An exploration of the density and genetics of Wolbachia in natural populations of Onchocerca volvulus – the causative agent of river blindness

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Life Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Thesis with publications.

Wolbachia are endosymbionts of Onchocerca volvulus and other parasitic nematodes that cause 150 million cases of filariases worldwide. These endosymbionts have become important drug targets, given that their removal by antibiotic treatment kills the worm. Wolbachia have also been implicated in the human host-induced immunopathologies that are associated with filariases. Crucial to understanding the role of Wolbachia in worm biology and to assessing the potential for the evolution of antibiotic resistance is an exploration of the abundance and genetic diversity of Wolbachia. This study sampled host adult O. volvulus worms from Côte d’Ivoire, Ghana and Mali, and employed quantitative real-time PCR and next-generation sequencing approaches to determine Wolbachia copy number and genetic diversity respectively. Significant heterogeneity in Wolbachia copy number was observed between and within natural populations of O. volvulus. For the first time, multiple genetically distinct Wolbachia genomes were observed in a single worm. This study proposes that Wolbachia population structure is influenced by the severe population bottleneck that most likely occurs during the maternal transmission of Wolbachia to microfilariae and the accumulation of new mutations during the subsequent population expansion from the larval to adult stage and the long adult life span (up to 14 years). There was high proportion of non-synonymous mutations, a phenomenon observed in other bacteria, which presents the potential for functional diversity in Wolbachia between individual worms. Particularly noteworthy is nucleotide polymorphism within the 16S rDNA, which encodes the 16S rRNA that is targeted by doxycycline and other antibiotics. This may indicate the potential for evolution of resistance to doxycycline and suggests that the development of novel anti-Wolbachia drugs should incorporate investigation of the likely sustainability of a single antibiotic treatment. In addition, non-synonymous SNPs in the Wolbachia surface protein, which is reported to drive human host immunopathology, could give an indication for the observed diversity in disease presentation among human populations.