All experimental procedures were conducted in accordance with the guidelines of the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology in China. All oyster experiments were performed in accordance with animal ethics guidelines approved by the Ethics Committee of Dalian Ocean University.

Gonadal-matured C. gigas oysters, with an average shell length of 13.0 cm ± 2 cm, were collected from a commercial shellfish farm (Changhai, Liaoning, China) during the spawning season. They were transferred from the farm to the hatchery for a two-weeks acclimatization period. Seawater was pumped directly from the sea of Heishijiao and filtered through a sand filter (40 μm) before draining the tanks. The oysters were fed Spirulina daily. To eliminate the bacteria carried out from the natural sea field, all oyster batches were subjected to a 5-day antibiotic treatment (10 mg/L of Flumequine, Shanghai Zhengyu Biotechnology Co., Ltd.) before subsequent experiments.

The pathogenic bacterial strain V. splendidus strain JZ6 was chosen to elicit a biparental immune response (45). A total of 100 μL of V. splendidus stored in 30% glycerol at −80°C, was inoculated into 100 ml 2216E media (tryptone, 5 g/L; yeast extract, 1 g/L; ferric phosphate 0.1 g/L; seawater, 1 L) at 16°C for 24 h. After centrifugation at 8,000×g at 4°C for 10 min, the bacterial pellets were washed three times and resuspended in filtered seawater to a final concentration of 2 × 10⁸ CFU/mL for subsequent experiments.

Two independent experiments were conducted (hereafter referred to as experiments 1 and 2), and the experimental design is shown in Figure 1 . For experiment 1, gonadal matured oysters (160 individuals) were randomly divided into two groups and subjected to the following immune priming treatments: either injection of 100 μL V. splendidus (Vs, the Vs primed group) or equal sterile seawater (Sw, the naïve group). Hemocytes and eggs were collected by dissection at 1, 3, 5, and 7-days post-priming stimulation and directly stored in liquid nitrogen for enzyme activity assay or suspended in 1 mL TRIzol Reagent (Invitrogen, USA) for qualitative RT-PCR analysis.

In vitro fertilization was performed between different treatment groups for the convenience of synchronizing gametic activation and fertilization. Briefly, spawning and fertilization were performed artificially seven days post-priming (46). After in vitro dissection, eggs from the females of each treatment group (control Sw and Vs primed group) were dissected and rinsed together with filtered seawater into a 10 L bucket, while sperm from the males of each treatment group were rinsed in a beaker. The egg suspension was passed through a 90 μm nylon screen, rinsed on a 25 μm screen, and resuspended on 5 L of filtered seawater to a density of approximately 50,000 eggs/mL. Egg and sperm quality were checked via observation (bright orange color, cell integrity, and sperm motility) under an Inverted Metallurgical Microscope (Axio Observer A1, Zeiss, Germany). Qualified sperm were added to the egg suspension at a ratio of 1:10–1:15 based on the following pairing strategy: (1) both parents were naïve (control group, ♀Sw and ♂Sw), (2) V. splendidus primed female and naïve male (maternal primed group, ♀Vs and ♂Sw), (3) naïve female and V. splendidus primed male (paternal primed group, ♀Sw and ♂Vs) ( Figure 1 ). Thus, approximately 250 million zygotes were obtained and incubated in 25 m³ sand-filtered seawater at 26°C. Salinity was around 30.

Experiment 2 was conducted after the embryos had developed to the blastula stage (4.5 h post-fertilization (hpf)). Briefly, embryos from the naïve group (n ≈ 3,000,000 embryos per tank) were divided into two groups, which remained unchallenged (nSw-Sw group, namely N group) and exposed to a challenge with V. splendidus (1 × 10⁷ CFU/mL, nSw-Vs group, namely N1 group). Embryos from the maternal-primed group (mothers were challenged with V. splendidus before spawning, N ≈ 3000,000 embryos per tank) were also divided into two groups and again exposed to a challenge with either V. splendidus (1 × 10⁷ CFU/mL, mVs-Vs (M1) group), or remained unchallenged (mVs-Sw (M) group). Embryos from the paternal primed group (fathers were challenged with V. splendidus before fertilization, N ≈ 3000,000 embryos per tank) were treated using the same method and two groups, pVs-Vs (P1) and pVs-Sw (P), were obtained ( Figure 1 ). Each group was maintained in a 10-L plastic bucket containing recirculating filtered seawater. Three replicates were used for each treatment. Larvae were collected at 6 h, 12 h, and 24 h after V. splendidus challenge, suspended in 1 mL TRIzol Reagent (Invitrogen, USA), and immediately stored in liquid nitrogen. Three parallel samples were pooled as one biological replicate, with three biological replicates for each time point.

