2. My research Current research : Caste conflict in social insects (stingless bees & termites)

3. Sex allocation queen 50% females workers 75% females INTRAGENOMIC CONFLICT autosome 50% females plasmagenes 100% females sperm 100% females PARENT- OFFSPRING CONFLICT

4. Wolbachia  Maternally transmitted symbiont that manipulates host to produce female biased broods  “Cytoplasmic sex ratio distorter”  Alpha-proteobacterium  Occurs mainly in arthropods (insects+Crustacea) + nematodes

5. Female Biased Sex-Ratios  Male Killing  Feminisation  Parthenogenesis Induction Effects on host reproduction

6. Normal Offspring Production  Reduces fitness of Uninfected Female x Infected Male Crosses  Gives an advantage to infected females  Sterility in diploids, but production of males only in haplo-diploids Cytoplasmic incompatibility Inviable + - - - - + + +

7. Phylogeny Other alpha proteobacteria Ehrlichieae Neorickettsia Gamma proteobacteria 0.1 Wolbachia Caedibacter MtK Mitochondria CMS Orientia MK Rickettsia MK

8. Aims  Does Wolbachia occur in ant societies and if so in what frequency?  What effects does it have? Three case studies : – Parthenogenetic species – Wood ant Formica truncorum – Leptothorax nylanderi  Host-parasite coevolution?

9.  Polymerase Chain Reaction using Specific Primers  Targets: ftsZ and wsp Wolbachia genes  Positive, negative and nuclear DNA (18S rDNA) controls  Negative samples retested twice Methodology: PCR Assay

11. High Incidence Worldwide Indonesia Wenseleers et al. (1998) Proceedings of the Royal Society of London # species=50 Florida Jeyaprakash & Hoy (2000) Insect Molecular Biology # species=10 Panama Van Borm et al. (2001) Journal of Evolutionary Biology # species=7 Europe # species=50 3451 samples

12. Morphological evidence  Present in trophocytes and oocytes  Electron and light microscopical (DAPI) evidence

13. Aims  Does Wolbachia occur in ant societies and if so in what frequency? YES, IN HIGH FREQUENCY  What effects does it have? Three case studies : – Parthenogenetic species – Wood ant Formica truncorum – Leptothorax nylanderi  Host-parasite coevolution?

14. Aims  Does Wolbachia occur in ant societies and if so in what frequency? YES, IN HIGH FREQUENCY  What effects does it have? Three case studies : – Parthenogenetic species – Wood ant Formica truncorum – Leptothorax nylanderi  Host-parasite coevolution?

15. Parthenogenesis induction? 6 Parthenogenetic AntsCape honeybee Cataglyphis cursor Apis mellifera capensis Cataglyphis piliscapa Pristomyrmex pungens Cerapachys biroi Messor capitatus Platythyrea punctata 250 samples, avg. 6 cols./species Grasso et al. (2000) Ethology, Ecology & Evolution 12:309-314 Wenseleers & Billen (2000) Journal of Evolutionary Biology 13:277-280 None infected. Wolbachia does not induce parthenogenesis in ants.

16. Wolbachia in F. truncorum With: Lotta Sundström University of Helsinki

17. Formica truncorum  Extensive variation in sex-ratio produced by different colonies  Linked to facultative sex-ratio biasing : – Workers kill brothers in colonies headed by singly mated queen – But not in colonies with double mated queen  Does Wolbachia affect the sex-ratio too?

18.  Effect on the sex-ratio : –Males should be infected less than queens –Sex-ratio should be correlated with infection rates  Incompatibility : –Males and queens should be infected equally –Uninfected colonies should not be able to survive Predictions

19. Formica truncorum  Males (96%) and queens (94%) infected equally  All colonies infected (total # 33) despite production of 6% uninfected queens by each colony  Consistent with an incompatibility effect : Uninfected queens do not survive past the founding stage due to incompatible matings Wenseleers, Sundström & Billen (2002) Proceedings of the Royal Society of London B, in press

20. GLM EffectsFp No. of mates4.880.04 Infection rate0.850.37 Colony size0.690.42 Infection and sex-ratio Wenseleers, Sundström & Billen (2002) Proceedings of the Royal Society of London B, in press

