The testes transcriptome of the New World Screwworm, Cochliomyia hominivorax

The New World Screwworm (NWS), Cochliomyia hominivorax, is a pest insect that is endemic to subtropical and tropical regions of the Western Hemisphere. The female lays eggs in open wounds or orifices of warm-blooded animals. Upon hatching, the resulting larvae feed upon the host׳s living tissues, which can become infected and death can occur. The sterile insect technique was developed to eradicate this pest from North America and new female conditional-lethal strains that generate only male individuals are being developed for use in the eradication program. To facilitate the identification of useful transcripts and gene promoters for these new strains, we used an Illumina Hi-Seq protocol to sequence the testes transcriptome of NWS. We report the assembly of 4149 transcripts (≥200 nt) from testes dissected from NWS males obtained from the J06 strain used in the screwworm production plant in Pacora, Panama. Functional annotation resulted in 2060, 2031, 558, and 325 transcripts with assigned BlastX, Gene Ontology, Enzyme Codes, and KEGG pathway information, respectively. In the Gene Ontology annotations, 6% and 3% of the transcripts in the Biological Process Ontology were noted as Developmental Process and Reproduction, respectively. This data set will serve as a resource to facilitate studies of sex determination in the NWS and the development of recombinant vectors that can be used to create new male-only strains of NWS.


Value of Data
Testes-specific transcript sequences to supplement the available NWS transcriptomes previously reported for embryos, larvae, adult male, and adult female [1].
Resource for investigations of sex-specific gene expression and sex determination pathways. Provides candidate protein coding regions and gene promoters for the development of recombinant vectors that can be used to create new male-only strains of NWS.

Data
Testes were dissected out of male NWS from Pacora, Panama. Following RNA isolation, 1 lane of 2 Â 54 paired end RNAseq reads were obtained, de novo assembled and annotated. The raw reads are accessible at NCBI's SRA through the direct link http://trace.ncbi.nlm.nih.gov/Traces/sra_sub/sub.cgi? subid ¼707563 or through SRA accession number SRP076734. The assembled transcriptome shotgun assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GEVJ00000000. The version described in this paper is the first version, GEVJ01000000. The overall BioProject ID is PRJNA324578 and the BioSample accession is SAMN05213682.

Testes tissue
One hundred male NWS adults were obtained from the Pacora, Panama screwworm production plant colony and testes dissected out and placed directly into a 1.5 ml microcentrifuge tube containing 100 μl of RNAlater at room temperature (Ambion Inc., Austin, TX, USA). The tissues were shipped on dry ice to the USDA-ARS laboratory for RNA isolation.

RNA isolation
RNA was extracted using the ToTALLY RNA Isolation Kit, following the manufacturer's protocols (Ambion). A disposable pellet pestle (Kontes Inc., Vineland, NY, USA) was used to grind the tissue in the microcentrifuge tube, using a final volume of 700 μl of the ToTALLY RNA kit's Denaturation Buffer. The optional LiCl precipitation step to selectively precipitate RNA was incorporated into our protocol and at this point 45 μg of nucleic acids resulted. Agarose gel electrophoresis determined the integrity of the nucleic acid sample was good, but DNA was still present. Following DNAse treatment with the Turbo DNA-free kit and protocols (Ambion), 40 μg of DNA-free RNA was obtained.

Sequencing and bioinformatics.
Sequencing was performed at the National Center for Genome Resources (Santa,Fe, NM, USA) using the standard Illumina RNAseq library preparation protocol and a single lane of the RNAseq 2 Â 54 paired-end approach. A total of 72750822 raw reads were produced and quality preassessment performed by the Illumina pipeline and the contaminant filtering pipeline developed at National Center for Genome Resources. De novo assembly of the transcriptome used an iterative k-mer strategy in ABySS [2] followed by gap closure and additional assembly using overlap methods in both MIRA [3] and Cap3 [4]. The assembled contigs were screened at submission to the NCBI Transcriptome Shotgun Assembly (TSA) database using the NCBI foreign contamination screen protocol. Supplementary File 1 contains the FastA sequences of the final assembled dataset of 4149 entries Z200 nt. The assembled transcripts were screened using Blast2GO PRO version 1.4 plugin CloudBlastX [5][6][7] on the CLC Genomics Workbench version 8.0.1 (http://www.clcbio.com, CLC Inc, Aarhu, Denmark) against the UniProtKB/Swiss-Prot database using 1.0E-10 e-Value cutoff. The transcripts with CloudBlast hits were mapped using Blast2GO PRO Mapping and GO Annotation performed using Blast2GO PRO Annotation [8]. KEGG pathway maps were determined using Blast2GO Basic version 3.1.3. Statistics from the different steps of annotation of the transcripts can be found in Table 1 and a hierarchy summary in Fig. 1. Fig. 2 shows the functional annotation of C. hominivorax testes transcripts for Gene Ontology Level 2 terms for biological process, cellular component and molecular function. Transcript information including CloudBlastX hits, GO terms, InterProScan, Enzyme Codes and KEGG pathway data can be found in Supplementary Table 1.