The Reference Transcriptome of the Adult Female Biting Midge (Culicoides sonorensis) and Differential Gene Expression Profiling during Teneral, Blood, and Sucrose Feeding Conditions

Abstract

Unlike other important vectors such as mosquitoes and sandflies, genetic and genomic tools for Culicoides biting midges are lacking, despite the fact that they vector a large number of arboviruses and other pathogens impacting humans and domestic animals world-wide. In North America, female Culicoides sonorensis midges are important vectors of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), orbiviruses that cause significant disease in livestock and wildlife. Libraries of tissue-specific transcripts expressed in response to feeding and oral orbivirus challenge in C. sonorensis have previously been reported, but extensive genome-wide expression profiling in the midge has not. Here, we successfully used deep sequencing technologies to construct the first adult female C. sonorensis reference transcriptome, and utilized genome-wide expression profiling to elucidate the genetic response to blood and sucrose feeding over time. The adult female midge unigene consists of 19,041 genes, of which less than 7% are differentially expressed during the course of a sucrose meal, while up to 52% of the genes respond significantly in blood-fed midges, indicating hematophagy induces complex physiological processes. Many genes that were differentially expressed during blood feeding were associated with digestion (e.g. proteases, lipases), hematophagy (e.g., salivary proteins), and vitellogenesis, revealing many major metabolic and biological factors underlying these critical processes. Additionally, key genes in the vitellogenesis pathway were identified, which provides the first glimpse into the molecular basis of anautogeny for C. sonorensis. This is the first extensive transcriptome for this genus, which will serve as a framework for future expression studies, RNAi, and provide a rich dataset contributing to the ultimate goal of informing a reference genome assembly and annotation. Moreover, this study will serve as a foundation for subsequent studies of genome-wide expression analyses during early orbivirus infection and dissecting the molecular mechanisms behind vector competence in midges.

Inducing RNA interference in the arbovirus vector, Culicoides sonorensis

“…Previous work demonstrated that a cloned C. sonorensis cell line was capable of environmental RNAi, and provided support for the potential application of reverse genetics in this species. As the first step to examine whether a functional siRNA pathway exists in adult C. sonorensis, we queried the recently published transcriptome (Nayduch et al, 2014b). Using Aedes aegypti siRNA pathway protein sequences as queries, multiple C. sonorensis transcripts with sequence similarity to AGO2, Dicer2 and R2D2 were identified by best reciprocal BLAST hits.…”

“…Transcript levels of CsIAP1 were determined by quantitative reverse transcription PCR (qRT-PCR) 5 days post-injection (dpi). Elongation factor 1b (EF1b, GAWM01010754) was used as the reference gene, as its expression remains constant during multiple physiological processes (Nayduch et al, 2014b). CsIAP1 expression was reduced by 40% in dsCsIAP1-treated midges relative to both dsGFP-injected and untreated midges (one-way ANOVA, P < 0.001, Newman−Keuls post test P < 0.05; Fig.…”

“…: + 1 785 532 0161; e-mail: kmichel@ ksu.edu and cell lines exist (Nayduch et al, 2014a). In addition, a number of transcriptome studies has been published (Campbell & Wilson, 2002;Campbell et al, 2005), most recently using RNAseq data (Nayduch et al, 2014b). Some of these C. sonorensis gene products are potential targets for new vector control strategies, but require gene function analysis at the molecular level.…”

Molecular ecology of insect pests of agricultural importance: the case of aphids

“…For example, it is now possible to study the responses to different environmental factors and identify the genetic bases of phenotypes by sequencing the transcriptomes of individuals, tissues or cells and analysing the genes expression levels (e.g. Nayduch et al, 2014;Keeling et al, 2016;Wu et al, 2016;Yu et al, 2016;Braden et al, 2017). Similarly, they have also revolutionised the area of phylogeography and evolution allowing the discovery of genome-wide molecular markers that could not be used before to study genetic variation in natural populations.…”

Management of North American Culicoides Biting Midges: Current Knowledge and Research Needs

“…Although the utilization of molecular methods for the study of Culicoides vector biology has expanded very recently (Nayduch et al 2014a), these tools have not yet been brought to bear on control tactics. However, the recent release of the C. sonorensis reference transcriptome (Nayduch et al 2014b) as well as a demonstration of RNA interference (RNAi) in vivo (Mills et al 2015) present new tools in the molecular biological toolbox to use in vector competence studies. These advancements will allow scientists to identify physiological and genetic mechanisms related to pathogen defense (Nayduch et al 2014c) and use reverse genetics approaches to elucidate their role in vector competence.…”