Characterization of Wolbachia infections in Culicoides midge cell lines and the comprised microbiome



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Culicoides midges are hematophagous insects that transmit arboviruses of veterinary importance. These viruses include bluetongue virus (BTV) and epizootic hemorrhagic fever virus (EHDV). The endosymbiont Wolbachia pipientis Hertig spreads rapidly through insect host populations and has been demonstrated to inhibit viral pathogen transmission in multiple mosquito vectors. Here, we have demonstrated a replication inhibitory effect on BTV and EHDV in a Wolbachia (wAlbB strain) infected Culicoides sonorensis Wirth and Jones W8 cell line. Viral replication was significantly reduced by day 5 for BTV and by day 2 for EHDV as detected by real- time polymerase chain reaction (RT-qPCR) of the non-structural NS3 gene of both viruses. Evaluation of innate cellular immune responses as a cause of the inhibitory effect showed responses associated with BTV but not with EHDV infection. Wolbachia density also did not play a role in the observed pathogen inhibitory effects, and an alternative hypothesis is suggested. Applications of Wolbachia-mediated pathogen interference to impact disease transmission by Culicoides midges are discussed. The microbiome of Culicoides midges has gone relatively unexplored, especially in comparison to their arthropod vector counterparts. Here, I explore the bacterial and fungal microbiome of C. haematopotus, C. furens, C. venestus, C. arboricola, and C. edeni. Pools of 8-14 males and females from each species were collected and DNA was extracted. The samples then underwent sequencing with 16S primers and ITS gene for bacteria and fungi, respectively. The bacterial samples contained four major Phyla: Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. In the Fungal samples, there were some interesting results with regards to phyla. One species. C. haematopotus, had Zoopagomycota in both sexes and C. venestus had a 99% composition of ‘other.’ Overall, the results provide knowledge that could lead to future studies to determine if the microbiome interactions could be used for population and/or disease control approaches.



Culicoides, Midge, Wolbachia