The C. hongkongensis used in the experiment were all of 2 years old, purchased from the seafood market in Xiashan District, Zhanjiang City, and temporarily reared for one week in the Shellfish Genetics and Enrichment Laboratory of Guangdong Ocean University. Chlorella was fed during the temporary incubation period, the water temperature was 28°C, the salinity was 20 ppt, continuous aeration was maintained. 108 C. hongkongensis were randomly selected, with a mean weight of 201.65 ± 22.20 g and a mean shell height of 117.03 ± 11.16 mm. They were divided into 4 groups, i.e., PBS group, Vibrio group, LPS group, and Poly(I:C) group, and each group was set up in 3 parallels, with 15 C. hongkongensis placed in each parallel group. C. hongkongensis in the Vibrio group were injected with 200 μL of V. harveyi solution at a concentration of 1×10⁸ CFU·mL^-1. The LPS group was injected with 200 μL of LPS (Solarbio, purity≥98%) solution at a concentration of 10 μg·mL^-1. The Poly(I:C) group was injected with 200 μL of Poly(I:C) solution at a concentration of 5 mg·mL^-1. The control group was injected with an equal amount of PBS. At 0, 12, 24, 48 and 72 h after injection, 2 C. hongkongensis were randomly selected from each parallel group and their hemolymph, gill, mantle, digestive diverticulum, adductor muscle, and gonad were sampled. All tissue samples were collected under strict sterile conditions. After collection, the samples were rapidly immersed in liquid nitrogen for snap-frozen to preserve their biological activity. They were then transferred to a -80°C low-temperature environment for storage in preparation for subsequent experiments.

At each time point within each group, three C. hongkongensis individuals were randomly selected, and RNA was extracted from the hemolymph, gill, mantle, digestive diverticulum, adductor muscle, and gonad. The extraction was performed using the TransZol Up Plus RNA Kit (TransGen Biotech), following the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer. RNA integrity was determined by agarose gel electrophoresis. All RNA samples were reverse transcribed using the EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech), following the instructions.

We screened the intermediate sequence of the ChPDIA3 gene based on the available transcriptome data (GenBank project accession PRJNA999463), and then used this sequence to design 5’RACE and 3’RACE specific primers for this gene using Primer Premier 5 ( Table 1 ). Full-length amplification was performed using the SMARTer^® RACE 5’/3’ Kit Protocol-At-A-Glance (TaKaRa), following the instructions. PCR products were detected by agarose gel electrophoresis, then cut and recovered using the NucleoSpin^® Gel and PCR Clean-Up kit (TaKaRa), and the recovered products were ligated into pEASY-Blunt (TransGen Biotech) vector. Trans1-T1 competent cell (TransGen Biotech) were used for transformation, and the products were placed in solid medium and incubated for 12 h in a 37°C incubator. Single colonies were screened and placed in liquid culture at 37°C, 200 rpm for 12 h. Positive cloning was performed, and the positive cloning products matching the target bands were sent to Sangon Biotech Ltd. for sequencing.

We used ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) to identify open reading frame as well as sequence translations. The physicochemical properties of the amino acids were queried by the online software ProtParam (https://web.expasy.org/protparam/). The conserved domains were analyzed according to the protein information resource InterPro (https://www.ebi.ac.uk/interpro). The full-length protein sequences of the PDIA3 homologues were downloaded at NCBI, and multiple sequence comparisons were performed using DNAMAN 5.0 with default parameters. Phylogenetic tree was constructed using Neighbor Joining in MEGA (v11.0.13).

Based on the sequences obtained from RACE cloning, primers were designed for RT-qPCR using Premier 5.0 ( Table 1 ). Reactions were performed in Light Cycler 96, as described in the instructions for the PerfectStart^® Green qPCR SuperMix kit (TransGen Biotech). Each sample was processed in triplicate in Light Cycler 96. The 2^-ΔΔCt method was used to calculate the relative expression of the ChPDIA3 gene in tissues of the C. hongkongensis such as hemolymph, gill, mantle, digestive diverticulum, adductor muscle, and gonad, with β-actin as the reference gene. The cDNAs of Vibrio group, LPS group, Poly(I:C) group and PBS group were taken as templates, and the rest of the conditions were the same as those mentioned above, to verify the expression of ChPDIA3 at different time points in each group.

