1. Tom Wenseleers University of Leuven, Belgium Ph.D. defence May 22nd, 2001 Conflict from Cell to Colony

2. Cooperation is Key Feature in Evolution of Life on Earth  Genes to Genomes  Prokaryotes to Eukaryotes  Unicellular to Multicellular Organisms  Organisms to Societies Major transitions in evolution

3.  Cooperation seems obvious to explain when viewed in terms of species-level benefits  But erroneous logic: non-cooperative ’free-riders’ outcompete altruists But potential for conflict Potential for Conflict in Most Societies  Conflicts may occur between organisms, but also between cells or genes (’intragenomic conflict’)

4. In what ratio should males and females be reared? F ½ M ½ ¼ M ¾ F Conflicts in insect societies Equal Sex-Ratio 3:1 Female Biased Sex-Ratio

5. Cytoplasmic sex-ratio distorters  Conflict also occurs at the genomic level: maternally transmitted genes favour more female biased sex- ratios than nuclear genes (“intragenomic conflict”)  Cytoplasmic genes such as mitochondria or some bacterial symbionts may manipulate host to produce female biased broods (“cytoplasmic sex-ratio distorters”)

6. Wolbachia  Example of a maternally transmitted symbiont  Alpha-proteobacterium  Occurs mainly in arthropods (insects+Crustacea) + nematodes  Manipulates host reproduction to favour own spread

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

8. 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

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

10. Aims of my thesis  Part I : empirical – Does Wolbachia occur in ant societies? – Alternative explanation for female biased sex-ratios in this group?  Part II : theoretical – What do animal and genomic conflicts have in common? – Can sociobiological theory be applied to both?

11. S e q u e n c e o f E v e n t s Modelling Make predictions DNA Analysis Measure key parameters Experiments Formally test hypotheses Ideas Hypotheses Molecular Data Experimental Data Integrated approach

12. Part I. Wolbachia - a cause of intragenomic conflict in ant colonies

13. Work plan  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?

14.  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 Sensitive & Reliable

15. High Incidence Worldwide Indonesia Chapter 1 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 Chapter 6 3451 samples

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

17. Work plan  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?

18. Work plan  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?

19. Parthenogenesis induction? Were not infected. Parthenogenesis not induced by Wolbachia. PCR Assay Grasso et al. (2000) Ethology, Ecology & Evolution 12:309-314 Wenseleers & Billen (2000) Journal of Evolutionary Biology 13:277-280 6 Parthenogenetic Ants and Cape Honey Bee N=250 36 cols.

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

21. 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?

22.  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

23. 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 series B, in press.

24. 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 series B, in press.

25. 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 series B, in press.

26. 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 series B, in press.

27. 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

28. 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

29.  2 = 10.51, p < 0.001 Antibiotics experiments 4 colonies N=70 7 colonies N=152

30. Work plan  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?

31.  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

32. 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

33. 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

34. 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!

35. 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

36. Work plan  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? NO, OCCASIONAL HORIZONTAL TRANSMISSION

37. Part II. Theoretical aspects of conflict and cooperation With: Francis Ratnieks and Kevin Foster University of Sheffield

38. Animal vs. intragenomic conflict  What do animal and intragenomic conflict have in common?  Is there a “general theory of conflict” that provides insight into the evolution of conflict at both levels?

39. Theories of conflict Two Approaches in the Study of Conflict Kin Selection Hamilton Game Theory von Neumann & Morgenstern Single method r.B > C Cost Depends on Social Context

40. Generalised Hamilton’s rule Wenseleers & Ratnieks submitted Hamilton’s rule (costs & benefits independent of social context) Terms that take into account social context Consequence of both cooperating Regression of genotype on joint behaviour

41. Animal vs. intragenomic conflict 0 -B B -C DOVEHAWK DOVE HAWK ANIMAL CONFLICT COOPERATEDRIVE COOPERATE DRIVE G DC.(1-k)1/2 G DC.kG DD /2 GENOMIC CONFLICT (MEIOTIC DRIVE)

42. Animal vs. intragenomic conflict  Shows that game theoretic logic of conflict at both levels is the same  But can genes also be related?  Yes, kinship measures genetic correlation and for 2 genes at a locus this is the inbreeding coefficient F IT  When genes are related they are selected to be altruistic !  Application of generalised Hamilton’s rule allows detailed analysis

43. Spite: Hamilton’s unproven theory  Medea killed her children to take away the smile from her husband’s face.  Example of a paradoxical behaviour that harms another at no benefit to self (“spite”)  We showed that some forms of intragenomic conflict qualify as spiteful behaviour (Maternal effect lethals, queen killing in the fire ant) Foster, Ratnieks & Wenseleers (2000) Trends in Ecology & Evolution 15:469-470 Foster, Wenseleers & Ratnieks (2001) Annales Zoologici Fennici, in press

44. Why become a worker?  Why do social insect females work for the benefit of others?  Usual explanation: indirect genetic benefit when altruism is directed towards relatives (’kin selection’)  But is relatedness in insect societies high enough?  E.g. honey bee: queen mates with several males so that workers mostly rear half-sisters (r=0.3)

45. New calculations  Female should become a queen with a probability of (1-R f )/(1+R m ) (self determination) –= 20% for stingless bees (singly mated) –= 56% for honey bees (polyandrous)  Too high for the colony as a whole, since queens are only needed for swarming (“tragedy of the commons”)  Adult workers and mother queen selected to prevent production of excess queens (“policing”)

46. Comparative predictions hold Self determination 20% queen production stingless bees Policing of caste fate 0.02% queen production honey bees Individual Freedom Causes a Cost to Society But females prefer to become queen with probability of 56% ! Efficient Society but No Individual Freedom THE SAME TENSION OCCURS IN HUMAN SOCIETY !

47. General conclusions  Part I : empirical – Does Wolbachia occur in ant societies? YES, IN HIGH FREQUENCY – Alternative explanation for female biased sex-ratios in this group? PROBABLY NOT – Other effects? INCOMPATIBILITY (SPECIATION?)  Part II : theoretical – What do animal and genomic conflicts have in common? SAME LOGIC – Can sociobiological theory be applied to both? YES (GENERALISED HAMILTOM’S RULE) – What do we learn from this more generally? DEEPER INSIGHT INTO THE FUNCTIONING OF HUMAN SOCIETIES (TOC)

48. The End

49. Acknowledgements Prof. Dr. J. BillenProf. Dr. R. Huybrechts Prof. Dr. J.J. BoomsmaDr. F. Ito Dr. K.R. Foster Dr. F.L.W. Ratnieks Prof. S.A. Frank Dr. L. Sundström Dr. D.A. Grasso Drs. S. Van Borm Prof. Dr. F. Volckaert Academy of Finland, British Council, FWO-Vlaanderen, Vlaamse Leergangen, EU Network “Social Evolution”