In marine aquaculture environments, microplastics (MPs) and cadmium (Cd) are widespread contaminants that may jointly affect host–microbe interactions. Here, we examined the combined effects of MPs (5 mg/L) and Cd (5 μg/L) on the intestinal microbial community of pearl oysters after a 48 h exposure, followed by a 5-day recovery period. Gut microbiota dynamics were characterized using 16S rRNA gene sequencing. Alpha diversity did not vary significantly, whereas beta diversity showed marked alterations in community composition among the different exposure treatments. LEfSe analysis revealed distinct microbial biomarkers and putative pathogens under each treatment: Sulfitobacter in the MPs-alone group; Vibrio and Candidatus_Megaira in the Cd-alone group; and Tenacibaculum, Roseibacillus, and Enterovibrio across different co-exposure and recovery groups. A brief recovery period partially decreased the abundance of certain pathogens (e.g., Vibrio), yet some taxa (e.g., Enterovibrio and Tenacibaculum) remained enriched. These results indicate that exposure to MPs and Cd, whether alone or in combination, disrupts gut microbial homeostasis in pearl oysters by reshaping community structure and promoting the proliferation of potential pathogens, with some disturbances persisting after exposure ceases. Generally, our findings will aid evaluation of the ecological risks of combined pollutants in marine aquaculture systems.

Transplantation surgery induces severe immune stress in pearl oysters Pinctada fucata martensii, yet effective immunomodulatory strategies remain limited. This study establishes a proteomics-guided strategy for immunomodulation by identifying cyclophilin as a candidate mediator through iTRAQ-based quantitative analysis and validating cyclosporin A (CsA) through serum immune indicator and transcriptome analysis. Serum iTRAQ analysis identified 1069 differentially expressed proteins following transplantation, among which cyclophilin exhibited significant up-regulation and was selected as a candidate target for therapeutic intervention. Dose-response assays determined 3.9 × 10-3 mol/L as the optimal concentration, which significantly suppressed TNF-α levels under LPS stimulation (P < 0.05). At this concentration, CsA pre-treatment markedly decreased circulating IL-17 concentrations, while it enhanced SOD and CAT enzymatic activities (P < 0.05). Transcriptome analysis revealed that CsA treatment was associated with upregulation of HIF-1 and Notch signaling pathway-related genes, concomitant with alleviation of LPS-induced downregulation in DNA replication and cell cycle pathways. Furthermore, CsA significantly inhibited hemocyte apoptosis compared to the control group (P < 0.05). These findings demonstrate that iTRAQ-based proteomic screening enables rational target identification for immunomodulation, and CsA exerts protective effects associated with altered expression of HIF-1/Notch pathway-related genes and apoptosis inhibition. These findings provide a molecular basis for developing immunomodulatory strategies in pearl oyster aquaculture.

Investigating the salinity tolerance of the freshwater mussel Hyriopsis cumingii is important for enhancing the ecological use of saline waters. This work integrated gill transcriptomics and metabolomics with assays of key hepatopancreatic enzyme activities to characterize the response of H. cumingii to chronic salinity stress (5 PSU) for 40 days). We detected 1011 DEGs and 385 DAMs. Mussels relied on several coordinated mechanisms to mitigate stress. An inorganic ion-regulatory network centered on calcium signaling is triggered; during prolonged exposure, energy allocation shifts by downregulating energetically expensive ion pumps (e.g., Na+/K+-ATPase). An organic osmoregulatory program is engaged that depends on free amino acids (e.g., alanine, taurine), betaine, and glycerol, supported by the marked upregulation of major transporter (Slc1a4, SLC1A3, Abcb1a) and synthase (gadl1) genes. Antioxidant protection is redirected away from the conventional SOD/CAT axis toward a more energy-saving route centered on glutathione metabolism, with GPX2 and chac1/chac2 acting in concert to maintain redox homeostasis. Energy-intensive functions, including muscle contraction (downregulation of Ttn) and biomineralization (downregulation of chs-2, Perlucin), are suppressed, whereas gluconeogenesis (upregulation of Pck1) and lipid mobilization (increased lipase activity) are strengthened. Thus, the salinity response of H. cumingii mounts an integrated response via energy trade-offs and metabolic rewiring. This study sheds new light on the adaptive capacity of H. cumingii across the 1-5 PSU salinity range and has implications for advancing its ecological aquaculture in saline-alkaline waters.