Based on the sequences obtained by RACE cloning, primers were designed using Premier 5.0 ( Table 1 ) and PCR amplification was performed using Seq Amp DNA Polymerase (TaKaRa). Cutting gel recovery, ligation, transformation, positive cloning, and bacteriophage sequencing were performed as in Section 2.3. After the bacterial fluids were sequenced, a pair of forward and reverse sequences were selected for amplification. Plasmids obtained from amplification were extracted using the GeneJET Plasmid Miniprep Kit (Thermo). The plasmids were digested using Past I-HF enzyme and the digested products were detected by agarose gel electrophoresis. Products from single bands were purified using the GeneJET PCR Purification Kit (Thermo). The purified products were transcribed in vitro using the T7 RNAi Transcription Kit (Vazyme Biotech) as described in its instructions. The products of in vitro transcription were extracted by phenol-chloroform-ethanol absolute, then precipitated by ethanol absolute and finally dissolved in DEPC water.

The C. hongkongensis used in the experiment were temporarily reared for one week. Chlorella was fed during the temporary incubation period, the water temperature was 28°C, the salinity was 20 ppt, continuous aeration was maintained. 90 C. hongkongensis of similar growth condition and free from disease and injury were randomly selected. They were divided into three groups, i.e., RNA interference group, Vibrio group and control group, and three parallels were set in each group. From the pre-experiment, dsRNA acted 24 h after injection, so the experimental group was first injected with 100 μL of dsRNA (at a concentration of 1 μg·μL^-1). After 24 h, 100 μL of V. harveyi was injected. The Vibrio group was injected with an equal amount of V. harveyi solution at a concentration of 1×10⁸ CFU·mL^-1, and the control group was injected with an equal amount of 5 × PBS. 2 C. hongkongensis were randomly selected from each parallel group for sampling 12, 24 48 and 72 h after the completion of injection, respectively. The samples were first snap-frozen in liquid nitrogen and later stored at -80°C for later RNA extraction.

At each time point in each group, three samples were randomly selected for RNA extraction and reverse transcription, which was performed in the same way as in Section 2.2. The cDNA at each time point was used as a template for RT-qPCR to detect the expression of ChPDIA3 in V. harveyi infection after RNA interference.

The relationship between ChPDIA3 and miR-126-x and miR-21-y binding sites were selected for vector construction, respectively. The vector was prepared by Wuhan Zhibo Biotechnology Co., Ltd. and its sequence accuracy was verified. HEK293T cells were inoculated in 96-well plates at 2×10⁴ cells/well, with 3 replicate wells per group. The medium was 100 μl of DMEM-High Sugar medium containing 10% FBS, which was incubated overnight in a 5% CO₂, 37°C incubator. The transfection system was configured according to Table 2 . Solution A and solution B were mixed and left to stand for 20 min at common temperature. 20 μL of transfection complex was added to each well and then placed in 5% CO₂, 37°C incubator. After 48 h of transfection, the old medium was aspirated and 100 μL of PLB (Passive Lysis Buffer) was added to each well of cells. Lysis was performed on a shaker at common temperature for 15 min. After adding 20 μL of cell lysate to the luminescent plate, background values were read for 2 s using Promega’s GloMax^®-Multi multifunctional enzyme marker. Add 20 μL of LAR II working solution to each well, mix quickly, and read values for 2 s. After the reading is complete, add another 20 μL of Stop & Glo^® Reagent to each sample and mix quickly. Place in a luminescence detector, run the program and read the fluorescence value for 2 s. After completing the reading, save the data.

All data analyses were performed using Origin 2024 and IBM SPSS Statistics 26.0. Samples from different groups at the same time were analyzed for statistical significance using Duncan’s Multiple Range Test, the significance level was set at 0.05.