21. GLM Effects F p F p No. of mates2.110.16 2.5 0.13 Infection rate2.890.11 10.2 0.005 Infection and colony fitness Wenseleers, Sundström & Billen (2002) Proceedings of the Royal Society of London B, in press

22. p<0.015 p<0.0001 Infection rates N=296N=158N=387 Adaptive clearance to reduce colony load? Wenseleers, Sundström & Billen (2002) Proceedings of the Royal Society of London B, 269: 623-629

23. p < 0.0001 Clearance in lab experiments N=89N=90 After 2 month exposure to 20°C Worker pupae 7 colonies

24. Conclusions  No effects on the sex-ratio  Probably causes incompatible matings  Deleterious effects on colony function, but partly mitigated by clearance of infection in adult workers

25. Leptothorax nylanderi  Test experimentally whether Wolbachia causes incompatible matings  Setup: antibiotic treatment as an artificial means of creating the uninfected queen x infected male crossing type  Prediction: male production (infertility) following antibiotic treatment

26.  2 = 10.51, p < 0.001 Antibiotics experiments 4 colonies N=70 7 colonies N=152

27. Aims  Does Wolbachia occur in ant societies and if so in what frequency? YES, IN HIGH FREQUENCY  What effects does it have? Three case studies : – Parthenogenetic species – Wood ant Formica truncorum – Leptothorax nylanderi  Host-parasite coevolution?

28.  Wolbachia surface protein wsp was sequenced (approx. 550 bp)  Direct cycle sequencing when ants were infected by single strain  Cloning and sequencing when ants were infected by multiple strains (TA-cloning kit, pUC57 vector) Methodology: Sequencing 28 sequences Aligned with previously sequenced relatives

29. Solenopsis invicta (imported) Coleomegilla maculata lengi Diaphorina citri Plutella xylostella Laodelphax striatellus Acraea encedon 1 Trichopria Tsp2 Dryinid wasp sp Porcellionides pruinosus Sphaeroma rugicauda Bactocera cucurbitae Tribolium madens Tribolium confusum Rhinophoridae unid Doronomyrmex kutteri B Doronomyrmex pacis B2 Trichogramma spp. Adalia bipunctata B Coleomegilla maculata Adalia bipunctata A Acromyrmex octospinosus B3 Acromyrmex insinuator B1 Acromyrmex echinatior B Solenopsis invicta (native) Acromyrmex octospinosus B1 Acromyrmex octospinosus B2 Acromyrmex insinuator B2 Myrmica sabuleti Telenomus nawai Encarsia formosa Diplolepis rosae Leptopilina australis Cadra cautella Tetranychus urticae Acraea encedon Culex quinquefasciatus Culex pipiens (ESPRO) Drosophila simulans (Watsonville) Aedes albopictus (Houston) Doronomyrmex pacis B1 Isopods Trichopria drosophilae Asobara tabida Myrmica sulcinodis (Samso D) Myrmica sulcinodis (Russia) Teleutomyrmex schneideri Neochrysocharis formosa Formica rufa Dacus destillatoria Doronomyrmex goesswaldi A2 Doronomyrmex pacis A4 Doronomyrmex kutteri A Formica fusca (Mols D) Formica fusca (SJW B) Formica fusca (KH B) Leptothorax acervorum Bactocera sp 1 AscD Cataglyphis iberica Glossina austeni Formica polyctena Formica truncorum Formica pratensis Asobara tabida 3 Drosophila sechellia Drosophila simulans (Hawaii) Cadra cautella 2 Doronomyrmex pacis A3 Gnamptogenys menadensis Phlebotomus papatasi (Israel) Doronomyrmex goesswaldi A1 Acromyrmex octospinosus A1 Solenopsis invicta A (native) Doronomyrmex pacis A2 Solenopsis richteri A Acromyrmex echinatior A1 Drosophila simulans (Riverside) Drosophila melanogaster (CantonS) Drosophila melanogaster (Cairns) Drosophila simulans (Coffs Harbour) Aedes albopictus (Houston) Nasonia vitripennis A Drosophila bifasciata Glossina morsitans centralis Leptopilina heterotoma 2 Trichogramma bourarachae Trichogramma kaykai (LC110) Muscidifurax uniraptor Acromyrmex insinuator A Plagiolepis pygmaea Myrmica sulcinodis (Pyrenees) Formica lemani Myrmica rubra Doronomyrmex pacis A1 0.050 (25 MY) AB High strain diversity

