Investigating the molecular basis for the maintenance of persistent infection by Wolbachia pipientis wAlbB strain in the dengue mosquito vector, Aedes aegypti

Abstract

Arthropod-borne viruses (arboviruses) pose a significant global health burden. Among the most medically important arboviruses, dengue, Zika, Yellow fever, and Chikungunya viruses are transmitted by two mosquito species, Aedes aegypti and Aedes albopictus, with Ae. aegypti being the primary vector. In the absence of safe and effective vaccines, efforts to limit disease have focused on vector-control, typically relying on the use of insecticides. Due to environmental concerns and the increasing problem of insecticide-resistance, alternative approaches to vector-control are being explored. One promising strategy involves the release of mosquitoes infected with the bacterial endosymbiont, Wolbachia pipientis. When transinfected into Ae. aegypti, certain strains of Wolbachia have the ability to rapidly invade populations and inhibit viral transmission or reduce population density. Field trials using the Drosophila-derived wMel strain have yielded positive results. However, recent evidence suggests that the Aedes albopictus-derived wAlbB strain may be more suitable due to its broader range of thermal tolerance compared to wMel. The usefulness of Wolbachia for arbovirus suppression is the result of a complex set of molecular interactions between host, symbiont, and pathogen. Some recent research has begun to unravel certain aspects of the relationship, including the elegant way in which Wolbachia manipulates host reproduction to facilitate its invasion into naive populations. However, much about the biology of Wolbachia infection in mosquitoes remains unknown, including, crucially, the mechanism of viral inhibition. To add further complication, in addition to vectoring arboviruses, Aedes mosquitoes also carry insect-specific viruses (ISVs), some of which have an effect on arbovirus transmission while also being affected by Wolbachia themselves. A thorough understanding of the biological underpinnings of Wolbachia maintenance and viral inhibition is required in order to optimize vector-control strategies and predict the future success of field applications. microRNAs (miRNAs) are small non-coding RNA molecules involved in regulating a range of biological processes in eukaryotes and viruses. Some miRNAs have been implicated in mediating host-microbe interactions, including between Wolbachia and mosquitoes. Using a wAlbB-transinfected Ae. aegypti mosquito line, we investigated the effect of wAlbB on the host miRNA expression profile, and used miRNA inhibitors of selected differentially expressed miRNAs to explore their effect on host longevity, innate immune function, and wAlbB maintenance. We also created a stably transinfected Ae. aegypti Aag2.wAlbB cell line using wAlbB from Ae. albopictus Aa23 cells to investigate the effect of transinfection on wAlbB gene expression. We observed a general downregulation of gene expression in wAlbB following transinfection, and identified several differentially expressed genes relating to membrane integrity, replication and biosynthesis, as well as several genes associated with prophage WOAlbB. Furthermore, we characterised a novel insect-specific positive-sense RNA negev-like virus, present in Aa23 cells and showed that it is inhibited by wAlbB in Aag2 cells but not in Aa23 cells. The results presented here serve to advance our current understanding of the intimate symbiotic relationship between wAlbB and Ae. aegypti.