The cDNA sequence of the PDIA3 gene of the C. hongkongensis, named ChPDIA3, has been cloned using the RACE technique. ChPDIA3 has a total of 2081 bp, containing 55 bp of 5’-untranslated region (UTR) and 547 bp of 3’-UTR. The sequence of ChPDIA3 has an ORF with a length of 1,479 bp, encoding 492 amino acids ( Figure 1A ). The molecular weight is 55530.92 Da, the theoretical pI is 5.48, and the grand average of hydropathicity is -0.551<0, which makes it a hydrophilic protein. The results of the phylogenetic tree showed that the C. hongkongensis was clustered with other shellfish and was genetically distant from crustaceans and vertebrates. The predicted conserved structural domains of the ChPDIA3 sequence showed that the PDI_thioredoxin-like_dom are located at 23-123 aa and 368-470 aa ( Figure 1B ). Thioredoxin_CS in the ChPDIA3 protein has the closest homology to the C.gigas and the Ostrea edulis compared to homologs from other species ( Figure 1C ).The cDNA sequence of ChPDIA3 has been submitted to NCBI (GenBank: PP530092).

The expression of ChPDIA3 in different tissues was examined using RT-qPCR, and the results are shown in Figure 2 . ChPDIA3 was expressed in tissues such as hemolymph, gill, mantle, digestive diverticulum, adductor muscle and gonad. And the highest expression was found in gill (P<0.05). Therefore, gills were targeted for subsequent studies.

The relative expression of ChPDIA3 in the gill tissues of the C. hongkongensis under PBS, V. harveyi, LPS and Poly (I:C) treatments, at different time points, was examined using RT-qPCR. The results are shown in Figure 3 , under V. harveyi and LPS treatments, the expression of ChPDIA3 was significantly higher that of the other groups (P<0.05). And the highest expression at 12h after treatment, after which the expression of ChPDIA3 gradually decreased. The expression of ChPDIA3 in the gills of C. hongkongensis treated with Poly(I:C) was significantly lower than that of the PBS group (P<0.05), but showed a gradually increasing trend.

The expression of ChPDIA3 after silencing by dsRNA was detected by RT-qPCR. The results showed that the expression of ChPDIA3 was significantly lower than that of C. hongkongensis injected with PBS and V. harveyi at all four time points (P<0.05). The inhibitory effect was most significant at 12 h of V. harveyi injection (P<0.05), and the inhibitory effect gradually failed after 72 h of injection, when the expression started to increase ( Figure 4A ). In the protein processing in endoplasmic reticulum pathway, the expression of its downstream genes was also downregulated when ChPDIA3 was silenced by dsRNA. Except for LMAN2, the expression levels of the other two genes did not show significant differences at 12 hours (P>0.05). It was not until 48 hours that the expression level of CALR exhibited significant differences (P<0.05). Furthermore, the expression levels of LMAN1 and LMAN2 became progressively less significant compared to the control group (P>0.05) ( Figures 4B–D ).

The luciferase activities of miR-126-x mimics, miR-21-y mimics and their pmirGLO-PDIA3-3’-UTR co-transfected HEK293T cell lines were assayed by dual luciferase experiments to validate the targeting relationship between miR-126-x, miR-21-y and ChPDIA3, respectively. The results showed that both miR-126-x and miR-21-y inhibited ChPDIA3. In the miR-126-x group, compared with mimics NC (control group), luciferase activity extremely significantly decreased (P<0.01), being 74.93% of the mimics NC (control) group’s level. Mutating the predicted site, the luciferase activity was restored to 99.13% of the control (P>0.05) ( Figure 5A ). In the miR-21-y mimics group, dual luciferase activity extremely significantly (P<0.01), dropping by 76.94% compared to the control group. Mutating the predicted site restored the luciferase activity to 100.68% of the control (P>0.05) ( Figure 5B ).

