An essential role of Rieske domain oxygenase Neverland in the molting cycle of black tiger shrimp, Penaeus monodon.

Molting is an important process for development and growth in arthropods. In crustaceans, molt is regulated by ecdysteroids or molting hormones that are synthesized in Y-organs. However, ecdysteroid biosynthesis pathway in crustaceans and its participating enzymes have not been well studied so far. In this study, a Rieske domain oxygenase, the enzyme that acts as cholesterol 7,8-dehydrogenase by converting cholesterol to 7-dehydrocholesterol in the first step of the ecdysteroid biosynthesis was characterized in black tiger shrimp, Penaeus monodon. A full-length cDNA of P. monodon's Rieske domain oxygenase Neverland (PmNvd) was successfully cloned. The expression of PmNvd was dominantly found in the Y-organ, and changed during molting period. The PmNvd mRNA level was low in intermolt and early premolt stages, then dramatically increased in the mid premolt stage suggesting its role in molt regulation. The function of PmNvd in the molting process was investigated by RNAi approach. Silencing of PmNvd transcript in shrimp by specific double-stranded RNA (dsNvd) led to prolonged molt duration with abnormal molting progression, i.e. the molting process got stuck at early premolt stage. In addition, 20-hydroxyecdysone titer in the hemolymph of dsNvd-injected shrimp was significantly reduced compared with that in NaCl-injected shrimp. These evidences suggested a crucial role of PmNvd in molt progression, particularly during the initiation of premolt phase via the regulation of ecdysteroid production.