any males are sighted: the dramatically female-biased sex ratio appears to have caused sex role reversal. Again, this idea requires more stringent analysis. It is true that in the experiments presented in Chapter 3 (section 3.12.) males are being compared with females, rather than like with like. However, this more controlled experiment may be impossible to perform due to the paucity and low adult survivorship of male H. bolina from Independent Samoa. If sex role reversal is occurring in high prevalence H. bolina populations, such as those of Independent Samoa, and females are competing for males, then it follows that eventually male choice would evolve (Randerson et a l, 2000). This phenomenon has been observed in a number of different species, for example certain fish, mice and insects (Amundsen & Forsgren, 2001; Katvala & Kaitala, 2001; Ryan & Altmann, 2001). In such a situation, with males choosing females, females would be expected to be monomorphic in order to maximise their chance of reproductive success (Krebs & Davies, 1993b). Of all the H. bolina populations studied, it is interesting that only female H. bolina on Independent Samoa display a far more limited range of wing patterns (only two) compared to their highly polymorphic counterparts. Although this is an interesting anecdote suggesting that it is possible that female polymorphism has decreased in response to male choice, another explanation is that the population has lost much genetic diversity due to the lack of males caused by the high prevalence of the male killer. Hence, genes for the multitude of H. bolina morphs have been lost over successive generations. Notably Mathew’s (1888) reports from Independent Samoa indicate diversity of female morphs, similar to the polymorphism seen mH. bolina in Fiji today. Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 241 6.2.2.3. Life History Parameters Examining life history parameters, for example body size and age at maturity, of both male and female H. bolina from different populations would reveal whether or not these correlate with variation in prevalence of the Wolbachia male killer. We might expect Independent Samoan H. bolina females to contain fewer eggs at maturity; hence their body size is smaller. There is no point in a female from Independent Samoa producing a maximum number of eggs, because, even if she does mate, she will not mate more than once, and all of her eggs are unlikely to be fertilised (see Chapter 3). 6.2.2.4. Heritability Heritability of the above traits can be examined in the H. bolina system. This is potentially unique as H. bolina populations are both discrete and diverse. This system would permit unique observation of the effects of different parameters on the host population. For example, due to high male killer prevalence Independent Samoan i/. bolina males would be expected to have no ‘antiaphrodisiacs’ (defences against female remating, a term first introduced by Happ, 1969). In contrast, in other populations in which multiple mating is more common, such as H. bolina in Australia, males might have these defences, reflecting a different degree of sperm competition. 6.2.3. Effects on genetic diversity Developing microsatellites for H. bolina would enable examination of the genetic diversity of different Ff. bolina populations. The general diversity within different single populations could be calculated and compared. This would enable assessment of the degree to which island bottlenecks have affected the genetic diversity: r%ardless of infection status, island populations would be expected to show less diversity compared with mainland populations, associated with a lower founder number. If male killer prevalence is high in a particular island population, with no evidence for evolution of resistance (for example. Independent Samoa), we would further predict that genetic Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 4 2 diversity in this population would be reduced in comparison with other island populations. It would be interesting to compare diversity on a large island with an exceedingly high prevalence male killer with the genetic diversity of ai/, bolina population from a very small isolated island where male killer prevalence is not so extreme. Such a comparison would reveal whether island size or existence of a high prevalence male killing element has the more significant effect on genetic diversity. Microsatellite variation can also be used to create a phylogeny of/i. bolina, which would permit a formal comparative analysis of allü. bolina study populations. The phylogeny could be used to examine and estimate the history of colonisation of the different Ü. bolina island populations. Other nuclear genes might be required in order to obtain the most robust phylogeny. As demonstrated in Chapter 5, mitochondrial genes confound phylogenies when a selfish genetic element has spread through the host populations (Ballard, 2002; Jiggins, 2002). The analysis carried out using genes from the mitochondrial COI block, indicates thatü. bolina is paraphyletic. It would be beneficial to resolve this by carrying out molecular examination, and fieldwork on the closely-related species H. alimena and Ü. deios to see if Wolbachia is present in these species, and if it is, to examine its action. 6.2.4. The Last Word Whether the H. bolinalWolbachia symbiosis is a unique system or whether the populations investigated in this thesis represent a general pattern applicable to other male killing interactions can only be speculated. There is reason to believe that theü. bolina situation may be unique due to the huge range of the butterfly and its island nature. However, it is true that thorough studies of other male killing symbioses, for example those of Drosophila bifasciata (Hurst et a l, 2001) and Xho Acraea butterflies (Jiggins et al, 2003) have provided no evidence suggesting resistance to the male killing element has evolved, even under intense selection. In these cases, prevalence and therefore Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 4 3 selection pressure is not as high as has been recorded inH. bolina in Independent Samoa. Whether or not certain populations of H. bolina (particularly those of Independent Samoa and Borneo) represent unique examples of male killer/ host interactions in natural populations, it seems obvious that the diversity of male killer prevalence levels within different populations of this host species will rarely, if ever, be repeated in other systems. As such the H. bolinalWolbachia symbiosis is a worthy candidate for intense future research. This thesis has concentrated on a select few of the many island populations of this species and has reported a number of interesting and novel facts concerning male killer symbioses. Investigating morei/. bolina populations, and carrying out more stringent comparative analysis of the populations already studied in this thesis cannot fail to further our knowledge of the evolution, action and population genetics of male killing symbionts in natural host populations. I would venture to suggest that the data presented here is simply the tip of the proverbial iceberg. Inherited parasites in the butterfly