In addition, larvae at different developmental stages, including zygote (1 hpf), blastula (4.5 hpf), gastrula (8.5 hpf), trochophore larvae (15 hpf), D-veliger stage 1 (24 hpf), D-veliger stage 2 (48 hpf), and early umbo larvae (120 hpf), were collected and directly stored in liquid nitrogen for enzyme activity assays or suspended in 1 mL of TRIzol Reagent for qualitative RT-PCR analysis. The experimental scheme is illustrated in Figure 1 .

Embryos developed into D-larvae (24 hpf) at a density of 100 larvae/mL. The tank water was changed every two days from this development stage onwards. The larvae were fed Chrysophyceae once daily. Larval density was assessed by means of microscope counts for each batch to further limit competition: larval concentration was progressively reduced from 10 to 5 and 3 larvae/mL on days 1, 5, and 7 post-fertilization, respectively. Sampling was performed at various stages of the oyster life cycle during the course of the experiment (46). The sieves (33 μm and 45 μm) were washed after each use and changed regularly depending on the spat growth.

Approximately 2 × 10⁶ larvae of blastula (4.5 hpf), D-veliger larvae 1 (24 hpf), and early umbo (120 hpf) stages were cultivated in three replicates with or without V. splendidus (1 × 10⁷ CFU/L). The larvae were collected at 6 h, 12 h, and 24 h after V. splendidus stimulation, suspended in 1 mL TRIzol Reagent, and immediately stored in liquid nitrogen for further mRNA extraction and transcriptome sequencing. Three parallel samples were pooled as one biological replicate, and there were three biological replicates for each time point.

The mortality and hatching rates of oyster larvae were distinguished by microscopy, according to a previous study (47), and calculated in samples of 100–200 individuals after the embryo developed into the D-veliger stage (24 hpf). For mortality statistics, the swimming capacity is generally used to measure larval viability. In addition, the cilium and velum of vibrant larvae would protrude the shell after being anesthetized, and thus, they were considered as the criteria to determine mortality. The hatching rate refers to the percentage of embryos reaching the D-larvae stage according to the initial number of oocytes used for fertilization. All larvae were collected from each tank by sieving through a 45 μm nylon mesh and then placed in a graduated beaker. Larvae were observed and counted under a binocular microscope (Olympus BX41, ×100). At this stage, larvae that were not D-shaped were considered to be abnormal. Larval samples were placed at a concentration of 50,000 individuals per liter of filtered seawater and fixed using 50 μL of 8% paraformaldehyde (Sangon, China). The percentage of abnormal D-shell larvae was scored for 3 × 100 individuals in each group. Abnormalities (D-larvae presenting mantle and/or shell abnormalities) were identified.

Total mRNA was isolated from D-veliger larvae of six experimental groups, including [nSw-Sw (N), nSw-Vs (N1), mVs-Sw (M), mVs-Vs (M1), pVs-Sw (P), and pVs-Vs (P1)], using TRIzol reagent following the manufacturer’s instructions. Qualified mRNA was converted into cDNA and then sequenced using the Ion Torrent Proton sequencing platform according to previous reports (48, 49). Three biological transcriptome sequencing replicates were used for each case. All bioinformatic analyses were performed according to a previous study (47), and the differentially expressed genes (DEGs) were further subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes Ortholog database (KEGG2) analyses.

