An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans

https://doi.org/10.1016/j.ibmb.2009.06.004Get rights and content

Abstract

Adult stable flies are blood feeders, a nuisance, and mechanical vectors of veterinary diseases. To enable efficient feeding, blood sucking insects have evolved a sophisticated array of salivary compounds to disarm their host's hemostasis and inflammatory reaction. While the sialomes of several blood sucking Nematocera flies have been described, no thorough description has been made so far of any Brachycera, except for a detailed proteome analysis of a tabanid (Xu et al., 2008). In this work we provide an insight into the sialome of the muscid Stomoxys calcitrans, revealing a complex mixture of serine proteases, endonucleases, Kazal-containing peptides, anti-thrombins, antigen 5 related proteins, antimicrobial peptides, and the usual finding of mysterious secreted peptides that have no known partners, and may reflect the very fast evolution of salivary proteins due to the vertebrate host immune pressure. Supplemental Tables S1 and S2 can be downloaded from http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T1/Sc-tb1-web.xls and http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T2/Sc-tb2-web.xls.

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

References (66)

  • T.J. Milne et al.

    Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

    J. Biol. Chem.

    (2003)
  • J.M. Ribeiro et al.

    An insight into the salivary transcriptome and proteome of the adult female mosquito Culex pipiens quinquefasciatus

    Insect Biochem. Mol. Biol.

    (2004)
  • J.M.C. Ribeiro et al.

    Salivary apyrase activity of some old world phlebotomine sand flies

    Insect Biochem.

    (1989)
  • B. Schlott et al.

    Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies

    J. Mol. Biol.

    (2002)
  • M.C. Schreiber et al.

    A novel cDNA from Drosophila encoding a protein with similarity to mammalian cysteine-rich secretory proteins, wasp venom antigen 5, and plant group 1 pathogenesis-related proteins

    Gene

    (1997)
  • K.R. Stark et al.

    Isolation and characterization of the gene encoding a novel factor Xa-directed anticoagulant from the yellow fever mosquito, Aedes aegypti

    J. Biol. Chem.

    (1998)
  • J.G. Valenzuela et al.

    Purification, cloning, and expression of an apyrase from the bed bug Cimex lectularius. A new type of nucleotide-binding enzyme

    J. Biol. Chem.

    (1998)
  • J.G. Valenzuela et al.

    Toward a description of the sialome of the adult female mosquito Aedes aegypti

    Insect Biochem. Mol. Biol.

    (2002)
  • J.G. Valenzuela et al.

    Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito

    Insect Biochem. Mol. Biol.

    (2003)
  • X. Xu et al.

    Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands

    Mol. Cell. Proteomics

    (2008)
  • Y. Yamazaki et al.

    Wide distribution of cysteine-rich secretory proteins in snake venoms: isolation and cloning of novel snake venom cysteine-rich secretory proteins

    Arch. Biochem. Biophys.

    (2003)
  • Y. Yamazaki et al.

    Structure and function of snake venom cysteine-rich secretory proteins

    Toxicon

    (2004)
  • D. Zhang et al.

    Thrombostasin: purification, molecular cloning and expression of a novel anti-thrombin protein from horn fly saliva

    Insect Biochem. Mol. Biol.

    (2002)
  • S.F. Altschul et al.

    Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

    Nucleic Acids Res.

    (1997)
  • M. Ameri et al.

    An immunoglobulin binding protein (antigen 5) of the stable fly (Diptera: Muscidae) salivary gland stimulates bovine immune responses

    J. Med. Entomol.

    (2008)
  • J.F. Andersen et al.

    The role of salivary lipocalins in blood feeding by Rhodnius prolixus

    Arch. Insect Biochem. Physiol.

    (2005)
  • J.F. Andersen et al.

    Insight into the sialome of the black fly, Simulium vittatum

    J. Proteome Res.

    (2009)
  • B. Arca et al.

    An updated catalogue of salivary gland transcripts in the adult female mosquito, Anopheles gambiae

    J. Exp. Biol.

    (2005)
  • M. Ashburner et al.

    Gene ontology: tool for the unification of biology. The Gene Ontology Consortium

    Nat. Genet.

    (2000)
  • A. Bateman et al.

    The Pfam protein families database

    Nucleic Acids Res.

    (2000)
  • J.D. Bendtsen et al.

    Feature-based prediction of non-classical and leaderless protein secretion

    Protein Eng. Des. Sel.

    (2004)
  • E. Calvo et al.

    The transcriptome of adult female Anopheles darlingi salivary glands

    Insect Mol. Biol.

    (2004)
  • E. Calvo et al.

    A novel secreted endonuclease from Culex quinquefasciatus salivary glands

    J. Exp. Biol.

    (2006)
  • Cited by (32)

    • An insight into the female and male Sabethes cyaneus mosquito salivary glands transcriptome

      2023, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      Cyaneus female SGs, antigen 5 and mucins have been detected in all mosquito species (Calvo et al., 2007a, 2008, 2009a, 2010a, 2010b; Chagas et al., 2013; Arcà et al., 2005; Francischetti et al., 2002; Valenzuela et al., 2002, 2003; Ribeiro et al., 2016; Lombardo et al., 2007). Antigen 5 family are widespread in insect and tick sialomes suggesting a unique association to hematophagy, including the inhibition of platelet aggregation in a tabanid salivary protein (Xu et al., 2008), immunoglobulin binding (Ameri et al., 2008), and putative inhibitor of the classic pathway of complement in the stable fly Stomoxys calcitrans (Wang et al., 2009). Mucins probably function by coating and lubricating the salivary channels and food canals, and may also have antimicrobial properties (Calvo et al., 2009a; Ribeiro et al., 2010).

    • Molecular tools used in medical and veterinary entomology

      2018, Medical and Veterinary Entomology
    • An insight into the sialome of the horse fly, Tabanus bromius

      2015, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      Further analysis of this family reveals members with additional disintegrin domains and agonist binding pockets indicative of functional differences acquired by gene expansion of the tablysin family. It should be remarked that these characteristics are different from those of other higher flies that evolved independently to hematophagy such as the tsetse Glossina morsitans morsitans (Alves-Silva et al., 2010), which do not sugar feed, have no salivary glycosidases and have large amounts on an inactive endonuclease with unknown function and various unique peptide families, or Stomoxys calcitrans (Wang et al., 2009) that have large amounts of a non-RGD antigen 5 family that binds immunoglobulins (Ameri et al., 2008) and have their own additional unique families. In common with other sialomes, apyrase, hyaluronidase, endonuclease, peroxidase, phospholipase, serine protease, dipeptidyl proteases, as well as protease inhibitors and immunity related transcripts are members of the salivary cocktail.

    • An insight into the sialome of blood-feeding Nematocera

      2010, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      Its members are associated with host defenses in plants, and various functions in animals, such as toxins in snake and lizard venoms (Nobile et al., 1996; Yamazaki et al., 2002), proteolytic activity in Conus snails (Milne et al., 2003), and platelet aggregation inhibition in a tabanid salivary protein (Xu et al., 2008), although this latter function results from the novel incorporation of an RGD domain that functions as a disintegrin. A protein member of this family, found expressed in the salivary glands of the stable fly Stomoxys calcitrans binds immunoglobulin (Ameri et al., 2008) and may function as an inhibitor of the classic pathway of complement (Wang et al., 2009). The variety of functions that this family is endowed with precludes assigning a function to any of the proteins listed in Supplemental file S1, none of which has been characterized functionally.

    View all citing articles on Scopus
    View full text