An annotated catalogue of salivary gland transcripts in the adult female mosquito, Aedes aegypti

Abstract

Background: Saliva of blood-sucking arthropods contains a cocktail of antihemostatic agents and immunomodulators that help blood feeding. Mosquitoes additionally feed on sugar meals and have specialized regions of their glands containing glycosidases and antimicrobials that might help control bacterial growth in the ingested meals. To expand our knowledge on the salivary cocktail of AEdes aegypti, a vector of dengue and yellow fevers, we analyzed a set of 4,232 expressed sequence tags from cDNA libraries of adult female mosquitoes.

Results: A nonredundant catalogue of 614 transcripts (573 of which are novel) is described, including 136 coding for proteins of a putative secretory nature. Additionally, a two-dimensional gel electrophoresis of salivary gland (SG) homogenates followed by tryptic digestion of selected protein bands and MS/MS analysis revealed the expression of 24 proteins. Analysis of tissue-specific transcription of a subset of these genes revealed at least 31 genes whose expression is specific or enriched in female SG, whereas 24 additional genes were expressed in female SG and in males but not in other female tissues. Most of the 55 proteins coded by these SG transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. An unexpected finding is the occurrence of four protein families specific to SG that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes.

Conclusion: Overall, this paper contributes to the novel identification of 573 new transcripts, or near 3% of the AE. aegypti proteome assuming a 20,000-protein set, and to the best-described sialome of any blood-feeding insect.

Figure 1

The D7 gene cassette in supercontig1.204 of Ædes ægypti. A, Overview of genomic region containing three short and two long D7 genes. B, Exon-intron structure for gene D7L1. C, Exon-intron structure for gene D7s1.

Figure 2

Additional transcripts observed mapping to the D7 gene region of Ædes ægypti on supercontig1,204.

Figure 3

Comparison of the D7 salivary proteins from Ædes ægypti (AEAE), Æ. albopictus (AEAL), Culex quinquefasciatus (CUQU), Anopheles gambiæ (ANGA), and Lutzomyia longipalpis (LULO). A, Clustal alignment. B, Phylogram showing the bootstrap values.

Figure 4

Two-dimensional gel electrophoresis of 50 μg salivary protein from adult female Ædes ægypti mosquitoes. Numbers on the left indicate molecular weight marker positions in the gel. The + and - signs indicate the anode or cathode side of the isoelectrophocusing dimension, which ranged from pH3-10. Gel bands that were identified to a protein (following tryptic digestion and mass spectrometry) are shown in the gel. In some cases, more than one band accounted for the same protein, possibly due to trailing or multiple isoforms. The proteins found were: D7l1 (gi: 16225992), PNase (purine nucleosidase, gi: 21654712) PDI (protein disulfide isomerase, gi: 94468800), HS70 (heat-shock protein 70 KDa, gi: 94468818), F0F1β (F0F1 ATPase beta subunit, gi: 94468834), Ang1 (angiopoietin-like protein, gi: 18568298), Serp1 (salivary serpin1, gi: 18568304), 62 k (62-kDa proteins gi: 18568300 and gi: 18568302), Serp2 (salivary serpin2, gi: 94469320), amylase (gi: 2190949), ADA (adenosine deaminase, gi: 18568326), apyrase (gi: 1703351), F0F1α (F0F1 ATPase, alpha subunit, gi: 94468442), D7l2 (gi: 118216), Ag5 (antigen5 protein, gi: 18568284), C-Lec2 (C-type lectin, gi: 94468370), 34k1 (34-kDa protein 1, gi: 94468642), Ang2 (angiopoietin-like protein2, gi: 94468352), 34 k2 (34-kDa protein2, gi: 18568296), C-Lec1 (C-type lectin 1, gi: 18568318), actin (gi: 94468486), 30.5 (30.5-kDa salivary protein, gi: 61742033), and 30ag (30-kDa antigen, gi: 18568322). For experimental details, see Methods.

Figure 5

Tissue and gender expression specificity of salivary gland genes of Ædes ægypti. RT-PCR results determining gene expression in female salivary glands (SG), carcass of female mosquitoes (C), and whole male mosquitoes (M) for selected genes in the sialotranscriptome of Ædes ægypti.

Figure 6

Alignment of members of the 5' nucleotidase family deriving from salivary glands of mosquitoes or from Drosophila melanogaster, D. pseudoobscura, Bos taurus, or Rattus rattus. A, Total aligment. B, Alignment on the carboxyterminal region. The numbers in the sequence titles indicate the NCBI accession number. Notice the conserved serine (where the inositol phosphate membrane anchor binds) surrounded by hydrophobic amino acids in the nonsalivary enzymes.

Figure 7

Clustal alignment of the 29/30-kDa protein family in hematophagous Diptera. The letters represent the species Ædes ægypti, Æ. albopictus, Culex pipiens, Culicoides sonorensis, Phlebotomus ariasi, Anopheles albimanus, An. gambiæ, An. stephensi, and An. dirus using the three first letters of the genus and two letters of the species name. The numbers are NCBI accession numbers.

Figure 8

Phylogenetic tree of the 29/30-kDa protein family in hematophagous Diptera. The numbers in the tree nodes indicate the bootstrap values. Tree branches in red represent mosquito species; in blue, sand fly; and in green, Culicoides. The bar at the bottom of B indicates amino acid divergence between sequences. See also legend of figure 7. The bar at the bottom of B indicates amino acid divergence between sequences.

Figure 9

The 34-kDa gene region in supercontig1.92.