Frozen gametes, larval samples and hemocytes were melted on ice and homogenized in 200 μL pre-cold PBS by using an electric homogenizer (T10 basic, IKA). The homogenate supernatants were collected as total protein extracts by centrifugation at 12,000×g at 4°C for 30 min, and the protein concentration was determined and quantified using the bicinchoninic acid method (BCA, Beyotime Biotechnology, China). The supernatants were divided into several aliquots and stored at −20°C.

The antibacterial activities of egg extracts from different groups were measured by detecting the bacterial growth curves. Prior to the assays, V. splendidus was grown overnight to stationary phase at 37°C in 5 mL of LB broth. A 1/50 dilution of the overnight culture was suspended in 5 mL fresh LB broth and grown for an additional 5 h at 37°C to obtain a mid-log-phase culture. The bacterial suspension was centrifuged, washed with PBS, and resuspended in PBS (1 × 10⁶ CFU/mL). Then, 100 μL of bacterial culture was mixed with 200 ng or 500 ng of egg extract (isolated from control and maternal primed groups, respectively, dissolved in PBS) and incubated at room temperature for 2 h. PBS and LB liquid medium were used as negative controls. Then, 20 μL of the mixture was added to a 96-well microliter plate with 200 μL of LB medium, which was placed in a microplate reader (Biotek, USA) at 37°C with shaking, and OD 600 was measured every 2 h to detect the growth of V. splendidus. Each experiment was repeated for three times.

The activities of superoxide dismutase (SOD), catalase (CAT), lysozyme (LYZ), and nitric oxide (NO) were measured using water-soluble tetrazolium salt (WST-1) and visible light and turbidimetry methods, respectively (Nanjing Jiancheng Bioengineering Institute, China).

Total mRNA was extracted using TRIzol reagent according to the manufacturer’s protocol. The quality and quantity of mRNA were assessed by electrophoresis on a 1% agarose gel and by Nanodrop 2000 measurement, respectively. Reverse transcription was performed with 1 mg of total mRNAs by using the Prime Script RT reagent Kit with gDNA Eraser (Takara, China) with oligo dT and random primers. The cDNAs from larvae at different developmental stages were used as templates after dilution to 1:50. The qRT-PCR reaction mixture (10 mL) consisted of 2×SYBR Green PCR Master mix, 0.4 mM each of the forward and reverse primers, and 1 mL of template cDNA. PCR amplification was performed under the following conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. Dissociation curve analysis was performed to determine target specificity. The specific primers for target genes and RS18 gene were prepared ( Table 1 ). The relative expression ratio of the target genes versus the RS18 gene was calculated using the comparative Ct method (2^△△Ct method), and all data were given in terms of relative mRNA expression.

The expression level in the control was regarded as 1.0; therefore, the expression ratio of the treatments was expressed in relation to the control. Significant differences in expression levels between the control and treatment groups were analyzed as previously described. The significance level was set at p <0.05.

To investigate the transfer of immune substances between mothers and offspring post-immune stimulation during the spawning season, the enzymatic activities of lysozyme and SOD, as well as NO and glucose levels, were examined in the eggs and hemocytes at 7-days post-priming from control (Sw) and Vs-primed females (experiment 1). In the eggs, compared with the control group (SW), the activities of lysozyme and SOD, and NO and glucose levels, all exhibited significant enhancement at 7-days post-priming stimulation (p <0.05, Figure 2 ). Briefly, the activity of lysozyme was 0.28 μg/mL in the control group (nSw) was 0.486 μg/mL in the maternally primed group (mVs), which was significantly upregulated (p <0.05, Figure 2A ). Similarly, the activity of SOD was also significantly increased in the maternally primed group (mVs), and was 0.877 U/gprot, while there was only 0.487 U/gprot in the control group (nSw) (p <0.05, Figure 2B ). For the NO production, compared with the control group (nSw), NO production was sharply increased in eggs from maternal primed group (mVs) at 7 days, which was 0.335 μmol/L in the maternal primed group (mVs), and only 0.073 μmol/L in the control group (nSw) (p <0.05, Figure 2C ). There were no significant differences in lysozyme, SOD activity, or NO production in hemocytes at 7 days between the control group (nSw) and maternally primed group (mVs) ( Figures 2A–C ). The glucose level in the eggs from the maternally primed group (mVs) was slightly higher than that in the eggs from the control group (nSw) at 7 days (p <0.05, Figure 2D ), whereas the glucose level in the hemocytes from the maternally primed group (mVs) was remarkably lower than that in the hemocytes from the control group (nSw) at 7 days (p <0.01, Figure 2D ). These results suggest that female oysters specifically transfer and store antioxidant enzymes in eggs upon bacterial priming during the spawning season.