PDIA3, also known as 1,25D3-MARRS or ERp57, is a member of the PDI family, which functions as an endoplasmic reticulum-based redox chaperone protein that interacts with various fibrosis-related proteins. In the endoplasmic reticulum, PDIA3 acts as a molecular chaperone and redox catalyst to regulate glycoprotein folding (Oliver et al., 1999). In our previous study, PDIA3 was significantly enriched in the protein processing in endoplasmic reticulum pathway (Hou et al., 2023). In this study, the cDNA sequence of PDIA3 from the C. hongkongensis, was obtained by RACE cloning technology. The homogeneous analysis demonstrated that the nucleic acid sequence and amino acid sequence of this gene are relatively conserved, especially conserved site Thioredoxin_CS (Fritz-Wolf et al., 2011; Yang et al., 2024). It was hypothesized that ChPDIA3 was identified as a homologous gene of PDIA3 in the C. hongkongensis. Therefore, the mechanism of ChPDIA3 might be similar to that of other species that have been studied. In addition, in shellfish and crustaceans, the ChPDIA3 sequence contains the PDI_thioredoxin-like_dom conserved domain, whereas the PDIA3_PDI_b conserved domain is also present in fish and mammals. This difference may indicate some evolution and differentiation in the structure and function of the PDI_thioredoxin-like_dom conserved domain in the PDIA3 sequences of different species. The PDI_thioredoxin-like_dom conserved domain is usually associated with the function of protein disulfide isomerase (PDI), which plays an important role in the endoplasmic reticulum, helping proteins to fold correctly and form disulfide bonds (Mahmood et al., 2021). However, differences in the conserved domains in the PDIA3 sequences of different species may reflect differences in their functional requirements or adaptive strategies for this conserved domain during evolution. Differences in the conserved domains in the PDIA3 sequences of different species may imply that they have diverged in functions such as protein folding and endoplasmic reticulum stress response. These species may have evolved specific PDIA3 structures and functions during adaptation to different environments and lifestyles (Liu et al., 2022; Trnková et al., 2013). When they are exposed to specific endoplasmic reticulum stress conditions or protein folding requirements, which contribute to the alteration of the PDIA3 sequence and the evolution of the conserved domains (Huang et al., 2009; Pinto et al., 2013).

Hydrophilicity plays an important role in the immune response (Boquet et al., 1995). It was found that there was a significant hydrophilic complementation between the complementary determining regions (CDR) of anti-neuropeptide substance monoclonal antibody (mAb SP31) and the C-terminal pentapeptide epitope of SP. Peptides that retained the hydrophilicity feature were recognized by mAb SP31 with reduced affinity even though the sequence differed from that of SP, whereas peptides with altered hydrophilicity feature showed a significant decrease in antibody binding affinity or even failed to bind. In addition, the degree of hydrophilic complementarity was closely related to the binding affinity, with peptides with similar ydrophilic profiles having a higher binding affinity to mAb SP31, while peptides with large differences in hydrophilicity showed significantly lower affinity (Hanin et al., 1997). Kinetic studies also suggest that hydrophilic complementarity is important in maintaining the stability of antigen-antibody complexes and may regulate binding affinity by affecting conformational changes and interaction stability after complex formation (Bhat et al., 1994; Van Oss, 1995). hydrophilicity also plays an important role in the immunomodulation of viral infections. The β-convex trigger loop region (QGEESND) of interleukin-1β (IL-1β) is hydrophilic and complementary to specific peptides (e.g., LITVLNI) in the interleukin-1 type I receptor (IL-1R1), and this complementarity influences viral binding to the receptor as well as the subsequent immune response (Heal et al., 1999). In the present study, ChPDIA3 is a hydrophilic protein, and we speculated that ChPDIA3 may play important roles in immune responses through various mechanisms, such as participating in antigen processing and presentation, regulating immune cell activity, influencing cytokine production and secretion, and interacting with other immune-related molecules, thus positively affecting the body’s immune defenses and maintenance of immune homeostasis. However, these roles still need further experimental validation and in-depth studies.

Due to its function as a molecular chaperone, PDIA3 expression rises when cells are stressed. It has been reported that PDIA3 can trigger Bak-dependent apoptosis by affecting mitochondrial outer membrane permeability (Zhao et al., 2015). Moreover, PDIA3 has functions such as regulating inflammation and oxidative stress (Wang, 2019) and inhibiting cancer cells (Yang et al., 2024). PDIA3 also plays an important role in immune activity in aquatic animals. Immunity-related proteins such as PDIA3 were significantly up-regulated in the intestine of Cynoglossus semilaevis in response to Shewanella algae infection (Han et al., 2020). The expression level of ERp57 protein in O. mykiss was elevated in peripheral blood leukocytes and RTS11 macrophage-like cell lines in response to stimulation with double-stranded RNA and phytohaemagglutinin, which also suggests a possible conserved function of ERp57 in the endoplasmic reticulum and activation of immune responses in O. mykiss (Sever et al., 2013). This may be due to the respiratory, filter feeding and immunological effects of the gills of the C. hongkongensis (Xie et al., 2023; Yue et al., 2024). This makes it possible for C. hongkongensis to be frequently exposed to externally supported disease-causing microorganisms such as bacteria and viruses. The gills of the C. hongkongensis, as respiratory organs, are the first to come into contact with the outside world, which also makes the gills of the C. hongkongensis play an irreplaceable role in its immune defense activities. The expression of ChPDIA3 was significantly higher in V. harveyi and LPS infections relative to controls.