Hypolimnas bolina Chapter 6: General Discussion 2 4 4 Inherited parasites in the butterfly Hypolimnas bolina References 2 4 5 References Inherited parasites in the butterfly Hypolimnas bolina References 2 4 6 References Agarwala, B. K. and Dixon, A. F. G. (1992). Laboratory study of cannibalism and interspecific predation in ladybirds. Ecological Entomology 17, 303-309. Agarwala, B. K. and Dixon, A. F. G. (1993). Kin recognition - egg and larval cannibalism in Adalia-bipunctata (Coleoptera, coccineWidac). European Journal of Entomology 90, 45-50. Altshul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., and Zhang, Z. (1997). Gapped BLAST and PSI-BLAST a new generation of protein database search programs. Nucleic Acids Research 3389-3402. Amundsen, T. and Forsgren, E. (2001). Male mate choice selects for female coloration in a fish. Proceedings of the National Academy of Sciences of the United States of America 98,13155-13160. Arakaki, N., Miyoshi, T., and Noda, H. (2001). Wb/bac/i/a-mediated parthenogenesis in the predatory thrips Fanklintothrips vespiformis (Thysanoptera : Insecta). Proceedings of the Royal Society of London Series B-Biological Sciences 268,1011-1016. Ashbumer,M. (1989). 'Drosophila, a laboratory manual.’ (University Press: Cold Spring Harbour). Atlan, A., Mercot, H., Landre, C., and Montchamp-Moreau, C. (1997). The sex-ratio trait in Drosophila simulans: Geographical distribution of distortion and resistance. Evolution 51,1886-1895. Avise, J. C., Arnold, J., Ball, R. M., Bermingham, E., Lamb, T., Neigel, J. E., Reeb, C. A., and Saunders, N. C. (1987). Intraspecific phylogeography- the mitochondrial-DNA bridge between population-genetics and systematics. Annual Review of Ecology and Systematics 18, 489-522. Ballard, J. W. O. (2000). When one is not enough: Introgression of mitochondrial DNA in Drosophila. Molecular Biology and Evolution 17,1126-1130. Ballard, J. W. O., Chernoff, B., and James, A. C. (2002). Divergence of mitochondrial DNA is not corroborated by nuclear DNA, morphology, or behavior in Drosophila simulans. Evolution 56,527-545. Ballard, J. W. O., Hatzidakis, J., Karr, T. L., and Kreitman, M. (1996). Reduced variation in Drosophila simulans mitochondrial DNA. Genetics 144,1519-1528. Bandi, C., Dunn, A. M., Hurst, G. D. D., and Rigaud, T. (2001). Inherited microorganisms, sex-specific virulence and reproductive parasitism. Trends in Parasitology 17, 88-94. Bandi, C., McCall, J. W., Genchi, C., Corona, S., Venco, L., and Sacchi, L. (1999). Effects of tetracycline on the filarial 'worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia. InternationalJournal for Parasitology 29, 357-364. Inherited parasites in the butterfly Hypolimnas bolina References 2 4 7 Baumann, P., Lai, C. Y., Baumann, L., Rouhbakhsh, D., Moran, N. A., and Clark, M. A. (1995). Mutualistic associations of aphids and prokaryotes - biology of the genus Buchnera. Applied and Environmental Microbiology 61,1-7. Bazzocchi, C., Jamnongluk, W., O'Neill, S. L., Anderson, T. J. C., Genchi, C., and Bandi, C. (2000). wsp gene sequences from the Wolbachia of filarial nematodes. Current Microbiology 41, 96-100. Beard, C. B., Hamm, D. M., and Collins, F. H. (1993). The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization and comparisons with mitochondrial sequences of other insects. Insect Molecular Biology 2,103-124. Behura, S. K., Sahu, S. C., Mohan, M., and Nair, S. (2001). Wolbachia in the Asian rice gall midge, Orseolia oryzae (Wood- Mason): correlation between host mitotypes and mitciioïi sXdXas. Insect Molecular Biology Bordenstein, S. R. and Werren, J. H. (1998). Effects of A and B Wolbachia and host genotype on interspecies cytoplasmic incompatibility mNasonia. Genetics 148,1833- 1844. Bordenstein, S. R. and Werren, J. H. (2000). Do Wolbachia influence fecundity in Nasonia vitripennisi Heredity 84, 54-62. Bouchon, D., Rigaud, T., and Juchault, P. (1998). Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization. Proceedings of the Royal Society of London Series B-Biological Sciences 265,1081- 1090. Bourtzis, K and Braig, H. R. (1999). The many faces of Wolbachia. In ‘Rickettsiae and Rickettsial diseases at the turn of the third millenium’. (Eds D. Raoult and P. Brouqui) pp. 199-219 (Elsevier Press, Paris). Bourtzis, K., Nirgianaki, A., Markakis, 0., and Savakis, C. (1996). Wolbachia infection and cytoplasmic incompatibility in Drosophila species. Genetics 144,1063-1073. Breeuwer, J. A. J. (1997). Wolbachia and cytoplasmic incompatibility in the spider mites Tetranychus urticae and T-turkestani. Heredity 79, 41-47. Breeuwer, J. A. J. and Jacobs, G. (1996). Wolbachia: Intracellular manipulators of mite reproduction. Experimental & Applied Acarology 20, 421-434. Brimacombe, L. C. (1980). All-female broods in field and laboratory populations of the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). Bulletin of Entomological Research 70,475-481. Brower, A. V. Z. (1994). Rapid morphological radiation and convergence among races of the butterfly Heliconius erato inferred from patterns of mitochondrial-DNA evolution. Proceedings of the National Academy of Sciences of the United States of America 91, 6491-6495. Inherited parasites in the butterfly Hypolimnas bolina References 2 4 8 Bmnton, C. F. A. and Hurst, G. D. D. (1998). Mitochondrial DNA phylogeny of brimstone butterflies (genus Gonepteryx) from the Canary Islands and Madeira. BiologicalJournal of the Linnean Society 63, 69-79. Buchner, P. (1965). 'Endosymbiosis of animals with plant microorganisms.' (Interscience: New York.) Bull, J. J. (1983). 'Evolution of sex determining mechanisms.' (Benjamin/ Cummings: Menlo Park, California.) Bulnheim, H. P. and Vavra, J. (1968). Infection by the microsporidian Octosporea effeminans. sp. N., and its sex determining influence in the amphipod Gammarus duebeni. Journal of Parasitology 54, 248. Burgdorfer, W. and Brinton, L. P. (1975). Mechanisms of transovarial infection of spotted fever rickettsiae in ticks. Annals of the New York Academy of Science 266, 61-72. Cabot, E. XESEE. The eyeball sequence editor DOS protected mode version of ESEE3. [3.1.]. 1997. New York, University of Rochester, Rochester. Casiraghi, M., Anderson, T. J. C., Bandi, C., Bazzocchi, C., and Genchi, C. (2001). A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology 122, 93-103. Cazemajor, M., Landre, C., and Montchamp-moreau, C. (1997). The sex-ratio trait in Drosophila simulans: Genetic analysis of distortion and suppression. Genetics 147, 635- 642. Chang, K. S., Shiraishi, T., Nakasuji, P., and Morimoto, N. (1991). Abnormal sex-ratio condition in the walnut leaf beetle, Gastrolina depressa (Coleoptera, Chrysomelidae). Applied Entomology and Zoology 26, 299-306. Charlat, S., Calmet, C., and Mercot, H. (2001). On the mod/resc model and the evolution of Wolbachia compatibility types. Genetics 159,1415-1422. Clarke, C., Johnston, G., and Johnston, B. (1983). All-female broods in Hypolimnas- bolina (L) - A re-survey of West Fiji after 60 y cdxs. BiologicalJournal of the Linnean Society 19, 221-235. Clarke, C. and Sheppard, P. M. (1975). The Genetics of the mimetic butterfly Hypolimnas bolina. Philosophical transactions o f the Royal Society o f London 272, 229- 265. Clarke, C. and Sheppard, P. M. (1977). Data suggesting absence of linkage between two loci in the mimetic butterfly Hypolimnas bolina (Nymphalidae). Jowr/iaZ o f the Lepidopterists’ Society. 