Abstract
BackgroundNitric oxide (NO) is presumed to be a 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.ResultsIn this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% 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, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. 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.ConclusionTogether, these results suggest that the NO pathway acts as 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.
Highlights
Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and 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
Competence of larvae was verified by exposing larvae to epinephrine (EPI) and MK-801 for 3 h at 10− 4 M, with both compounds resulting in induction percentages of 96.9 ± 0.6% for epinephrine hydrochloride (EPI) and 62.1 ± 7.3% for MK-801 in dpf larvae, and 98.1 ± 0.4% for EPI and 81.0 ± 1.0% for MK-801 in dpf larvae, respectively
Compared to the non-treatment control, significant induction of metamorphosis was achieved in 18 dpf competent larvae after 24 h continuous exposure to smethylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-NI at different concentrations with most effective concentrations for SMIS at 10− 4 M with 75.1 ± 3.1%, for AGH at 10− 3 M with 75.9 ± 2.1% and 7NI at 10− 4 M with 83.0 ± 0.3% metamorphosis
Summary
Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and 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. Cloning and characterisation of NMDA receptors in the Pacific oyster Crassostrea gigas as well as localisation of NMDA receptor subunit 1, CgNR1, in key structures of competent larvae such as the apical sensory organ (ASO), the underlying apical/ cerebral ganglia and the nerve network of the foot [5], established the existence of functional NMDA receptors in bivalve nervous systems. Both the ASO and the foot are structures specific to larval stages, that disappear after metamorphosis, which are assumed to be involved in sensing the environment for settlement cues [7,8,9]. Based on the combined findings of our previous work, it is apparent that NMDA receptors are part of the regulatory mechanism of bivalve metamorphosis and that opening of NMDA receptors initiates intracellular signalling and cells specific responses that negatively regulate metamorphosis
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