Transovarial transmission fuels persistent infections of a core virome in the Chagas disease vector Rhodnius prolixus

Triatomine assassin bugs comprise hematophagous insect vectors of Trypanosoma cruzi , the causative agent of the Chagas disease. Although the microbiome of these species has been investigated to some extent, only one virus infecting Triatoma infestans has been identified to date. Here, we describe for the first time seven (+) single-strand RNA viruses (RpV1-7) infecting Rhodnius prolixus , a primary vector of the Chagas disease in Central and South America. We show that the RpVs belong to the Picorna-Calici, Permutotetra and Luteo-Sobemo clades and are vertically transmitted from the mothers to the progeny via transovarial transmission. Consistent with this, all the RpVs, except RpV2 that is related to the entomopathogenic Slow bee paralysis virus, established persistent infections in our R. prolixus colony. Furthermore, we show that R. prolixus ovaries express 22-nucleotide viral siRNAs (vsiRNAs), but not viral piRNAs, that originate from the processing of dsRNA intermediates during viral replication of the RpVs. Interestingly, the Permutotetra and Luteo-Sobemo viruses display shared pools of visRNAs that might provide the basis for a cross-immunity system. The vsiRNAs are maternally deposited in the eggs, where they likely contribute to reduce the viral load and protect the developing embryos. Our results unveil for the first time a complex core virome in R. prolixus and begin to shed light on the RNAi-based antiviral defenses in triatomines. Author summary Rhodnius prolixus is a triatomine insect and a primary vector of Trypanosoma cruzi , the etiologic agent of the Chagas disease, in Central and South America. Despite the medical relevance, very little is known about the viruses that infect these so-called assassin bugs. In this study, we show for the first time that triatomines can support the concomitant infection of a variety of RNA viruses belonging to distantly related viral families. Remarkably, we show that the viruses are vertically transmitted from the mothers to the progeny via transovarial transmission. The detection of 22-nucleotide viral small interfering RNAs in mature eggs strongly suggests that RNAi mechanisms contribute to reduce the viral load during oogenesis and embryogenesis in R. prolixus , thus safeguarding the development of embryos and nymphs. In agreement with these findings, all the viruses, except one, could establish persistent infections in our colony. Our results substantially expand the knowledge of the virus complexity in triatomine species. This viral toolkit might be harnessed to develop novel insect population control strategies to reduce the diffusion of the Chagas disease. ### Competing Interest Statement The authors have declared no competing interest.