Insight into the sialome of the Black Fly, Simulium vittatum

Abstract

Adaptation to vertebrate blood feeding includes development of a salivary "magic potion" that can disarm host hemostasis and inflammatory reactions. Within the lower Diptera, a vertebrate blood-sucking mode evolved in the Psychodidae (sand flies), Culicidae (mosquitoes), Ceratopogonidae (biting midges), Simuliidae (black flies), and the frog-feeding Corethrellidae. Sialotranscriptome analyses from several species of mosquitoes and sand flies and from one biting midge indicate divergence in the evolution of the blood-sucking salivary potion, manifested in the finding of many unique proteins within each insect family, and even genus. Gene duplication and divergence events are highly prevalent, possibly driven by vertebrate host immune pressure. Within this framework, we describe the sialome (from Greek sialo, saliva) of the black fly Simulium vittatum and discuss the findings within the context of the protein families found in other blood-sucking Diptera. Sequences and results of Blast searches against several protein family databases are given in Supplemental Tables S1 and S2, which can be obtained from http://exon.niaid.nih.gov/transcriptome/S_vittatum/T1/SV-tb1.zip and http://exon.niaid.nih.gov/transcriptome/S_vittatum/T2/SV-tb2.zip .

Figure 1

1D gel electrophoresis of Simulium vittatum salivary gland homogenates. The left lane shows the protein MW markers (kDa). The right lane shows the fractionated salivary gland proteins, with the proteins identified from various locations within the gel indicated. More detailed information about the proteins can be found in Supplemental Table S2 by appending the number shown to the prefix SV-. Abbreviations for the protein class are, from top to bottom: PK, protein kinase; UC, unknown conserved; 5^/Nuc, 5^/ nucleotidase/apyrase; Q rich, glutamine-rich protein family; TIF, translation initiation factor; RPL5, ribosomal protein L5; Col like, collagen like; AKR, aldo-keto reductase; SerProt, serine protease; Ag5, antigen 5; HPQE, HPQE-rich family; OBP, odorant-binding protein; STkinase, serine/threonine kinase; OP, orphan protein; SVEP, S. vittatum erythema protein; D7, D7 protein family; KU, Kunitz protein; Dest, destabilase; Serf, similar to SERF-like protein; 7.8 kDa, 7.8-kDa family.

Figure 2

The S. vittatum erythema protein (SVEP) family. (A) ClustalW alignment of SVEP (gi|3319215) with eight novel proteins of the same family. (B) Dendrogram derived from the alignment. The mature (signal peptide removed) protein sequences were aligned by the Clustal program, and the dendrogram was done with the Mega package after 10,000 bootstraps with the neighbor-joining (NJ) algorithm. The numbers on the tree bifurcations indicate the percentage bootstrap support above 50%. The bar at the bottom represents 5% AA substitution.

Figure 3

The S. vittatum GHPQ-rich protein family. (A) Clustal alignment of the S. vittatum proteins indicates five domains: 1) signal peptide indicative of secretion, 2) arginine-rich region, 3) HG repeat region, 4) HPH repeat region, and 5) QPE repeat region. (B) Alignment of regions 2, 3, 4, and part of 5 with salivary proteins from Culicoides sonorensis (CULSO) and Phlebotomus argentipes (PHLAR). (C) Bootstrapped dendrogram from the alignment in (B). The number following the non-Simulium protein names refers to the GenBank GI number.

Figure 4

The S. vittatum QE-rich protein family. Clustal alignment indicates at least two genes coding for the shorter proteins, named SV-76 and SV-77, and two larger proteins, named SV-37 and SV-38. The bar at the top of the alignment indicates the signal peptide region, the mature proteins starting with the Phe-Phe doublet. The arrows point to leucine repeats spaced by 6 AA.

Figure 5

The S. vittatum collagen-like protein family. Clustal alignment indicates a mature amino terminal region of higher complexity and rich in acidic residues (1), then a basic carboxyterminal region with Pro-Gly-Lys repeats. The alignment excludes the signal peptide region and the carboxy termini of the proteins, which are divergent.

Figure 6

The GE-rich/30-kDa antigen family of mosquitoes and S. vittatum. (A) Clustal alignment. Notice the first half of the alignment is dominated by Gly, Asp, and Glu, and the second part is dominated by hydrophobic as well as by lysine residues. The signal peptide region is not shown. The S. vittatum protein is represented by SV-163. The remaining proteins are from Aedes aegypti, Ae. albopictus, Anopheles albimanus, An. gambiae, An. stephensi, An. dirus, An. funestus, and Culex pipiens, recognized by the three letters of the genus name followed by the first two letters of the species name, followed by the NCBI GI number, except for the An. gambiae protein, which was obtained from Arca et al. The symbols above the alignment indicate identity (*), high similarity (:), and similarity (.) of residues in the indicated alignment position. (B) Phylogram following 10,000 bootstraps; numbers indicate the percentage of bootstrap support.