Characterising the bacterial microbiota of Phlebotomus and Glossina insects : vectors of parasitic diseases

As the technology used to examine bacteria using culture-independent methods has advanced, so too has our knowledge of the bacterial microbiome of insect vectors and its effect on the pathogens they carry. Trypanosomiasis and leishmaniasis are neglected tropical diseases caused by kinetoplast parasites and transmitted between human hosts by biting flies. In this thesis the bacterial microbiota of tsetse flies and sand flies, the vectors of trypanosomiasis and leishmaniasis respectively, were examined using molecular biology and next generation sequencing to understand their distribution in wild populations and their impact on pathogen transmission. An experimentally evolved isolate of the tsetse secondary endosymbiont Sodalis glossinidius (SgGMMC1*), which is believed to increase tsetse susceptibility to trypanosome infection, was sequenced and compared to an ancestor S. glossinidius strain (SgGMMB4). SgGMMC1* contained a single large 17 kb deletion as well as 57 small indels and SNPs resulting in 12 putatively pseudogenised genes, supporting the theory that the S. glossinidius genome is undergoing evolutionary reduction. The distribution of known tsetse symbionts including Sodalis and Spiroplasma, as well as the trypanosome parasite, was explored in wild Ugandan tsetse populations. Sodalis was detected in 39% of wild-caught tsetse, Wolbachia in 1.7%, Spiroplasma in 88% and Trypanosoma in 12%, providing essential information about the ecology of these symbiotic organisms in wild populations. Lab-reared tsetse flies were experimentally infected with trypanosomes to examine metatranscriptional differences of commensal bacteria between trypanosome-refractory and successfully infected flies. Wigglesworthia expression differed over time but not between refractory and infected tsetse; in Sodalis virulence genes were upregulated in trypanosome infected flies compared to refractory flies; and little effect of trypanosome infection on Serratia or Wolbachia transcription was observed. These results suggest that increased Sodalis virulence may increase tsetse susceptibility to trypanosome infection. Finally, a novel 16S sequencing pipeline for low-biomass individual sand flies was designed and implemented on wild Phlebotomus argentipes, the vector of visceral leishmaniasis in the Bihar region of India, to discover variation in individual sand fly microbiota within and between houses, villages, and larger districts. A pipeline was optimised to reduce the effect of contamination in low-biomass samples, allowing the description of a core microbiome of seven genera including Corynebacterium, Acinetobacter, and Staphylococcus. A variable peripheral microbiome was discovered with no effect of geographical location, and included the important human pathogen Orientia, implying a potential role for P. argentipes in Orientia transmission