The salivary gland transcriptome of the eastern tree hole mosquito, Ochlerotatus triseriatus

Abstract

Saliva of blood-sucking arthropods contains a complex mixture of peptides that affect their host's hemostasis, inflammation, and immunity. These activities can also modify the site of pathogen delivery and increase disease transmission. Saliva also induces hosts to mount an antisaliva immune response that can lead to skin allergies or even anaphylaxis. Accordingly, knowledge of the salivary repertoire, or sialome, of a mosquito is useful to provide a knowledge platform to mine for novel pharmacological activities, to develop novel vaccine targets for vector-borne diseases, and to develop epidemiological markers of vector exposure and candidate desensitization vaccines. The mosquito Ochlerotatus triseriatus is a vector of La Crosse virus and produces allergy in humans. In this work, a total of 1,575 clones randomly selected from an adult female O. triseriatus salivary gland cDNA library was sequenced and used to assemble a database that yielded 731 clusters of related sequences, 560 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 159 protein sequences, 66 of which code for putative secreted proteins. Supplemental spreadsheets containing these data are available at http://exon.niaid.nih.gov/transcriptome/Ochlerotatus_triseriatus/S1/Ot-S1.xls and http://exon.niaid. nih.gov/transcriptome/Ochlerotatus_triseriatus/S2/Ot-S2.xls.

Fig. 1

The basic tail family of mosquito salivary proteins. (A) Clustal alignment. The O. triseriatus protein is named OT-131. 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 NCBI protein accession number. The symbols above the alignment indicate the following: (*) identical sites; (:) conserved sites; (.) less conserved sites. (B) NJ bootstrapped phylogram of the alignment in (A), showing culicine and anopheline clades. The numbers on the branches represent the percentage of bootstrap support. The bar on the bottom represents 10% amino acid divergence. For more details, see text. (Online figure in color.)

Fig. 2

Members of the GGHGQQ family of culicines. (A) Clustal alignment of two O. triseriatus proteins with a hypothetical protein from Cx. quinquefasciatus. The signal sequence indicative of secretion is marked with a bar; remaining markings refer to repeat structure, indicated in (B). (Online figure in color.)

Fig. 3

The Aedes glycine-rich family of proteins. (A) Clustal alignment. The O. triseriatus protein is represented by OT-157. 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 NCBI protein accession number. The symbols above the alignment indicate the following: (*) identical sites; (:) conserved sites; (.) less conserved sites. (B) NJ bootstrapped phylogram of the alignment in A. The numbers on the branches represent the percentage of bootstrap support. The bar on the bottom represents 5% amino acid divergence. For more details, see text. (Online figure in color.)

Fig. 4

The 6.2-kDa family of mosquito salivary peptides. Clustal alignment of the mature versions (signal peptide removed) of the O. triseriatus protein OT-547 with those of Ae. aegypti, An. gambiae, Anopheles funestus, and Anopheles darlingi. These sequences are named with the first three letters from the genus name, followed by two letters from the species name and by their NCBI protein accession number. The symbols below the alignment indicate the following: (*) identical sites; (:) conserved sites; (.) less conserved sites. (Online figure in color.)

Fig. 5

The SG3/WWW family of mosquito salivary peptides. Clustal alignment of the O. triseriatus protein OT-175 with those of Ae. aegypti, An. gambiae, and An. stephensis. These sequences are named with the first three letters from the genus name, followed by two letters from the species name and by their NCBI protein accession number. The symbols below the alignment indicate the following: (*) identical sites; (:) conserved sites; (.) less conserved sites. (Online figure in color.)

Fig. 6

The WPCWW family of mosquito salivary peptides. Clustal alignment of the mature versions (signal peptide removed) of the O. triseriatus protein OT-86 with those of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus. These sequences are named with the first three letters from the genus name, followed by two letters from the species name and by their NCBI protein accession number. The symbols below the alignment indicate the following: (*) identical sites; (:) conserved sites; (.) less conserved sites. The cysteine residues are in black background. (Online figure in color.)

Fig. 7

The KKK circle family of aedine proteins. Clustal alignment of OT-492, an O. triseriatus protein, with Ae. aegypti and Ae. albopictus homologs. The bar under the alignment indicates a triple lysine surrounded by cysteines (arrows). (Online figure in color.)