Chemical reagents
The catecholamine epinephrine hydrochloride (EPI), the NOS inhibitors S-methylisothiourea hemisulfate salt (SMIS) and aminoguanidine hemisulfate salt (AGH) as well as the NO donor sodium nitroprusside dihydrate (SNP) were purchased from Sigma-Aldrich. The irreversible inhibitor of soluble guanylyl cyclase 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ) and the NO donor 3-morpholinosydnonimine chloride (SIN-1) were obtained from AdipoGen Life Sciences and the NOS inhibitors L-NG-nitroarginine methyl ester (L-NAME) hydrochloride, L-NG-nitroarginine (L-NNA) and 7-nitroindazole (7-NI) were purchased from Cayman chemicals. Additional chemicals were Levodopa (L-DOPA), obtained from Santa Cruz Biotechnology, as well as (+)-MK 801 maleate (MK-801) and Ifenprodil (+)-tartrate salt (ifenprodil), both obtained from Selleckchem. Stock solutions at 10-1M for SMIS, AGH and SNP or at 10-2M for remaining compounds were either prepared with autoclaved Milli-Q dH2O or with DMSO (Sigma-Aldrich) for 7-NI and ODQ. Working solutions (10x concentrate of final concentration of treatment) for each compound were prepared prior to experiments with 1 µm filtered fresh seawater (FSW).
Animal husbandry and metamorphosis assay
Pacific oysters, C. gigas, were cultured at the South Australian Research and Development Institute in Adelaide, South Australia with larvae derived from several independent spawning events were used for experiments. Larvae from nine family lines were obtained from the selective breeding program in January 2018 (C. gigas 2018) and are described in (4); larvae from six females and nine males were similarly obtained twelve months later in December 2018, and finally larvae from nineteen family lines in March 2019 (C. gigas 2019). All larvae were reared in FSW, maintained at 24.5±0.5 °C with a salinity of 36.5±0.5 ppt and fed with a microalgal mixture of Tisochrysis lutea, Pavlova lutheri, Chaetoceros calcitrans and Chaetoceros muelleri.
All metamorphosis assays were conducted following a general protocol as previously described (2-4). In brief, competent pediveliger larvae (shell length 300-330 µm, visible eyespot, crawling behaviour) were placed in FSW in glass shell vials, continuously exposed to chemical compounds for either 1 h, 3 h, 6 h or 24 h; the chemicals were then removed by pipetting, and 10 ml FSW including microalgae was added to each vial. For the 24 h continuous exposures, the microalgal mixture was added to the vials at the beginning of exposures as a proportion of the total volume of 10 ml in the vial. Differences in total FSW volumes during exposure were necessary in order to provide sufficient water volume to maintain an appropriate environment for the duration of exposure, while keeping chemical usage to a minimum. Controls, including a no-treatment control, as well as a DMSO control with maximum volume of DMSO equal to the highest volume solvent in working solutions were used. After 24 h, animals were assessed under an inverted microscope and early spat, live and dead larvae were counted. Mortality percentages varied from 0% - 7.6 % in individual vials after 24 h, but did not differ significantly between treatments.
NO inhibitor and donor experiments - (A) Single exposures: approximately 80-115 larvae (December 2018; 18 dpf & 19 dpf) were exposed to EPI and MK-801 at 10-4M for 3 h in a total volume of 2.5 ml, and to SMIS, AGH, 7-NI, ODQ, L-NAME and L-NNA for 24 h continuously at final concentrations ranging from 10-7 M to 10-3 M. (B) Co-exposures: approximately 90-120 larvae (March 2019; 18 dpf) were exposed to single treatments of EPI, MK-801 and SMIS at 10-4 M for 3 h in a volume of 2.5 ml, and to SIN-1 and SNP at 10-6 M to 10-4 M for 24 h continuously, as well as to co-treatments with either EPI, MK-801 or SMIS at 10-4 M together with either SNP or SIN-1 at 10-6 M to 10-4 M for 3 h in a volume of 2.5 ml with a subsequent single exposure to either SNP or SIN-1 for the remaining 21 h continuously in a total volume of 10 ml.
