Molting behavior in insects is controlled by the ecdysis triggering hormone (ETH), eclosion hormone (EH) and the crustacean cardioactive peptide (CCAP). At present, the regulation of molting behavior in crustaceans remains unclear. Here, we studied the roles of ETH, EH, and CCAP in the molt regulation of the crab, Scylla paramamosain from their expression pattern and in vivo assays. The results showed that transcripts of ETH, EH, and CCAP were mainly localized in thoracic ganglia and fluctuated periodically with the molting cycle. When ETH or CCAP was knockdown at early premolt stage (D0), molting of crabs was interrupted and all animals died at late premolt stage (D2). While the EH gene was knock-down, most crabs were dead before D2. Injection of synthetic peptide for ETH or CCAP rescued ETH- or CCAP-gene knock-down crabs separately. However, none of peptides could rescue dsEH-injected crabs. At D0 stage, knockdown of ETH down-regulated the transcriptions of EH and CCAP; while ETH was up-regulated when EH was knockdown. At D2 stage, ETH transcripts levels were reduced with the injection of dsEH but increased with the same dose of dsETH as crabs at D0 stage. Co-injection of dsETH and dsEH down-regulated ETH at D2 stage. Results showed that ETH, EH, and CCAP play essential but different roles in molt regulation in mud crab. In summary, the result of this study contributes to the discovery of different molecular mechanisms between Insecta and Crustacea and may provide insight to develop fishery drugs that helps aquacultured crustaceans to molt successfully.

Ecdysis triggering hormone (ETH) plays an important role in molting, reproduction, and courtship behavior in insects. To investigate the potential downstream pathways and genes of ETH in Scylla paramamosain, RNA interference (RNAi) was conducted on crabs at early (D0) and late (D2) premolt substages, and the transcriptome profiles of each group were compared by RNA sequencing. Real-time quantitative polymerase chain reaction (RT-qPCR) and semiquantitative polymerase chain reaction (RT-PCR) results showed a significant knockdown of ETH at D0 stage, whereas a significant increase was shown conversely in crabs at D2 substage after the injection of dsETH. A total of 242,979 transcripts were assembled, and 44,012 unigenes were identified. Transcriptomic comparison between crabs at D2 and D0 substages showed 2,683 differentially expressed genes (DEGs); these genes were enriched in ribosome and pathways related to transcription factor complex and cell part. Twenty DEGs were identified between dsETH-injected and dsGFP-injected crabs at D0 substage; these DEGs were involved in carbohydrate metabolism, one carbon pool by folate, and chitin binding. Twenty-six DEGs were identified between dsETH-injected and dsGFP-injected crabs at D2 substage; these DEGs were involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction. RT-qPCR verified the differential expression of the selected genes. In conclusion, crabs at D0 substage are more active in preparing the macromolecular complex that is needed for molting. Moreover, ETH has potential roles in carbohydrate metabolism, one carbon pool by folate, and chitin binding for crabs at D0 substage, while the role of ETH turns to be involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction at D2 substage to facilitate the occurrence of molting. The selected DEGs provide valuable insight into the role of ETH in the regulation of crustacean molting.

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