Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae

Abstract

Background: Two sibling members of the Anopheles gambiae species complex display notable differences in female blood meal preferences. An. gambiae s.s. has a well-documented preference for feeding upon human hosts, whereas An. quadriannulatus feeds on vertebrate/mammalian hosts, with only opportunistic feeding upon humans. Because mosquito host-seeking behaviors are largely driven by the sensory modality of olfaction, we hypothesized that hallmarks of these divergent host seeking phenotypes will be in evidence within the transcriptome profiles of the antennae, the mosquito's principal chemosensory appendage.

Results: To test this hypothesis, we have sequenced antennal mRNA of non-bloodfed females from each species and observed a number of distinct quantitative and qualitative differences in their chemosensory gene repertoires. In both species, these gene families show higher rates of sequence polymorphisms than the overall rates in their respective transcriptomes, with potentially important divergences between the two species. Moreover, quantitative differences in odorant receptor transcript abundances have been used to model potential distinctions in volatile odor receptivity between the two sibling species of anophelines.

Conclusion: This analysis suggests that the anthropophagic behavior of An. gambiae s.s. reflects the differential distribution of olfactory receptors in the antenna, likely resulting from a co-option and refinement of molecular components common to both species. This study improves our understanding of the molecular evolution of chemoreceptors in closely related anophelines and suggests possible mechanisms that underlie the behavioral distinctions in host seeking that, in part, account for the differential vectorial capacity of these mosquitoes.

Figure 1

Chemosensory gene repertoires of An. gambiae and An. quadriannulatus. The numbers of An. gambiae and An. quadriannulatus chemosensory genes annotated in this study (ovals). The estimated numbers of chemosensory genes in the most recent common ancestor (MRCA) of the two species (boxes). The numbers along vertical arrows indicate the estimated numbers of gene gain (+) and loss (-) events. The red and blue schematic arrows indicate the host preferences (red: anthropophagic, blue: zoophagic) of An. gambiae and An. quadriannulatus.

Figure 2

Evolutionary rates of anopheline chemosensory genes. Box plots of (A) protein distances and (B) dN/dS ratios between orthologous pairs of OR, GR, IR, and OBP genes. Orthologous gene pairs delineated from An. gambiae genome and An. quadriannulatus transcriptome assembly were used as control. Wilcoxon rank sum tests showed that evolutionary rates were significantly different between chemosensory gene families and the control (* denotes p-value < 0.001). Outliers (circles) are shown for chemosensory gene families but not for the transcriptome background due to their large numbers. Ortholog pairs having a dN/dS value greater than one are noted in red.

Figure 3

Differential antennal abundances of chemosensory genes. Heat maps for the three most abundant chemoreceptor gene families in the antenna. Only transcripts that were detectable one or both species are displayed. Colors indicate a normalized, GFOLD score (reliable log2 fold difference in transcript abundance) denoting enrichment in either An. gambiae (red) or An. quadriannulatus (blue); genes showing no discernible expression differences between the species (GFOLD=0) are shown in grey. The gene names are ordered (from high to low, top to bottom) based upon their relative abundance in An. gambiae. Co-receptors are displayed in bold above the OR and IR gene family heat maps. Chemoreceptor names are color coded if they were only classified as being detectable in An. gambiae (red) or in An. quadriannulatus (blue).

Figure 4

Distribution of differentially abundant antennal Ors and their relative abundance levels in An. gambiae and An. quadriannulatus. Individual tuning Or orthologs are represented by bubbles with areas scaled to their respective abundance (RPKM) in either An. gambiae (red) or An. quadriannulatus (blue). Or orthologs are arranged horizontally based upon their enrichment (GFOLD value) in either An. gambiae (left) or An. quadriannulatus (right). Total RPKMs for each quadrant are indicated in the center. The asterisk denotes the larger than expected proportion of Or abundance in An. gambiae ascribable to Ors that are also enriched in An. gambiae (Fisher’s Exact Test, p=2.2x10^-16).

Figure 5

Differential expression of antennal ORs plotted against dN/dS.X-axis represents the absolute GFOLD score (reliable log2 fold difference in transcript abundance) for Ors enriched in either An. gambiae (left half) or in An. quadriannulatus (right half). Ors displaying no significant difference in transcript abundance are plotted at zero. Y-axis is the interspecific dN/dS for each Or. Ors are color coded as follows: grey: conserved in sequence and in transcription, blue: conserved in sequence but diverged in transcription, yellow: diverged in sequence but conserved in transcription, green: diverged in sequence and in transcription. Horizontal dashed line denotes the top 10% of transcriptome wide dN/dS value and the vertical dashed line denotes the top 10% of transcriptome wide, absolute fold change.

Figure 6

Differences in OR mediated odorant receptivity between An. gambiae and An. quadriannulatus antennae. Vertical axis represents computed, interspecific differences in antennal receptivity to a panel of odors. Displayed results are sorted left to right based upon the level of each odor’s relative receptivity enhancement in either An. gambiae (positive values) or An. quadriannulatus (negative values). The grey region around zero denotes an absolute change in relative receptivity of 10% or less. Chemical names are color coded by chemical class and asterisks denote chemical classes whose receptivity is disproportionately represented in one species (Fishers Exact Test, p<0.05). Red points denote odors that have been detected in human-associated skin emanations.