Exploring changes in microplastic-associated bacterial communities with time, location, and polymer type in Liusha Bay, China.

Microplastics have become a widespread concern within marine environments and are particularly evident in aquaculture regions that are characterized by plastic accumulation. This study employed 16 S rDNA sequencing to investigate the dynamic succession of microbial communities colonizing polyvinyl chloride (PVC), polystyrene (PS), and polyamide (PA) microplastics in seawater, when subjected to varying exposure durations in the Liusha Bay aquaculture region. Results revealed that the composition of microplastics microbial communities varied remarkably across geographical locations and exposure times. With an increase in exposure duration, both the diversity and richness of bacterial communities colonizing microplastics significantly increased, microbial communities show adaptations to the plastisphere. The type of microplastics had a significant effect on the community structure characteristicsof bacteria attached to their surfaces, with inconsistent trends in the relative abundance of different genera on different substrates. Notably, microplastic surfaces harbored a significant abundance of hydrocarbon-degrading bacteria, exemplified by Erythrobacter. These findings underscore the potential of microplastics as unique microbial niches. Meanwhile, long-term exposure experiments also offer the possibility of screening for plastic-degrading bacteria. In addition, the presence of the pathogenic bacterium Vibrio was detected in all microplastic samples, implying that microplastics could serve as carriers for pathogenic dissemination. This underscores the urgency of addressing the risk posed by the proliferation of harmful bacteria on microplastic surfaces. Overall, this study enhances our understanding of microbial community dynamics on microplastics under diverse conditions. It contributes to the broader comprehension of plastisphere microbial ecosystems in the marine environment, thereby addressing critical environmental implications.

Effects of titanium dioxide nanoparticle exposure on the gut microbiota of pearl oyster (Pinctada fucata martensii).

With the advancement of nanotechnology and the growing utilization of nanomaterials, titanium dioxide (TiO2) has been released into aquatic environments, posing potential ecotoxicological risks to aquatic organisms. In this study, the toxicological effects of TiO2 nanoparticles were investigated on the intestinal health of pearl oyster (Pinctada fucata martensii). The pearl oysters were subjected to a 14-day exposure to 5-mg/L TiO2 nanoparticle, followed by a 7-day recovery period. Subsequently, the intestinal tissues were analyzed using 16S rDNA high-throughput sequencing. The results from LEfSe analysis revealed that TiO2 nanoparticle increased the susceptibility of pearl oysters to potential pathogenic bacteria infections. Additionally, the TiO2 nanoparticles led to alterations in the abundance of microbial communities in the gut of pearl oysters. Notable changes included a decrease in the relative abundance of Phaeobacter and Nautella, and an increase in the Actinobacteria, which could potentially impact the immune function of pearl oysters. The abundance of Firmicutes and Bacteroidetes, as well as the expression of genes related to energy metabolism (AMPK, PK, SCS-1, SCS-2, SCS-3), were down-regulated, suggesting that TiO2 nanoparticles exposure may affect the digestive and energy metabolic functions of pearl oysters. Furthermore, the short-term recovery of seven days did not fully restore these levels to normal. These findings provide crucial insights and serve as an important reference for understanding the toxic effects of TiO2 nanoparticles on bivalves.

The physiological responses to titanium dioxide nanoparticles exposure in pearl oysters (Pinctada fucata martensii).