31,139-143. Clarke, C., Sheppard, P. M., and Scali, V. (1975). All female broods in the butterfly Hypolimnas bolina. Proceedings of the Royal Society of London Series B-Biological Sciences 189, 29-37. Inherited parasites in the butterfly Hypolimnas bolina References 2 4 9 Cordaux, R., Michel-Salzat, A., and Bouchon, D. (2001). Wolbachia infection in crustaceans: novel hosts and potential routes for horizontal transmission. Journal of Evolutionary Biology 14, 237-243. Counce, S. J. and Poulson, D. F. (1962). Developmental effects of the sex-ratio agent in embryos of Drosophila willistoni. Journal of Experimental Zoology 151,17-31. Crow, J. F. and Kimura, M. (1988). ’An Introduction to Population Genetics.' (Harper and Row: New York, Evanston, London.) De Bary, A. (1879). "Die Erscheinung der Symbiose.' (Karl J. Trubner: Strassburg.) Dedeine, F., Vavre, F., Fleury, F., Loppin, B, Hochberg, M. E., and Bouletreau, M. (2001). Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp. Proceedings of the National Academy of Sciences of the United States of America 98, 6247-6252. Dhem, J. F. and Feyt, N. (2001). Hardware and software symbiosis helps smart card evolution. le Micro 21,14-25. Dobson, S. L., Marsland, E. J., and Rattanadechakul, W. (2002). Mutualistic Wolbachia Infection in Aedes albopictus: Accelerating cytoplasmic drive. Genetics 160,1087-1094. Dunn, A. M., Adams, J., and Smith, J. E. (1993). Transovarial transmission and sex ratio distortion by a microsporidian parasite in a shrimp. Journal of Invertebrate Pathology 61, 252. Dunn, A. M., Hatcher, M. J., Terry, R. S., and Tofts, C. (1995). Evolutionary ecology of vertically transmitted parasites: Transovarial transmission of a microsporidian sex ratio distorter in Gammarus duebeni. Parasitology 111, S91-S109. Dunn, A. M. and Rigaud, T. (1998). Horizontal transfer of parasitic sex ratio distorters between crustacean hosts. Parasitology 117,15-19. Dyson, E. A., Kamath, M. K., and Hurst, G. D. D. (2002). Wolbachia infection associated with all-female broods in Hypolimnas bolina (Lepidoptera: Nymphalidae). Evidence for horizontal transmission of a butterfly male kiWtx. Heredity 88,166-171. Ebbert, M. A. (1991). The interaction phenotype in Drosophila willistoni- Spiroplasma symbiosis. Evolution 45, 971-988. Ewald, P. W. (1987). Transmission modes and the evolution of the parasitism-mutualism contmuum. Annals of the New York Academy of Science 503, 295-306. Excoffier, L., Smouse, P., and Quattro, J. (1992). Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479-491. Fialho, R. F. and Stevens, L. (2000). Male-killing Wolbachia in a flour beetle. Proceedings of the Royal Society of London Series B-Biological Sciences 267,1469- 1473. Inherited parasites in the butterfly Hypolimnas bolina References 2 5 0 Fine, P. E. M. (1975). Vectors and vertical transmission: an epidemiologic perspective. Annals of the New York Academy of Science 266,173-194. Fisher, R. A. (1930). The genetical theory of natural selection.' (Oxford University Press: Oxford.) Frankish, H. (2002). Wolbachia bacteria have a key role in the pathogenesis of river blindness. Lancet 359, 856-856. Geier, P. W., Briese, D. T., and Lewis, T. (1978). The light-brown apple moth, Epiphyas postvittana (Walker) 2. Uneven sex ratios and a condition contributing to them in the field. Australian Journal of Ecology 3,467-488. Gillespie, R. G. (2002). Biogeography of spiders on remote oceanic islands of the Pacific: archipelagoes as stepping stones? Journal of Biogeography 29, 655-662. Ginsburger-Vogel, T. (1991). Intersexuality in Orchestia mediterranea Costa, 1853, and Orchestia aestuarensis Wildish, 1987 (Amphipoda) - a consequence of hybridisation or parasite infection? Journal of Crustacean Biology 11, 530-539. Giordano, R., O’Neill, S. L., and Robertson, H. M. (1995). Wolbachia infections and the expression of cytoplasmic incompativility m Drosophila-sechellia and D-mauritiana. Genetics 140,1307-1317. Girin, C. and Bouletreau, M. (1995). Microorganism-associated variation in host infestation efficiency in a parasitoid wasp, Trichogramma-bourarachae (Hymenoptera, Trichogrammatidae). 51, 398-401. Grant, P. R. (1998). Patterns on islands and microevolution. In Evolution on Islands.' (Ed P. R. Grant.) pp. 1-17. (Oxford University Press Inc.: New York.) Haldane, J.B.S. (1922). Sex ratio and unisexual sterility in hybrid animals. of Genetics 12,101-109. Hamilton, W. D. (1967). Extraordinary sex ratios. Science 156, 477-488. Happ, G. M. (1969). Multiple sex pheromones of the mealworm beetle Tenebrio molitor h. Nature 222, 180-181. Hariri, A. R., Werren, J. H., and Wilkinson, G. S. (1998). Distribution and reproductive effects of Wolbachia in stalk- eyed flies (Diptera : Diopsidae). Heredity 81, 254-260. Hassell, M. P., Lawton, J. H., and May, R. M. (1976). Patterns of dynamical behaviour in single-species populations. Journal of Animal Ecology 45, 471-486. Hatcher, M. J. and Dunn, A. M. (1995). Evolutionary consequences of cytoplasmically inherited feminizing factors. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 348,445-456. Hentschel, U. and Steinert, M. (2001). Symbiosis and pathogenesis: common themes, different outcomes. Trends in Microbiology 9,585-585. Inherited parasites in the butterfly Hypolimnas bolina References 251 Hiroki, M., Kato, Y., Kamito, T., and Miura, K. (2002). Feminization of genetic males by a symbiotic bacterium in a butterfly, Eurema hecabe (Lepidoptera : Pieridae). Naturwissenschaften 89,167-170. Hoffmann, A. A., Clancy, D., and Duncan, J. (1996). Naturally-occurring Wolbachia infection in Drosophila simulans that does not cause cytoplasmic incompatibility. Heredity 76,1-8. Hoffmann, A. A., Clancy, D. J., and Merton, E. (1994). Cytoplasmic incompatibility in Australian populations of Drosophila-melanogaster. Genetics 136, 993-999. Hoffmann, A. A. and Turelli, M. (1997). Cytoplasmic incompatibility in insects. In Influential Passengers.' (Eds S. L. O'Neill, A. A. Hoffmann, and J. H. Werren.) pp. 42- 80. (Oxford University Press: Oxford.) Hoffmann, A. A., Turelli, M., and Harshman, L. G. (1990). Factors affecting the distribution of cytoplasmic incompatibility in Drosophila-simulans. Genetics 126, 933- 948. Hopkins, 0 . H. E. (1927). Part III: Butterflies of Samoa and some neighbouring island groups. In Insects of Samoa and other Samoan terrestrial arthropoda.' pp. 25-33. (Oxford University Press: London.) Huger, A. M., Skinner, S. W., and Werren, J. H. (1985). Bacterial-infections associated with the son-killer trait in the parasitoid wasp Nasonia-(= Mormoniella)-vitripennis (Hymenoptera, Pteromalidae). Journal of Invertebrate Pathology 46, 272-280. Hughes, L., Chang, B. S. W., Wagner, D., and Pierce, N. E. (2000). Effects of mating history on ejaculate size, fecundity, longevity, and copulation duration in the ant-tended lycaenid butterfly, evagoras. Behavioral Ecology and Sociobiology 47,119- 128. Huigens, M. E., Luck, R. F., Klaassen, R. H. G., Maas, M. F. P. M., Timmermans, M. J. T. N., and