Mollusks typically possess a relatively unsophisticated adaptive immune system, thus relying predominantly on their innate immunity to counteract against foreign pathogens (Chakroun et al., 2021; Tincu and Taylor, 2004; Yang et al., 2021). The innate immune response is characterized by its swift activation, commencing promptly during the initial phase of acute infections(L. Li et al., 2024; Olasard et al., 2024). V. harveyi, a Gram-negative bacterium, and LPS, a component of the Gram-negative bacterial cell wall, are both potent activators of the innate immune response (Shi et al., 2024; Yao et al., 2024). They can stimulate pattern recognition receptors (PRR), such as Toll-like receptors (TLRs) (Kawai and Akira, 2010). This recognition mechanism can trigger a rapid inflammatory response, which may include the upregulation of ChPDIA3 as part of the cellular stress response to invading pathogens (Mahmood et al., 2021).In our study, focusing on the C. hongkongensis, we had observed the highest expression levels of ChPDIA3 at 12 hours post-infection with V. harveyi and LPS. This had suggested that during the incipient stages of infection, C. hongkongensis may have bolstered its defense mechanisms by enhancing the proper folding and maturation of proteins critical for immune function, such as antimicrobial peptides (Yoo et al., 2019). Additionally, the early phase of bacterial invasion was likely to have provoked oxidative stress, which in turn, may have activated the unfolded protein response (UPR). Given that ChPDIA3 had played a role in modulating oxidative stress through its interactions with other oxidoreductases (Chamberlain et al., 2019), its heightened expression during the early infection stages may have served as a cellular protective response to oxidative challenges. However, our sampling regimen, initiated at 12 hours post-infection, precluded a more precise determination of the peak expression timing of ChPDIA3. In future related studies, we will consider collecting samples at earlier time points after infection. This will help us more accurately depict the changing trend of the expression level of ChPDIA3, so as to evaluate the importance of ChPDIA3 in the early immune response. Furthermore, we observed a significant downregulation of ChPDIA3 expression in C. hongkongensis that had been treated with Poly(I:C). This phenomenon may have been associated with the immune pathways activated by viral infections, which might not have relied on the function of ChPDIA3. For instance, RNA sensing pathways played a primary role in response to viral infections (Kourko et al., 2023; Yue et al., 2023), while ChPDIA3 may have been more involved in the immune response to bacterial infections. These findings suggested that ChPDIA3 may have played distinct roles in response to different immune challenges, and its expression regulation could have been closely linked to signaling pathways specific to certain pathogens.

The endoplasmic reticulum protein processing pathway is of crucial significance in the folding, modification, and transport of proteins within cells. PDIA3, being a vital member of this pathway, interacts with downstream genes that are indispensable for maintaining the normal physiological functions of cells. In this study, when the ChPDIA3 was silenced, the expression of the downstream genes of the ChPDIA3 in the endoplasmic reticulum protein processing pathway was successively down-regulated. We hypothesized that in response to V. harveyi infection, ChPDIA3 might have adjusted the protein processing and folding process by regulating the expression of downstream genes to enhance cellular defenses or adapt to infection-induced stress responses. For example, CALR (calreticulin), which was involved in protein folding and quality control, and changes in its expression might have affected the correct folding and stability of intracellular proteins, thereby influencing the cellular immune response and stress resistance (Galluzzi and Kroemer, 2022; Ziffels et al., 2019). LMAN1 (mannose-binding lectin-associated serine protease 1) and LMAN2 (mannose-binding lectin-associated serine protease 2), which played a role in innate immunity, and the down-regulation of their expression might have affected the ability of oysters to recognize and clear pathogens (Kwon et al., 2016; Zhang et al., 2023; Zheng et al., 2010). This further emphasized the importance of ChPDIA3 in the immune defense system of the C. hongkongensis and its critical position in the complex regulatory network in response to V. harveyi infection.