NOS gene expression experiments – (A) C. gigas 2018: approximately 250-320 larvae (January 2018, 18 dpf) were exposed to EPI at 10-4 M for 1h, and to MK-801 at 10-4 M, L-DOPA at 10-5 M and ifenprodil at 10- 6M for 3 h in a total volume of 2.5 ml as previously described in (4). The larvae were either sampled at 4 hpe and 6 hpe as well as spat 24 hpe for further analysis, or kept in the vials for 24 h and assessed under an inverted microscope. (B) C. gigas 2019: approximately 250-320 larvae (March 2019, 17 dpf & 18 dpf) were exposed to EPI and MK-801 at 10-4 M for 3 h in a volume of 2.5 ml, as well as to SMIS and 7-NI at 10-4 M, AGH at 10-3 M and ODQ at 5x10-5 M for either 3 h or 6 h in a total volume of 5 ml, or for 24 h continuously. Larvae were either sampled at 3 hpe, 6 hpe or as spat 24 hpe or kept for 24 h assessment of metamorphosis. Larvae and spat attached to glass vials were gently detached with a sharp spatula. Three separate samples were taken at each sampling point with each sample consisting of three biological replicates.
Gene expression analysis
C. gigas larvae of different developmental larval stages, larvae after exposure to metamorphosis inducers, as well as spat were analysed for the gene expression study: C. gigas 2018 animals at mid-veliger (9 dpf), late veliger (14 dpf), pediveliger larvae (16 dpf), competent pediveliger larvae (17 dpf & 18 dpf), larvae exposed to EPI, L-DOPA, MK-801 and ifenprodil at 4 hpe, 6 hpe and spat 24 hpe, were preserved in RNAlater (Sigma-Aldrich); C. gigas 2019 animals at 14 dpf -18 dpf, larvae exposed to SMIS, 7-NI, AGH and ODQ at 3 hpe, 6 hpe and spat at 24 hpe, were preserved in PaxGene Tissue system (PreAnalytix).
An NOS homolog, CgNOS (GenBank ID: XM_011421861), was identified in the Pacific oyster genome (ASM29789v2, Annotation Release 101 (85)) by a tBLASTn search using protein sequences to human nNOS (GenBank ID: AAA36376), eNOS (GenBank ID: AAA36364) and iNOS (GenBank ID: AAA59171). A primer pair for CgNOS with an amplicon length of 185 bp was designed with Primer Blast at NCBI (86): forward primer 5'-GAAGATGACCTCGGAGCAGG-3', reverse primer 5'-TGACCACTTCATCAGTCCGC-3'. Relative gene expression for CgNOS was assessed for all samples using quantitative PCR based on a modified comparative Ct model (87) following the protocol as previously described with the elongation factor-1 α, ribosomal protein S18, ribosomal protein L7 as reference genes (4). The amount of cDNA template was 0.5 µg in 2018 experiments and 1 µg in 2019 experiments. Optimal primer efficiency for CgNOS was reached at an annealing temperature of 62°C and a final primer concentration of 0.2 µM.
DAF-FM diacetate assay
For NO detection in competent Pacific oyster larvae, eyed pediveliger larvae (derived from two males and two females) were obtained from the Roem van Yerseke hatchery in Yerseke, the Netherlands, and held at 23°C in 100 L static tanks at the Ghent University in UV treated FSW with salinity and pH of 33.5 ±0.5 ppt and pH 8.1±0.1, respectively. Larvae were fed daily after daily water change with a microalgal mixture (1:1:2) of T. lutea, C. muelleri and P. lutheri.
Nitric oxide in oyster larvae was detected using the 4-amino-5-methylamino-2’,7’- difluorofluorescein (DAF-FM) diacetate, a non-fluorescent, cell-permeant compound that after hydrolysis to DAF-FM, reacts with NO to produce a relatively impermeable fluorescent triazolofluorescein derivative. Twenty to fifty competent larvae (24-26 dpf, 290-310 µm) were placed a 12 well plate in a total volume of 2.5 ml FSW, untreated or exposed to EPI, MK-801, SMIS and 7NI at 10-4 M or AGH at 10-3 M for 3 h, chemicals were then removed and 5 ml of FSW with microalgal feed was added to each well. Stock solution of 5 mM DAF-FM diacetate (Sigma Aldrich) was prepared with DMSO (Sigma-Aldrich) a few days prior to experiments and stored at -20°C until usage. After 1 h, water was changed again without microalgal feed and larvae were exposed to a final concentration of 7.5 µM DAF-FM diacetate in 2 ml FSW for 60 min in the dark. Spat from a previous day exposure to EPI were also incubated with 7.5 µM DAF-FM diacetate. Animals were washed twice with 1 ml FSW and incubated for 30 min in 2 ml FSW to allow complete de-esterification of intracellular diacetates. Animals were assessed alive using a Zeiss Axioskop 2 plus fluorescent microscope equipped with a FLUO filter set 38 (Ex: 470±40 BP, Em: 525±50 BP;) and pictures were taken using a monochrome Retiga3 Camera (QImaging) with Ocular V 2.0 Software (Digital Optics Limited) for imaging. To aid taking pictures of swimming larvae, animals were occasional anaesthetised with 7.5% MgCl2 in FSW. Animals not exposed to DAF-FM diacetate were also assessed, but exhibited only very weak fluorescence at the wavelength employed.