Stouthamer, R. (2000). Infectious parthenogenesis. Aari/rc 405,178-179. Hurst, G. D. D., Bandi, C., Sacchi, L., Cochrane, A. G., Bertrand, D., Karaca, I., and Majerus, M. E. N. (1999b). Adonia variegata (Coleoptera: Coccinellidae) bears maternally inherited Flavobacteria that kill males ovXy.Parasitology 118,125-134. Hurst, G. D. D., Hammarton, T. C., Bandi, C., Majerus, T. M. O., Bertrand, D., and Majerus, M. E. N. (1997b). The diversity of inherited parasites of insects: the male- killing agent of the ladybird beetle Coleomegilla maculata is a member of the Flavobacteria. Genetical Research 70,1-6. Hurst, G. D. D., Hammarton, T. C., Obrycki, J. J., Majems, T. M. O., Walker, L. E., Bertrand, D., and Majerus, M. E. N. (1996b). Male-killing bacterium in a fifth ladybird beetle, Coleomegilla maculata (Coleoptera: Coccinellidae). Heredity 77,177-185. Hurst, G. D. D., Hurst, L. D., and Majerus, M. E. N. (1997a). Cytoplasmic sex-ratio distorters. In Influential Passengers.' (Eds S. L. ONeill, A. A. Hoffmann, and J. H. Werren.) pp. 125-154. (Oxford University Press: New York). Inherited parasites in the butterfly Hypolimnas bolina References 2 5 2 Hurst, G. D. D. and Jiggins, F. M. (2000). Male-killing bacteria in insects: mechanisms, incidence and implications. Emerging infectious diseases 6, 329-336. Hurst, G. D. D., Jiggins, F. M., Schulenburg, J. H. G. v. d., Bertrand, D., West, S. A., Goriacheva, 1.1., Zakharov, I. A., Werren, J. H., Stouthamer, R., and Majerus, M. E. N. (1999a). Male-killing Wolbachia in two species of insect. Proceedings of the Royal Society of London Series B-Biological Sciences 266, 735-740. Hurst, G. D. D., Johnson, A. P., Schulenburg, J. H. G. v. d., and Fuyama, Y. (2000). Male-killing Wolbachia in Drosophila: a temperature sensitive trait with a threshold bacterial density. Genetics 156, 699-709. Hurst, G. D. D. and Majerus, M. E. N. (1993). Why do maternally inherited microorganisms kill males? Heredity 71, 81-95. Hurst, G. D. D., Majerus, M. E. N., and Walker, L. E. (1992). Cytoplasmic male killing elements in Adalia-bipunctata (Linnaeus) (Coleoptera, coccinellidae).69, 84- 91. Hurst, G. D. D., Majerus, M. E. N., and Walker, L. E. (1993). The importance of cytoplasmic male killing elements in natural- populations of the 2 spot ladybird, bipunctata (Linnaeus) (Coleoptera, cocc\ntWid 2i€).BiologicalJournal oftheLinnean Society 49,195-202. Hurst, G. D. D., Purvis, E. L., Sloggett, J. J., and Majerus, M. E. N. (1994). The effect of infection with male-killing rickettsia on the demography of iemdXe Adalia-bipunctata 1. (2-Spot ladybird). Heredity 73, 309-316. Hurst, G. D. D., Sloggett, J. J., and Majerus, M. E. N. (1996a). Estimation of the rate of inbreeding in a natural population of Adalia bipunctata (Coleoptera: Coccinellidae) using a phenotypic indicator. European Journal of Entomology 93,145-150. Hurst, G. D. D., Schulenburg, J. H. G. v. d., Majerus, T. M. O., Bertrand, D., Zakharov, I. A., Baungaard, J., Volkl, W., Stouthamer, R., and Majerus, M. E. N. (1999c). Invasion of one insect species, Adalia bipunctata, by two different male-killing bacteria. Insect Molecular Biology 8,133-139. Hurst, L. D. (1991). The incidences and evolution of cytoplasmic male killers. Proceedings of the Royal Society of London Series B-Biological Sciences 244, 91-99. Hurst, L. D. (1993). The incidences, mechanisms and evolution of cytoplasmic sex-ratio distorters in animals. Biological Reviews of the Cambridge Philosophical Society 68, 121-194. Ikeda, H. (1970). The cytoplasmically inherited 'sex-ratio' condition in natural and experimental populations of Drosophila bifasciata. Genetics 311-333. Jiggins, F. M. (2002). Male-killing Wolbachia and mitochondrial DNA: selective sweeps, hybrid introgression and the maintenance of diversity in uninfected individuals. Genetics. In Press. Inherited parasites in the butterfly Hypolimnas bolina References 2 5 3 Jiggins, F. M., Bentley, J. K., Majerus, M. E. N., and Hurst, G. D. D. How many species are infected with Wolbachia"! Cryptic sex ratio distorters revealed to be common by intensive sampling (2001c). Proceedings of the Royal Society of London Series B- Biological Sciences 267,1123-1126. Jiggins, F. M., Bentley, J. K., Majerus, M. E. N., and Hurst, G. D. D. (2002). Recent changes in phenotype and patterns of host specialization in Wolbachia bacteria. Molecular Ecology 11,1275-1283. Jiggins, F. M., Hurst-, G. D. D., Dolman, C. E., and Majerus, M. E. N. (2000a). High prevalence male-killing Wolbachia in the butterfly Acraea encedana. Journal of Evolutionary Biology 13, 495-501. Jiggins, F. M., Hurst, G. D. D., Jiggins, C. D., Schulenburg, J. H. G. v. d., and Majerus, M. E. N. (2000b). The ecology of male-killer hosts: the huWtrüy Danaus chrysippus vs infected by a male-killing Spiroplasma bacterium. Parasitology 120, 439-446. Jiggins, F. M., Hurst, G. D. D., and Majerus, M. E. N. (2000c). Sex-ratio-distorting Wolbachia causes sex-role reversal in its butterfly host Proceedings of the Royal Society of London Series B-Biological Sciences 267, 69-73. Jiggins, F. M., Hurst, G. D. D., Majems, M. E. N., Jiggins, F. M., Hurst, G. D. D., and Majems, M. E. N. (1998). Sex ratio distortion mAcraea encedon (Lepidoptera: Nymphalidae) is caused by a male-killing bacterium. Heredity 81, 87-91. Jiggins, F. M., Hurst, G. D. D., Schulenburg, J. H. G. v. d., and Majems, M. E. N. (2001b). Two male-killing Wolbachia strains coexist in a population of the butterfly Acraea encedon. Heredity 86,161-166. Jiggins, F. M., Hurst, G. D. D., and Yang, Z. (2002). Host-symbiont conflicts: positive selection on outer membrane proteins of parasitic but not mutualistic rickettsiaceae. Molecular Biology and Evolution 19,1341-1349. Jiggins, F.M., Randerson, J.P., Hurst, G. D. D., Majems, M.E.N. (2003). How can sex ratio distorters reach extreme prevalence? A natural population in which male killing Wolbachia have near perfect vertical transmission efficiency and are not suppressed. Evolution. In Press. Jiggins, F. M., Schulenburg, J. H. G. v. d., Hurst, G. D. D., and Majems, M. E. N. (2001a). Recombination confounds interpretations of Wolbachia evolution. Proceedings of the Royal Society of London Series B-Biological Sciences 268,1423-1427. Johnstone, R. A. and Hurst, G. D. D. (1996). Maternally inherited male-killing microorganisms may confound interpretation of mitochondrial DNA variability. BiologicalJournal oftheLinnean Society 58, 453-470. Juchault, P., Rigaud, T., and Mocquard, J. P. (1993). Evolution of sex determination and sex-ratio variability in wild populations of Armadillidium-vulgare (Latr) (Cmstacea, Isopoda) - a case-study in conflict-resolution. Acta Oecologica-International Journal of Ecology 14,547-562. Inherited parasites in the butterfly Hypolimnas bolina References 2 5 4 Kageyama, D., Nishimura, G., Hoshizaki, S., and Ishikawa, Y. (2002). Feminizing Wolbachia in an insect, Ostrinia furnacalis (Lepidoptera : Crambidae). Heredity 88, 444- 449. Katvala, M. and Kaitala, A. (2001). Male choice for current female fecundity in a polyandrous egg- carrying hug. Animal Behaviour 62,133-137. Kellen, W. R., Chapman, H. C., Clark, T. B., and