An insight into the sialome of Anopheles funestus reveals an emerging pattern in anopheline salivary protein families

Abstract

Anopheles funestus, together with Anopheles gambiae, is responsible for most malaria transmission in sub-Saharan Africa, but little is known about molecular aspects of its biology. To investigate the salivary repertoire of this mosquito, we randomly sequenced 916 clones from a salivary-gland cDNA library from adult female F1 offspring of field-caught An. funestus. Thirty-three protein sequences, mostly full-length transcripts, are predicted to be secreted salivary proteins. We additionally describe 25 full-length housekeeping-associated transcripts. In accumulating mosquito sialotranscriptome information--which includes An. gambiae, Anopheles stephensi, Anopheles darlingi, Aedes aegypti, Aedes albopictus, Culex pipiens quinquefasciatus, and now An. funestus--a pattern is emerging. First, ubiquitous protein families are recruited for a salivary role, such as members of the antigen-5 family and enzymes of nucleotide and carbohydrate catabolism. Second, a group of protein families exclusive to blood-feeding Nematocera includes the abundantly expressed D7 proteins also found in sand flies and Culicoides. A third group of proteins, only found in Culicidae, includes the 30 kDa allergen family and several mucins. Finally, 10 protein and peptide families, five of them multigenic, are exclusive to anophelines. Among these proteins may reside good epidemiological markers to measure human exposure to anopheline species such as An. funestus and An. gambiae.

Fig. 1

Phylogram of the salivary D7 proteins of anopheline mosquitoes showing the short and long D7 clades. An. gambiae proteins are marked with an oval box. An. gambiae sequences (starting with ANGA) originate from the annotation given by Arcà et al. (2005) and can be accessed online here. An. funestus sequences from this work can be recognized by the AF prefix. An. stephensi, An. dirus, and An. darlingi sequences can be recognized by the four letters originating from the genus and species name and by NCBI accession numbers. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 20% amino acid divergence in the sequences. The three different background shades indicate the three robust clades in the family.

Fig. 2

(A) Clustal alignment of the unique SG2 family of anopheline salivary peptides. The sequences shown are from An. darlingi (AD), An. stephensi (AS), An. funestus (AF), and An. gambiae (AG). (B) Neighbor-joining phylogram. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The vertical bars show the Cellia and Nyssorhynchus subgroups. The bar at the bottom indicates 10% amino acid divergence in the sequences.

Fig. 3

Clustal alignment of the unique SG6 family of anopheline salivary peptides. The ten cysteines are shown in black background. Identities are marked with ‘*’, strong amino acid conservations with ‘:’, and other conserved amino acids with ‘.’. The sequences shown are from An. stephensi (AS), An. funestus (AF), and An. gambiae (AG).

Fig. 4

(A) Clustal alignment of the unique SG7 family of anopheline salivary peptides. The sequences shown are from An. darlingi (AD), An. stephensi (AS), An. funestus (AF), and An. gambiae (AG). The signal peptide region is not shown. Cysteines have black background, conserved amino acids (aa) yellow background. Positions of similar aa are shown with bold characters on white background. (B) Neighbor-joining phylogram. The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 10% aa divergence in the sequences.

Fig. 5

Clustal alignment of the anopheline family of antithrombin peptides. The sequences shown are from An. gambiae (AG), An. funestus (AF), An. stephensi (AS), An. darlingi (AD), and An. albimanus (AA). The signal peptide region is not shown. Acidic amino acids (aa) are red on yellow background. Identical aa are marked in yellow background. Other symbols are as in Figs. 3 and 4.

Fig. 6

Clustal alignment of the 8.2-kDa family of peptides. The sequences shown are from An. gambiae (AG), An. funestus (AF), An. stephensi (AS), and An. darlingi (AD). The signal peptide region is not shown. Serine and threonine amino acids (aa) are marked in red on yellow background. Identical aa are marked in yellow background. Other symbols are as in Figs. 3 and 4.

Fig. 7

Clustal alignment of the 6.2-kDa family of peptides. The sequences shown are from An. gambiae (AG), An. funestus (AF), An. stephensi (AS), and An. darlingi (AD). Symbols above sequences are explained in Figs. 3 and 4.

Fig. 8

Clustal alignment of hypothetical family 15/17 of salivary peptides. The sequences shown are from An. gambiae (AG), An. funestus (AF), and An. darlingi (AD). Symbols above sequences are explained in Figs. 3 and 4.

Fig. 9

Clustal alignment of hypothetical family 10/12 of salivary secreted peptides. The sequences shown are from An. gambiae (AG), An. funestus (AF), and An. stephensi (AS). Cysteines are marked in black background. Conserved amino acids (aa) are shown in yellow background. Conserved aa in either the 10 or 12 amily are shown in red over yellow background. Symbols below sequences are explained in Figs. 3 and 4.