An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans
Introduction
The habit of blood feeding evolved independently several times in Diptera (Grimaldi and Engel, 2005), including the Nematocera and Brachycera. Evolution to blood feeding is associated with the expression of specialized saliva that is pharmacologically active against vertebrate hemostasis and inflammation. Because inflammation and hemostasis are complex and redundant phenomena, the salivary potion of blood feeders is also complex, containing vasodilators, anti-clotting substances and enzymes that destroy vertebrate agonists and matrix components (Ribeiro, 1995). Sialotranscriptome analysis (from the Greek, Sialo = Saliva) of mosquitoes, sand flies, biting midges and black flies are revealing a vast repertoire of polypeptides recruited to serve this function (Andersen et al., 2009, Arca et al., 2007, Arca et al., 2005, Calvo et al., 2004, Campbell et al., 2005, Ribeiro et al., 2004, Valenzuela et al., 2003).
It has been proposed that all blood sucking Nematocera, except for sand flies, have a common blood-feeding ancestor (Grimaldi and Engel, 2005). While the blood-feeding Nematocera salivary proteins have some common protein families, such as the very divergent D7 protein family also found in sand flies (Valenzuela et al., 2002a), most other protein families are family or genus specific. For example, the salivary anti-clotting protein of Aedes aegypti is a member of the serpin family (Stark and James, 1998), while a novel peptide named anophelin is an anti-thrombin in Anopheles albimanus (Valenzuela et al., 1999). As another example, while salivary apyrase activity, responsible for ADP hydrolysis, is found in both sand flies and mosquitoes, the gene families recruited for these functions are completely different in these two organisms (Champagne et al., 1995, Valenzuela et al., 2001). While the Brachycera contain diverse families of blood sucking arthropods of medical and veterinary importance including the tsetse, tabanids, keds and stable flies, no detailed transcriptome analysis has been done so far from any of these flies, although a detailed sialoproteome analysis has been made from the tabanid Tabanus yao (Xu et al., 2008).
The stable fly, Stomoxys calcitrans is an important pest of cattle, their larvae growing in decaying faeces and straw, the adults biting and blood feeding on mammals (Campbell et al., 2001, Taylor and Berkebile, 2006). They can also vector pathogens by mechanical transmission. It is the aim of this study to provide a preliminary description of the sialome of S. calcitrans.
Section snippets
Insects
Stable flies were reared from a colony collected in Manhattan, KS in 1990. Eggs were collected with a wet black cloth wick. The larvae were reared in a fermented mixture of wheat bran (Farmers' Cooperative Association, Frederick, MD), vermiculite (Hummert International, St. Louis, MO), and Calf Manna (Manna PRO Corporation, Chesterfield, MO). Adult flies were fed with 1 M sucrose or Gatorade. For reproduction, the flies were fed daily with citrated bovine blood supplied in saturated cotton pads.
Overview of the assembled salivary EST set
A total of 820 cDNA clones were used to assemble a database (Supplemental Table S1) that yielded 240 clusters of related sequences, 187 of which contained only one EST. The 240 clusters were compared, using the programs blastx, blastn, or RPS-BLAST (Altschul et al., 1997), to the nonredundant (NR) protein database of the National Center of Biological Information (NCBI), National Library of Medicine, NIH, to a gene ontology database (Ashburner et al., 2000), to the conserved domains database of
Conclusions
From the analysis of previous sialotranscriptomes, it is becoming clear that the “generic” salivary potion of any blood-feeding arthropod, even those not descending from a common blood sucker ancestor, consists of the somewhat unrelated classes of enzymes, protease inhibitors, vasodilator agonists, serotonin- and histamine-binding proteins, antigen 5 proteins, antimicrobial peptides and a large group of mysterious, unknown secreted proteins. This disjointed list is found in triatomines,
Acknowledgements
This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, NIH Grant GM41247, and Kansas State University Agricultural Experimental Station Grant, 418343. We thank Kent Hampton for insect rearing.
Because JMCR is a government employee and this is a government work, the work is in the public domain in the United States. Notwithstanding any other agreements, the NIH
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