To evaluate the physiological responses to titanium dioxide nanoparticles exposure in pearl oysters (Pinctada fucata martensii), pearl oysters were exposed for 14 days to different levels (0.05, 0.5, and 5 mg/L) of nano-TiO2 suspensions, while a control group did not undergo any nano-TiO2 treatment. And then recovery experiments were performed for 7 days without nano-TiO2 exposure. At days 1, 3, 7, 14, 17, and 21, hepatopancreatic tissue samples were collected and used to examine the activities of protease, amylase, lipase, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), lysozyme (LYS), alkaline phosphatase (AKP), and acid phosphatase (ACP). The microstructure of the nacreous layer in shell was also analyzed by scanning electron microscopy. Results showed that pearl oysters exposed to 5 mg/L of TiO2 nanoparticles had significantly lower protease, amylase, and lipase activities and significantly higher CAT, SOD, GPx, LYS, ACP, and AKP activities than control pearl oysters did even after 7-day recovery (P-values <0.05). Pearl oysters exposed to 0.5 mg/L or 0.05 mg/L of TiO2 nanoparticles had lower protease, amylase, and lipase activities and higher CAT, SOD, GPx, LYS, ACP, and AKP activities than control pearl oysters did during the exposure period. After 7-day recovery, no significant differences in protease, lipase, SOD, GPx, CAT, ACP, AKP, or LYS activities were observed between pearl oysters exposed to 0.05 mg/L of TiO2 nanoparticles and control pearl oysters (P-values >0.05). In the period from day 7 to day 14, indistinct and irregular nacreous layer crystal structure in shell was observed. This study demonstrates that TiO2 nanoparticles exposure influences the levels of digestion, immune function, oxidative stress, and biomineralization in pearl oysters, which can be partially and weakly alleviated by short-term recovery. These findings contribute to understanding the mechanisms of action of TiO2 nanoparticles in bivalves. However, studies should evaluate whether a longer recovery period can restore to their normal levels in the future.

Integrated transcriptomic and metabolomic analysis reveals the response of pearl oyster (Pinctada fucata martensii) to long-term hypoxia.

The frequency at which organisms are exposed to hypoxic conditions in aquatic environments is increasing due to coastal eutrophication and global warming. To reveal the effects of long-term hypoxic stress on metabolic changes of pearl oyster, commonly known as Pinctada (Pinctada fucata martensii), the present study performed the integrated analysis of transcriptomics and metabolomics to investigate the global changes of genes and metabolites following 25 days hypoxia challenge. Transcriptome analysis detected 1108 differentially expressed genes (DEGs) between the control group and the hypoxia group. The gene ontology (GO) analysis of DEGs revealed that they are significantly enriched in functions such as "microtubule-based process", "histone (H3-K4, H3-K27, and H4-K20) trimethylation", "histone H4 acetylation", "kinesin complex", and "ATPase activity", and KEGG pathway functions, such as "DNA replication", "Apoptosis", and "MAPK signaling pathways". Metabolome analysis identified 68 significantly different metabolites from all identified metabolites, and associated with 25 metabolic pathways between the control and hypoxia groups. These pathways included aminoacyl-tRNA biosynthesis, arginine and proline metabolism, and phenylalanine metabolism. Our integrated analysis suggested that pearl oysters were subject to oxidative stress, apoptosis, immune inhibition, and neuronal excitability reduction under long-term hypoxic conditions. We also found a remarkable depression in a variety of biological functions under long-term hypoxia, including metabolic rates, biomineralization activities, and the repression of reorganization of the cytoskeleton and cell metabolism. These findings provide a basis for elucidating the mechanisms used by marine bivalves to cope with long-term hypoxic stress.

Effects of diel-cycling hypoxia and salinity on lipid metabolism and fatty acid composition of the oyster Crassostrea hongkongensis.

For marine animals living in estuarine, coastal, and intertidal areas, salinity changes and periodic hypoxia are typical stressors; however, how the varying salinity and dissolved oxygen affect the quality and nutrition of marine aquaculture species, such as oysters remains unknown. In this study, we evaluated the diel-cycling hypoxia under different salinities on fatty acid composition and lipid metabolism in oyster Crassostrea hongkongensis digestive glands. After 28 days of exposure, both hypoxia and elevated salinity caused a decrease in the saturated fatty acid (SFA)/polyunsaturated fatty acid (PUFA) ratio of C. hongkongensis, salinity mainly causes changes in C17:0, C17:1, C18:1n9, C20:1n9, C20:4n6, C21:5n3, C22:5n3, with high salinity being more damaging to the fatty acid fractions. Also, Hypoxia accelerates the synthesis of C18:1n9 and C20:4n6. Fatty acid synthase (FAS) synthesis is increased by reduced salinity or hypoxia, but Acetyl CoA carboxylase (ACC) only weakly promotes fatty acid synthesis. Under hypoxic conditions, the activity of both hepatic lipase (HL) and lipoprotein lipase activity (LPL) decreases, which is contrary to the results for dissolved oxygen. The increase in salinity under dissolved oxygen leads to a decrease in LPL activity and an increase in HL activity. Our findings highlighted that exposure to a combination of salinity and hypoxia stressors, can disrupt the protective mechanisms of the oyster and affect the function of its lipid metabolism. Therefore, long-term exposure to periodic hypoxia with salinity changes poses a risk to the nutritional quality of C. hongkongensis, affecting oyster aquaculture and the coastal ecosystem.

