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.