Genome analysis and expression patterns of odorant-binding proteins from the Southern House mosquito Culex pipiens quinquefasciatus

Abstract

Olfactory-based behaviors in mosquitoes are mediated by odorant-binding proteins (OBPs). They form a multigenic family involved in the peripheral events in insect olfaction, specifically the transport of odorants to membrane-bound odorant receptors. OBPs contribute to the remarkable sensitivity of the insect's olfactory system and may be involved in the selective transport of odorants.We have employed a combination of bioinformatics and molecular approaches to identify and characterize members of the "classic" OBP family in the Southern House mosquito Culex pipiens quinquefasciatus ( = Cx. quinquefasciatus), a vector of pathogens causing several human diseases. By taking advantage of the recently released genome sequences, we have identified fifty-three putative Cx. quinquefasciatus OBP genes by Blast searches. As a first step towards their molecular characterization, expression patterns by RT-PCR revealed thirteen genes that were detected exclusively and abundantly in chemosensory tissues. No clear differences were observed in the transcripts levels of olfactory-specific OBPs between antennae of both sexes using semi-quantitative RT-PCR. Phylogenetic and comparative analysis revealed orthologous of Cx. quinquefasciatus OBPs in Anopheles gambiae and Aedes aegypti. The identification of fifty-three putative OBP genes in Cx. quinquefasciatus highlights the diversity of this family. Tissue-specificity study suggests the existence of different functional classes within the mosquito OBP family. Most genes were detected in chemosensory as well as non chemosensory tissues indicating that they might be encapsulins, but not necessarily olfactory proteins. On the other hand, thirteen "true" OBP genes were detected exclusively in olfactory tissues and might be involved specifically in the detection of "key" semiochemicals. Interestingly, in Cx. quinquefasciatus olfactory-specific OBPs belong exclusively to four distinct phylogenetic groups which are particularly well conserved among three mosquito species.

Figure 1. Amino acids alignment of Cx. quinquefasciatus putative OBPs.

Residues conservation is indicated by different levels of shading: dark grey: 90% conservation; medium grey: 60% conservation; light gray: 40% conservation. The conserved cysteine residues are indicated by the letter C below the alignment. GenBank accession numbers are available in Table 1.

Figure 2. Phylogenetic relationships of mosquito “classic” OBPs.

The unrooted consensus tree was generated with 1000 bootstrap replicates using the neighbor joining method. Cx. quinquefasciatus OBPs are in black, A. gambiae OBPs are in blue and A. aegypti OBPs are in red. A. gambiae and A. aegypti OBPs follow the nomenclature established in and . Robust groupings identified by high bootstrap values at nodes are indicated in bold.

Figure 3. Amino acids alignments of five groups of mosquito OBPs.

(A) OS-E/OS-F-like OBPs; (B) PBPRP1-like OBPs; (C) LUSH-like OBPs; (D) OBP19a-like OBPs; (E) PBPRP4-like OBPs. Residues conservation is indicated by different levels of shading: dark grey: 100% conservation; medium gray: 80% conservation; light gray: 60% conservation.

Figure 4. Expression patterns of OBP genes in various tissues of adults Cx. Quinquefasciatus.

Specific primers of forty-seven putative OBP genes have been used in non quantitative RT-PCR experiments using thirty-four cycles of amplification. (A) OBP genes can be subdivided into three main categories. Olfactory-specific genes were detected exclusively in antennae, maxillary palps or proboscis. (B) Distribution profiles of olfactory-specific genes in olfactory tissues. Details are available in Table 4.

Figure 5. Expression of OBP genes in female and male antennae.

Expression ratios (FA/MA) of thirteen olfactory-specific OBP genes and two control genes (RpL8, OR7) were calculated after quantification of bands intensities in semi-quantitative RT-PCR experiments. Antennal CDNAs of both sexes were normalized to the expression levels of CquiRpL8 (purple) and CquiOR7 (blue). Bars represent standard deviations.

Figure 6. PCR amplification in female and male antennae.

Amplification of thirteen olfactory-specific OBP genes and two control genes (RpL8, OR7) in female antennae (FA) and male antennae (MA) cDNAs. (A) cDNAs normalized to the expression levels of CquiRpL8; (B) cDNAs normalized to the expression levels of CquiOR7.