Lindegren, J. E. (1965). Host-parasite relationships of some Thelohania from mosquitoes (Nosematidae: Microsporidia). Journal of Invertebrate Pathology 7,161-166. Kemp, D. J. (1998). Oviposition behaviour of post-diapause Hypolimnas bolina (L.) (Lepidoptera: Nymphalidae) in tropical Australia. Australian Journal of Zoology 46, 451-459. Kemp, D. J. (2000). Contest behavior in territorial male butterflies: does size matter? Behavioral Ecology 11, 591-596. Kemp, D. J. (2001). Age-related site fidelity in the territorial huXXerlly Hypolimnas bolina (L.) (Lepidoptera : 'Hym^hdXiddit). Australian Journal of Entomology 40, 65-68. Kemp, D. J. (2002). ‘Territoriality and male contest behaviour in the butterfly Hypolimnas bolina’ (Ph.D. Thesis, James Cook University, Queensland, Australia) Kemp, D. J. and Rutowski, R. L. (2001). Spatial and temporal patterns of territorial mate locating behaviour in Hypolimnas bolina (L.) (Lepidoptera : Nymphalidae). of Natural History 35,1399-1411. Kimura, M. (1983). The neutral theory of molecular evolution.' (Cambridge University Press: Cambridge.) Krabill, R. (2001). Symbiosis: mass media and the Truth and Reconciliation Commission of South Africa. Media Culture & Society 23, 567-571. Krebs, J. R. and Davies, N. B. (1993a). Predators versus prey: evolutionary arms races. In 'An Introduction to Behavioural Ecology.' (Eds J. R. Krebs and N. B. Davies.) pp. 77- 101. (Blackwell Science Ltd.: Oxford.) Krebs, J. R. and Davies, N. B. (1993b). Sexual conflict and sexual selection. In 'An introduction to behavioural ecology.' (Eds J. R. Krebs and N. B. Davies.) pp. 175-207. (Blackwell Science Ltd.: Oxford.) Langworthy, N. G., Renz, A., Mackenstedt, U., Henkle-Duhrsen, K., Bronsvoort, M. B. D., Tanya, V. N., Donnelly, M. J., and Trees, A. J. (2000). Macrofilaricidal activity of tetracycline against the filarial nematode Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent xeXuiionshv^. Proceedings o f the Royal Society of London Series B-Biological Sciences 267,1063-1069. Lawson, E. T., Mousseau, T. A., Klaper, R., Hunter, M. D., and Werren, J. H. (2001). Rickettsia associated with male-killing in a buprestid beetle. Heredity 86, 497-505. Inherited parasites in the butterfly Hypolimnas bolina References 2 5 5 Lipsitch, M., Nowak, M. A., Ebert, D., and May, R. M. (1995). The population-dynamics of vertically and horizontally transmitted ^dXdiSiXts. Proceedings of the Royal Society of London Series B-Biological Sciences 260, 321-327. Longstaff, G. B. (1912). Butterfly hunting in many lands: Notes of a field naturalist.' (Longmans and Co.) Lundrigan, B. L, Jansa, S. A., and Tucker, P. K. (2002). Phylogenetic relationships in the genus Mus, based on paternally, maternally, and biparentally inherited characters. Systematic Biology 51, 410-431. Lyons, D. B. (1996). Oviposition and fecundity of pine false webworm (Hymenoptera: Pamphiliidae). Canadian Entomologist 128, 779-790. Lyttle, T. W. (1977). Experimental population genetics of meiotic drive systems L Pseudo-T chromosomal drive as a means of eliminating cage populations oiDrosophila melanogaster. Genetics 86, 413-455. Majerus, T. M. O., Majerus, M. E. N., Knowles, B., Wheeler, J., Bertrand, D., Kuznetzov, V. N., Ueno, H., and Hurst, G. D. D. (1998). Extreme variation in the prevalence of inherited male-killing microorganisms between three populations of Harmonia axyridis (Coleoptera: Coccinellidae). Heredity 81, 683-691. Majerus, T. M. O., Schulenburg, J. H. G. v.d., Majerus, M. E. N., and Hurst, G. D. D. (1999). Molecular identification of a male-killing agent in the ladybird/farmow/a axyridis (Pallas) (Coleoptera: Coccinellidae). Insect Molecular Biology 8, 551-555. Malogolowkin, C. and Rodriguez-Pereira M.A.Q (1975). Sexual drive of normal and SR flies oi Drosophila nebulosa. Evolution 29,579-580. Marcade, I., Souty-Grosset, C., Bouchon, D., Rigaud, T., and Raimond, R. (1999). Mitochondrial DNA variability and Wolbachia infection in two sibling woodlice species. Heredity 83, 71-78. Mathew, G. F. (1888). Life-histories of the Rhopalocera from the Australian region. Transactions of the Entomological Society of London 137-188. Matsuka, M., Hashi, H., and Okada, I. (1975). Abnormal sex-ratio found in the lady beetle, Harmonia axyridis Pallas (Coleoptera: Coccinellidae). Applied Entomology and Zoology 10, 84-89. McCall, J. W., Jun, J. J. and Bandi, C. (1999). Wolbachia and the antifilarial properties of tetracycline, an untold sioiy .Italian Journal of Zoology 66, 7-10. Montchamp-moreau, C., Ferveur, J. F., and Jacques, M. (1991). Geographic-distribution and inheritance of 3 cytoplasmic incompatibility typQ S in Drosophila-simulans. Genetics 129, 399-407. Montchamp-Moreau, C. and Joly, D. (1997). Abnormal spermiogenesis is associated with the X-linked sex- ratio trait in Drosophila simulans. Heredity 79, 24-30. Inherited parasites in the butterfly Hypolimnas bolina References 2 5 6 Moran, N. A. and Telang, A. (1998). Bacteriocyte-asscciated symbionts of insects - A variety of insect groups harbor ancient prokaryotic tnàosyvabionXs.Bioscience 48, 295- 304. Morimoto, S., Nakai, M., Ono, A., and Kunimi, Y. (2001). Late male-killing phenomenon found in a Japanese population of the oriental tea tortrix, Homona magnanima (Lepidoptera : T o rtric id a e ).87,435-440. O'Neill, S. L., Hoffmann, A. A., and Werren, J. H. (1997). Influential Passengers.' (Oxford University Press: New York.) O'Neill, S. L. and Werren, J. H. (1997). The evolution of heritable symbionts. In 'Influential Passengers.' (Eds S. L. O'Neill, A. A. Hoffmann, and J. H. Werren.) pp. 1-41. (Oxford University Press: New York.) Osawa, N. (1992). Sibling cannibalism in the ladybird h ttû t Harmonia-axyridis- fitness consequences for mother and offspring. Researches on Population Ecology 34, 45-55. Page, R. D. M. (1996). TreeView: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357-358. Poinsot, D. and Mercot, H. (1997). Wolbachia infection in Drosophila simulans: Does the female host bear a physiological costal Evolution 51,180-186. Posada, D. and Crandall, K. A. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817-818. Poulton, E. B. (1923). All female families oi Hypolimnas bolina , bred in Fiji by H.W. Simmonds. Proceedings of the Royal Entomological Society of London 9-12. Poulton, E. B. (1927). Sex-ratio oi Hypolimnas bolina, L., in Viti Le vu, Fiji. Proceedings of the Entomological Society of London 2, 5-10. Poulton, E. B. (1928). Mr H.W. Simmonds' conclusion that all-female producing females form a persistant strain in Suva. Proceedings of the Royal Society of London. 