Hypoxia is a major constraint in aquaculture, and improving tolerance is a practical priority. HPD has been proposed as a strategy to improve hypoxia tolerance in the pearl oyster Pinctada fucata martensii. In this study, we applied HPD to the pearl oyster P. fucata martensii and profiled the regulatory mechanisms by wholetranscriptome sequencing. Oysters in the EG received HPD consisting of eight repeated cycles of moderate hypoxia (DO, 2.0 mg L- 1) for 24 h followed by 48 h recovery under normoxia, while the CG received no HPD. Both groups were then subjected to a 15-day hypoxic challenge (1.5 mg L- 1). HPD preserved gill architecture, with intact filaments, uniform cells, and maintained lacunae. We identified 1165 differentially expressed mRNAs, 676 lncRNAs, 81 circRNAs, and 40 miRNAs, and constructed a ceRNA network comprising 23 lncRNAs, 5 circRNAs, 6 miRNAs, and 10 mRNAs. The network linked the MSTRG.19199.1/novel-m0158-3p axis and the MSTRG.22411.1/miR-363-y axis to modulation of Cu/Zn-SOD and CuAO. It also connected the novel_circ_002334/ novel-m0103-5p axis and the novel_circ_002335/miR-363-y axis to the repression of MED10, with Wnt-associated anti-apoptotic signaling supported by Bcl-2 and ATF6 dynamics. Concomitant suppression of an E3 ubiquitin ligase suggests a key hub coupling hypoxia sensing to immune reprogramming. Additional signatures suggested immune recalibration, metabolic switching involving FAT4 and PDHB, hypoxia-inducible factor 1 alpha related upregulation of PDK1, and reduced transcriptional load through coordinated suppression of HSP70, PIF1, Tcb2, and Dis3L. Together, HPD promotes a pre-adapted, lower-cost homeostatic state that enhances hypoxia tolerance and provides molecular targets for improving oyster resilience in aquaculture.

Hypoxia frequently triggers mass mortality events in pearl oysters during the summer months. Hypoxic preconditioning (HP), repeated exposure to sublethal low-oxygen conditions, has been proposed as a potential strategy to enhance stress resistance. Here, we investigated how HP affects hypoxia tolerance in the pearl oyster Pinctada fucata martensii, with emphasis on host apoptotic and immune regulation and the gut microbiota. Pearl oysters assigned to HP (experimental group, EG) and to a non-preconditioned control group (CG) were subjected to sustained hypoxic challenge (1.5 +/- 0.1 mg/L DO for 15 days). HP significantly increased the expression of apoptosis- and immunity-related genes (MyD88, I kappa K, NF-kappa B) while suppressing JNK expression in gills after extended hypoxia (MyD88: EG 2.26 +/- 0.65 vs. CG 0.96 +/- 0.29, p < 0.05, similar to 2.3-fold increase; NF-kappa B: EG 1.50 +/- 0.20 vs. CG 0.81 +/- 0.31, p < 0.05, similar to 1.8-fold increase; I kappa K: EG 1.55 +/- 0.38 vs. CG 0.65 +/- 0.12, p < 0.05, similar to 4.0-fold increase; JNK: EG 0.49 +/- 0.25 vs. CG 1.44 +/- 0.51, p < 0.05, similar to 0.34-fold), consistent with a pre-activated yet controlled stress response. In parallel, HP markedly reshaped the intestinal microbial community under hypoxia, increasing alpha diversity (Ace, Chao, and Sobs indices) and enriching potentially beneficial bacterial phyla such as Planctomycetota, Nitrospirota, and Fusobacteriota, groups often linked to nutrient cycling and short-chain fatty acid production. Collectively, these results suggest that HP-enhanced hypoxia tolerance in P. f. martensii is associated with coordinated modulation of host apoptotic and immune signaling and concomitant shifts in gut microbiome diversity. These findings highlight the role of the host-microbiota axis in environmental acclimation and suggest that HP may be a practical tool for improving bivalve performance under hypoxic stress in aquaculture.

