A genome-wide analysis in Anopheles gambiae mosquitoes reveals 46 male accessory gland genes, possible modulators of female behavior

Abstract

The male accessory glands (MAGs) of many insect species produce and secrete a number of reproductive proteins collectively named Acps. These proteins, many of which are rapidly evolving, are essential for male fertility and represent formidable modulators of female postmating behavior. Upon copulation, the transfer of Acps has been shown in Drosophila and other insects to trigger profound physiological and behavioral changes in females, including enhanced ovulation/oviposition and reduced mating receptivity. In Anopheles gambiae mosquitoes, the principal vectors of human malaria, experimental evidence clearly demonstrates a key role of MAG products in inducing female responses. However, no Acp has been experimentally identified to date in this or in any other mosquito species. In this study we report on the identification of 46 MAG genes from An. gambiae, 25 of which are male reproductive tract-specific. This was achieved through a combination of bioinformatics searches and manual annotation confirmed by transcriptional profiling. Among these genes are the homologues of 40% of the Drosophila Acps analyzed, including Acp70A, or sex peptide, which in the fruit fly is the principal modulator of female postmating behavior. Although many Anopheles Acps belong to the same functional classes reported for Drosophila, suggesting a conserved role for these proteins in mosquitoes, some represent novel lineage-specific Acps that may have evolved to perform functions relevant to Anopheles reproductive behavior. Our findings imply that the molecular basis of Anopheles female postmating responses can now be studied, opening novel avenues for the field control of these important vectors of human disease.

Fig. 1.

Chromosomal organization of 46 genes expressed in the MAGs of An. gambiae. These genes are localized on chromosome arms 2L, 2R, and 3R, which are indicated by gray bars with the centromere marked by a circle. Chromosome arms are drawn to scale. Direction of the triangles reflects orientation on the chromosomal strands, and the color is indicative of pattern of expression, as specified in the key. T, testes; RB, male carcasses without male reproductive organs; F, whole females. The shaded boxes next to chromosome arms 2L and 3R highlight the presence of gene clusters. The five-digit numbers refer to the vector base identifier (AgamP3.4 gene build), omitting the AGAP code and the first digit (i.e., 06418 refers to AGAP006418). (See Table 1 legend.)

Fig. 2.

Phylogenetic analysis of serine protease inhibitors in three insect species. (A) Multiple protein sequence alignment for the D. melanogaster (Acp62F and Acp63F), An. gambiae (06587, 06586, 06585, 06583, and 06581), and Ae. aegypti (identified by the last six digits of their Ensembl entry codes omitting the AAEL code: 012194, 000374, 000323, 012204, 000317, 000369, 000333, 000361, and 000363) homologues generated by using Clustal X and Clustal W. Conserved cysteine residues are indicated by an asterisk. The red box in the sequence of 06585 specifies the region used for the 3D model shown in C. (B) Unrooted phylogenetic tree was constructed by the neighbor-joining method based on the sequence alignment as above. Anopheles genes are shaded in pink, Drosophila genes are shaded in blue, and Aedes genes are shaded in orange. The bootstrap values of 1,000 replicates are indicated. The scale bar represents the amino acid divergence. (C) Three-dimensional model of the Anopheles putative serine protease inhibitor 06585. The cysteine residues engaged in disulfide bridges and the free Cys-5g are shown in ball-and-stick form in yellow.