3, 43-44. Randerson, J. P. and Hurst, L. D. (1999). Small sperm, uniparental inheritance and selfish cytoplasmic elements: a comparison of two moéoXs. Journal o f Evolutionary Biology 12,1110-1124. Randerson, J. P., Jiggins, F. M., and Hurst, L. D. (2000). Male killing can select for male mate choice: a novel solution to the paradox of the lek. Proceedings of the Royal Society of London Series B-Biological Sciences 267, 867-874. Reznick, D., Baxter, R. J., and Endler, J. (1994). Long-term studies of tropical stream fish communities - the use of field notes and museum collections to reconstruct communities of the past. American Zoologist 34,452-462. Rigaud, T. (1997). Inherited microorganisms and sex determination of arthropod hosts. In Influential Passengers.' (Eds S. L. O'Neill, A. A. Hoffmann, and J. H. Werren.) pp. 81-101. (Oxford University Press: New York.) Inherited parasites in the butterfly Hypolimnas bolina References 2 5 7 Rigaud, T., Bouchon, D., Souty-Grosset, C., and Raimond, P. (1999). Mitochondrial DNA polymorphism, sex ratio distorters and population genetics in the isopod Armadillidium vulgare. Genetics 152,1669-1677. Rigaud, T. and Juchault, P. (1993). Conflict between feminizing sex-ratio distorters and an autosomal masculinizing gene in the terrestrial isopod Armadillidium-vulgare Latr. Genetics 133, 247-252. Roehrdanz, R. L., Degrugillier, M. E., and Black, W. C. (2002). Novel rearrangements of arthropod mitochondrial DNA detected with Long-PCR: Applications to arthropod phytogeny and owoiuXion. Molecular Biology and Evolution 19, 841-849. Rosenbaum, J. R. and Sepkowitz, K. A. (2002). Infectious disease experimentation involving human volunteers. Clinical Infectious Diseases 34, 963-971. Rothbarth, F. (2001). Perfect optical symbiosis - Perspectives on the paint finish of automobile external parts made of plastic. Kunststoffe-Plast Europe 91, 94-99. Rothman, L. D. and Myers, J. H. (1996). Debilitating effects of viral diseases on host Lepidoptera. JowrAza/of Invertebrate Pathology 67,1-10. Rousset, P., Bouchon, D., Pintureau, B., Juchault, P., and Solignac, M. (1992). Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods. Proceedings of the Royal Society of London Series B-Biological Sciences 250, 91-98. Rousset, F. and Solignac, M. (1995). Evolution of single and double Wolbachia symbioses during spéciation in Xhe Drosophila-simulans complex. Proceedings of the National Academy of Sciences of the United States of America 92, 6389-6393. Royama, T. O. (2001). Measurement, analysis, and interpretation of mortality factors in insect survivorship studies, with reference to the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera : Tortricidae). Population Ecology 43,157-178. Ryan, K. K. and Altmann, J. (2001). Selection for male choice based primarily on mate compatibility in the oldfield mouse, Peromyscus polionotus rhoadsi. Behavioral Ecology and Sociobiology 50, 436-440. Sabater, B., Van Ham, R. C. H. J., Martinez-Torres, D., Silva, P., Latorre, A., and Moya, A. (2001). Molecular evolution of aphids and their primary (Buchnera sp.) and secondary endosymbionts: Implications for the role of symbiosis in insect evolution. Interciencia 26, 508-516. Saint-Andre, A. V., Blackwell, N. M., Hall, L. R., Hoerauf, A., Brattig, N. W., Volkmann, L., Taylor, M. J., Ford, L., Hise, A. G., Lass, J. H., Diaconu, E., and Pearlman, E. (2002). The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science 295,1892-1895. Sakaguchi, B. and Poulson, D. F. (1963). Interspecific transfer of the 'sex ratio' condition from Drosophila willistoni to D. melanogaster. Genetics 48, 841-861. Inherited parasites in the butterfly Hypolimnas bolina References 258 Sasaki, T. and Ishikawa, H. (1999). Wolbachia infections and cytoplasmic incompatibility in the almond moth and the mediterranean flour moth. Zoological Science 16, 739-744. Schneider, S., Kueffer, J. M., Roessli, D., and Excoffier, L. ARLEQUIN, version 1.1: a software for population genetic data analysis. 1997. University of Geneva, Switzerland, Genetics and Biometry Laboratory. Schulenburg, J. H. G.v.d., Habig, M., Sloggett, J. J., Webberley, K. M., Bertrand, D., Hurst, G. D. D., and Majerus, M. E. N. (2001). Incidence of male-killing spp. (alpha- proteobacteria) in the ten-spot ladybird Adalia decempunctata L. (Coleoptera : Coccinellidae). Applied and Environmental Microbiology 67, 270-277. Schulenburg, J. H. G. v. d., Hurst, G. D. D., Huigens, T. M. E., van Meer, M. M. M., Jiggins, F. M., and Majerus, M. E. N. (2000). Molecular evolution and phylogenetic utility of Wolbachia ftsZ and wsp gene sequences with special reference to the origin of male-killing. Molecular Biology and Evolution 17,584-600. Schulenburg, J. H. G. v. d., Hurst, G. D. D., Tetzlaff, D., Booth, G. E., Zakharov, I. A., and Majerus, M. E. N. (2002). History of infection with different male-killing bacteria in the two-spot ladybird hetile Adalia bipunctata revealed through mitochondrial DNA sequence analysis. Genetics 160,1075-1086. Schutt, C. and Nothiger, R. (2000). Structure, function and evolution of sex-determining systems in Dipteran insects. Development 127, 667-677. Simmonds, H. W. (1926). Sex ratio oi Hypolimnas bolina in Viti Levu, Fiji. Proceedings of the Royal Entomological Society of London 1, 29-32. Simon, C., Frati, F., Beckenbach, A., Crespi, B. J., Liu, H., and Flook, P. (1994). Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. o f the Entomological Society of America 87, 651-701. Sinkins, S. P., Braig, H. R., and O’Neill, S. L. (1995). Wolbachia superinfections and the expression of cytoplasmic incompatibility. Proceedings of the Royal Society of London Series B-Biological Sciences 261, 325-330. Skinner, S. W. (1985). Son-killer: a third extrachromosomal factor affecting sex ratios in the parasitoid v/aspNasonia vitripennis. Genetics 109, 745-754. Smith, D. C. (2001). Symbiosis research at the end of the millenium. Hydrobiologia 461, 49-54. Smith, J. E. and Dunn, A. M. (1991). Transovarial transmission. Parasitology Today 7, 146-148. Stolk, C. and Stouthamer, R. (1996). Influence of a cytoplasmic incompatibility-inducing Wolbachia on the fitness of the parasioid vjaspNasonia vitripennis. Proceedings of the Royal Society of London Series B-Biological Sciences 7, 33-37. Inherited parasites in the butterfly Hypolimnas bolina References 2 5 9 Stouthamer, R. (1997). Wolbachia-méuctà. parthenogenesis. In Influential Passengers.' (Eds S. L. O'Neill, A. A. Hoffmann, and J. H. Werren.) pp. 102-124. (Oxford University Press: New York.) Stouthamer, R., Breeuwer, J. A. J., and Hurst, G. D. D. (1999). Wolbachia pipientis: Microbial manipulator of arthropod reproduction. Annual Review of Microbiology 53, 71-102. Stouthamer, R., Hurst, G. D. D., and Breeuwer, J. A. J. (2000). Sex ratio distorters. In The sex ratio handbook.' (Ed I. C. W. Hardy.) (Cambridge University Press) Stouthamer, R. and Kazmer, D. J. (1994). Cytogenetics of microbe-associated parthenogenesis and its consequences for gene flow in Trichogramma wasps. Heredity 73, 317-327. Stouthamer, R., Luck, R. P., and Hamilton, W. D. (1990). Antibiotics cause parthenogenetic Trichogramma (Hymenoptera/Trichogrammatidae) to revert to sex. Proceedings of the National Academy of Sciences of the United States of America 87, 2424-2427. Stouthamer, R. and Luck, R. F. (1993). Influence of microbe-associated parthenogenesis on the fecundity of Trichogramma-deion and T-pretiosum. Entomologia Experimentalis etApplicata 67,183-192. Streelman, J. T., Alfaro, M., Westneat, M. W., Bellwood, D. R., and Karl, S. A. (2002). Evolutionary history of the parrotfishes: Biogeography, ecomorphology, and comparative diversity. Evo/wrio/z 56, 961-971. Swofford, D. L. (1993). PAUP - A computer-program for phylogenetic inference using maximum parsimony. Journal