Hypoxia is a prevalent environmental stressor in both natural and aquaculture environments. Pinctada fucata martensii, a commercially valuable pearl oyster species, is particularly susceptible to hypoxic stress in aquaculture. To explore its molecular response to short-term hypoxia, whole-transcriptome sequencing was performed on gill tissues under normoxic and hypoxic conditions. A total of 721 differentially expressed mRNAs (DEmRNAs), 259 long non-coding RNAs (DElncRNAs), 55 circular RNAs (DEcircRNAs), and 17 microRNAs (DEmiRNAs) were identified. Based on predicted interactions among these differentially expressed RNAs, a competing endogenous RNA (ceRNA) network was constructed, consisting of 9 DElncRNAs, 10 DEcircRNAs, 3 DEmiRNAs, and 6 DEmRNAs. Six DElncRNAs and three DEcircRNAs were predicted to interact with miR-152, and the axis-mediated activation of target genes—ALDH5A1 and SLC23A2, FAT4 and CCDC97, and LAC—was implicated in the modulation of metabolic pathways, enzyme activity, antioxidant defenses, thereby promoting antioxidant capacity and suppressing cell growth as part of an adaptive survival strategy. This ceRNA network reveals a key molecular hub that links hypoxia sensing to immunomodulatory mechanisms. Moreover, the co-repression of stress-response genes HSP20 and HSP70 in this network enhances hypoxic tolerance increased energy utilization efficiency and regulation of RNA. Functional analysis of the network revealed enrichment in pathways related to energy metabolism, antioxidant defense, and RNA splicing regulation. These findings provide novel insights into transcriptomic regulation and adaptive mechanisms in bivalves under hypoxic stress.

As primary producers in aquatic ecosystems, microalgae function not only as a natural source of nourishment for several economically important aquatic species but also as reservoirs of bioactive molecules. Microalgae can secrete exosome-like nanoparticles that transport functional biomolecules, such as proteins and nucleic acids, into the extracellular milieu, thereby mediating intercellular signaling and eliciting ecological or biomedical responses. Although plant-derived exosome-like nanoparticles have attracted attention for their utility in drug delivery and dermatology, the functional properties of microalgae-derived nanoparticles—particularly from species extensively applied in aquaculture—remain inadequately characterized. In this study, exosome-like nanovesicles were isolated from Nannochloropsis oculata (N-ELNs), a microalgal species widely used in aquaculture, and their skin-whitening potential was evaluated using the B16-F10 mouse melanoma cell model. The highest N-ELN yield was observed during the adaptation, exponential, and stationary growth phases. Uptake analyses confirmed the efficient internalization of N-ELNs by B16-F10 cells. Cell counting kit-8 assays indicated that N-ELNs exhibited no cytotoxic effects on melanoma cells or normal human dermal fibroblasts (HFF-1). Scratch wound healing assays revealed that N-ELNs exerted no significant effect on cellular migration. In B16-F10 cells, N-ELNs suppressed tyrosinase activity by downregulating Mitf and its downstream genes Tyr and Tyrp1, resulting in a substantial reduction in melanin synthesis (p < 0.05). The inhibitory effects of N-ELNs on melanin production, tyrosinase activity, and gene expression of Tyr, Tyrp1, and Mitf were comparable to those of the positive control, arbutin. Collectively, these findings suggest that N. oculata exhibits promising skin-whitening properties, providing a novel perspective for clinical applications and supporting the high-value utilization of the microalgae aquaculture industry.

