Bivalves are NO Different: Nitric Oxide as Negative Regulator of Metamorphosis in the Pacific Oyster, Crassostrea Gigas.

Abstract

Background Nitric oxide (NO) is a presumed regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish.Results In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas, demonstrating that the nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induce metamorphosis in a concentration-dependent manner ranging from 75%, 76–83% respectively. Additional induction resulted from exposures to 1H-[1, 2, 4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot’s smooth muscle relaxation.Conclusion Together, these results suggest that NO is a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.