Location: Arthropod-Borne Animal Diseases Research
Title: Update on the Culicoides sonorensis transcriptome project: a peek into the molecular biology of the midge Authors
|Saski, Christopher -|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 7, 2013
Publication Date: N/A
Technical Abstract: Next Generation Sequencing is transforming the way scientists collect and measure an organism’s genetic background and gene dynamics, while bioinformatics and super-computing are merging to facilitate parallel sample computation and interpretation at unprecedented speeds. Analyzing the complete gene catalogue (or transcriptome) expressed under particular circumstances affords researchers the ability to identify key genetic elements that are critical to a successful response to the stimulus, as well as an opportunity to peek into the molecular biological operations of the organism under those conditions. Sequencing the entire gene catalogue (RNAseq) is an explosive technology that has become more affordable and as a result transformed the way we view the extent and complexity of eukaryotic transcriptomes. RNAseq comprehensively measures even the rarest genes at nucleotide resolution and is now a routine approach to cataloging and exploring the functional elements of the genome. Using RNAseq, we explored the adult female Culicoides sonorensis transcriptome (gene structure and gene expression patterns) to dissect and quantify the genes involved in the teneral state as well as blood and sugar feeding over time. Culicoides sonorensis is a primary vector for bluetongue and epizootic hemorrhagic disease which both impact cervids, sheep and cattle in North America. Our study is the first of its kind for this midge, whose genome has not yet been published, and is aimed at understanding the expression of genes associated with the midge’s function on a cellular, physiological and molecular level. Through these studies we hope identify key genes can help reveal potential “intervention points” involved in the midge biological processes as well as vector competence. We sequenced the transcriptome of the adult female Culicoides and identified a total of 19,041 genes. To annotate and determine potential functional roles, we BlastX aligned the complete unigene set to the closest well-annotated phylogenetic species (Aedes aegypti and Culex quinquefasciatus) and the non-redundant (NR) peptide database at NCBI (E-value cutoff of = 1e-5); this resulted in14,512 (76%), 14,601 (76%), and 16,000 (84%) homologous sequences in Aedes, Culex, and NR, respectively. There were 3,041 unigenes that showed no homology to sequences deposited in these databases. Gene Ontology (GO) terms were assigned to the complete unigene set with the Blast2GO software, which allowed us to broadly look at biological processes. Comparative analysis across several different transcriptomes (teneral, and early and late blood or sugar feeding; two biological replicates of each) identified 10,000 genes that were differentially expressed in blood-fed compared to unfed/teneral midges and the expression of ~1,200 genes differed between teneral and sugar fed midges. The greatest differences in gene expression were seen between the teneral and early blood fed libraries (8,414 genes), teneral and late blood fed libraries (5,413 genes) and early and late blood fed libraries (7,334 genes). Our analysis also revealed that teneral and early sugar gene expression was very similar, with only 140 genes differing in expression between these two conditions. Similarly, early and late sugar expression profiles only differed by 114 genes. Many of the genes we identified in the overall unigene were associated with housekeeping processes, blood or sugar meal digestion and transport. Remarkably, we also were able to identify genes associated with hematophagy (e.g. salivary gland proteins), vitellogenesis, reproduction, and antimicrobial defense. Additionally, differential expression analysis revealed the temporal changes in expression of these genes in response to the type of ingested meal (sugar or blood) which gave us insight into the role feeding alone has on the biology of this important vector species.