To investigate the transfer of immune substances between mothers and offspring after immune stimulation during the spawning season, the expression of immune-related genes (TLR2, MACPF, and FBG) was examined at different time points (1 day, 3 days, 5 days, and 7 days) in the eggs and hemocytes from the control group (nSw) and maternal primed group (mVs) (experiment 1). Compared with the control group (nSw), the mRNA transcripts of the three candidate genes were significantly increased in hemocytes and eggs from the maternally primed group (mVs) ( Figure 3 ). Briefly, the transcripts of CgTLR2 were only significantly upregulated at 3 days and 5 days in the hemocytes after bacterial priming (p <0.05, Figure 3A ), while they exhibited a significant increase at all the tested time points (1 day, 3 days, 5 days, and 7 days) in the eggs, and showed the highest expression at 3 days in the maternally primed group (mVs), which was 11.93-fold of that in the control group (nSw) (p <0.05, Figure 3B ). Similarly, the mRNA level of CgMACPF was upregulated only at 3 days in hemocytes after bacterial priming (p <0.01, Figure 3C ). In contrast, the transcripts of CgMACPF were upregulated from days 1 to 7 in eggs after bacterial priming (p <0.01, Figure 3D ). Compared with the control group (nSw), the expression of CgFBG was both significantly induced in the hemocytes and eggs from the maternally primed group (mVs) (p <0.05, Figures 3E, F ). The expression of CgFBG in the hemocytes of the maternally primed group (mVs) was higher at 1 day and 3 days post-priming, which was 2.34- and 2.13-fold higher than that in the control group (nSw), respectively (p <0.05, Figure 3E ). Notably, the transcripts of CgFBG in the eggs were upregulated from 3 days to 7 days post-bacterial priming and exhibited the highest expression at 7 days, which was 14.8-fold higher than that in the control group (nSw) (p <0.01, Figure 3F ). These results collectively confirmed the transfer of immunity between mothers and offspring after immune stimulation during the spawning season.

To investigate the antibacterial activity of egg proteins extracted from control and V. splendidus-primed mature female oysters, a classical bacterial inhibition assay was performed. As shown in Figure 4 , compared with the control group (PBS), egg extracts from either the control group (nSw) or maternal primed group (mVs) at all the time points (1 day, 3 days, 5 days, and 7 days) all showed inhibitory effects on the growth of V. splendidus in a dose-dependent manner ( Figures 4A–D ). In addition, compared with the egg extracts from the control group (nSw), equal amounts of egg extracts from the maternally primed group (mVs) at 1 day ( Figure 4A ), 3 days ( Figure 4B ), 5 days ( Figure 4C ), and 7 days ( Figure 4D ) all showed more remarkable antibacterial activities from 10 h to 14 h (p <0.05), confirming the induced anti-bacterial activity of eggs after V. splendidus priming.