of General Physiology 102, A9-A9. Tamas, I., Klasson, L. M., Sandstrom, J. P., and Andersson, S. G. E. (2001). Mutualists and parasites: how to paint yourself into a (metabolic) corner. Febs Letters 498,135-139. Tamura, K. and Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial-DNA in humans and chimpanzees. Molecular Biology and Evolution 10, 512-526. Taylor, D. R. (1990). Evolutionary consequences of cytoplasmic sex ratio distorters. Evolutionary Ecology 4, 235-248. Terry, R. S., Smith, J. E., Bouchon, D., Rigaud, T., Duncanson, P., Sharpe, R. G., and Dunn, A. M. (1999). Ultrastructural characterisation and molecular taxonomic identification oiNosema granulosis n. sp., a transovarially transmitted feminising ( LI P) microsporidium. Journal of Eukaryotic Microbiology 46,492-499. Thomas, C. D. and Hanski, I. A. (1997). Butterfly metapopulations. In 'Metapopulation biology: ecology, genetics and evolution.' (Eds I. A. Hanski and M. E. Gilpin.) pp. 359- 386. (Academic Press: London.) Thomas, C. D., Jordano, D., Lewis, O. T., Hill, J. K., Sutcliffe, O. L., and Thomas, J. A. (1998). Butterfly distributional patterns, processes and conservation. In 'Conservation in Inherited parasites in the butterfly Hypolimnas bolina References 2 6 0 a changing world.' (Eds G. M. Mace, A. Balmford, and J. R. Ginsberg.) pp. 107-138. (Cambridge University Press: Cambridge.) Tram, U. and Sullivan, W. (2002). Rote of delayed nuclear envelope breakdown and mitosis in Wolbachia-mdnc&d cytoplasmic incompatibility. 5c/e«ce 296,1124-1126. Turelli, M. (1994). Evolution of incompatibility-inducing microbes and their hosts. Evolution 48,1500-1513. Turelli, M. and Hoffmann, A. A. (1995). Cytoplasmic incompatibility m Drosophila- simulans - dynamics and parameter estimates from natural-populations. Genetics 140, 1319-1338. Vane-Wright, R. I., Ackery, P. R., and Smiles, R. L. (1977). The polymorphism, mimicry and host plant relationships of Hypolimnas butterflies. BiologicalJournal of the Linnaean Society 9, 285-297. Wade, M. J. and Chang, N. W. (1995). Increased male-fertility in Tribolium-confusum beetles after infection with the intracellular parasite Wolbachia. Nature 373, 72-74. Wahlberg, N. and Zimmermann, M. (2000). Pattern of phylogenetic relationships among members of the tribe Melitaeini (Lepidoptera : Nymphalidae) inferred from mitochondrial DNA sequences. Cladistics-the International Journal o f the Willi Hennig Society 16, 347-363. Watson, R. A. and Pollack, J. B. (1999). How symbiosis can guide evolution. in Artificial Life, Proceedings 1674, 29-38. Wedell, N. (2001). Female remating in butterflies: interaction between female genotype and nonfertile sperm. Journal of Evolutionary Biology 14, 746-754. Wedell, N. and Cook, P. A. (1999). Strategic sperm allocation in the Small White butterfly rapae (Lepidoptera: Pieridae). Functional Ecology 13, 85-93. Weeks, A. R. and Breeuwer, J. A. J. (2001). Wolbachia-mdwotd parthenogenesis in a genus of phytophagous mites. Proceedings of the Royal Society of London Series B- Biological Sciences 268, 2245-2251. Weir, B. S. and Cockerham, C. C. (1984). Estimating F-statistics for the analysis of population structure. Evolution 38,1358-1370. Wernegreen, J. J. and Moran, N. A. (2001). Vertical transmission of biosynthetic plasmids in aphid endosymbionts (Buchnera). Journal o f Bacteriology 183, 785-790. Werren, J. H. (1987). The coevolution of autosomal and cytoplasmic sex-ratio factors. Journal of Theoretical Biology 124, 317-334. Werren, J. H. (1997). Biology of Wolbachia. Annual Review of Entomology 42,587-609. Werren, J. H. and Beukeboom, L. W. (1998). Sex determination, sex ratios, and genetic conflict. Annual Review of Ecology and Systematics 29, 233-261. Inherited parasites in the butterfly Hypolimnas bolina References 261 Werren, J. H. and O'Neill, S. L. (1997). The evolution of heritable symbionts. In Influential Passengers.' (Eds S. L. O'Neill, A. A. Hoffmann, and J. H. Werren.) pp. 1-41. (Oxford University Press: Oxford.) Werren, J. H., Skinner, S. W., and Huger, A. M. (1986). Male-killing bacteria in a parasitic wasp. Science 231, 990-992. Werren, J. H., Zhang, W., and Guo, L. R. (1995). Evolution and phytogeny ofWolbachia - reproductive parasites of arthropods. Proceedings of the Royal Society of London Series B-Biological Sciences 261, 55-63. Whistler, W. A. (1995). Wayside plants of the islands: A guide to the lowland flora of the Pacific Islands.' (Isle Botanica: Honolulu, Hawaii.) Williamson, D. L. and Poulson, D. F. (1979). Sex ratio organisms (Spiroplasmas) of Drosophila. In The My coplasmas.' (Eds R. F. Whitcomb and J. G. Tully.) pp. 175-208. (Academic Press: New York.) Zchori-Fein, E., Gottlieb, Y., Kelly, S. E., Brown, J. K., Wilson, J. M., Karr, T. L., and Hunter, M. S. (2001). A newly discovered bacterium associated with parthenogenesis and a change in host selection behavior in parasitoid wasps. Proceedings of the National Academy of Sciences of the United States of America 98,12555-12560. Zchori-Fein, E., Rousch, R. T., and Hunter, M. S. (1992). Male production induced by antibiotic treatment in Encarsia formosa. Experientia 48,102-105. Zhou, W. G., Rousset, F., and ONeill, S. (1998). Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proceedings of the Royal Society of London Series B-Biological Sciences 265, 509-515. Inherited parasites in the butterfly Hypolimnas bolina References Inherited parasites in the butterfly Hypolimnas bolina Appendix I 2.63 Appendix I Inherited parasites in the butterfly Hypolimnas bolina Appendix I 2 6 4 Appendix I A.I. DNA Preparation: Chelex Method 1. Each//, bolina specimen was dissected under a light microscope and a small amount (approximately Imm^) of abdominal tissue removed to a fresh 1.5ml Eppendorff tube and the sample heated gently to drive off excess ethanol. 2. Following drying, 200pl 5% w/v Chelex, Ipl proteinase K (20mg/ml) and 7pi DTT (Dithiothreitol) were added to each tissue sample and the samples well mixed using sterilised cocktail sticks. 3. Samples were incubated for one hour at 56°C in a water bath, and then at 95°C for ten minutes. 4. Samples were put on ice to cool and then centrifuged for five minutes. 