Immunomodulatory effects of one novel microRNA miR-63 in pearl oyster Pinctada fucata martensii.

Novel microRNA miR-63 (novel-miR-63) from pearl oyster Pinctada fucata martensii (Pm-novel-miR-63) is a species-specific miRNA. Our previous research has shown that the expression of Pm-novel-miR-63 was significantly downregulated at 24 h after nucleus transplantation. In this study, we analyzed the function and regulatory role of Pm-novel-miR-63 in the immune response of pearl oysters. The results showed that Pm-novel-miR-63 expression increased after the stimulation of pathogen associated molecular patterns at 6-12 h, and the activity of immune and antioxidant enzymes in the serum decreased after Pm-novel-miR-63 overexpression. Transcriptome analysis revealed that Pm-novel-miR-63 participated in regulating transplantation immunity through the Notch and mRNA surveillance signaling pathways. Target prediction and dual luciferase analysis revealed that Pm-GDP-FucTP, Pm-CysLTR2, and Pm-RLR were the target genes of Pm-novel-miR-63. These results suggested that Pm-novel-miR-63 participated in regulating the immune response in pearl oysters and can serve as a new interference target to reasonably control excessive immune rejection in pearl culture.

Whole Transcriptome Analysis Reveals the Global Molecular Responses of mRNAs, lncRNAs, miRNAs, circRNAs, and Their ceRNA Networks to Salinity Stress in Hong Kong Oysters, Crassostrea hongkongensis.

The Hong Kong oyster, Crassostrea hongkongensis, is an estuarine bivalve with remarkable commercial value in South China, and the increase of salinity in estuaries during the dry season has posed a major threat to the oyster farming. To explore the global transcriptional response to salinity stress, a whole-transcriptome analysis was performed with the gills of oysters in 6‰, 18‰, and 30‰ filtered seawater. Overall, 2243, 194, 371, and 167 differentially expressed mRNAs (DEmRNAs), differentially expressed long non-coding RNAs (DElncRNAs), differentially expressed circular RNAs (DEcircRNAs), and differentially expressed microRNAs (DEmiRNAs) were identified, respectively. Based on GO enrichment and KEGG pathway analysis, these important DEmRNAs, DElncRNAs, DEcircRNAs, and DEmiRNAs were predicted to be mainly involved in amino acids metabolism, microtubule movement, and immune defense. This demonstrated the complexity of dynamic transcriptomic profiles of C. hongkongensis in response to salinity fluctuation. The regulatory relationships of DEmiRNAs-DEmRNAs, DElncRNAs-DEmiRNAs, and DEcircRNAs-DEmiRNAs were also predicted, and finally, a circRNA-associated competing endogenous RNA (ceRNA) network was constructed, consisting of six DEcircRNAs, eight DEmiRNAs, and five DEmRNAs. The key roles of taurine and hypotaurine metabolism and phenylalanine metabolism were highlighted in this ceRNA network, which was consistent with the major contribution of free amino acids to intracellular osmolality and cell volume regulation. Collectively, this study provides comprehensive data, contributing to the exploration of coding and non-coding RNAs in C. hongkongensis salinity response. The results would benefit the understanding of the response mechanism of bivalves against salinity fluctuation, and provide clues for genetic improvement of C. hongkongensis with hyper-salinity tolerance.

Bacterial community profiling associated with pearl culture facilities of Liusha Bay, the largest marine pearl culture base on the western Guangdong coast, South China.

