1. Wolbachia – a Heritable Endosymbiont Patricia Sidelsky Symbiosis http://tools.neb.com/wolbachia /http://tools.neb.com/wolbachia /

2. Wolbachia  Classified in a monophyletic clade in Domain Bacteria  A member of Alphaproteobacteria – Related to Rickettsia  Sequencing of 16sRNA connects Wolbachia to Ehrlichia and Rickettsia  16srRNA sequences used to determine relationships between Rickettsia  Estimated to infect 15-20% of arthropods. New estimates include as many as 50% of insect species  Broad host range – Insects, isopods, mites, and arachnids. They have been isolated from crustaceans. More recently found in Nematodes  Manipulate the reproduction in their hosts in a number of unusual ways

3. http://www.bacterialphylogeny.com/ branching_order_al...www.bacterialphylogeny.com/ branching_order_al...

4. Host - Endosymbiont  Wolbachia are found in the reproductive tissues of their hosts  The bacterial obligatory endosymbionts are passed vertically in the cytoplasm of the eggs of their hosts  The Wolbachia affects sex determining factors in insects – affects the sex of the insect progeny  The host exerts population control of bacteria  A force in speciation in arthropods  Viewed as a possibility for the evolution of insects Wolbachia infected ovaries of insect host Amy Hise, Science

5. Wolbachia and Reproduction  Vertical transmission – cytoplasmic inheritance  Causes male killing and sterility in males  Induces parthenogenesis  Cytoplasmic incompatability( conflict between cytoplasmic and nuclear components) Insect egg containing Wolbachia http://www.rochester.edu/.../ images/Wolb_egg.jpg

6. Symbiont related Sexual Anomalies  Thelytoky occurs in Trichogramma wasps  In wasps there is a haplofiploid situation with males haploid and females diploid.  Virgin Females produce diploid females without fertilization. The n( haploid) is converted to 2n( diploid)  This occurs early in the first mitotic division  Feminization of males  Occurs in Isopods  Suppresses the androgenic gland and causes males to become functional females http://www.biconet.com/flies/FC3.html http://www.biodiversity.org.uk/.../images/isopods.jpg

7. Cytoplasmic Incompatability and vertical transmission  If both male and female insects are infected with Wolbachia – the progeny will be infected  If the female is infected and the male is not infected, the progeny will all be infected.  If the female is not infected and the male is infected there will not be any progeny http://www.cdc.gov/ncidod/eid/vol4no4/beard6t.gif

8. Examples of symbiont – host interactions  Male killing mechanism has been studied in two species of insect  In Adalia bipunctata and Acraea encedon the presence of Wolbachia halves the hatch size and changes the female/male ratio  Microbial nature of infection has been verified by treatment with rifampin and sulphamethoxazole  After treatment with antibiotics the hatch size returns to normal and the with a normal sex ratio  Condition can be artificially induced by injection of macerated infected insect into pupae of uninfected ( Hurst et al) Adalia bipunctata – http :bugguide.net/ node/view/13976bugguide.net/ node/view/13976 Acraea encedon- www2.nrm.se/.../e/ acraea_encedonfumosa.htmlwww2.nrm.se/.../e/ acraea_encedonfumosa.html

9. Why Male Killing ?  Reduces competition for food ( H)  Reduces cannibalism in siblings (H)  Improves female fitness for breeding- selective advantage (H)  Insures propagation of Wolbachia endosymbionts to future generations ( S + H) H= Host selective advantage S= Symbiont selective advantage

10. Mechanism of Action  Bacterial modification of sperm by bacterial interactions  Bacteria present in testes interact with DNA binding proteins – These may be similar to H1, histones may be similar to H1, histones  The same bacterial strain must be present in the female in order to rescue the egg  Mitotic defects appear in the early divisions of the fertilized egg( during cleavage) HI – Protein database

11. Wolbachia – Evidence for specificity of infection  PCR for 16sRNA from infected lines  Product purified and ligated into pGEM vector  Plasmids were transformed into DH5a  Colonies with insert were detected  39 /42 positive for Wolbachia insert and not other bacteria 16sRNA Transformants are recognized by blue color on agar plate

12. Wolbachia and genetics  Genome recently sequenced  Loss of genes due to obligatory nature of relationship between bacteria and host  Loss of genes over time due to dependency on host  Many repetitive elements and insertion sequences in the genome  Imply high level of intergenomic and intragenomic recombination Genomic map of Wolbachia NCBI http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.govhttp://www.ncbi.nlm.nih.gov