The Runt family of transcription factors is evolutionarily conserved. Although its role in vertebrate endoskeleton formation is established, its function in invertebrate exoskeleton biomineralization remains unclear. Here, we show that PmRunt, the sole Runt homolog in pearl oyster Pinctada fucata martensii, is a master regulator of both biomineralization and immunity. Knockdown of PmRunt severely inhibited calcification and growth by disrupting ion homeostasis, reducing CA and inverting the expression balance of crystallization regulators. Transcriptomic exhibited that the interruption of PmRunt expression in mantle induced disorder of ion transportation and absorption of Ca2 + and HCO3-, protein/phosphonate synthesis as well as cell proliferation and differentiation. And CUT&Tag analyses revealed that PmRunt may directly bind RUNX2/3-type motifs (GAAACC) to regulate key genes (including matrix protein, cell cycle proteins and mineralization-related genes), while also associating with immune-related genes likely reflecting an ancestral RUNX1-like function in resident hemocytes. Our findings elucidate a conserved genetic pathway for biomineralization and provide novel insights into the evolutionary deep homology of skeletal systems.

Hypoxia poses a significant threat to aquaculture. Enhancing hypoxia tolerance in aquaculture animals is therefore critically important. Here, we examined the protective effects of HPD on the pearl oyster Pinctada fucata martensii and investigated the underlying mechanisms using transcriptome sequencing and DNA methylation profiling. The experimental groups comprised HPG (eight cycles at 2.0 mg/L for 24 h followed by 48 h of recovery) and CG (without HPD), both of which were exposed to a 15-d hypoxic challenge (1.5 mg/L). The survival rate was 36.96 % higher in the HPG than in the CG after 15 d of hypoxic stress. Physiological analyses revealed enhanced activities of antioxidant enzymes, digestive enzymes, and energy metabolism-related enzymes and lower malondialdehyde levels in HPG compared with CG. Transcriptomic profiling revealed that 1253 DEGs were enriched in pathways such as glutathione metabolism, lipid metabolism, and serotonergic synapse. DNA methylation analysis revealed that CG-type methylation was predominant in pearl oysters, with 21,430 CG-type DMRs identified between HPG and CG. DMR-associated genes were significantly enriched in 55 KEGG pathways, including the MAPK signaling pathway, protein processing in the endoplasmic reticulum, and glycolysis/gluconeogenesis. The 217 genes shared between CG-type DMR-associated genes and DEGs were enriched in tryptophan metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, and the synaptic vesicle cycle. Our findings suggest that hypoxic preconditioning enhances hypoxia tolerance by reinforcing antioxidant defenses, shifting energy metabolism from glycolysis to lipid and amino acid utilization, regulating apoptosisrelated pathways, and preserving neural function, thereby enhancing the resilience of oysters to hypoxic stress in aquaculture systems.

This study investigated the effects of nanoplastic (NP) exposure (0.15, 1.5 and 15 mg/L for 60 days) on Pinctada fucata martensii using integrated biochemical and transcriptomic analyses following exposure and a 7-day recovery period. Results showed that NP exposure induced significant oxidative stress, characterized by concentration- and time-dependent suppression of SOD, GPx, and CAT activities, alongside dynamic alterations in MDA content. A transient rebound in antioxidant enzyme activities was observed on day 15, followed by a significant decline by day 60. Concurrently, immunoenzyme (ACP, AKP) and digestive enzyme (amylase, protease) activities were significantly inhibited. A 7-day recovery period mitigated these disturbances in lower concentration groups (0.15 and 1.5 mg/L), but significant effects persisted in the 15 mg/L group. The differentially expressed genes (DEGs) between CG and EGs at 60 days post-exposure were determined by transcriptome data analysis, which identified 212 upregulated and 95 downregulated DEGs. Similarly, 491 downregulated and 577 upregulated DEGs were identified between the EGs and CG at 67 days post-exposure. Functional analysis revealed that exposure to NPs exerted adverse effects on detoxification, immune response, apoptosis, cytoskeletal dynamics, protein synthesis, and homeostasis in P. f. martensii at 60 days post-exposure. Short-term recovery experiments induced the aberrant expression of an increased number of genes, suggesting that NP exposure exerts long-term effects at a molecular level, potentially resulting in the establishment of a new equilibrium in P. f. martensii. These findings indicate that NP exposure causes substantial physiological and molecular dysregulation, with incomplete recovery at high concentrations, elucidating the underlying toxic mechanisms and potential long-term ecological risks.