30. Solenopsis invicta (imported) Coleomegilla maculata lengi Diaphorina citri Plutella xylostella Laodelphax striatellus Acraea encedon 1 Trichopria Tsp2 Dryinid wasp sp Porcellionides pruinosus Sphaeroma rugicauda Bactocera cucurbitae Tribolium madens Tribolium confusum Rhinophoridae unid Doronomyrmex kutteri B Doronomyrmex pacis B2 Trichogramma spp. Adalia bipunctata B Coleomegilla maculata Adalia bipunctata A Acromyrmex octospinosus B3 Acromyrmex insinuator B1 Acromyrmex echinatior B Solenopsis invicta (native) Acromyrmex octospinosus B1 Acromyrmex octospinosus B2 Acromyrmex insinuator B2 Myrmica sabuleti Telenomus nawai Encarsia formosa Diplolepis rosae Leptopilina australis Cadra cautella Tetranychus urticae Acraea encedon Culex quinquefasciatus Culex pipiens (ESPRO) Drosophila simulans (Watsonville) Aedes albopictus (Houston) Doronomyrmex pacis B1 Isopods Trichopria drosophilae Asobara tabida Myrmica sulcinodis (Samso D) Myrmica sulcinodis (Russia) Teleutomyrmex schneideri Neochrysocharis formosa Formica rufa Dacus destillatoria Doronomyrmex goesswaldi A2 Doronomyrmex pacis A4 Doronomyrmex kutteri A Formica fusca (Mols D) Formica fusca (SJW B) Formica fusca (KH B) Leptothorax acervorum Bactocera sp 1 AscD Cataglyphis iberica Glossina austeni Formica polyctena Formica truncorum Formica pratensis Asobara tabida 3 Drosophila sechellia Drosophila simulans (Hawaii) Cadra cautella 2 Doronomyrmex pacis A3 Gnamptogenys menadensis Phlebotomus papatasi (Israel) Doronomyrmex goesswaldi A1 Acromyrmex octospinosus A1 Solenopsis invicta A (native) Doronomyrmex pacis A2 Solenopsis richteri A Acromyrmex echinatior A1 Drosophila simulans (Riverside) Drosophila melanogaster (CantonS) Drosophila melanogaster (Cairns) Drosophila simulans (Coffs Harbour) Aedes albopictus (Houston) Nasonia vitripennis A Drosophila bifasciata Glossina morsitans centralis Leptopilina heterotoma 2 Trichogramma bourarachae Trichogramma kaykai (LC110) Muscidifurax uniraptor Acromyrmex insinuator A Plagiolepis pygmaea Myrmica sulcinodis (Pyrenees) Formica lemani Myrmica rubra Doronomyrmex pacis A1 0.050 (25 MY) AB No match with host phylogeny Acromyrmex insinuator A Plagiolepis pygmaea Myrmica sulcinodis (Pyrenees) Formica lemani Myrmica rubra Doronomyrmex pacis A1 Hosts diverged 35 MY ago, but share a recently evolved W. strain (1.7 MY old) Doronomyrmex kutteri B Doronomyrmex pacis B2 Doronomyrmex pacis B1 Doronomyrmex goesswaldi A2 Doronomyrmex pacis A4 Doronomyrmex kutteri A Doronomyrmex pacis A3 Doronomyrmex goesswaldi A1 Doronomyrmex pacis A2 Doronomyrmex pacis A1