5. All samples were kept at -70°C when not in use. Inherited parasites in the butterfly Hypolimnas bolina Appendix I 265 A.2. Primer Sequences Prim er Name Used to Assay for Primer Sequence Reference wspSlf Wolbachia: wsp 5’-TGGTCCAAT (Zhou et a l, gene AAG TGATGAAGA 1998) AAC (Zhou et a l, wsp691r 5’-AAAAATTAA 1998) ACG CTA CTC CA wsp522r Wolbachia: wsp 5’-ACCAGCTTT (Thon et al, gene, B group TGC TTG ATA 1998) specific 16SAf Wolbachia: 16 S 5’-TTC GGC CGG (Werren et al, subunit, A group GTT TCA CAC AG 1995) specific 16SAr 5’-TAAGGG ATT (Werren et a l, AGC TTA GCC TC 1995) ftsZfl Wolbachia: ftsZ 5’-GTT GTC GCA (Werren et a l, gene AATACCGATGC 1995) ftsZrl 5’-CTTAAGTAA (Werren et al, GCTGGTATATC 1995) ftsZ-Intf Wolbachia: ftsZ 5’ -ATA TTG GCA Designed by gene, internal TAA GAG GAG F. Jiggins primers (unpublished) ftsZ-Intr 5’ - ATC GGC GAG Designed by TTG AAA TGC F. Jiggins (unpublished) CO If Mitochondrial 5-GGATCACCTGAT (Brunton & CO 1 block ATA GCA TTC CC Hurst, 1998) CO Ir 5’CCG GTAAAATTA (Brunton & AAATATAAACTTC Hurst, 1998) Inherited parasites in the butterfly Hypolimnas bolina Appendix I 2 6 6 A3. PCR Protocols PCR was performed on a PTC-100 programmable thermal cycler (MJ Research Inc.) using cycling conditions specific to the particular assay, as detailed below. All primer sequences are detailed in the previous section. For each protocol, DNA was amplified in a volume of 25 pil, consisting of: 1 pi DNA sample 2.5 pi NH4^ buffer (lOx) 2.5 pi magnesium chloride (15mM stock solution) 2 pi nucleotide mix (dNTP, 2.5mM stock solution) 0.5 pi each primer (20pmol/pl stock solution) 0.1 pi Taq polymerase (Biotaq: 5 units/ pi stock solution) 15.9 pi MilliQ grade water. Primers were ordered from Helena Biosciences (http://www.helena-biosciences.com), and other reagents (except water) from Bioline (http://www.bioline.com). The PCR reaction mix was prepared in one batch and then added to each sample. In all cases the remainder was run as a negative control. A positive control was also run in every PCR. A.3.1. Assay for Wolbachia via amplification of the surface protein gene, wsp The same protocol (with different primer combinations) was used to assay, both for general Wolbachia presence, and for Wolbachia B-group. Primers 81f and 691r were used to assay for general Wolbachia presence. Primers 81f and 522r were used to assay specifically for B-group Wolbachia. Inherited parasites in the butterfly Hypolimnas bolina Appendix I 2 6 7 I. 94° C for two minutes II. 35 cyles of: i. 94° C for 23 seconds ii. 55° C for one minute iii. 72° C for 50 seconds III. 72° C for ten minutes A.3.2. Amplification of mitochondrial cytochrome oxidase, subunit one: COL Primers used: CO lf and COlr. I. 94° C for two minutes II. 35 cyles of: i. 94° C for 25 seconds ii. 53° C for 45 seconds iii. 72° C for 50 seconds III. 72° C for ten minutes A.3.3. Assay for Wolbachia A-group using amplification of the ribosomomal 16S subunit Primers used: 16SAf and 16SAr. I. 94° C for one minute 40 seconds II. 35 cyles of: i. 94° C for 22 seconds ii. 55° C for one minute iii. 72° C for 40 seconds III. 72° C for ten minutes Inherited parasites in the butterfly Hypolimnas bolina Appendix I 2 6 8 A.3.4. Assay for Wolbachia using amplification of the bacterial cell cycle gene,^sZ Primers used: ftsZfl and ftsZrI. I. 94° C for one minute II. 55° C for one minute III. 72° C for three minutes IV. 35 cyles of: i. 94° C for 15 seconds ii. 55° C for one minute iii. 72° C for three minutes V. 94° C for 15 seconds VI. 55° C for one minute VII. 72° C for ten minutes A 4. Assessing PCR Results PCR products were visualised using horizontal agarose gel electrophoresis. One percent agarose gels were set up in 1 x TAE-buffer incorporating 1 pi ethidium bromide (lOmg/ml). Each sample was mixed with 4 pi bromo-phenol-blue buffer, and then loaded onto the gel. PCR products were assessed in comparison to the X-DNA size marker, cut with BamHl, EcoBJ, and Hindlll. Gels were run in 1 x TAErbuffer for 25 minutes at 85 volts and the results visualised under UV light. A photograph was taken for documentation of results (for example, see Figure A.I.). Inherited parasites in the butterfly Hypolimnas bolina Appendix I 2 6 9 A.5.Sequence Analysis A.5.1. Purification of PCR Product 100 \û PCR product was obtained using one of the protocols detailed above. Product was purified using Amicon® Microcon®-PCR centrifugal filter devices (Millipore) following the manufacturer’s protocol. The Amicon® column removes residual nucleotides, salt and primers, concentrating the PCR product in 20 pi of solution. A.5.2. Calculation of DNA Concentration 2 pi of concentrated PCR product was run out on a 1% agarose gel in TAB against quantification standards, XHind III (50 ng/pl). This enabled estimation of the concentration of DNA in the purified product, as well as ensuring that the primers had been removed. A.5.3. Preparation of Purified PCR Product for Sequence Analysis For sequencing purposes, 20ng of purified PCR product is required for every 100 base pairs of sequence. Hence, the quantity of sample required varied depending on the length of the PCR product. For example, the wsp gene is ca. 500 base pairs in length, i.e. lOOng of product would be required to obtain this sequence. Depending on the material to be sequenced, the relevant quantity of purified product was placed in a 1.5 ml Eppendorff tube together with 20 pmol of the relevant primer. In all cases except ftsZ (where internal primers were also used for sequencing purposes, see section A.2.) the primer used was one of the original primers used in the PCR assay. The tube contents were then vacuum dried and sequenced commercially by MWG biotech (http;// mwg-biotech.com/). In all cases, sequences were completed through both strands, except where mentioned in the text. Inherited parasites in the butterfly Hypolimnas bolina Appendix I 270 A.6. Restriction Digest Restriction digests were carried out to ascertain whether the infecting Wolbachia was a member of the A or B clade. PCR assay for the wsp gene using the general wsp primers (81f and 691r) was carried out as above. 1 pi of the restriction enzyme Dral was added to each 25 pi sample of PCR product in the original PCR buffer and the samples incubated at 37 C for 90 minutes in the PCR machine. Samples were run out on a 1% agarose gel and visualised as above. The restriction enzyme Dral cuts at ‘TTTAAA’. This sequence motif is not present in the wsp gene in the A-clade Wolbachia, but is present in the B-clade Wolbachia. Incubation with Dral would therefore cut the B-strain PCR product into two segments of size 70bp and 550bp, and not the A-strain. An example of a gel obtained from a mixture of A- and B- strain samples cut with Dral is given below (Figure A.I.). Figure A.I. Example results gel from Dral cut PCR products from wsp PCR. Samples were all Wolbachia infected. From left to right two B-infected individuals, then five A- infected individuals. Inherited parasites in the butterfly Hypolimnas bolina Appendix I 271 Inherited parasites in the butterfly Hypolimnas bolina Appendix II: Photographs 2 7 2 Appendix II: Photographs Inherited parasites in the butterfly Hypolimnas bolina Appendix II: Photographs 273 Appendix II: Photographs Figure A2.1. Laboratory workbench in \ Colo-I-Suva, Fiji Islands showing hanging cages Figure A2.2. Specially built mating cage, Colo-I-Suva, Fiji Islands Figure A2.3. ‘Laboratory’ in Independent Samoa, 2000 Inherited parasites in the butterfly Hypolimnas bolina Appendix II: Photographs 2 7 4 Figure A2.4. Hypolimnas bolina mating pair. The female is labelled ‘4’ using Tipp-Ex and permanent marker pen Figure A2.5. Mosquito net mating cage, used to fly H. bolina in Independent Samoa Figure A2.6. Oviposition cages. Independent Samoa Inherited parasites in the butterfly Hypolimnas bolina Appendix II: Photographs 275 'Anyway, I shall never forget the impression produced by my first sighting of its fHypolimnas bolina’s) truly oriental splendour; it was like Kingsley *s 'At Last! ’ ” G.B. Longstaff ‘Butterfly hunting in many lands: notes o f a field naturalist’, 1912 Inherited parasites in the butterfly Hypolimnas bolina 2 7 6 Inherited parasites in the butterfly Hypolimnas bolina