RAR-related orphan receptor alpha (RORα), a core member of the NR1F subgroup within the nuclear receptor superfamily, critically regulates lipid metabolism and immune responses. This study identified the Pm-RORα (Pm-RORα1 and Pm-RORα2) gene and its alternative splicing isoforms in Pinctada fucata martensii and investigated their functions through bioinformatic analysis, quantitative PCR (qPCR), and bimolecular fluorescence complementation (BiFC). Phylogenetic analysis revealed high homology between Pm-RORα and the Pacific oyster Crassostrea gigas ROR. Pm-RORα1_1 and Pm-RORα1_2 were generated by exon skipping of exons 2 and 6 in Pm-RORα1, respectively, while intron retention in exon 7 of Pm-RORα2 produced Pm-RORα2_1. Domain analysis showed that Pm-RORα1_1 and Pm-RORα1_2 lacked the ZnF_C4 and HOLI domains, respectively, while Pm-RORα2_1 was deficient in the HOLI domain. qPCR analysis demonstrated the expression patterns following LPS stimulation, Pm-RORα1_1 and Pm-RORα2_1 were significantly upregulated at 12 h, whereas Pm-RORα1_2 peaked at 6 h. Following nucleus transplantation, stimulation induced significant upregulation of Pm-RORα1, Pm-RORα1_1 and Pm-RORα1_2 at 6 h, while both of Pm-RORα2 and Pm-RORα2_1 was significantly downregulated from 6 to 24 h. Bimolecular fluorescence complementation assays confirmed protein interactions between Pm-RORα1 and the three isoforms (Pm-RORα1_1, Pm-RORα1_2, and Pm-RORα2_1). These findings indicate that Pm-RORα and its splicing isoforms potentially form protein interactions to modulate dynamic immune regulation in P. f. martensii, providing molecular insights into molluscan innate immunity and pearl cultivation optimization.

Mytella strigata, a bivalve mollusk native to the Atlantic coast of South America, has recently become a globally significant marine invasive species, posing serious threats to native ecosystems and aquaculture operations. Here, we report a haplotype-resolved, chromosome-level genome assembly of M. strigata (2n = 30), generated using high-fidelity (HiFi) long-read sequencing and high-throughput chromosome conformation capture (Hi-C). Two haplotypes were independently assembled: haplotype 1 (Hap1) spans 692.37 Mb with a contig N50 of 6.93 Mb, and haplotype 2 (Hap2) spans 683.91 Mb with a contig N50 of 7.61 Mb. Both assemblies were anchored to 15 chromosomes, achieving anchoring rates of 93.84% (Hap1) and 97.08% (Hap2). Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis revealed high completeness, identifying 92.33% and 93.22% of expected single-copy orthologs in Hap1 and Hap2, respectively. We annotated 27,887 protein-coding genes and conducted analyses of gene functions. This high-quality genomic resource provides a foundation for investigating the genetic mechanisms underlying invasiveness and environmental adaptability in M. strigata.