31. Solenopsis invicta (imported) Coleomegilla maculata lengi Diaphorina citri Plutella xylostella Laodelphax striatellus Acraea encedon 1 Trichopria Tsp2 Dryinid wasp sp Porcellionides pruinosus Sphaeroma rugicauda Bactocera cucurbitae Tribolium madens Tribolium confusum Rhinophoridae unid Doronomyrmex kutteri B Doronomyrmex pacis B2 Trichogramma spp. Adalia bipunctata B Coleomegilla maculata Adalia bipunctata A Acromyrmex octospinosus B3 Acromyrmex insinuator B1 Acromyrmex echinatior B Solenopsis invicta (native) Acromyrmex octospinosus B1 Acromyrmex octospinosus B2 Acromyrmex insinuator B2 Myrmica sabuleti Telenomus nawai Encarsia formosa Diplolepis rosae Leptopilina australis Cadra cautella Tetranychus urticae Acraea encedon Culex quinquefasciatus Culex pipiens (ESPRO) Drosophila simulans (Watsonville) Aedes albopictus (Houston) Doronomyrmex pacis B1 Isopods Trichopria drosophilae Asobara tabida Myrmica sulcinodis (Samso D) Myrmica sulcinodis (Russia) Teleutomyrmex schneideri Neochrysocharis formosa Formica rufa Dacus destillatoria Doronomyrmex goesswaldi A2 Doronomyrmex pacis A4 Doronomyrmex kutteri A Formica fusca (Mols D) Formica fusca (SJW B) Formica fusca (KH B) Leptothorax acervorum Bactocera sp 1 AscD Cataglyphis iberica Glossina austeni Formica polyctena Formica truncorum Formica pratensis Asobara tabida 3 Drosophila sechellia Drosophila simulans (Hawaii) Cadra cautella 2 Doronomyrmex pacis A3 Gnamptogenys menadensis Phlebotomus papatasi (Israel) Doronomyrmex goesswaldi A1 Acromyrmex octospinosus A1 Solenopsis invicta A (native) Doronomyrmex pacis A2 Solenopsis richteri A Acromyrmex echinatior A1 Drosophila simulans (Riverside) Drosophila melanogaster (CantonS) Drosophila melanogaster (Cairns) Drosophila simulans (Coffs Harbour) Aedes albopictus (Houston) Nasonia vitripennis A Drosophila bifasciata Glossina morsitans centralis Leptopilina heterotoma 2 Trichogramma bourarachae Trichogramma kaykai (LC110) Muscidifurax uniraptor Acromyrmex insinuator A Plagiolepis pygmaea Myrmica sulcinodis (Pyrenees) Formica lemani Myrmica rubra Doronomyrmex pacis A1 0.050 (25 MY) AB Multiple infections Doronomyrmex pacis B2 Doronomyrmex pacis B1 Doronomyrmex pacis A4 Doronomyrmex pacis A3 Doronomyrmex pacis A2 Doronomyrmex pacis A1 Multi infections may drive speciation events!

32. Acromyrmex echinatior A1 Acromyrmex octospinosus A1 Acromyrmex octospinosus B3 Acromyrmex octospinosus B2 Acromyrmex echinatior Bc Acromyrmex octospinosus B1 Wolbachia in Leafcutter Ants Van Borm, Wenseleers, Billen & Boomsma, Mol. Phyl. Evol., in press Van Borm, Wenseleers, Billen & Boomsma (2001) J. Evol. Biol. 13: 277-280. Neochrysocharis Eulophidae Dacus destillatoria Drosophila simulans Drosophila melanogaster Aedes albopictus Acromyrmex insinuator A1 Muscidifurax uniraptor Nasonia vitripennis Drosophila bifasciata Drosophila simulans Formica truncorum Asobara tabida Solenopsis richteri Solenopsis invicta Armadillidium vulgare Culex pipiens Encarsia Formosa Diplolepis rosae Acromyrmex insinuator B2 Telenomus nawai Adalia bipunctata Trichogramma kaykai Tribolium madens Acraea encedon Solenopsis invicta Acromyrmex insinuator B1 Solenopsis invicta A B InvA InvB Naw InsA 56 65 100 74 100 76 100 87 88 100 70 96 99 85 96 79 85 71 93 69  Two free-living species (A. octospinosus, A. echinatior) have multiple “Solenopsis- like” Wolbachia infections  Infection males < gynes + workers  possibly partial male killers  No evidence for abnormal sex ratios

