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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/19178717
The salivary gland transcriptome of the neotropical malaria vector Anopheles darlingi reveals accelerated evolution of genes relevant to hematophagy - PubMed Skip to main page content
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. 2009 Jan 29:10:57.
doi: 10.1186/1471-2164-10-57.

The salivary gland transcriptome of the neotropical malaria vector Anopheles darlingi reveals accelerated evolution of genes relevant to hematophagy

Eric Calvo  1 , Van M Pham, Osvaldo Marinotti, John F Andersen, José M C Ribeiro
Affiliations

The salivary gland transcriptome of the neotropical malaria vector Anopheles darlingi reveals accelerated evolution of genes relevant to hematophagy

Eric Calvo et al. BMC Genomics. .

Abstract

Background: Mosquito saliva, consisting of a mixture of dozens of proteins affecting vertebrate hemostasis and having sugar digestive and antimicrobial properties, helps both blood and sugar meal feeding. Culicine and anopheline mosquitoes diverged ~150 MYA, and within the anophelines, the New World species diverged from those of the Old World ~95 MYA. While the sialotranscriptome (from the Greek sialo, saliva) of several species of the Cellia subgenus of Anopheles has been described thoroughly, no detailed analysis of any New World anopheline has been done to date. Here we present and analyze data from a comprehensive salivary gland (SG) transcriptome of the neotropical malaria vector Anopheles darlingi (subgenus Nyssorhynchus).

Results: A total of 2,371 clones randomly selected from an adult female An. darlingi SG cDNA library were sequenced and used to assemble a database that yielded 966 clusters of related sequences, 739 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 183 protein sequences, 114 of which code for putative secreted proteins.

Conclusion: Comparative analysis of sialotranscriptomes of An. darlingi and An. gambiae reveals significant divergence of salivary proteins. On average, salivary proteins are only 53% identical, while housekeeping proteins are 86% identical between the two species. Furthermore, An. darlingi proteins were found that match culicine but not anopheline proteins, indicating loss or rapid evolution of these proteins in the old world Cellia subgenus. On the other hand, several well represented salivary protein families in old world anophelines are not expressed in An. darlingi.

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Figures

Figure 1
Figure 1
Distribution of the transcripts from the salivary gland cDNA library of An. darlingi according to functional class.
Figure 2
Figure 2
The D7 protein family of An. darlingi and An. Gambiae. (A) Clustal alignment. (B) Phylogram based on the alignment in (A). The numbers on the tree nodes represent the percent bootstrap support in 10,000 trials. The bar at the bottom indicates 20% amino acid divergence. The An. gambiae sequence names start with D7 followed by s or L for short and long forms; the number following s or L represents the order of the gene in the D7 chromosomal region, following its transcription direction. The An. darlingi sequences start with AD, followed by a number derived from the cluster number, as determined in Supplemental Table S1. For more details, see text.
Figure 3
Figure 3
The 30-kD/GE-rich/Aegyptin protein family of mosquitoes. (A) Clustal alignment. (B) Phylogram based on the alignment in (A). The numbers on the tree nodes represent the percent bootstrap support in 10,000 trials (only values above 50% are shown). The bar at the bottom indicates 10% amino acid divergence. The sole An. darlingi sequence is identified by AD-26 and a filled circle symbol. 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. For more details, see text.
Figure 4
Figure 4
The salivary basic tail family of mosquito proteins. (A) Clustal alignment. The sole An. darlingi sequence is identified by AD-217. 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. Conserved cysteines are shown in black, hydrophobic conserved amino acids (aa) in light blue, conserved Pro and Gly in yellow, conserved bulky non-charged aa (Asn, Gln, Ser, Thr) in grey, conserved Ser + Thr in brown, conserved negatively charged aa in red, identical positively charged aa in violet, conserved charged aa in green. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. (B) Phylogram derived from the alignment in (A). The numbers on the tree nodes represent the percent bootstrap support in 10,000 trials (only values above 50% are shown). The bar at the bottom indicates 10% aa divergence.
Figure 5
Figure 5
The salivary 4.3-kDa family of mosquito proteins. (A) Clustal alignment. The sole An. darlingi sequence is identified by AD-476. 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. Conserved cysteines are shown in black, hydrophobic conserved amino acids (aa) in light blue, conserved Pro and Gly in yellow, conserved bulky non-charged aa (Asn, Gln, Ser, Thr) in grey, conserved Ser + Thr in brown, identical negatively charged aa in red, identical positively charged aa in violet, conserved charged aa in green. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. (B) Phylogram derived from the alignment in (A). The numbers on the tree nodes represent the percent bootstrap support in 10,000 trials (only values above 50% are shown). The bar at the bottom indicates 5% aa divergence.
Figure 6
Figure 6
Clustal alignment of the 41.9-kDa family of mosquito proteins. The sole An. darlingi sequence is identified by AD-114. 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. For more details, see text. Conserved cysteines are shown in black, hydrophobic conserved amino acids (aa) in light blue, conserved Pro and Gly in yellow, conserved bulky non-charged aa (Asn, Gln, Ser, Thr) in grey, conserved Ser + Thr in brown, conserved negatively charged aa in red, identical positively charged aa in violet, conserved charged aa in green. The symbols above the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites.
Figure 7
Figure 7
The expanded 41.9-kDa family. Phylogram based on the alignment of sequences derived from the use of the PSI-BLAST tool to retrieve sequences on the NR database from the NCBI using as seed the An. darlingi sequence AD-114. The numbers on the tree nodes represent the percent bootstrap support in 10,000 trials (only values above 50% are shown). The bar at the bottom indicates 20% amino acid divergence. Except for the An. darlingi sequence, 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. For more details, see text.
Figure 8
Figure 8
The G1 protein family of anopheline mosquitoes. A) Clustal alignment. (B) Phylogram based on the alignment in (A). The numbers on the tree nodes represent the percent bootstrap support in 10,000 trials (only values above 50% are shown). The bar at the bottom indicates 20% amino acid divergence. The An. darlingi sequences are identified by AD and a filled square symbol. The An. gambiae sequences are identified by a circle and are named as reported before [7]. 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. For more details, see text.
Figure 9
Figure 9
The 2WIRRP family of Anopheline proteins. Clustal alignment of the An. darlingi proteins with the An. gambiae homologue. Background colour follows convention as in Figure 6. Bar labelled I indicates region of Ser [Asp/Glu] [Asp-Glu] repeats. Bar labelled II identifies the WIRRP repeats notable on the An. gambiae sequence.
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