In-situ hybridisation
Expression of CgNOS and the previously identified NMDA receptor subunit CgNR1 were localised in pediveliger larvae by in-situ hybridisation (ISH). Competent larvae, untreated 17 dpf and 3 hpe of EPI, as well as spat 24 hpe of EPI from the March 2019 spawning event were anaesthetised with 7.5 % MgCl2 in FSW prior fixing in PaxGene Tissue system (PreAnalytix). Sectioning of larvae and execution of ISH followed the protocol previously described (4). Riboprobes were produced from cDNA fragments from CgNOS (344 bp) and CgNR1 (323 bp). Antisense riboprobes for DIG labelling were generated with specific forward primers and reverse primers with a T7 antisense extension for CgNOS (forward primer 5’-GGAATGTGGGACGTGTTGCC-3’, reverse primer + T7 extension 5’-TAATACGACTCACTATAGGGGCTCGGTCCTTCCACAGTGA-3’) and CgNR1 (forward primer 5’-TGCAACTGGGACAAGAACGA-3’, reverse primer + T7 extension 5’- TAATACGACTCACTATAGGGTGCAACTGGGACAAGAACGA-3’) by PCR using MyTag PCR mix (Bioline) at 95°C for 1 min, 35 cycles of 95°C for 15 sec, 65°C (CgNOS) or 64°C (CgNR1) for 15 sec and 72°C for 30 sec with a final extension of 2 min at 72°C. Sense riboprobes for CgNOS used as non-specific control in the ISH assay were amplified using the specific forward primer with a T7 sense extension and the reverse primer during the PCR. DIG labelling of all probes was conducted using the DIG RNA labelling kit (Roche) using an RNA T7 polymerase. After successful hybridisation, sections were mounted using a Vectamount (Vector Laboratories) and examined and photographed using an epi-fluorescent Arcturus XT Laser Capture Microdissection system (ThermoFisher Scientific) build on a Nikon Eclipse Ti-E microscope with triple-band DAPI-FITC-Texas Red excitation filter and a QImaging MicroPublisher Color RTV-5.0 CCD Camera (QImaging Corp.) for imaging.
Additional conventional hematoxylin and eosin (H&E) staining was performed for anatomy identification with the sections used for sense probes of CgNOS or CgNR1 after removal of Vectamount. Unfortunately, some individuals on sections were lost during this procedure and consecutive H&E stained section were used instead.
Immunofluorescent assay
For localisation of cGMP in larva, immunofluorescent assays were carried out using a rabbit anti-cGMP polyclonal antibody PAb (Sigma-Aldrich, 09-101) on sections of larvae and in whole mount. Before fixation larvae were relaxed with 7.5% MgCl2 and then fixed in PaxGene Tissue system (PreAnalytix).
Sections of competent larvae were prepared as for the ISH analysis previously described (4). Sections were dewaxed in xylene for 2x 5 min and gradually rehydrated through graded alcohol bath with 100% ethanol followed by 70% ethanol, each for 5 min, and a final 5 min bath in 1x PBS (0.02 M phosphate, 0.15M NaCl, pH 7.1). Sections were washed in 1xPTA (PBS + 4% Triton X-100 (Sigma-Aldrich)) for 10 min and then pre-treated with blocking buffer (5% goat serum in 1xPTA) for 45 min in the dark. Blocking buffer was removed and sections were incubated overnight in the dark at 4°C in blocking buffer with primary antibodies, either 1:500 anti-cGMP PAb or 1:200 rabbit anti-Salmonid MHC II PAb (Vertebrate Antibodies Ltd., UK) as rabbit antibody control for non-specific binding, or without a primary antibody functioning as control for non-specific secondary antibody control (Additional file 4). Sections were washed three times with 1xPTA for 10 min and then incubated for 2 h in the dark in blocking buffer with 1:100 secondary antibody Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody DyLight 550 (Invitrogen, SA5-10033). Slides were rinsed three times with 1xPTA for 10 min.
