Characterization of Shed genes encoding ecdysone 20-monooxygenase (CYP314A1) in the Y-organ of the blackback land crab, Gecarcinus lateralis.

Molting in decapod crustaceans is controlled by ecdysteroid hormones synthesized and secreted by the molting gland, or Y-organ (YO). Halloween genes encode cytochrome P450 enzymes in the ecdysteroid synthetic pathway. The current paradigm is that YOs secrete an inactive precursor (e.g., ecdysone or E), which is hydroxylated at the #20 carbon to form an active hormone (20-hydroxyecdysone or 20E) by a mitochonrial 20-monooxygenase (CYP314A1) in peripheral tissues. 20-Monooxygenase is encoded by Shed in decapods and Shade in insects. We used eastern spiny lobster Shed sequences to extract six orthologs in the G. lateralis YO transcriptome. Phylogenetic analysis of the deduced amino acid sequences from six decapod species organized the Sheds into seven classes (Sheds 1-7), resulting in the assignment of the G. lateralis Sheds to Gl-Shed1, 2, 4A, 4B, 5A, and 5B. The mRNA levels of the six Gl-Sheds in the YO of intermolt animals were comparable to those in nine other tissues that included hepatopancreas and muscle. qPCR was used to compare the effects of molt induction by multiple leg autotomy (MLA) and eyestalk ablation (ESA) on Gl-Shed mRNA levels in the YO. Molt stage had little effect on Gl-Shed1 and Gl-Shed5B expression in the YO of MLA animals. Gl-Shed5A was expressed at the highest mRNA levels in the YO and was significantly increased during early and mid premolt stages. By contrast, ESA ± SB431542 had no effect on Gl-Shed expression at 1, 3, 5, and 7 days post-ESA. SB431542, which inhibits Transforming Growth Factor-ß/activin signaling and blocks YO commitment, decreased Gl-Shed2 and Gl-Shed4A mRNA levels at 14 days post-ESA. A targeted metabolomic analysis showed that YOs cultured in vitro secreted E and 20E to the medium. These data suggest that the YO expresses 20-monooygenases that can convert E to 20E, which may contribute to the increase in active hormone in the hemolymph during premolt.

Characterization of G-protein coupled receptors from the blackback land crab Gecarcinus lateralis Y organ transcriptome over the molt cycle.

BACKGROUND: G-protein coupled receptors (GPCRs) are ancient, ubiquitous, constitute the largest family of transducing cell surface proteins, and are integral to cell communication via an array of ligands/neuropeptides. Molt inhibiting hormone (MIH) is a key neuropeptide that controls growth and reproduction in crustaceans by regulating the molt cycle. It inhibits ecdysone biosynthesis by a pair of endocrine glands (Y-organs; YOs) through binding a yet uncharacterized GPCR, which triggers a signalling cascade, leading to inhibition of the ecdysis sequence. When MIH release stops, ecdysone is synthesized and released to the hemolymph. A peak in ecdysone titer is followed by a molting event. A transcriptome of the blackback land crab Gecarcinus lateralis YOs across molt was utilized in this study to curate the list of GPCRs and their expression in order to better assess which GPCRs are involved in the molt process. RESULTS: Ninety-nine G. lateralis putative GPCRs were obtained by screening the YO transcriptome against the Pfam database. Phylogenetic analysis classified 49 as class A (Rhodopsin-like receptor), 35 as class B (Secretin receptor), and 9 as class C (metabotropic glutamate). Further phylogenetic analysis of class A GPCRs identified neuropeptide GPCRs, including those for Allatostatin A, Allatostatin B, Bursicon, CCHamide, FMRFamide, Proctolin, Corazonin, Relaxin, and the biogenic amine Serotonin. Three GPCRs clustered with recently identified putative CHH receptors (CHHRs), and differential expression over the molt cycle suggests that they are associated with ecdysteroidogenesis regulation. Two putative Corazonin receptors showed much higher expression in the YOs compared with all other GPCRs, suggesting an important role in molt regulation. CONCLUSIONS: Molting requires an orchestrated regulation of YO ecdysteroid synthesis by multiple neuropeptides. In this study, we curated a comprehensive list of GPCRs expressed in the YO and followed their expression across the molt cycle. Three putative CHH receptors were identified and could include an MIH receptor whose activation negatively regulates molting. Orthologs of receptors that were found to be involved in molt regulation in insects were also identified, including LGR3 and Corazonin receptor, the latter of which was expressed at much higher level than all other receptors, suggesting a key role in YO regulation.