A large number of aquaculture facilities produced during the farming process are made of plastics. These plastics can be a distinct habitat for bacteria due to their unique materials. Therefore, this paper focuses on plastic aquaculture facilities and investigates the impact of bacterial accumulation on plastic surfaces. In this study, the high-throughput sequencing of 16S rRNA was conducted to investigate bacterial community profiling associated with the pearl culture facilities (cultured net cages and foam buoys) and surrounding water of Liusha Bay. Alpha diversity analysis showed that the richness and diversity indexes of bacterial communities in pearl culture facilities were higher than those in the aquatic environment. The richness and diversity indexes of bacterial communities were different between cultured net cages and foam buoys. Spatially influenced bacterial communities attached to pearl culture facilities varied between aquaculture areas. Thus, plastic has become a habitat for bacteria, floating in the marine environment and providing a favorable living environment for marine microorganisms and specific preferences for different substrate types. The relative abundance of certain functions on the attached bacterial community of the culture facility was high, which suggested that plastics did not only alter community structure but also influenced bacterial function. In addition, we detected small amounts of pathogenic bacteria, such as Vibrio and Bruegeria, in pearl culture facilities and surrounding seawater, suggesting that plastics can act as vectors for potentially pathogenic bacteria that may have an impact on the development of aquaculture. Our understanding of plastic ecology has been enriched by the discovery of the various microbial assemblages that can occur in aquaculture facilities.

Combined effect of salinity and hypoxia on digestive enzymes and intestinal microbiota in the oyster Crassostrea hongkongensis.

Anthropologic activities caused frequent eutrophication in coastal and estuarine waters, resulting in diel-cycling hypoxia. Given global climate change, extreme weather events often occur, thus salinity fluctuation frequently breaks out in these waters. This study aimed to evaluate the combined effects of salinity and hypoxia on intestinal microbiota and digestive enzymes of Crassostrea hongkongensis. Specifically, we sequenced 16 S rRNA of intestinal microbiota and measured the digestive enzymes trypsin (TRS), lipase (LPS) and amylase (AMY) in oysters exposed for 28 days to three salinities (10, 25 and 35) and two dissolved oxygen conditions, normoxia (6 mg/L) and hypoxia (6 mg/L for 12 h, 2 mg/L for 12 h). Oysters in normoxia and salinity of 25 were treated as control. After 28-day exposure, for microbial components, Fusobacteriota, Firmicutes, Bacteroidota, Proteobacteria and Actinobacteriota comprised the majority for all experimental groups. Compared with the control group, the diversity and structure of intestinal microbiota tended to change in all treated groups. The species richness in C. hongkongensis intestine also changed. It was the most significant that high salinity increased Proteobacteria proportion while low salinity and hypoxia increased Fusobacteriota but decreased Proteobacteria, respectively. Additionally, Actinobacteriota was sensitive and changed under environmental stressor (P < 0.01). The prediction results on intestinal microbiota showed that, all functions of oysters were up-regulated to distinct degrees under low/high salinity with hypoxia. According to the KEGG prediction, cellular processes were more active and energy metabolism upregulated, indicating the adaptation of C. hongkongensis to environmental change. Periodical hypoxia and low/high salinity had complex effect on the digestive enzymes, in which the activity of TRS and LPS decreased while AMY increased. High/low salinity and periodical hypoxia can change the secretion of digestive enzymes and influence intestinal microbial diversity and species richness of C. hongkongensis, deducing the chronic adverse effects on the digestive physiology in long-term exposure.

The first high-quality chromosome-level genome of the Sipuncula Sipunculus nudus using HiFi and Hi-C data.

