Amino acid sequence alignment of the dipteran AST-A mature peptides with the vertebrate KISS, GAL and SPX family members.

<p>The highly conserved FGL motif between AST-A and KISS peptides is indicated in bold and red and conserved N residues in bold and blue. Sequence conservation of GAL and SPX is indicated in italics and totally conserved are in italics and bold. The vertebrate predicted peptide sequences were obtained from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref074" target="_blank">74</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref042" target="_blank">42</a>] and the <i>Xenopus laevis</i> mature galanin peptide deduced from EU446417.1.</p

Number of predicted AST-AR and peptide genes identified in arthropods and <i>C</i>. <i>elegans</i>.

<p>Accession numbers are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.s002" target="_blank">S1 Table</a>. The number of AST-A peptides is indicated within brackets and references are provided. The <i>T</i>. <i>urticae</i>, <i>D</i>. <i>plexippus</i>, <i>H</i>. <i>melpomene</i>, <i>S</i>. <i>invicta</i> and <i>A</i>. <i>darlingi</i> AST-A peptides were predicted by comparison with the insect homologues and identification of the C-terminal FGL-amide motif. * indicates species in which a putative <i>AST-AR</i> pseudogene (orthologue of the third Culicidae <i>AST-AR</i> gene) was identified. Data from <i>D</i>. <i>pulex</i> and <i>A</i>. <i>cephalotes</i> obtained from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref115" target="_blank">115</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref116" target="_blank">116</a>].</p

Gene organisation of the AST-A receptors in <i>Anopheles</i> and <i>D</i>. <i>melanogaster</i>.

<p>The structure of the <i>Anopheles</i> receptor genes was deduced from the consensus organisation obtained from several mosquito genomes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.s006" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.s007" target="_blank">S6</a> Tables). The <i>D</i>. <i>melanogaster AST-ARs</i> gene organizations were obtained from ENSEMBL. In the <i>A</i>. <i>gambiae</i> PEST genome duplicated exons highly similar in sequence to <i>GPRALS1</i> (exon 1) and <i>GPRALS2</i> (exon 2, 3 and 4) are predicted and are not represented. Closed boxes represent exons and dashed lines introns. Mosquito exons are numbered and exons encoding the UTR are represented by pink boxes. Gene structures were built using FancyGene 1.4 software. The figure is not drawn to scale.</p

Phylogeny of the AST-AR with the KISSR and GALR.

<p>Phylogenetic analysis was performed using the ML method and three subsets of the same phylogenetic tree showing the expansion of the different family members (A, B and C) are represented to facilitate interpretation. Only bootstrap support values above 50% are indicated. In the most important receptor family nodes statistical support was established using the aLRT SH-like test and is indicated (bootstrap method/ aLRT SH-like test). The deduced <i>A</i>. <i>darlingi</i> (Scaffold_325) was not used, as the receptor sequence was very incomplete and only 3 TM domains were predicted. The phylogenetic tree was rooted with the vertebrate GPR151 cluster (12 sequences). Species names and accession numbers of the receptor genes are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.s002" target="_blank">S1 Table</a>.</p

Proposed model for the origin and evolution of the <i>AST-AR</i> genes.

<p>Circles with different colours represent the AST-AR (light blue), KISSR (green) and GALR (pink) family members. The tetraploidization events basal to vertebrate radiation (1R, 2R) and the teleost specific genome duplication (3R) are indicated. The circle with a cross indicates gene loss during evolution. Numbers within the circles indicate predicted gene numbers of each family. Gene number from early deuterostome and lophotrochozoa representatives were obtained from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref041" target="_blank">41</a>]. For simplicity, lineage-specific duplications are not indicated and the time line is not drawn to scale. Receptor mapping for the vertebrate ancestral chromosomes (VAC) was obtained from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref042" target="_blank">42</a>].</p

Sequence conservation of the duplicate dipteran AST-ARs with the insect and human orthologues.

<p>The predicted seven transmembrane domains are boxed in red and numbered. Potential sites for N-glycosylation are underlined in the N-terminal region and two conserved motifs D-R-Y/F localized after TM3 and NSxxNPxxY within TM7 are annotated with asterisks [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref087" target="_blank">87</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref088" target="_blank">88</a>]. Two conserved cysteine residues that may form a disulphide bond were identified are connected by a line; predicted residues involved in protein kinase C phosphorylation are indicated by a blue square and potential protein A phosphorylation sites are annotated by a green diamond; C-terminal cysteine residues for potential palmitoylation after TM7 are denoted in italics and indicated with an orange pentagon. Amino acids important for binding of human galanin to GALR1 are indicated in red. The arginine residue important for the function of human KISSR1 that is proximate to the end of TM7 is indicated in bold and circled. Shading denotes amino acid conservation and dark grey means 80% and black 100% conservation. Shading after TM4 was manually edited and did not considered the incomplete receptor regions * indicate incomplete mosquito receptor sequences. Accession numbers of receptor genes are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.s002" target="_blank">S1 Table</a>.</p

AST-A peptide precursor in <i>A</i>. <i>gambiae</i>.

<p>The deduced sequence of AST-A in <i>A</i>. <i>gambiae</i> (Aga, PEST) was obtained from the AGAP003712 gene and confirmed using EST data. The <i>A</i>. <i>aegypti</i> (Aae, AAEL015251,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref081" target="_blank">81</a>]) and <i>D</i>. <i>melanogaster</i> (Dme, FBgn0015591,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130347#pone.0130347.ref048" target="_blank">48</a>]) orthologues were used for comparisons. The predicted mature peptides are highlighted in bold and the Gly residues processed to the C-terminal amide in mature AST-A’s are indicated in italics.</p

Capacity of the insect AST-A peptides to activate the <i>A</i>. <i>coluzzii</i> GPRALS.

<p>The mosquito Ano_AST-A1 and Ano_AST-A2 peptides and the cockroach BLAST-2 peptide were tested at several different concentrations and the response of the receptor monitored by measuring concentrations of intracellular calcium (RFU). The <i>D</i>. <i>melanogaster</i> DAR-2-RA receptor was used as a positive control: A) Response to BLAST-2 peptide (0.5 μM to 0.005 μM); B) Receptor response to the presence of decreasing concentrations of Ano_AST-A1 and Ano_AST-A2 peptides (1 μM to 1nM). A Kruskal-Wallis test with a Dunn’s Multiple Comparison test was performed using Prism GraphPad version 5 software. No significant differences were found.</p

Sequence identity and similarity of insect AST-ARs with human GALR1 and KISSR1.

<p>Percentages were calculated using the full-length amino acid sequence of the receptors.</p><p>Sequence identity and similarity of insect AST-ARs with human GALR1 and KISSR1.</p