Heat and Cd2+ stress are major environmental challenges for marine benthic invertebrates. This study examined their combined effects on growth, physiology, and transcriptomic responses in the peanut worm (Sipunculus nudus). After 30 days, Cd2+ reduced survival at 26 °C without significantly affecting growth, whereas at 32 °C, both survival and growth declined with increasing Cd2+ concentration, indicating that heat stress exacerbates Cd2+ toxicity. Cd accumulation increased with exposure concentration but was not affected by temperature. Heat stress increased immune (AKP) and antioxidant (SOD, CAT) enzyme activities, although significant increases in SOD and CAT were observed only under Cd2+ exposure. AKP activity rose at low Cd2+ concentrations and fell at high Cd2+ concentrations at 26 °C, whereas no significant difference occurred at 32 °C between 0 and 0.25 mg/L Cd2+. At the same temperature, SOD and CAT activities were significantly higher under high Cd2+ exposure than under low Cd2+ exposure. Transcriptome analysis showed that Cd2+ exposure activated longevity-related pathways, protein processing, and translation initiation. Heat stress activated Jak-STAT signaling and endoplasmic reticulum protein processing while inhibiting the ribosome pathway. Under combined stress, pathways related to xenobiotic metabolism, nutrient digestion and absorption, and amino acid derivative metabolism were broadly suppressed. These results highlight that heat stress exacerbates Cd2+ toxicity, affecting growth, enzyme activity, and transcriptomic responses, and provide insights into the adaptive strategies of marine benthic organisms under the combined pressures of climate change and heavy metal pollution.

Whether marine bivalves, as osmoconformers, actively transport inorganic ions to counter osmotic stress remains unclear due to insufficient direct evidence. The estuarine bivalve Crassostrea hongkongensis offers an ideal model to address this issue, as our previous ultrastructural study hinted at the existence of ionocyte‑like cells in the gill. Here, single‑cell RNA sequencing revealed that the homologs of key ion transporters in oyster gill, Na⁺/K⁺-ATPase α subunit (CHNKAα), Na⁺/K⁺/2Cl⁻ cotransporter 1 (CHNKCC1) and Na⁺/H⁺ exchanger 3 (CHNHE3), lack the cluster‑specific expression typical of teleost homologs. Instead, their transcripts are broadly distributed across multiple gill epithelial cells, confirmed by RNA fluorescence in situ hybridization. At 8 h post‑salinity exposure, CHNKAα and CHNHE3 transcripts increased at salinity 30 (positively correlated with gill Na⁺, K⁺ and Cl⁻ contents), whereas CHNKCC1 was upregulated at salinity 6 (negatively correlated), implicating their involvement in active ion transport. Multiplex immunofluorescence revealed an interesting membrane distribution: CHNKAα retained a conserved basal membrane pattern in gill epithelial cells, CHNKCC1 showed an unusual apical localization, and CHNHE3 displayed a cell‑type‑specific dual (apical and basal) membrane distribution. RNA interference-mediated knockdown further indicated that CHNKAα drives Cl⁻ uptake while CHNKCC1 mediates Cl⁻ secretion, directly linking their active transport to salinity‑induced ion content changes. Notably, these observations, particularly membrane distribution and functional contribution to ion transport, do not completely conform to any existing ion transport model in aquatic animals. Our findings provide direct evidence for active ion transport in a euryhaline bivalve under salinity stress and indicate a potential ion transport system distinct from that observed in most teleosts, opening new avenues for understanding osmoregulation in marine invertebrates.