33. Acromyrmex echinatior A1 Acromyrmex octospinosus A1 Acromyrmex octospinosus B3 Acromyrmex octospinosus B2 Acromyrmex echinatior Bc Acromyrmex octospinosus B1 Wolbachia in Leafcutter Ants Neochrysocharis Eulophidae Dacus destillatoria Drosophila simulans Drosophila melanogaster Aedes albopictus Acromyrmex insinuator A1 Muscidifurax uniraptor Nasonia vitripennis Drosophila bifasciata Drosophila simulans Formica truncorum Asobara tabida Solenopsis richteri Solenopsis invicta Armadillidium vulgare Culex pipiens Encarsia Formosa Diplolepis rosae Acromyrmex insinuator B2 Telenomus nawai Adalia bipunctata Trichogramma kaykai Tribolium madens Acraea encedon Solenopsis invicta Acromyrmex insinuator B1 Solenopsis invicta A B InvA InvB Naw InsA 56 65 100 74 100 76 100 87 88 100 70 96 99 85 96 79 85 71 93 69  Several unrelated Wolbachia infections in inquiline A. insinuator  Equally common in males and females  cytoplasmic incompatibility? Van Borm, Wenseleers, Billen & Boomsma, Mol. Phyl. Evol., in press Van Borm, Wenseleers, Billen & Boomsma (2001) J. Evol. Biol. 13: 277-280.

34. No match with host phylogeny pratensis lemani fusca rufa O 100 99 polyctena truncorum 84 100 0.02 (10 MY)...and their symbionts rufa polyctena pratensis truncorum lemani fusca O Formica hosts... Gyllenstrand, unpublished

35. Sequencing conclusions  No host-parasite coevolution  But distinct ant Wolbachia clades – implies degree of host specialisation  Frequent horizontal transmission  Single ants may be infected with up to 6 different strains  Different populations usually, but not always, infected by same strains

36. Conclusions  Does Wolbachia occur in ant societies? YES, IN HIGH FREQUENCY  Alternative explanation for female biased sex- ratios? NO STRONG EVIDENCE Other effects? INCOMPATIBILITY (SPECIATION?)  Host-parasite coevolution? NO, OCCASIONAL HORIZONTAL TRANSMISSION

37. References S. Van Borm, T. Wenseleers, J. Billen and J.J. Boomsma (2002) Cloning and sequencing of wsp encoding gene fragments reveals a diversity of co-infecting Wolbachia strains in Acromyrmex leafcutter ants. Molecular Phylogenetics and Evolution, in press. T. Wenseleers, L. Sundström and J. Billen (2002) Deleterious Wolbachia in the ant Formica truncorum. Proceedings of the Royal Society of London Series B-Biological Sciences, 269: 623-629. S. Van Borm, T. Wenseleers, J. Billen and J.J. Boomsma (2001) Wolbachia in leafcutter ants: a widespread symbiont that may induce male killing or incompatible matings. Journal of Evolutionary Biology, 14: 805-814. T. Wenseleers (2001) Conflict from Cell to Colony. Ph.D. thesis, University of Leuven, Belgium, 205 pp. Advisor: Prof. Dr. J. Billen. D. Grasso, T. Wenseleers, A. Mori, F. Le Moli and J. Billen (2000) Thelytokous worker reproduction and lack of Wolbachia infection in the harvesting ant Messor capitatus. Ethology, Ecology & Evolution, 12 : 309-314. T. Wenseleers and J. Billen (2000) No evidence for Wolbachia-induced parthenogenesis in the social Hymenoptera. Journal of Evolutionary Biology, 13 : 277-280. T. Wenseleers, F. Ito, S. Van Borm, R. Huybrechts, F. Volckaert and J. Billen (1998) Widespread occurrence of the micro-organism Wolbachia in ants. Proceedings of the Royal Society of London Series B-Biological Sciences, 265: 1447-1452. PDFs at www.shef.ac.uk/uni/projects/taplab/twpub.html

38. Acknowledgements Prof. Dr. J. BillenDr. F. Ito Prof. Dr. J.J. BoomsmaDr. F.L.W. Ratnieks Dr. D.A. Grasso Dr. L. Sundström Prof. Dr. R. HuybrechtsS. Van Borm Prof. Dr. F. Volckaert Academy of Finland, British Council, FWO-Vlaanderen, Vlaamse Leergangen, EU “Social Evolution” & INSECTS networks, Marie Curie