Sipuncula is a class of exocoelomic unsegmented animals whose evolutionary relationships are unresolved. The peanut worm Sipunculus nudus is a globally distributed, economically important species belonging to the class Sipuncula. Herein, we present the first high-quality chromosome-level assembly of S. nudus based on HiFi reads and high-resolution chromosome conformation capture (Hi-C) data. The assembled genome was 1,427 Mb, with a contig N50 length of 29.46 Mb and scaffold N50 length of 80.87 Mb. Approximately 97.91% of the genome sequence was anchored to 17 chromosomes. A BUSCO assessment showed that 97.7% of the expectedly conserved genes were present in the genome assembly. The genome was composed of 47.91% repetitive sequences, and 28,749 protein-coding genes were predicted. A phylogenetic tree demonstrated that Sipuncula belongs to Annelida and diverged from the common ancestor of Polychaeta. The high-quality chromosome-level genome of S. nudus will serve as a valuable reference for studies of the genetic diversity and evolution of Lophotrochozoa.

Integrated transcriptomic and metabolomic analysis sheds new light on adaptation of Pinctada fucata martensii to short-term hypoxic stress.

Analyses of the transcriptome and metabolome were conducted to clarify alterations of key genes and metabolites in pearl oysters following exposure to short-term hypoxic treatment. We totally detected 209 DEGs between the control and hypoxia groups. Enrichment analysis indicated the enrichment of GO terms including "oxidation-reduction process", "ECM organization", "chaperone cofactor-dependent protein refolding", and "ECM-receptor interaction" KEGG pathway by the DEGs. In addition, between the two groups, a total of 28 SDMs were identified, which were implicated in 13 metabolic pathways, such as "phenylalanine metabolism", "D-amino acid metabolism", and "aminoacyl-tRNA biosynthesis". Results suggest that pearl oysters are exposed to oxidative stress and apoptosis under short-term hypoxia. Also, pearl oysters might adapt to short-term hypoxic treatment by increasing antioxidant activity, modulating immune and biomineralization activities, maintaining protein homeostasis, and reorganizing the cytoskeleton. The results of our study help unveil the mechanisms by which pearl oysters respond adaptively to short-term hypoxia.

Transcriptomic responses reveal impaired physiological performance of the pearl oyster following repeated exposure to marine heatwaves.

Marine heatwaves are predicted to become more intense and frequent in the future, possibly threatening the survival of marine organisms and devastating their communities. While recent evidence reveals the adaptability of marine organisms to heatwaves, substantially overlooked is whether they can also adjust to repeated heatwave exposure, which can occur in nature. By analysing transcriptome, we examined the fitness and recoverability of the pearl oyster (Pinctada maxima) after two consecutive heatwaves (24 °C to 32 °C for 3 days; recovery at 24 °C for 4 days). In the first heatwave, 331 differentially expressed genes (DEGs) were found, such as AGE-RAGE, MAPK, JAK-STAT, FoxO and mTOR. Despite the recovery after the first heatwave, 2511 DEGs related to energy metabolism, body defence, cell proliferation and biomineralization were found, where 1655 of them were downregulated, suggesting a strong negative response to the second heatwave. Our findings imply that some marine organisms can indeed tolerate heatwaves by boosting energy metabolism to support molecular defence, cell proliferation and biomineralization, but this capacity can be overwhelmed by repeated exposure to heatwaves. Since recurrence of heatwaves within a short period of time is predicted to be more prevalent in the future, the functioning of marine ecosystems would be disrupted if marine organisms fail to accommodate repeated extreme thermal stress.

Genome-wide association study analysis to resolve the key regulatory mechanism of biomineralization in Pinctada fucata martensii.

Biomineralization-controlled exo-/endoskeleton growth contributes to body growth and body size diversity. Molluscan shells undergo ectopic biomineralization to form the exoskeleton and biocalcified "pearl" involved in invading defence. Notably, exo-/endoskeletons have a common ancestral origin, but their regulation and body growth are largely unknown. This study employed the pearl oyster, Pinctada fucata marntensii, a widely used experimental model for biomineralization in invertebrates, to perform whole-genome resequencing of 878 individuals from wild and breeding populations. This study characterized the genetic architecture of biomineralization-controlled growth and ectopic biomineralization. The insulin-like growth factor (IGF) endocrine signal interacted with ancient single-copy transcription factors to form the regulatory network. Moreover, the "cross-phylum" regulation of key long noncoding RNA (lncRNA) in bivalves and mammals indicated the conserved genetic and epigenetic regulation in exo-/endoskeleton growth. Thyroid hormone signal and apoptosis regulation in pearl oysters affected ectopic biomineralization in pearl oyster. These findings provide insights into the mechanism underlying the evolution and regulation of biomineralization in exo-/endoskeleton animals and ectopic biomineralization.