Molluskan aquaculture is increasingly constrained by low-temperature stress, which adversely affects growth and survival rates. To enhance aquaculture efficiency, it is crucial to identify molecular markers associated with growth performance and low-temperature resistance. In this study, we investigated the association between insertions and deletions (InDels) in the LncRunt gene and key phenotypic traits in Pinctada fucata martensii, while also exploring genotype distribution and the molecular mechanisms underlying growth and cold adaptation using transcriptome analysis. Results revealed a key InDel variant (InDel-16) significantly associated with shell growth traits, with individuals carrying the dominant ATATATAT genotype exhibiting significantly greater shell length, width, height, total weight, and shell dry weight (p < 0.05) compared to other genotypes. This genotype likely enhances somatic growth by modulating the flexibility of LncRunt's protein interactions. Three alleles (A, B, and C) were identified at the LncRunt locus, with the C allele characterized by fragment deletions. Genotyping across four populations-including low-temperature resistance lines RF4 and RF5-revealed a strong enrichment of the CC genotype among low-temperature resistance individuals, while it was infrequent in other populations. Expression analysis under 17 degrees C low-temperature stress showed a significant downregulation of LncRunt expression in individuals from the base stock (p < 0.05). To investigate its functional role, RNA interference was employed to knockdown LncRunt expression in mantle tissue, leading to the identification of 882 differentially expressed genes. Enrichment analysis revealed that these genes were primarily involved in the PI3K-AKT-mTOR pathways. Moreover, LncRunt knockdown led to reduced expression of key growth-related genes, including PmRunt, structural matrix proteins (MMP, Pif, and N23), and genes associated with the PI3K-mTOR signaling pathway. Collectively, these findings highlight LncRunt as a promising molecular marker for growth and low-temperature resistance, with significant potential for marker-assisted selection and genomic breeding in pearl oyster aquaculture.

Temperature significantly affects the survival, growth, and physiological functions of marine organisms. The bay scallop southern subspecies, Argopecten irradians concentricus, well known for its high temperature tolerance, has not been deeply explored in terms of the molecular regulatory mechanism underlying its heat stress tolerance trait. In this study, we investigated the mechanisms underlying heat stress tolerance in two geographically distinct populations of A. i. concentricus, the southern population (SP) from Zhanjiang in southern China and the northern population (NP), which was originally transplanted from Zhanjiang and reared for 4 generations in Yantai waters. The upper incipient lethal temperature (LT50) of SP (31.81 +/- 0.14 degrees C) was significantly higher than that of NP (29.25 +/- 0.06 degrees C), indicating a significant decline in heat tolerance in NP following its transplantation and breeding in the northern waters. Comparative transcriptomic and metabolomic analyses, along with physiological measurements, revealed distinct regulatory mechanisms in two populations. NP predominantly up-regulated energy and metabolic pathways, including carbohydrate, amino acid, and lipid metabolism, whereas SP enhanced the expression in immune-related pathways such as NF-kappa B, FoxO, AMPK, and MAPK signaling. However, the cGMP-PKG signaling pathway, the calcium signaling pathway, and the C-type lectin receptor signaling pathway were down-regulated in both the NP and SP groups. Metabolomic profiling revealed that SP exhibited more diversified metabolic regulations than the NP populations. Weighted Gene Co-expression Network Analysis (WGCNA) identified key genes strongly associated with heat stress, including GPX1, CYP2B5, and PLA2G4A within the magenta module, all of which are involved in the arachidonic acid pathway. Integrated co-analysis further pinpointed biomarkers and genes linked to heat stress adaptation. Physiological analyses demonstrated that SP possessed stronger antioxidant defenses than NP, as evidenced by higher activities of antioxidant enzymes (CAT, POD, SOD) and lower MDA levels. Additionally, SP displayed more robust immune responses, including higher haemocyte counts and phagocytosis rates, and lower apoptosis rates, to heat stress. In conclusion, our study comprehensively reveals the different tolerance mechanisms of the two A. i. concentricus populations to heat stress. The findings may enhance our understanding of the heat tolerance mechanisms in scallops, providing new insights for optimizing aquaculture practices, such as selective breeding and environmental management.

