Expression patterns of OBP genes in various tissues of adults <i>Cx. Quinquefasciatus</i>.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006237#pone-0006237-t004" target="_blank">Table 4</a>.</p

Amino acids alignments of five groups of mosquito OBPs.

<p>(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.</p

Phylogenetic relationships of mosquito “classic” OBPs.

<p>The unrooted consensus tree was generated with 1000 bootstrap replicates using the neighbor joining method. <i>Cx. quinquefasciatus</i> OBPs are in black, <i>A. gambiae</i> OBPs are in blue and <i>A. aegypti</i> OBPs are in red. <i>A. gambiae</i> and <i>A. aegypti</i> OBPs follow the nomenclature established in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006237#pone.0006237-Xu2" target="_blank">[21]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006237#pone.0006237-Zhou2" target="_blank">[23]</a>. Robust groupings identified by high bootstrap values at nodes are indicated in bold.</p

Structural characteristics of <i>Cx. quinquefasciatus</i> putative OBPs.

<p>The number of amino acids is indicated for complete/mature proteins. Molecular weights (MW) and isoelectric points (pI) values were predicted for mature proteins using ExPASy server. Cysteine spacing patterns were determined manually. The signal peptides probabilities were predicted using SignalP 3.0 server. Conserved protein motifs result from Blast in NCBI Conserved Domain Database (CDD) with associated E-values. Asterisks indicate when new GenBank accessions have been submitted. Corresponding GenBank accessions: CquiOBP1 (XP_001848926), CquiOBP2 (XP_001848939), CquiOBP3 (XP_001848933), CquiOBP4 (XP_001843595), CquiOBP5 (XP_001848930), CquiOBP6 (XP_001850448), CquiOBP7 (XP_001843143), CquiOBP8 (XP_001851195), CquiOBP9 (XP_001867234), CquiOBP11 (XP_001848048), CquiOBP12 (XP_001867235), CquiOBP13 (XP_001867238), CquiOBP14 (XP_001851213), putative salivary OBP1 AAR18408 (XP_001861427), putative salivary OBP2 AAR18456 (XP_001867923).</p

Expression of OBP genes in female and male antennae.

<p>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.</p

Amino acids alignment of <i>Cx. quinquefasciatus</i> putative OBPs.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006237#pone-0006237-t001" target="_blank">Table 1</a>.</p

Expression patterns of OBP genes by RT-PCR in adult tissues.

<p>Specific primers of forty-seven putative OBP genes were used in non quantitative RT-PCR experiments using thirty-four cycles of amplification. Yes: a PCR product of the expected size has been detected in a given tissue; No: absence of band. The same primer pairs have been used for CquiOBP35 and CquiOBP39, and for CquiOBP36 and CquiOBP38. Expression patterns are as follows. Olfactory-specific: detected only in antennae, palps or proboscis; non olfactory-specific: detected in antennae, palps or proboscis as well as in legs and/or bodies; ubiquitous: detected in every tissue; not detected. Olfactory-specific OBPs are in bold.</p

PCR amplification in female and male antennae.

<p>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.</p

Tissue distribution in male and female.

<p>Tissue distribution in male and female.</p

Protein expression profile in male and female antennae.

<p>Protein samples (30 µg equal concentration/lane) were extracted from the antennae of <i>A. transitella</i> male and female adults, and analyzed by 7.5% Native-PAGE as described above. The arrow on the right indicates the position of AtraAOX2. Recombinant AtraAOX2 showed a slightly higher position than native AtrAOX2 because of 6×His-tagged.</p