Lipidomic insights into the immune response and pearl formation in transplanted pearl oyster Pinctada fucata martensii.

During pearl culture, the excess immune responses may induce nucleus rejection and death of pearl oysters after transplantation. To better understand the immune response and pearl formation, lipidomic analysis was applied to investigate changes in the serum lipid profile of pearl oyster Pinctada fucata martensii following transplantation. In total, 296 lipid species were identified by absolute quantitation. During wound healing, the content of TG and DG initially increased and then decreased after 3 days of transplantation with no significant differences, while the level of C22:6 decreased significantly on days 1 and 3. In the early stages of transplantation, sphingosine was upregulated, whereas PC and PUFAs were downregulated in transplanted pearl oyster. PI was upregulated during pearl sac development stages. GP and LC-PUFA levels were upregulated during pearl formation stage. In order to identify enriched metabolic pathways, pathway enrichment analysis was conducted. Five metabolic pathways were found significantly enriched, namely glycosylphosphatidylinositol-anchor biosynthesis, glycerophospholipid metabolism, alpha-linolenic acid metabolism, linoleic acid metabolism and arachidonic acid metabolism. Herein, results suggested that the lipids involved in immune response, pearl sac maturation, and pearl formation in the host pearl oyster after transplantation, which might lead to an improvement in the survival rate and pearl quality of transplanted pearl oyster.

Circulating exosome miRNA, is it the novel nutrient molecule through cross-kingdom regulation mediated by food chain transmission from microalgae to bivalve?

MicroRNAs (miRNAs) can efficiently regulate gene expression at intracellular and extracellular levels. Plant-derived miRNAs are highly enriched in animal haemolymph and regulate mammalian gene expression. However, evidence for food-derived miRNAs in Mollusca species is lacking. In this study, we fed the microalga Nannochloropsis oculata to the pearl oyster Pinctada fucata martensii and detected dietary miRNAs in exosomes isolated from the haemolymph by RNA-seq. In total, 273 endogenous miRNAs were identified in all biological replicates. We identified 23 microalgae-derived miRNAs in the exosomes of pearl oyster haemolymph. Most microalgae-derived miRNAs showed high expression levels in both exosomes and microalgae and exhibited apparent variation among individuals. These food-derived miRNAs were predicted to participate in endocytosis, apoptosis, signal transduction, energy metabolism, and biomineralization by targeting multiple genes. These findings demonstrated the cross-kingdom transport of miRNAs from microalgae to bivalves and provide insights into novel nutrient transmission through the food chain.

Effects of microplastics on physiological performance of marine bivalves, potential impacts, and enlightening the future based on a comparative study.

This review aims to explore the effects of microplastics and their corresponding additives on the physiological performances of marine bivalves together with their related genes. We identified gaps based on studies that were conducted on other organisms, and we conducted a comparative study on similar and relevant aspects for exploring future potential areas of study and interest. Microplastics are widely dispersed in all forms of media (solid, liquid, and gas). Exposure to an organism (including humans) is inevitable. However, impacts depend on the concentration of exposure, location of a biomarker being observed, and treatment involved. Different shapes, colors, and polymer types are reported and the transfer of microplastics along the food chain are recorded. The impacts of microplastics intensify when coupled with other chemicals or additives (referred to as xenobiotics) in a treated group. Thus, the degree of inhibition or enhancement of a physiological response magnifies when a coexposure of microplastic and a xenobiotic occurs. Microplastics have been observed to reduce immune system functionality by reducing hemocytes count, distorting oxidative system, respiration, and increasing energy consumption in bivalves due to physiological modulations that result from ingestion of microplastics or their additives. We found knowledge gaps and suggested future research directions to fully understand the impact of microplastics and their additives on marine bivalves.

Characterization of transcription factor activator pretein-1 (AP-1) and its association with cold tolerance in Pinctada fucata martensii.

