Ecdysis Triggering Hormone, Eclosion Hormone, and Crustacean Cardioactive Peptide Play Essential but Different Roles in the Molting Process of Mud Crab, Scylla paramamosain

Abstract

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.

Transcriptome responses of RNAi-mediated ETH knockdown in Scylla paramamosain at different premolt substages

“…In the previous study, the expression level of ETH showed a periodic fluctuation with molting, and ETH exists not only at the E stage but also at the other stages ( 18 ). Functional diversity of ETH has been proven in insects (not just triggering ecdysis and working at the E stage) ( 14 – 16 ).…”

“…To balance, the injection of dsETH at the D2 substage led to an increase of ETH when we did sampling for transcriptome analysis. Our previous study showed that ETH was regulated by different models in S. paramamosain , and eclosion hormone was important to the regulation of ETH at the D2 substage ( 18 ).…”

“…The consistent trend of DEGs related to ribosome and transcription factor further supports the hypothesis that crab at the D0 substage is more active in preparing macromolecular complexes that are needed for molting. In S. paramamosain , the injection of dsETH with a dose of 5 µg/g could knock down ETH of crabs at the D0 substage but failed with crabs at the D2 substage ( 18 ). The expression level of ETH has a significant increase from the D0 substage to the D2 substage and then comes to a dramatic increase from the D2 substage to the E stage ( 18 ).…”

“…Primers flanked by the T7 promoter sequence were designed and used to synthesize the double-stranded RNA (dsRNA) for ETH and control gene ( GFP ) ( 18 ). The dsRNA templates were amplified with the procedure: 95°C for 5 min; followed by 30 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s; then 72°C for 10 min.…”

“…In Scylla paramamosain , we found that the knockdown of ETH leads to failure of molting at the early premolt substage. Interestingly, the transcript level of ETH had a significant increase conversely when crabs at the late premolt substage (D2) were injected with the same amount of dsETH as at the early premolt stage (D0), which suggests different means of regulation of ETH at the D0 and D2 substages in S. paramamosain ( 18 ).…”