To evaluate the effects of parental immune priming on larval oysters, the activities of two antioxidant enzymes, SOD and CAT, as well as lysozyme, were detected in the zygote (1 hpf), blastula (4.5 hpf), and gastrula (8.5 hpf) stages in the three groups [nSw-Sw (N), mVs-Sw (M), and pVs-Sw (P) groups in experiment 2, which were unchallenged with V. splendidus during ontogeny]. Briefly, compared with the control group nSw-Sw (N), the activities of the three investigated enzymes were significantly enhanced in the mVs-Sw (M) group (i.e., maternally primed group, p <0.05, Figure 5 ). In particular, the highest enzyme activities of SOD and CAT both appeared at the blastula stage (4.5 hpf) (p <0.01, Figures 5A, C ), and the highest enzyme activities of lysozyme were detected at the gastrula stage (8.5 hpf) (p <0.01, Figure 5B ). Compared with the control group, nSw-Sw (N), the activities of SOD and lysozyme showed almost no significant difference between nSw-Sw (N) and pVs-Sw (P) (paternal primed group) ( Figures 5B, C ). Additionally, the CAT activity in the pVs-Sw (P) group was lower than that in the control group (p <0.05, Figure 5A ). These results collectively indicate that maternal immune priming induced immunological enzyme activity of the offspring during early ontogeny, while paternal immune priming showed little or no immunological effects on the larvae offspring.

To further investigate the effects of parental immune priming on the larval oyster, three immune-related genes (Cggalectin, Cgmyd88, and CgLBP) were selected based on the transcriptomic analysis, and the expression levels were quantified in the three groups [nSw-Sw (N), mVs-Sw (M), and pVs-Sw (P) groups in experiment 2]. For the Cggalectin gene, the relative expression of Cggalectin in the maternal immune priming [mVs-Sw (M)] and paternal immune priming [pVs-Sw (P)] was significantly upregulated at the zygote, blastula, and gastrula stages compared with the control group (nSw-Sw (N)) (p <0.05, Figure 6A ). In particular, the expression level of Cggalectin at the gastrula stage in the mVs-Sw (M) group was 7.95-fold of that in the nSw-Sw (N) group (p <0.01, Figure 6A ). For Cgmyd88, higher expression was observed at the blastula and gastrula stages in both the mVs-Sw (M) and pVs-Sw (P) groups (p <0.05, Figure 6B ). In particular, the expression level of Cgmyd88 at the blastula and gastrula stages in the paternal group was 3.6- and 4.8-fold higher than that in the nSw-Sw (N) group, respectively (p <0.01). In the mVs-Sw (M) group, the expression of CgLBP exhibited a slight upregulation at the gastrula stage compared to the nSw-Sw (N) group. The mRNA transcripts of CgLBP in the pVs-Sw (P) group were significantly upregulated at the zygote, blastula, and gastrula stages compared with those in the control group (p <0.05, Figure 6C ).

The performance of oyster larvae was observed using microscopy. For mortality statistics, the swimming capacity of D-veliger larvae and the position of cilium and velum after anesthetization were used to evaluate larval vitality. Based on observations and statistics, the mortality rate in the nSw-Vs (N1) group (parental oysters were normal, but larvae were bacterial challenged) was 15.3%, which was significantly higher (p <0.05) than that in the nSw-Sw (N) control group (parental and larval oysters were both normal) (7.6%, Figure 7A ). The mortality rates in the parental-primed groups [mVs-Sw (M) and pVs-Sw (P)] (either parental oysters were bacterial challenged, but larvae were normal) were 5.1% and 6.3%, respectively ( Figure 7A ), both lower than those in the nSw-Sw (N) control group. Notably, the mortality rates were significantly reduced in the two TGIP groups [mVs-Vs (M1) and pVs-Vs (P1)] (parental and larval oysters were both bacterial challenged) compared to the nSw-Vs (N1) group (parental oysters were normal, but larvae were bacterial challenged), which were 3.25% and 4.1%, respectively (p <0.01, Figure 7A ).

Hatching rate refers to the percentage of embryos reaching the D-larvae stage according to the initial number of oocytes used for fertilization. Based on the observations and statistics, it was 78.1% in the nSw-Sw (N) control group (parental and larval oysters were both normal). The hatching rate in the nSw-Vs (N1) group (parental oysters were normal, but larvae were bacterially challenged) (55.17%, p <0.05) was lower than that in the control group, which was consistent with the higher mortality rate in the nSw-Vs (N1) group ( Figure 7B ). Similarly, the hatching rates in the two parental primed groups (mVs-Sw (M) and pVs-Sw (P)) (either parental oysters were bacterially challenged, but larvae were normal) fit the low mortality rate and were slightly higher than that of the nSw-Sw (N) control group ( Figure 7B ). The hatching rates in the two TGIP groups [mVs-Vs (M1) and pVs-Vs (P1)] (parental and larval oysters were both bacterial challenged) were significantly higher than those in the nSw-Vs (N1) group (p <0.01), and the highest hatching rate (82.33%) was observed in the maternal TGIP group mVs-Vs (M1).

