LAUSR

Aedes aegypti is the principal mosquito vector of dengue, yellow fever, Zika and chikungunya viruses. The wMel endosymbiotic bacteria Wolbachia pipientis has been introduced into this vector as a novel biocontrol strategy to stop transmission of these viruses. Mosquitoes with Wolbachia have been released in the field in North Queensland, Australia since 2011, at various locations and over several years, with populations remaining stably infected. Wolbachia infection is known to alter gene expression in its mosquito host, but whether (and how) this changes over the long-term in the context of field releases remains unknown. We sampled mosquitoes from Wolbachia-infected populations with different release histories along a time gradient and performed RNAseq to investigate gene expression changes in the insect host. We observed a significant impact on gene expression in Wolbachia-infected mosquitoes versus uninfected controls, but fewer genes had altered expression in the older releases (e.g. the year 2011) versus the more recent releases (e.g. 2017). Nonetheless, a fundamental signature of Wolbachia infection on host gene expression was observed across all releases, comprising upregulation of immunity and metabolism genes. There was limited downregulation of gene expression in the older releases, but significantly more in the most recent release. Our findings indicate that at > 8 years post-introgression into field populations, Wolbachia continues to profoundly impact host gene expression, particularly genes involved in insect immune response. We suggest that if gene expression changes underlie blocking of virus replication in Wolbachia-infected Ae. aegypti, then refractoriness of these mosquitoes to arboviruses may remain stable over the long-term. Author summary The Aedes aegypti mosquito is the main species responsible for urban transmission of dengue, Zika and chikungunya viruses. Control measures, including source reduction and insecticide treatment, have historically struggled to provide sustained control of this species to limit disease. An alternative approach involves releasing mosquitoes harbouring Wolbachia bacteria. Wolbachia inhibits virus transmission by Ae. aegypti and preliminary evidence indicates that dengue incidence is reduced in locations where it has been deployed. In this study, we found that Wolbachia significantly upregulates gene expression in Ae. aegypti at least 8 years after field deployment compared with uninfected controls, although some gene downregulation was also observed. We observed a more ‘muted’ response in mosquitoes from populations with older release histories, with far fewer genes being differentially regulated versus those from the most recent release. Irrespective of release history, immune response and metabolism genes were significantly upregulated, and to a lesser extent genes related to behaviour. Our results, combined with previous studies that have revealed few changes in the Wolbachia genome post release, provide further evidence of the long-term stability of the Wolbachia-mosquito relationship in the field.