An insight into the sialotranscriptome of Simulium nigrimanum, a black fly associated with fogo selvagem in South America

Abstract

Pemphigus foliaceus is a life threatening skin disease that is associated with autoimmunity to desmoglein, a skin protein involved in the adhesion of keratinocytes. This disease is endemic in certain areas of South America, suggesting the mediation of environmental factors triggering autoimmunity. Among the possible environmental factors, exposure to bites of black flies, in particular Simulium nigrimanum has been suggested. In this work, we describe the sialotranscriptome of adult female S. nigrimanum flies. It reveals the complexity of the salivary potion of this insect, comprised by over 70 distinct genes within over 30 protein families, including several novel families, even when compared with the previously described sialotranscriptome of the autogenous black fly, S. vittatum. The uncovering of this sialotranscriptome provides a platform for testing pemphigus patient sera against recombinant salivary proteins from S. nigrimanum and for the discovery of novel pharmacologically active compounds.

Figure 1.

Functional class distribution of expressed sequence tags (EST) or assembled contigs (Contigs) deriving from a salivary gland cDNA library from adult Simulium nigrimanum black flies. This figure appears in color at www.ajtmh.org.

Figure 2.

The 5′-nucleotidase/apyrase family of black flies, mosquitoes, and prokaryotes. (A) Clustal alignment. The Simulium nigrimanum proteins are Sim-214 and Sim187. The remaining sequences are named with the first three letters from the genus name followed by two letters from the species name and by their National Center for Biotechnology Information (NCBI) protein accession number. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. (B) Neighbor joining bootstrapped phylogram of the alignment in A, showing 4 clades. The numbers on the branches represent the percent bootstrap support. The bar in the bottom represents 10% amino acid divergence. Mosquito proteins are indicated by a triangle, Simulium proteins by a square, and bacterial proteins by a circle. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 3.

The 5′-nucleotidase/apyrase family of black flies, mosquitoes, tabanids compared with Drosophila and mammalian enzymes. The Simulium nigrimanum proteins are Sim-214 and Sim187. The remaining sequences are named with the first three letters from the genus name followed by two letters from the species name and by their National Center for Biotechnology Information (NCBI) protein accession number. The box shows the deletion of the membrane anchor region in the salivary apyrases of Diptera. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. This figure appears in color at www.ajtmh.org.

Figure 4.

The Simulium Kunitz salivary protein family. (A) Clustal alignment of 4 S. nigrimanum proteins (starting with Sim-) with 2 Simulium vittatum proteins, indicated by SIMVI_X where X is their National Center for Biotechnology Information (NCBI) accession numbers. The tick anti-thrombin peptide named boophilin (BOOMI_17529564) is included as an out-group. The symbols above the alignment indicate identity (*), high similarity (:), and similarity (.) of residues in the indicated alignment position. (B) Phylogram derived from the alignment in A. Simulium nigrimanum sequences are marked with a square. The numbers on the tree bifurcations indicate the percentage bootstrap support above 50%. The bar at the bottom represents 10% amino acid substitution. Protein sequences were aligned by the Clustal program39 and the dendrogram was done with the Mega package41 after 10,000 bootstraps with the neighbor joining (NJ) algorithm. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 5.

The Simulium D7 salivary protein family. Phylogram deriving from the alignment of S. nigrimanum proteins (starting with Sim-) with those of Simulium vittatum, indicated by SIMVI_X where X is their National Center for Biotechnology Information (NCBI) accession numbers. The numbers on the tree bifurcations indicate the percentage bootstrap support above 75%. The bar at the bottom represents 20% amino acid substitution. Members of the short D7 family are marked with a circle, those from the long family with a square. Protein sequences were aligned by the Clustal program39 and the dendrogram was done with the Mega package41 after 10,000 bootstraps with the neighbor joining (NJ) algorithm. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 6.

The 30 kDa antigen/Aegyptin family of black flies and mosquitoes. The Simulium nigrimanum proteins are indicated by Sim-XX where XX is the number indicated in supplemental file S2. The remaining sequences are named with the first three letters from the genus name followed by two letters from the species name and by their National Center for Biotechnology Information (NCBI) protein accession number. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 7.

The Hyp16 family of blood-sucking Diptera. The Simulium nigrimanum protein is indicated by Sim-692. The remaining sequences are named with the first three letters from the genus name followed by two letters from the species name and by their National Center for Biotechnology Information (NCBI) protein accession number. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 8.

The Simulium vittatum erythema protein (SVEP) superfamily of Simulium. (A) Clustal aligment. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. (B) Bootstrapped phylogram. The Simulium nigrimanum proteins are indicated by a square. The S. vittatum sequences are named SIMVI followed by their National Center for Biotechnology Information (NCBI) protein accession number. The vasodilator SVEP is marked with a triangle. The numbers on the tree bifurcations indicate the percentage bootstrap support. The bar at the bottom represents 10% amino acid substitution. Protein sequences were aligned by the Clustal program39 and the dendrogram was done with the Mega package41 after 10,000 bootstraps with the neighbor joining (NJ) algorithm. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 9.

The collagen-like superfamily of Simulium. (A) Clustal aligment. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. (B) Bootstrapped phylogram. The Simulium nigrimanum proteins are indicated by Sim- and their identification number. The Simulium vittatum sequences are named SIMVI followed by their National Center for Biotechnology Information (NCBI) protein accession number. The numbers on the tree bifurcations indicate the percentage bootstrap support above 75%. The bar at the bottom represents 5% amino acid substitution. Protein sequences were aligned by the Clustal program39 and the dendrogram was done with the Mega package41 after 10,000 bootstraps with the neighbor joining (NJ) algorithm. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 10.

The Sv 7kDa family of black flies. The Simulium nigrimanum proteins are indicated by Sim-XX where XX is the number indicated in supplemental file S2. The remaining sequences are from Simulium vittatum, with indicated National Center for Biotechnology Information (NCBI) protein accession number. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 11.

The Sv 4.8kDa family of black flies. The Simulium nigrimanum proteins are indicated by Sim-XX where XX is the number indicated in supplemental file S2. The remaining sequences are from Simulium vittatum, with indicated National Center for Biotechnology Information (NCBI) protein accession number. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. For more details, see text. This figure appears in color at www.ajtmh.org.

Figure 12.

The 8-10CysW family of Simulium nigrimanum. The signal peptide region is not shown. The symbols below the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. Cysteines, tryptophanes, and conserved residues are marked with different backgrounds Protein sequences were aligned by the Clustal program39. For more details, see text. This figure appears in color at www.ajtmh.org.