D-veliger larvae (24 hpf) were sampled from six groups [nSw-Sw (N) and nSw-Vs (N1), mVs-Sw (M) and mVs-Vs (M1), pVs-Sw (P), and pVs-Vs (P1)], and RNA-Seq data were generated from 18 transcriptome libraries with three replicates in each group. A total of 28,027 genes were identified. By performing pairwise analysis of differentially expressed genes (DEGs) in each group, a total of 1,029 DEGs with a fold-change of gene FPKM value <0.5 or >2 were obtained ( Tables 2 . 3 ). Among them, only 24 and 44 DEGs (upregulated) responded to the first stimulation of V. splendidus in female [nSw-Sw (N) and mVs-Sw (M)] and male [nSw-Sw (N) and pVs-Sw (P)] adult oysters 7 days before fertilization, respectively, and 479 DEGs responded to the secondary stimulation of V. splendidus that occurred at the blastula stage [nSw-Sw (N) and nSw-Vs (N1)], including 265 upregulated and 214 downregulated. Similarly, 321 DEGs were identified between the mVs-Sw (M) and mVs-Vs (M1) groups, including 190 upregulated and 131 downregulated genes, and 229 DEGs were identified between the pVs-Sw (P) and pVs-Vs (P1) groups, including 151 upregulated and 78 downregulated genes ( Tables 2 , 3 ), these DEGs were mainly induced because of the secondary challenge at the blastula stage.

First, to measure the ontogeny of larval innate immune system, the transcriptome of oyster D-veliger larvae in the present study [nSw-Sw (N)] was compared with the transcriptome of different tissues of oyster adults from previous reports (46, 50). More than 1,300 innate immune genes were identified in the transcriptome of nSw-Sw (N), and these genes were distributed in multiple immune signaling pathways, such as the Toll-like and TNF signaling pathways ( Supplementary Figure 1 ). Second, to explore the potential molecular mechanism in the maternal TGIP, 84 DEGs were screened by comparing nSw-Sw (N) and nSw-Vs (N1) with mVs-Sw (M) and mVs-Vs (M1), which responded to the secondary bacterial stimulation occurring at the blastula stage ( Figure 8A ). These genes were further analyzed to explore DEGs between the unstimulated group [nSw-Sw (N)] and the maternally primed group mVs-Sw (M), as well as the first response group nSw-Vs (N1) and TGIP group mVs-Vs (M1) ( Figures 8B, C ). However, only seven DEGs, including Peroxidasin, Selenium-binding protein 1, were differentially expressed in the first response group nSw-Vs (N1) and TGIP group mVs-Vs (M1), may might be responsible for the enhanced immune response during maternal TGIP ( Figure 8C ). A total of 84 DEGs were assigned into groups related to the “signal transduction,” “infection disease,” “translation,” “cancers,” and “endocrine system.” In addition, upregulated genes appeared to be assigned to “immune system” and “cell communication” ( Figure 8D ).

A similar analysis was performed in the paternal TGIP, and 24 DEGs were screened by comparing nSw-Sw (N) and nSw-Vs (N1) with pVs-Sw (P) and pVs-Vs (P1) ( Figure 9A ). To explore DEGs between the unstimulated group (nSw-Sw (N)) and primed group pVs-Sw (P), as well as the first response group nSw-Vs (N1) and TGIP group pVs-Vs (P1) ( Figures 9B, C ), 17 DEGs (such as E3 ubiquitin–protein ligase NEDD1, laminin) were identified in the first response group nSw-Vs (N1) and TGIP group mVs-Vs (M1), which might be responsible for the enhanced immune response during maternal TGIP ( Figure 9C ). The GO term distributions of the 24 DEGs were analyzed, and the biological processes of the GO terms are shown in Figure 9D , showing that they were mainly distributed in the oxidation–reduction process.

