Characterization of Wolbachia infections from native Australian mosquitoes

Abstract

Wolbachia are maternally transmitted intracellular bacteria that naturally infect over 40% of all insect species. Wolbachia have become promising biocontrol agents against mosquito-borne diseases due to their unique effects on mosquito reproduction and immunity. However, the phenotypes mediated by Wolbachia vary between strains and host-pathogen systems. Discovery of novel Wolbachia strains is essential for broadening the application of Wolbachia for mosquito control. In this study, I identified and characterized the anti-pathogen effects of natural Wolbachia infections in Australian mosquitoes. I identified three previously uncharacterized Wolbachia strains based on detection and sequencing of wsp, 16S rRNA and ftsZ genes. Wolbachia sequences were also detected from Coquillettidia xanthogaster, however, it remained unclear if the detection was resulted from a genuine infection. I established colonies of two local species that are naturally infected with Wolbachia; Aedes notoscriptus and Culex sitiens, and discovered a unique pattern of infection rates fluctuating between 15% and 60% in both colonies. Study on the maternal transmission of Wolbachia in Cx. sitiens revealed high transmission rates (99.7%) but low cytoplasmic incompatibility (9.9% mortality). I orally inoculated Wolbachia positive and negative Ae. notoscriptus and Cx. sitiens with the alphavirus Ross River virus (RRV; 103.31 – 6.48 and 105.41 – 6.36 CCID50 per mosquito, respectively). RRV infection rates varied between 17% - 85% for Ae. notoscriptus and 0% - 18% for Cx. sitiens with no significant differences between infection rates for Wol+ and Wol- mosquitoes. However, mean virus load in Wol+ Cx. sitiens was 1000× lower than in Wol- mosquitoes, suggesting that natural Wolbachia infection suppresses virus proliferation in that species. I report on progress towards transinfecting cells (Aag2 and Aa20) and mosquitoes (Aedes aegypti) with Wolbachia, including oral ingestion trials. Inoculations using the Shell vial technique resulted in temporal in vitro infections for up to three passages, however, stable infections were not yet achieved. I observed evidence that Wolbachia infected cells within the epithelial lining of the crop and potentially the midgut by fluorescence microscopy, however, the infection was transient. These discoveries broadened the current understanding on Wolbachia-host-pathogen interactions and shed light on future mosquito control strategies. The behaviour of Wolbachia in new hosts can be difficult to predict, therefore transinfection and further characterization is required to determine if these strains can induce anti-pathogen effects in the context of mosquito control.