Analysis of expressed sequence tags from a significant livestock pest, the stable fly (Stomoxys calcitrans), identifies transcripts with a putative role in chemosensation and sex determination

Abstract

The stable fly, Stomoxys calcitrans L. (Diptera: Muscidae), is one of the most significant pests of livestock in the United States. The identification of targets for the development of novel control for this pest species, focusing on those molecules that play a role in successful feeding and reproduction, is critical to mitigating its impact on confined and rangeland livestock. A database was developed representing genes expressed at the immature and adult life stages of the stable fly, comprising data obtained from pyrosequencing both immature and adult stages and from small-scale sequencing of an antennal/maxillary palp-expressed sequence tag library. The full-length sequence and expression of 21 transcripts that may have a role in chemosensation is presented, including 13 odorant-binding proteins, 6 chemosensory proteins, and 2 odorant receptors. Transcripts with potential roles in sex determination and reproductive behaviors are identified, including evidence for the sex-specific expression of stable fly doublesex- and transformer-like transcripts. The current database will be a valuable tool for target identification and for comparative studies with other Diptera.

Discovery of MicroRNAs of the Stable Fly (Diptera: Muscidae) by High-Throughput Sequencing

“…cgi), and additional manual curation of available S. calcitrans nucleotide and EST data (http://www. ncbi.nlm.nih.gov/nuccore/?termϭstomoxys_calcitrans; Wang et al 2009, Olafson et al 2010. For analysis of strict conservation of identiÞed mature stable ßy miRNAs with the closest matching miRBase entries for other species (i.e., beyond seed conservation), stable ßy miRNA sequences identiÞed and clustered from the analysis were further subjected to BLAST analysis (word size ϭ 16; CLC Main Workbench v6.6.2, CLC bio, Aarhus, Denmark) against all mature miRNA sequences (ftp://mirbase.org/pub/mirbase/CURRENT/ mature.fa.gz) contained in miRBase 18.…”

Use of Pyrosequencing and Denaturing Gradient Gel Electrophoresis to Examine the Effects of Probiotics and Essential Oil Blends on Digestive Microflora in Broilers Under MixedEimeriaInfection

“…Bacterial tag-encoded FLX amplicon pyrosequencing was performed as described previously using Gray28F 5¢TTTGATCNTGGCTCAG and Gray519r 5¢GTNTTACNG CGGCKGCTG (Bailey et al, 2010a,b;Callaway et al, 2010;Finegold et al, 2010;Gontcharova et al, 2010a,b;Olafson et al, 2010;Pitta et al, 2010;Smith et al, 2010;Stephenson et al, 2010;Williams et al, 2010;Andreotti et al, 2011;Handl et al, 2011;Ishak et al, 2011) with primers numbered relative to Escherichia coli 16S rDNA. Initial generation of the sequencing library utilized a one-step polymerase chain reaction (PCR) with a total of 30 cycles, a mixture of HotStart and HotStar high fidelity taq polymerases (Qiagen), and amplicons originating and sequencing extending from the 28F with average read length of 400 bp.…”

“…After sequencing, all failed sequence reads, low quality sequence ends and tags, and primers were removed and sequence collections depleted of any nonbacterial ribosome sequences and chimeras using B2C2 (Gontcharova et al, 2010b) as previously described (Bailey et al, 2010a,b;Finegold et al, 2010;Gontcharova et al, 2010a;Olafson et al, 2010;Pitta et al, 2010;Smith et al, 2010;Stephenson et al, 2010;Williams et al, 2010;Andreotti et al, 2011;Handl et al, 2011;Ishak et al, 2011). To identity the remaining bacterial sequences, sequences were denoised, assembled into clusters, and queried using a distributed BLASTn .NET algorithm (Dowd et al, 2005) against a high-quality 16S bacterial sequence database (based upon similar criteria utilized by RDP ver 9 (Cole et al, 2009)) derived and curated monthly from NCBI (01-01-11).…”

“…To identity the remaining bacterial sequences, sequences were denoised, assembled into clusters, and queried using a distributed BLASTn .NET algorithm (Dowd et al, 2005) against a high-quality 16S bacterial sequence database (based upon similar criteria utilized by RDP ver 9 (Cole et al, 2009)) derived and curated monthly from NCBI (01-01-11). Using a .NET and C# analysis pipeline, the resulting BLASTn outputs were compiled, validated using taxonomic distance methods, and data reduction analysis performed (Bailey et al, 2010a,b;Callaway et al, 2010;Finegold et al, 2010;Gontcharova et al, 2010a,b;Olafson et al, 2010;Pitta et al, 2010;Smith et al, 2010;Stephenson et al, 2010;Williams et al, 2010;Andreotti et al, 2011;Handl et al, 2011;Ishak et al, 2011).…”

“…Sequences with identity scores, to known or well-characterized 16S sequences, > 97% identity ( < 3% divergence) were resolved at the species level, between 95% and 97% at the genus level, between 90% and 95% at the family, and between 85% and 90% at the order level, 80% and 85% at the class, and 77% to 80% at phyla. Evaluations presented at each taxonomic level, including percentage compilations, represent all sequences resolved to their primary identification or their closest relative (Bailey et al, 2010a,b;Callaway et al, 2010;Finegold et al, 2010;Gontcharova et al, 2010a,b;Olafson et al, 2010;Pitta et al, 2010;Smith et al, 2010;Stephenson et al, 2010;Williams et al, 2010;Andreotti et al, 2011;Handl et al, 2011;Ishak et al, 2011).…”