Molecular characterization and expression analysis of prothoracicotropic hormone in the flesh flies, Sarcophaga bullata and S. crassipalpis

Prothoracicotropic hormone (PTTH) is an essential neuropeptide that regulates ecdysteroid production in insects, and thus it has an important impact on pupal diapause. It also regulates Drosophila melanogaster developmental timing, body size, and light preference as documented recently. In the present study, we cloned and identified ptth cDNAs from both Sarcophaga bullata and S. crassipalpis, two closely related species that are diapause research models. One of the S.bullata ptth cDNAs we cloned is 1308 bp long and encodes 294 amino acid (aa) residues, and it has multiple polyadenylation signals. Sabul-PTTH putatively produces a mature PTTH with 111 aa residues. The cloned S. crassipalpis ptth cDNA is 1047 bp long and encodes 296 aa residues. The putative mature Sacra-PTTH also consists of 111 aa residues, and there are only 5 different amino acid residues in the mature PTTHs of these two species. Sequence alignment indicates that Sarcophaga PTTH has the conserved 7 Cysteines motif as shown in other PTTHs. Phylogenetic analysis using PTTH sequences from different species well segregates Lepidoptera from Diptera, and Sarcophaga PTTH shares the clade with tsetse fly, but is well separated from all the fruit flies and mosquitoes. We also blasted our S. bullata ptth cDNA sequence against the unpublished genome sequencing data and confirmed that the ptth gene contains 3 exons and 2 introns. Our preliminary results indicate that there are alternative splicing PTTH cDNA isoforms in adult flies.  Expression data suggests that ptth mRNA mainly resides in the brain.  At the pupal stage, ptth mRNA content increases from day 1 of the prepupa until day 4 when it is the highest level, then decreases until day 10. Ptth mRNA first slightly increased 0-6 h after application of hexane, which acts to terminate diapause, then declined by 48 h. We also expressed the mature PTTH protein in E. coli; the diapause termination activity assays are ongoing.