2013 Annual Report
1a.Objectives (from AD-416):
Generate and analyze transcriptomes for female Culicoides sonorensis in order to assess the functional role of genes in important biological processes including blood and sugar feeding and digestion, and blue tongue virus infection outcome. This type of RNA catalogue will be the first of its kind for C. sonorensis and an invaluable resource for exploiting the functional elements of the midge.
1b.Approach (from AD-416):
De novo sequencing, assembly and annotation of transcriptomes for female Culicoides sonorensis including: (1) teneral state (2) blood fed (3) sugar fed (4) blue tongue virus infected (5) blue tongue virus recovered. Two-day-old unmated females will remain unfed (teneral), or will be fed blood, sugar water, or blue tongue virus (in blood). Fed flies will be killed for RNA extraction 3 days post feeding, and for virus infection studies, virus presence or absence (“infected” vs. “recovered”) will be determined before pooling midges accordingly.
A paired-end (PE) library will be constructed from RNA of whole, pooled female midges (minimum n=10) subjected to each condition above, with two biological replicates for each. In the absence of a reference genome for transcript mapping, trimmed PE sequences will be subject to de novo clustering and assembly into a set of contigs and singletons with suitable software such as Oases, Trinity, Velvet, Trans-ABySS, or RNnotator. This assembly will represent the unigene for each sample. Each individual sample/treatment will be analyzed for unique functional signatures; the unigenes will be assigned gene ontology (GO) terms, and the enzyme codes and pathways determined as follows. The unigene assembly will be the subject for functional annotation using REFseq gene models. Annotation will be performed using the Blast2GO software package, where unigene sequences will be BLASTed to the non-redundant protein database (GenBank), mapped to GO term database, and a GO annotation returned. Enzyme codes will be obtained by mapping GO terms associated with each unigene, and functional protein signatures determined by using the InterProScan service. Enzyme codes will be highlighted on KEGG biochemical pathway maps. Thus, from the transcriptome sequencing we will identify proteins involved in the biological processes of interest, and their function in cellular-biochemical pathways (i.e., functional transcriptomics).
Comparative transcriptomics will be used to compare (1) teneral to blood to sugar feeding processes and (2) blood to virus infected to virus recovered processes. These analyses will allow us to look at the differential expression of genes across conditions compared to their appropriate control state. For comparative transcriptomics, all biological replicates will be binned, normalized, and compared to identify and quantify differentially expressed transcripts as a result of various treatments. Quantitative differential expression analysis, and determination of spliceforms and paralogs will be used to assess genes and cellular processes involved in the biology of this vector when comparing the conditions/states described above. Gene expression levels will be quantified and labeled as upregulated, downregulated, or no change when compared to control level gene expression (teneral for blood and sugar fed; blood fed for virus infected or recovered) using EdgeR software. Statistical assessment of enrichment of GO terms between the sequence datasets will be determined with the Gossip software, which employs a Fisher’s exact test.
The objectives of the Culicoides sonorensis transcriptome project are two-fold: The first objective is to generate a comprehensive adult female gene catalogue, and secondly, to analyze on a global scale the functional role and gene dynamics responsible for critical biological processes such as blood and sugar feeding, digestion, reproduction, and early responses to virus infection. This type of gene expression catalogue is the first of its kind for Culicoides sonorensis and an invaluable resource for exploiting the functional elements and biochemical properties of the female midge. Objective 1 is complete and significant progress on objective 2 has been made. Briefly, total RNA was extracted and sequenced (in duplicate) from teneral, early/late sugar, and early/late blood fed midges and subject to complete transcriptome sequencing. Cumulatively, we collected 257 million read pairs of sequence (52 gigabases total) among the treatment conditions and replicates, which was subject to gene annotation and differential expression analysis. Assembly of the first adult female Culicoides unigene was performed and resulted in a total of 19,041 genes ranging from 300bp to 7.8kb with an average size of 1.3kb. These unigenes were annotated and treatment transcriptomes were mapped back from (teneral, early/late blood, and early/late sugar) to determine global gene responses to the various feeding conditions. As expected, we identified key genes involved in important biological processes including digestion and transport of blood or sugar meals, reproduction (such as egg production), hematophagy (such as salivary gland specific sequences), and defense responses. This project is ongoing and the immediate results will be a critical milestone and turning point for vector biology studies such as this.