For whole-mount localisation of cGMP, larvae were decalcified in 10% EDTA in 1xPBS for 5 h, washed with 1xPBS for 5 min and 1xPTA for 10 min and then incubated for 24 h at 4°C with blocking buffer. Next, larvae were incubated with primary antibody as above and incubated for 45-48 h in the dark at 4°C with occasional shaking. Larvae were then washed three times over a period of 5 h in the dark on a shaking plate and then incubated for 21-24 h at 4°C in the dark with the secondary antibody. After incubation, larvae were washed twice for 1 h in the dark on a rocking platform.
Section and individual whole larvae were mounted using Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories) and were examined and photographed using an epi-fluorescent Arcturus XT Laser Capture Microdissection system (ThermoFisher Scientific) built on a Nikon Eclipse Ti-E microscope with G-2A filter for the DyLight 550 secondary antibody and UV-2A filter for the DAPI staining. An additional conventional H&E staining of a section was performed for anatomy identification after mounting medium was removed.
In addition to the ISH detection of CgNOS, an immunofluorescent assay was performed using a universal uNOS polyclonal antibody (PA1-38835, Invitrogen) at 1:100 and 1:200 dilutions in larval section and whole-mount larvae samples, but successful binding could not be obtained, not recommending this antibody for NOS detection in the Pacific oyster (Supplementary file 4).
A Western blot analysis was conducted to confirm successful binding of anti-cGMP PAb to oyster larvae protein. Thirty milligrams of 17 dpf oyster larvae were homogenized in 0.5 ml 1xPBS using a bead beater. The supernatant was aliquoted and protein concentration was quantified using a Pierce BCA Protein Assay Kit (ThermoScientific) following the manufacture’s protocol. Undiluted protein extracts were diluted 4:1 with 5x sodium dodecyl sulphate (SDS) sample buffer (0.5 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 0.2 M dithiothreitol, 0.02% bromophenol blue), boiled for 10 min and centrifuged for 2 min at 16,000g. Fifteen microliter of prepared samples were loaded into a 10% Mini-PROTEAN TGX Precast Gel (Bio-Rad, UK) and subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then electro-transferred to a nitro-cellulose membranes (Amersham Hybond ECL, GE Healthcare) using a Pierce G2 Fast blotter (ThermoScientific) for 7 min at 25V. Membrane was blocked with 3% skimmed milk powder (SMP) in tris buffer saline (TBS, 0.02 M Trisma base, 0.5 M NaCl, pH 7.5) overnight at 4°C. Membranes were then washed three times in TBST (TBS + 0.05% Teeen20) for 5 min and cut into strips. The membrane strips were individually incubated in 1:500 anti-cGMP PAB, 1:100 anti-uNOS PAB or without antibody (negative control) in TBS + 1% SMP for 2h at RT and washed three times in TBST. Membranes were then incubated again with anti-rabbit IgG Peroxidase Conjugate (Sigma Aldrich) diluted 1:250 in TBS+1%SMP for 1h at RT. After a final wash with TBS positive bands showing antigen-PAb complexes in the membrane were developed with ImmPact DAB Peroxide Substrate Peroxidase (Vector Laboratories) following the manufacturer’s instructions.
Data analysis
Metamorphosis success was calculated as percentages of larvae completing metamorphosis based on the total number of individuals in each vial presenting the average and standard error of three biological replicates per treatment. The non-parametric Kruskal–Wallis H-test was applied to analyse the effects between different treatments, followed by pairwise comparison using a generalisation of the Dunnett's T3 method to trimmed means (88).
The gene expression patterns of all genes were analysed for three independent replicates for each sampling point using a one-way ANOVA follows by multiple pairwise comparisons using a Tukey’s Honestly Significant Difference Test. All statistical tests were run using the R software (R version 3.5.1) and the probability level of 0.05 was chosen as being significant.