Peptide human hormones and growth factors bind to membrane receptors and regulate a myriad of processes in insects and other metazoans. around the evolutionary history and function of certain genes. These analyses also indicate that several peptide hormones and GPCRs remain orphans (6, 9, 16, 18, 21, 22). In some cases, one-to-one orthologs of known ligand-receptor pairs in the genomes of related species have led to predicted pairing to an uncharacterized receptor (23). For others though, ligand-receptor associations remain unclear because gene duplication or loss events either create uncertainties about the functional homology of ARQ 197 IC50 in- and out-paralogs, or have resulted in clades that contain no characterized orthologs (7, 21, 24, 25). We also note that no lists have been published for RGCs or PKRs and their associated ligands in any insect. Our interests primarily focus on mosquitoes (Diptera: Culicidae), which are critically important insects because of their ability to vector several disease-causing pathogens to humans and other mammals. Peptide hormones and growth factors are key regulators of many physiological processes in mosquitoes that affect disease transmission. Three vector ARQ 197 IC50 species of mosquitoes have been sequenced: (26), (27), and (28). Annotation of the and genomes identify 35 and 43 peptide hormone genes respectively that are expressed in the nervous system, isolated glands, or midgut endocrine cells (9, 10). The function of several of these hormones is known (Table S1 in Supplementary Material). Homology-based searches were previously used to characterize the GPCR superfamily in (24) and analysis of the genome also identified some predicted peptide hormone GPCRs (27). No recent collation of GPCRs or other receptor types, however, is available. Here, we analyzed the GPCRs, PKRs, and RGCs that bind peptide hormones and growth factors in mosquitoes and select other Diptera to discern phylogenetic patterns of receptor evolution. Our results provide a number of new insights including the identification of several orphan receptors. Ligand binding studies are the only means to deorphanize a given receptor definitively. However, our results could greatly assist deorphanization studies because they identify evolutionary associations between receptors and thus narrow the spectrum of candidate ligands that a given orphan most likely binds (29, 30). Materials and Methods Identification of peptide hormone receptors Peptide hormone receptors were identified from previously published surveys of (7), (24), and (27). These sequences were downloaded from NCBI and used to plumb the genomes of and using BLASTp. Each set of peptide hormone receptors from an organism was searched against the other genomes. The program HMMscan (31) was used to independently verify the completeness of our gene sets. Each genome was scanned using the Pfam models for rhodopsin-like GPCRs (7tm_1, PF00001), secretin-like GPCRs (7tm_2, PF00002), and protein kinases (Pkinase, PF00069) for RGCs and PKRs. These protein sequences were also used to search OrthoDB (32) for orthologs that may have been missed by our homology-based searches. The lists of receptors identified by Pfam searches were compared to the lists generated by BLAST searches. Genes were retained for further analysis if they were identified in Pfam searches or had a BLAST hit that had >50% amino acid identity with a known GPCR, RGC, or PKR in one of the examined genomes. In some instances, multiple annotated ARQ 197 IC50 genes were found to encode parts of a single receptor. We used publicly available RNAseq data where available to join individual genes. In other cases, multiple predicted genes aligned to successive regions of orthologs in genomes with superior annotations (as described for the other GPCRs and visualized in Jalview (38). Previous targeted mutagenesis studies distinguished residues important for ligand binding (40C43) that were then identified and highlighted in the GPCR sequences. Results Database mining and phylogenetic analysis of dipteran receptors Prior studies provide strong support for the monophyly of the Diptera while also showing that mosquitoes (Culicomorpha: Culicidae) are an early lineage that evolved ca. 225?Mya and drosophilids like are a derived lineage (Ephydroidea: Drosophilidae) that emerged concurrently with other cyclorrhaphan flies 40C65?Mya (44). Prior studies also support the monophyly of the Culicidae, which consists of two subfamilies, the Anophelinae (as represented by and and one other drosophilid (are provided PKCA in Table S1 in Supplementary Material. VectorBase or FlyBase accessions are.