As a unique endothelial cell-associated miRNA, as well as a novel tumour suppressor gene, miR-126 plays a key role in the onset, progression and metastasis of various types of cancers, including hepatocellular carcinoma, colorectal carcinoma, melanoma and lung cancer. For example, cellular processing of MPM-derived spheroids by exosomal delivery of miR-126 leads to massive cell death and prevents tumour growth in vivo (Monaco et al., 2022). Overexpression of miR-126-3p in ovarian cancer cells inhibited cell proliferation and invasion as well as phosphorylation of AKT and ERK1/2 (Xiang and Cheng, 2018). The miR-21 family includes members such as miR-21-5p and miR-21-3p. These miRNAs play important regulatory roles in cell growth, differentiation, apoptosis and other processes, and play an important role in the treatment of cancer. The exosome miR-21-5p in hepatocellular carcinoma cells can affect hepatocellular carcinoma cell development and patient prognosis by regulating SP1/XBP1 and promoting M2 polarization in TAMs (Hu et al., 2024). miR-21 is elevated in canine mammary tumors and positively correlates with gene expression of IL-6 and TNF-α and also with the proliferation index (Ki67 index) of tumour cells (Abbate et al., 2023). In this study, the binding sites of miR-126-x, miR-21-y and ChPDIA3 were detected using dual luciferase experiments, respectively. The results showed that both miR-126-x and miR-21-y inhibited the 3’-UTR region of ChPDIA3. This suggested that they might be involved in the regulation of ChPDIA3 gene expression. And the expression of ChPDIA3 may be related to the response of C. hongkongensis to V. harveyi infection. When faced with V. harveyi infection, the organism may affect the expression of ChPDIA3 by regulating the expression levels of miR-126-x and miR-21-y, thereby modulating the immune response or other relevant physiological processes of the organism in response to the infection. This regulatory mechanism helps the C. hongkongensis to better resist V. harveyi infection and maintain the health and homeostasis of the organism.

Our findings on the role of ChPDIA3 in C. hongkongensis and its regulation by miR-126-x and miR-21-y offer potential applications for the aquaculture industry, particularly in enhancing disease resistance in oysters. One promising application is through selective breeding programs. By identifying and selecting oysters with higher levels of ChPDIA3 expression or those with genetic variants that enhance the gene’s function, breeders can develop lines with greater resistance to bacterial infections, such as those caused by V. harveyi. This approach will enable the breeding of oyster strains that are more resilient to disease outbreaks and reduce reliance on antibiotics, thereby promoting more sustainable and environmentally friendly aquaculture practices (Chamberlain et al., 2019; Wang et al., 2023). Additionally, the manipulation of miRNA expression levels, such as miR-126-x and miR-21-y, can be explored as a genetic engineering strategy. By regulating these miRNA levels, it is possible to increase the expression of ChPDIA3 and other immune-related genes, thereby enhancing the overall immune response in oysters (Chu et al., 2021; Pan, 2024; Qin et al., 2024). This could be particularly useful in hatcheries and nurseries, where oysters are most susceptible to infections. In conclusion, our study may contribute to the selection of disease-resistant oyster strains and disease control. However, the results of the current study are still some distance from direct application in production and require further in-depth research.

While our study provides new insights into the role of ChPDIA3 and its regulation by miR-126-x and miR-21-y in the immune response of C. hongkongensis against V. harveyi infection, several limitations should be acknowledged. First, the experimental design primarily focused on laboratory conditions, which may not fully replicate the complex environmental factors encountered in natural aquaculture settings. Factors such as water temperature, salinity, and the presence of other pathogens can significantly influence the expression and function of immune-related genes. Second, the study utilized a limited number of time points for sampling, which may not capture the complete dynamics of the immune response over the entire course of infection. Future studies would likely necessitate more frequent sampling to better understand the temporal changes in gene expression and the potential involvement of other immune pathways. Additionally, while the dual luciferase experiments provided evidence of miRNA regulation, the study did not explore the downstream effects of ChPDIA3 on other components of the immune system in detail, such as signaling pathways. Further research is needed to elucidate the broader network of interactions and pathways involved in the immune response mediated by ChPDIA3.