To investigate the potential regulation of DNA methylation in TGIP, the expression of three families of DNMTs was detected in the control group nSw-Sw (N), maternal primed group mVs-Sw (M), and paternal primed group pVs-Sw (P) at different developmental stages. In the maternally primed group mVs-Sw (M), the mRNA transcripts of the three DNMTs were low in the newly fertilized eggs (zygote, 1 hpf), but increased gradually to a relatively high level during oyster ontogeny ( Figure 10A ). Specially, the highest expressions of CgDNMT1 appeared at the gastrula stage (8.5 hpf, p <0.01), and also higher at the blastula (4.5 hpf) and D-veliger stage 2 (48 hpf), which were 2.75- and 3.53-fold of that in the control group (p <0.05) ( Figure 10A ). The expression level of CgDNMT2 was highest at D-veliger stage 1 (24 hpf), which was 9.17-fold higher than that in the control group, while it was also higher at the gastrula stage (8.5 hpf, p <0.01) ( Figure 10B ). The mRNA transcripts of CgDNMT3 increased gradually to a relatively higher level in the trochophore (15 hpf) and D-veliger stage 1 (24 hpf), which were 2.78- and 2.96-fold higher, respectively than those in the control group (p <0.01) ( Figure 10C ).

In the paternal primed group pVs-Sw (P), the expression patterns of the three DNMTs exhibited similar trends and showed lower or no significant difference during the early ontogeny (from zygote to gastrula) compared with the control group. In particular, the expression of CgDNMT1 and CgDNMT2 sharply increased after developing into D-veliger stage 1 (24 hpf), which was 30.64- and 5.44-fold higher than that in the in the control group, respectively (p <0.01), and reached their highest levels at D-veliger stage 2 (48 hpf), which were 30.64- and 5.44-fold higher than that in the in the control group, respectively (p <0.01) ( Figures 10A, B ). The expressions of CgDNMT3 was significantly upregulated in trochophore larvae (15 hpf, 2.17-fold, p <0.05) and reached its highest level at D-veliger stage 1 (24 hpf, 21.50-fold, p <0.01) ( Figure 10C ).

Based on transcriptome data, several E3 ligases were significantly induced after V. splendidus stimulation during ontogeny. Thus, to investigate the potential immune role of ubiquitination, the expression of four E3 ligases (CgWWP1, Cgsmurf2, CgNedd4, and CgMarch5) was examined at different time points after V. splendidus stimulation during early ontogeny. At the blastula stage (4.5 hpf), except for CgMarch5, the expression levels of the other three E3 ligases (CgWWP1, Cgsmurf2, and CgNedd4) were significantly downregulated at 6 h and 12 h post V. splendidus stimulation (p <0.05, Figures 11A–C ). At D-veliger stage 1 (24 hpf), except for Cgsmurf2, the expression of the other three E3 ligases (CgWWP1, CgNedd4, and CgMarch5) was significantly upregulated at 12 h post V. splendidus stimulation, which were 3.82-, 4.42-, and 3.53-fold of that in the control group (p <0.01, Figures 11A, C, D ). In the early umbo larvae (120 hpf), all four E3 ligases exhibited upregulation but distinct patterns after V. splendidus stimulation. Briefly, the mRNA transcripts of CgWWP1 were significantly increased at 6 h, 12 h, and 24 h after V. splendidus stimulation (p <0.05, Figure 11A ). The expressions of CgMarch5 were upregulated at 6 h and 24 h after V. splendidus stimulation (p <0.05, Figure 11D ), whereas the expression of Cgsmurf2 and CgNedd4 was upregulated at 6 h, and 24 h post V. splendidus stimulation, respectively (p <0.05, Figures 11B, C ). These results indicate that E3 ligases might function in immunoregulation after the larvae develop into the D-veliger stage, when the immune system of oyster larvae has been developed to some extent.