AP-1 is an important transcription factor for cell proliferation/differentiation and animal immunity/development; however, its role in research in shellfish is poorly understood. Here, the cDNA of AP-1 gene from Pinctada fucata martensii was characterized. Its expression was detected in all six examined tissues, and a high level was observed in the gill and hepatopancreas. Analysis of the developmental transcriptomes showed that the PmAP-1 gene expression levels were high during D-stage larval and spat stages. The gene also exhibited a significantly high expression under cold tolerance stress. SNP analysis of the exon region and 5' flanking region of PmAP-1 revealed 19 SNPs of which 8 showed significant differences between cold tolerance selection line and base stock. Furthermore, three haplotypes generated by the SNPs of PmAP-1 were significantly associated with cold tolerance, respectively.These results suggest that the PmAP-1 gene plays an important role in the response of P. f. martensii to low temperature stress. These SNPs and haplotypes of PmAP-1 may be related to the cold tolerance of P. f. martensii, and could be candidate markers potentially for further selective breeding.

Integrated GC-MS- and LC-MS-Based Untargeted Metabolomics Studies of the Effect of Vitamin D3 on Pearl Production Traits in Pearl Oyster Pinctada fucata martensii.

Pearl oyster Pinctada fucata martensii is widely recognized for biomineralization and has been cultured for high-quality marine pearl production. To ascertain how dietary vitamin D3 (VD3) levels affect the features of pearl production by P. f. martensii and discover the mechanisms regulating this occurrence, five experimental diets with variable levels of VD3 were used with inclusion levels of 0, 500, 1,000, 3,000, and 10,000 IU/kg. The distinct inclusion levels were distributed into five experimental groups (EG1, EG2, EG3, EG4, and EG5). All the experimental groups were reared indoors except the control group (CG) reared at the sea. Pearl oysters, one year and a half old, were used in the grafting operation to culture pearls. During the growing period that lasted 137 days, EG3 had the highest survival rate, retention rate, and high-quality pearl rate. A similar trend was found for EG3 and CG with significantly higher pearl thickness and nacre deposition rates than other groups, but no significant differences were observed between them. A metabolomics profiling using GC-MS and LC-MS of pearl oysters fed with low quantities of dietary VD3 and optimal levels of dietary VD3 revealed 135 statistically differential metabolites (SDMs) (VIP > 1 and p < 0.05). Pathway analysis indicated that SDMs were involved in 32 pathways, such as phenylalanine metabolism, histidine metabolism, glycerophospholipid metabolism, alanine aspartate and glutamate metabolism, arginine and proline metabolism, glycerolipid metabolism, amino sugar and nucleotide sugar metabolism, and tyrosine metabolism. These results provide a theoretical foundation for understanding the impacts of VD3 on pearl production traits in pearl oyster and reinforce forthcoming prospects and application of VD3 in pearl oyster in aquaculture rearing conditions.

Identification of a long non-coding RNA (LncMSEN2) from pearl oyster and its potential roles in exoskeleton formation and LPS stimulation.

Long non-coding RNAs (lncRNAs) play regulatory roles in various biological processes, including exoskeleton formation and immune response. The exoskeleton-based mantle-shell defense system is an important defense mechanism in shellfish. In this study, we found a novel lncRNA, herein formally named, LncMSEN2, from the pearl oyster Pinctada fucuta martensii, and its sequence was validated via polymerase chain reaction (PCR). LncMSEN2 was highly expressed in mantle tissues, especially in the central region (P < 0.05), and was also expressed in the pearl sac as detected by quantitative real-time PCR. In situ hybridization experiments revealed that LncMSEN2 had a strong positive signal in the inner and outer epidermal cells of the mantle pallial and central regions. RNA interference experiments showed that interference of LncMSEN2 expression with dsRNA in mantle tissues led to an abnormal crystal structure of the nacre. In addition, LncMSEN2 expression significantly increased 6 h after lipopolysaccharide stimulation in mantle tissues (P < 0.05). These results indicated that LncMSEN2 may be a novel regulator of the mantle-shell defense system of pearl oyster.