13. Genetics of Wolbachia  Wolbachian genome consists of one circular chromosome   1.26Mbp – 1195 proteins – GC content 35%  Contains genes with Ankyrin repeats – Ankyrins connect endosymbionts to the cell cytoskleton which is an adaptation for intracellular existence Ankyrin repeats – Ankyrins connect endosymbionts to the cell cytoskleton which is an adaptation for intracellular existence  Type Four Secretion System – required for persistence of endosymbiont in host – conserved in Rickettsia Female Drosophila melanogaster – laying egg P. Sidelsky – Motic Camera

14. Lateral Transfer of Genes  Evidence of lateral( horizontal) transfer of genes in Wolbachia species in Drosophila melanogaster and Nasonia vitripennis.  Drosophila and Nasonia can maintain infection by two strains of Wolbachia  Exchange between bacteria that coinfect in the same cells ( density dependent) ( density dependent)  Exchange between endosymbiont and host( gene loss)  IS sequences in Wolbachia suggest Bacteriophage transfer between Wolbachia strains.  Recent research has detected 3 prophage sequences in the Wolbachia genome. ( Wu et al)

15. Importance of study  Wolbachia does not infect humans – studying Wolbachia can lead to a better understanding of Rickettsial – host interactions for medical applciations  Understanding Wolbachia can lead to vector control in infections such as Rocky Mountain Spotted Fever ( Rickettsia and tic) ( Rickettsia and tic)  Since Wolbachia infects diverse species of insects including beetles, fruit flies, and mosquitoes, it could be evaluated for a form of biological control  Used as a model to study speciation Mediterranean Fruit Fly http://creatures.ifas.ufl.edu/fruit/mediterranean_fruit_fly.htm

16. Wolbachia and River Blindness  Wolbachia has also been identified in filarial worms ( Nematoda) ( Nematoda)  One of these worms causes Riverblindness(Onchoceriasis)  Wolbachia provides metabolic support through many pathways for the worm. for the worm.  Contributes to inflammatory responses  Instead of antihelminth drug protocols – will antibiotics be a cure?????? http://tools.neb.com/wolbachia /

17. References Anderson,Cort and Timothy Karr. Wolbachia:Evolutionary Novelty in a Rickettsial Bacteria. BMC Evolutionary Biology, 2001:1-10. Huigens,M.E. On the Evolution of Wolbachia Induced Parthenogenesis in Trichogramma Wasps. http://www.gcw.nl/dissertations/3389/dis3389.pdf http://www.gcw.nl/dissertations/3389/dis3389.pdfhttp://www.gcw.nl/dissertations/3389/dis3389.pdf Hurst, Gregory et al. Male-Killing Wolbachia in Two Species of Insect. National Center for Biotechnology Information – ncbi. http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/ Johanowicz,Denise et Marjorie Foy.Wolbachia Endosymbionts. Florida Entomologist.1998: 310-316. Protein Data Bank - http://www.rcsb.org/pdb/ http://www.rcsb.org/pdb/ Rigaud, T., Juchault, P., and Mocquard, J. P. 1991. Experimental study of the sex ratio of broods in terrestrial Crustacea Armadillium vulgare. Possible implications in natural populations. J. Evol. Biol. 1991: 603 - 607. Sun,Ling. Et al. Gene Organization in the dnaA Region of Wolbachia. Jornal of Bacterioology. 199.18115):4708-4710.

18. References( con) Wernegreen,Jennifer Endosymbiosis: Lessons in Conflict Resolution. Plos Biology. March 2004, 2:307-311. Werren,John H. Biology of Wolbachia. Annual Review of Entomology. 1997.42:587-609. Werren, John H. and Leo Beukboom. Sex Determination, Sex Ratios, and Genetic Conflict.Annual Review of Ecological Systematics. 1998,29:233- 261. Werren, John H. and David Windsor. Wolbachia Infection Frequencies in Insects: Evidence of a Global Equilibrium. Proceedings of the Royal Society. 200 267: 1277-1285 Werren, John H. Wolbachia Run Amok. Proceedings of the National Academy of Science. 1997. 92: 11154-11155 Wu,Martin et al. Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMeI: A Streamlined Genome Overrun by Mobile Genetic Elements. Plos Biology. 2004,2:327-333.