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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » ABADRU » Research » Publications at this Location » Publication #308018
Title: Inducing RNA interference in the arbovirus vector, Culicoides sonorensis

Author
item MILLS, MARY - Kansas State University
item Nayduch, Dana
item MICHEL, KRISTIN - Kansas State University

Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2014
Publication Date: 2/1/2015
Citation: Mills, M.K., Nayduch, D., Michel, K. 2015. Inducing RNA interference in the arbovirus vector, Culicoides sonorensis. Insect Molecular Biology. Feb;24(1):105-14. DOI: 10.1111/imb.12139. Epub 2014 Oct 7.

Interpretive Summary: Culicoides midges transmit arboviruses that cause significant diseases in ruminant livestock and wildlife in the US and worldwide. Research on these important insect vectors has been hindered by lack of molecular tools and resources such as a sequenced genome and ways to manipulate gene expression. One tool widely used in studying gene function is RNA-interference or RNAi, which can be used to effectively silence gene expression in vitro so that the gene’s function can be assessed. Here, we report the establishment of RNAi as a research tool for the adult female biting midge Culicoides sonorensis. A database from our previous study was mined to identify the genes involved in the RNAi machinery, and key components were found. A successful delivery mechanism for double-stranded RNA (used in the RNAi technique) was developed, and was subsequently used to “silence” the expression an inhibitor of apoptosis (IAP, involved in cell death) to demonstrate proof of concept. Successful silencing of the IAP gene was confirmed by both molecular methods (knockdown of mRNA confirmed by qRTPCR) and by an observed phenotype (increased mortality in RNAi-treated midges). mRNA transcript level decreased significantly by 40% in RNAi-treated midges compared to normal levels. Median survival in RNAi-treated midges was shortened to less than 8 days compared to 30 days in appropriate controls. These results demonstrate RNAi as an exploitable method in the molecular toolset used to study midges. This tool can help in future studies aimed at understanding genetic components underlying the vector competence of C. sonorensis.

Technical Abstract: Biting midges in the genus Culicoides are important vectors of arboviral diseases, including Epizootic Hemorrhagic Disease, Bluetongue, and likely Schmallenberg, which cause significant economic burden worldwide. Research on these vectors has been hindered by the lack of a sequenced genome, the difficulty of consistent culturing of certain species, and the absence of molecular techniques such as RNA interference (RNAi). Here, we report the establishment of RNAi as a research tool for the adult midge, Culicoides sonorensis. Based on previous research and transcriptome analysis, which revealed putative siRNA pathway member orthologs, we hypothesized that adult C. sonorensis midges have the molecular machinery needed to preform RNA silencing. The development of an injection protocol allowed for double-stranded (ds)RNA microinjection into the hemocoel of adult female midges. Using CO2 exposure as a means to immobilize adult midges, injection of control dsRNA, dsGFP, into 2-3 day old adult female midges resulted in survival curves that support virus transmission. DsRNA injection targeting the newly identified C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog, resulted in increased mortality rates, with median survivals shortened to less than eight days compared to 30 days in dsGFP-injected controls. CsIAP1 transcript levels were decreased significantly by 40% five days post injection. These results reveal the conserved function of IAP1. Importantly, they also demonstrate the feasibility of RNAi by dsRNA injection in adult midges, which will greatly facilitate studies of the underlying mechanisms of vector competence in C. sonorensis.