Functional validation of Apoptosis Genes IAP1 and DRONC in midgut tissue of the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) by RNAi

Authors: MILLS, MARY - Kansas State University; Nayduch, Dana; McVey, David; MICHEL, KRISTIN - Kansas State University

Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2016
Publication Date: 2/8/2017
Citation: Mills, M.K., Nayduch, D., McVey, D.S., Michel, K. 2017. Functional validation of Apoptosis genes IAP1 and DRONC in midgut tissue of the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) by RNAi. Journal of Medical Entomology. 54:559-557.

Interpretive Summary: Culicoides biting midges transmit multiple ruminant viruses, including bluetongue virus and epizootic hemorrhagic disease virus, that cause significant economic burden worldwide due to trade restrictions and production loss. To limit the spread of these viruses, control strategies have focused on vaccine development, livestock management, and vector control. Understanding vector-virus interactions within the midge is critical to developing new and innovative strategies for blocking disease transmission. In a previous study, we developed a double-stranded (ds)RNA delivery methods to induce RNA interference (RNAi) for targeted gene knockdown in adult Culicoides sonorensis using the midge inhibitor of apoptosis gene 1 (IAP1) ortholog (CsIAP1). In the current study, we confirm that CsIAP1 and a second gene, initiator caspase DRONC (CsDRONC), can be knocked down by injection of dsRNA into the midge body cavity. Further, this knockdown can be targeted in the midgut in C. sonorensis. We observed a statistically significant reduction of CsIAP1 gene transcripts in whole midges at two and five days after injection, and the highest transcript reduction was in midgut tissues. This knockdown resulted in a three-fold increase in overall caspase activity, which indicates that apoptosis (cell death) was taking place in these tissues. We visualized midge midguts to determine the effect of this cell death, and noted that tissue integrity and gut size was severely compromised, presumably being caused by cell loss. This observation as well as shortened life span previously reported was partially reverted by co-injection of dsRNA against CsDRONC, showing that we could reverse this effect with a "double knock down". Our results demonstrate that injected dsRNA and RNAi can be directed to the midge midgut, which is the initial site of virus infection . We also provide evidence that the core cell death pathway in midges be experimentally activated in the midgut to shorten the life span. Both of these results pave the way for future analyses midgut-virus interactions in midges, including the studies of antiviral defenses in the gut.

Technical Abstract: Background: Culicoides biting midges transmit multiple ruminant viruses, including bluetongue virus and epizootic hemorrhagic disease virus, causing significant economic burden worldwide due to trade restrictions and production loss. To limit the spread of these viruses, control strategies focus on vaccine development, livestock management, and vector control. To further enhance these control techniques, understanding vector-virus interactions within the midge is critical. Results: We previously developed a double-stranded (ds)RNA delivery methods to induce RNA interference (RNAi) for targeted gene knockdown in adult Culicoides sonorensis using the midge inhibitor of apoptosis gene 1 (IAP1) ortholog (CsIAP1). Here, we confirm CsIAP1 as the functional ortholog of IAP1, identify the ortholog of the initiator caspase DRONC (CsDRONC), and demonstrate that injection of dsRNA into the hemocoel can be used for targeted knockdown in the midgut in C. sonorensis. We observed a statistically significant reduction of CsIAP1 transcripts in whole midges at two and five days post injection, with highest transcript reduction in midgut tissues. IAP1knockdown (kd) resulted in a three-fold increase in overall caspase activity and detection of cleaved caspase-3 in midgut tissues. In IAP1kd midges, midgut tissue integrity and size was severely compromised, presumably caused by cell loss through apoptosis. This phenotype, as well as shortened life span previously reported for IAP1kd, was partially reverted by co-injection of dsRNA against CsDRONC and CsIAP1. Conclusions: This study revealed that RNA interference in C. sonorensis mainly, if not solely relies on environmental RNAi, where the RNAi trigger has to be taken up by each target cell, and RNAi trigger dose correlates positively with transcript kd levels and severety of phenotype. Our data further demonstrate that RNAi can be directed to the midge midgut, the initial site of virus infection using long dsRNA injection into the hemocoel. Finally, we provide evidence that the core apoptosis pathway is conserved in C. sonorensis, and can be experimentally activated in the midgut to shorten the life span in C. sonorensis. This study thus paves the way for future reverse genetic analyses of midgut-virus interactions in C. sonorensis, including the putative antiviral properties of RNAi and apoptosis pathways.