PHYLOGENY AND POTENTIAL TRANSMISSION ROUTES OF MIDGUT-ASSOCIATED ENDOSYMBIONTS OF TSETSE (DIPTERA, GLOSSINIDAE)

Many tsetse species (Diptera: Glossinidae) harbour two morphologically different intracellular endosymbiotic microorganisms associated with gut tissue: primary (P) and secondary (S) endosymbionts. The P-endosym bionts of tsetse (Wigglesworthia glossinidia) are sequestered in speci alized epithelial cells, bacteriocytes, which form a structure (bacter iome) in the anterior portion of the gut. Phylogenetic characterizatio n of P-endosymbionts from the three subgenera of genus Glossina has sh own that these organisms constitute a distinct lineage within the gamm a-subdivision of Proteobacteria and have evolved concordantly with the ir insect host species, suggesting an evolutionarily ancient associati on for this symbiosis. The S-endosymbiont is a smaller (1-2 mu m) gram -negative rod and is harboured in midgut epithelial cells. Its phyloge netic characterization from Glossina morsitans morsitans had shown tha t it is a member of the family Enterobacteriaceae within the gamma-3 s ubdivision of the Proteobacteria, closely related to enteric bacteria. Some tsetse species harbour a third bacterium in their reproductive t issue, which was shown phylogenetically to belong to the Wolbachia pip lentis assemblage of microorganisms. Here, we show that S-endosymbiont s from five tsetse species, representing all three subgenera, form a c luster of closely related microorganisms, based on their almost identi cal 16S rRNA gene sequences. This high similarity provides strong evid ence of recent independent acquisition of S-endosymbionts by individua l tsetse species, unlike Wigglesworthia which displays concordant evol ution with host insect species. A PCR-based assay and restriction frag ment length polymorphism (RFLP) analysis was developed to localize the S-endosymbionts and Wigglesworthia in ovary, egg, milk-gland and sper matheca tissues in order to investigate the potential routes for the v ertical transmission of these symbionts to the intrauterine larvae. On ly S-endosymbionts were found to infect milk gland tissue, suggesting that milk gland secretions represent a route of transmission for these symbionts into the developing larva. The ovary tissue was found to ha rbour only Wolbachia, confirming its transovarial transmission, wherea s the mode of transmission of Wigglesworthia remains unknown.