Oyster aquaculture in South China is persistently challenged by seasonal salinity increases, primarily due to the limited high-salinity tolerance of the dominant species, Crassostrea hongkongensis. Osmotic stress, a major consequence of salinity fluctuation, is often mitigated by marine bivalves through employing free amino acids (FAA) as compatible intracellular osmolytes. To elucidate the regulatory mechanisms underlying FAA-mediated osmotic response in C. hongkongensis, we conducted an integrative analysis of whole-transcriptome sequencing data and FAA profiles from gill tissues of oysters under different salinity stresses. Weighted gene co-expression network analysis identified the turquoise mRNA module, which not only exhibited a strong negative correlation with FAA levels but was also significantly enriched in amino acid metabolic pathways. Subsequent ncRNA-mRNA interaction analysis identified several miRNA-mediated regulatory relationships targeting genes involved in amino acid metabolism. Notably, three miRNAs (miR-425-x, novel-m0160-5p and novel-m0161-3p) were predicted to downregulate Mho_042430, which encodes a functionally characterized SHMT1 homolog critical for serine-glycine interconversion during hyperosmotic stress in C. hongkongensis. This study reveals potential ncRNA-mRNA interactions that regulate amino acid metabolism during osmotic stress, providing novel mechanistic insights into FAA-mediated osmoregulation in C. hongkongensis.

The accumulation of cadmium (Cd) and microplastics (MPs) can have major deleterious effects on the health of marine ecosystems and organisms, including the pearl oyster Pinctada fucata martensii. Here, we characterized the effects of Cd and MPs on key biochemical parameters of P. f. martensii via an experiment with various treatments. Pearl oysters were exposed to either only Cd (5 or 50 μg/L), only MPs (5 mg/L), or both Cd and MPs for 2 d, and this was followed by a 5-day recovery period. Measurements of the activities of lipase, amylase, protease, T-ATPase, catalase, glutathione peroxidase, acid phosphatase, and alkaline phosphatase enzymes, as well as the malondialdehyde content in the hepatopancreas, were made at various time points during the experiment. Metabolomics analysis of the gills was also performed. Significant interactions between time and treatment on lipase, protease, and catalase activities were observed. However, no significant effect of time–treatment interactions on amylase and T-ATPase activities was observed. Enzyme activities varied among groups both during the exposure period (6 to 48 h) and the recovery period. The malondialdehyde content was also increased throughout the experiment. Pathway analysis indicated that the purine metabolism, glycerophospholipid metabolism, nucleotide metabolism, arachidonic acid metabolism, neuroactive ligand–receptor interaction, and linoleic acid metabolism pathways were the most commonly affected under different treatments. The findings of our study revealed the differential effects of exposure time and treatment on enzyme activities and metabolites and their respective pathways. Our findings enhance our understanding of the biochemical responses of the pearl oyster P. f. martensii to environmental stressors, particularly Cd and MPs.

PVC microplastics (PVC MPs) and copper (Cu) are pervasive marine pollutants with significant ecological impacts. This study analyzed individual and combined effects of PVC microplastics (PVC MPs) at concentrations of 1 and 5 mg/L and copper (Cu) at concentration of 5 μg/L on immune response, oxidative balance, detoxification capacity, and hepatopancreas metabolomics in Pinctada fucata martensii. Pearl oysters were exposed to these stressors for 13 days, followed by a 7 days recovery phase. Immune enzymes: acidic phosphatase (ACP) and alkaline phosphatase (AKP); antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx); and detoxification enzyme: glutathione S-transferase (GST) were assessed, alongside hepatopancreas metabolomic profiling. Results showed that combined exposure (MPs + Cu) significantly elevated ACP and AKP activities compared to individual treatments, indicating enhanced immune activation. Oxidative stress biomarkers (CAT, SOD, GPx) were significantly higher in co-exposed groups, suggesting synergistic toxicity. Moreover, metabolomic analysis revealed significant alterations in key pathways, including glycerophospholipid metabolism, pentose and glucuronate interconversions, arachidonic acid metabolism, and phosphatidylinositol signaling, reflecting disruptions in membrane integrity, energy metabolism, and inflammatory responses. During recovery, enzyme activities and metabolite trends indicated partial physiological restoration, though co-exposed oysters exhibited prolonged metabolic disturbances. This study highlights the compounded toxicity of MPs and Cu, emphasizing their role in impairing bivalve health through oxidative stress, immune modulation, and metabolic disruption. The findings highlight the need for comprehensive risk assessments of marine pollutant interactions to mitigate ecological and aquacultural impacts.