Bivalves are NO different: nitric oxide as negative regulator of metamorphosis in the Pacific oyster, Crassostrea gigas

Abstract

Background: Nitric 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.

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. 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.

Conclusion: Together, 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.

Keywords: Bivalves; Crassostrea gigas; Metamorphosis; Nitric oxide; Nitric oxide synthase NOS; Pacific oyster; cGMP.

Fig. 1

Percentage (%) of metamorphosis in Pacific oyster larvae after 24 h continuous exposure to single treatments (black bars) of NO pathways inhibitors SMIS, AGH, 7-NI, L-NAME, L-NNA and ODQ, at different concentrations, as well as known inducers epinephrine (EPI; light grey bars) and MK-801 (MK; grey bars) at 10^− 4 M for 3 h, a DMSO (black-stripe bars) and a no treatment control (open bars). Data were collected 24 h post exposure start. C. gigas larvae from December 2018 experiment with competent larvae 18 dpf were used for all treatments except for ODQ, for which 19 dpf larvae were used. Error bars represent standard error. Different lower-case letters represent significant differences with p < 0.05

Fig. 2

Percentage (%) of metamorphosis in Pacific oyster larvae after 24 h continuous exposure to single treatments (black bars) of NO donors SIN-1 and SNP at 10^− 6 M to 10^− 4 M or as co-exposures (hashed bars) with epinephrine (Epi, light grey bars), MK-801 (MK, grey bars) and SMIS (dark grey bars) at 10^− 4 M for 3 h exposure followed by continuous single exposure to SIN-1 or SNP at 10^− 6 M to 10^− 4 M, and a non-treatment control. Data were collected 24 h post exposure start. C. gigas larvae from March 2019 experiment with competent larvae 18 days post fertilisation were used for all treatments. Error bars represent standard error. Different lower-case letters represent significant differences with p < 0.05

Fig. 3

Gene expression of CgNOS in Pacific oysters at different larval stages, larvae exposed to different metamorphosis inducers and spat 24 h after metamorphosis induction as well as metamorphosis percentages (%) in Pacific oyster larvae after exposure to inducers. A & C) C. gigas larvae, 18 dpf (d) in January 2018 experiments, exposed to epinephrine (EPI) and MK-801 (MK) at 10^− 4 M, L-DOPA at 10^− 5 M and ifenprodil (IP) at 10^− 6 M and sampled at 4 hpe and 6 hpe. B & D) C. gigas larvae, 17 dpf or 18 dpf for ODQ in March 2019 experiments, exposed to EPI, MK-801, SMIS and 7-NI at 10^− 4 M, AGH at 10^− 3 M and to ODQ at 5 × 10^− 5 M and sampled 3 hpe and 6 hpe. S: spat, C: control spat. Error bars represent standard error. Different lower-case letters represent significant differences with p < 0.05; *: significance not calculated due to differences in sampling days

Fig. 4

Nitric oxide (NO) detection in competent Pacific oyster larvae and spat using the NO-specific fluorescent indicator DAF-FM diacetate. Fluorescent and bright-field images of fluorescent NO signals in competent larvae swimming a left side view, b anterior view on foot, c ventral view on velum, d ventral view on velum with visible apical sensory organ; in larvae with predominant foot crawling on the bottom e left side view after AGH exposure, f anterior view and g left side view after MK-801 exposure; h larvae displaying organ contraction behaviour signalling metamorphosis after EPI exposure; j spat 24 hpe to EPI; control animals without DAF-FM diacetate treatment k in competent larvae without treatment and l spat hpe to EPI. aso: apical sensory organ, a.sh: adult shell growth, ci: cilia of the velum, f: foot, g: gills, gr: gill rudiments, l.sh: larval shell growth, ve: velum, ve.r: rim of velum. Scale bar: 100 μm

Fig. 5

CgNOS (a, c, e-g) and CgNR1 (b, d) expression localisation in competent Pacific oyster larvae and spat by in-situ hybridisation using digoxigenin labelled riboprobes (orange staining) with fluorescent signals visualised using a triple-band DAPI-FITC-Texas Red excitation filter. Frontal serial sections of foot area of the same larva 6 hpe to EPI with (a) CgNOS and (b) CgNR1, both with subsequent H&E staining of sections. Frontal sections of larvae (c) untreated with CgNOS and subsequent H&E staining of section, and (d) 6 hpe of EPI with CgNR1 and H&E staining of consecutive section. Transverse section with CgNOS probe of competent larvae (e) untreated and (f) 6 hpe EPI treatment. (g) Sagittal section whole spat and H&E staining of consecutive section. White arrows and arrow heads: signal for successful probe binding. aam: anterior adductor muscle; ci: cilia of the velum; e: oesophagus; f: foot; fg C: foot glands C; fg D: foot glands D; g: gills; gr: gill rudiments; m: mantle; mo: mouth; pam: posterior adductor muscle; pg: pedal ganglia; r. f: remnants foot; r. fg C: remnants foot glands C; vm: vellum membrane; *: Fast red dye unspecific binding mostly in remains of periostracum. Scale bar: 50 μm

Fig. 6

cGMP immunostaining in Pacific oyster larvae (a-f) and spat (g-k). Fluorescent and bright-field images of whole-mount stained individuals: 16 dpf larva (A, flattened with cover slip), 17 dpf larvae (c), 17 dpf larva 3 hpe EPI (e), spat with visible foot remnants (g), spat without visible foot remnants (j); fluorescent images of sectioned individuals accompanied with superimposed DAPI/cGMP signals as well as H&E staining: transverse section 17 dpf larva (b), sagittal section 17 dpf larva (d), frontal section 17 dpf larva 6hpe EPI (f), sagittal sections spat with large (h) and small foot remnants (k). e: oesophagus; f: foot; fg C: foot glands C; fg D: foot glands D; g: gills; gr: gill rudiments; m: mantle; pam: posterior adductor muscle; pg: pedal ganglia; r. f: remnants foot; r. fg C: remnants foot glands C; ve: velum. Scale bar: 50 μm