The First High-Quality Genome Assembly of Freshwater Pearl Mussel Sinohyriopsis cumingii: New Insights into Pearl Biomineralization.

China leads the world in freshwater pearl production, an industry in which the triangle sail mussel (Sinohyriopsis cumingii) plays a pivotal role. In this paper, we report a high-quality chromosome-level genome assembly of S. cumingii with a size of 2.90 Gb-the largest yet reported among bivalves-and 89.92% anchorage onto 19 linkage groups. The assembled genome has 37,696 protein-coding genes and 50.86% repeat elements. A comparative genomic analysis revealed expansions of 752 gene families, mostly associated with biomineralization, and 237 genes under strong positive selection. Notably, the fibrillin gene family exhibited gene family expansion and positive selection simultaneously, and it also exhibited multiple high expressions after mantle implantation by transcriptome analysis. Furthermore, RNA silencing and an in vitro calcium carbonate crystallization assay highlighted the pivotal role played by one fibrillin gene in calcium carbonate deposition and aragonite transformation. This study provides a valuable genomic resource and offers new insights into the mechanism of pearl biomineralization.

Integrated analysis of physiological, transcriptome, and metabolome analyses of the gills in Solenaia oleivora under ammonia exposure.

Ammonia is a common toxicant in aquatic systems and one of the key factors affecting aquaculture. However, data on mollusks' toxic response and coping mechanisms to ammonia nitrogen, especially freshwater mollusks, are still lacking. In this study, we evaluated the tolerance of a freshwater mollusk Solenaia oleivora to ammonia and investigated its coping mechanisms by combining physiological, metabolic, and transcriptomic analyses in the gills. The acute toxicity test revealed that the LC50-96 h (temperature-20 â„ƒ, pH-7.4) of ammonia in S. oleivora was 63.29 mg/L. The physiological and TUNEL results showed that although 10 mg/L ammonia exposure increased the activities of antioxidant, immune and ammonia detoxification-related enzymes, it still caused oxidative damage and cell apoptosis of gill tissues. A total of 97 differential metabolites (DMs) and 3431 differential expressed genes (DEGs) were identified after ammonia stress. Among them, most DMs and DEGs were involved in immune response, antioxidant, cell apoptosis, carbohydrate metabolism, amino acid metabolism, and lipid metabolism. The enhancement of glycolysis and lipid metabolisms may provide energy for immune response and ammonia detoxification. In addition, glutamine synthesis, alanine synthesis and urea cycle were involved in ammonia nitrogen detoxification in the gill tissue of S. oleivora. Our results indicate that ammonia leads to individual death in S. oleivora, as wells as oxidative damage, cell apoptosis, immune response, and metabolic changes of gill tissues. The findings will provide valuable information to assess the potential ecological risk of environmental ammonia to freshwater mollusks and theoretical guidance for the healthy aquaculture of S. oleivora.

A Novel Kunitz-Type Serine Protease Inhibitor (HcKuSPI) is Involved in Antibacterial Defense in Innate Immunity and Participates in Shell Formation of Hyriopsis cumingii.

Serine protease inhibitors (SPIs) are abundantly reported for its inhibition against specific proteases involved in the immune responses, but SPI data related to calcareous shells are scarce. Previously, our research group has reported the proteome analysis of non-nucleated pearl powder, and a candidate matrix protein containing two Kunitz domains in the acid soluble fraction caught our attention. In the present study, the full-length cDNA sequence of HcKuSPI was obtained from Hyriopsis cumingii. HcKuSPI was specifically expressed in the mantle, with hybridization signals mainly concentrated to dorsal epithelial cells at the mantle edge and weak signals at the mantle pallium, suggesting HcKuSPI was involved in shell formation. HcKuSPI expression in the mantle was upregulated after Aeromonas hydrophila and Staphylococcus aureus challenge to extrapallial fluids (EPFs). A glutathione S transferase (GST)-HcKuSPI recombinant protein showed strong inhibitory activity against the proteases, trypsin and chymotrypsin. Moreover, HcKuSPI expression in an experimental group was significantly higher when compared with a control group during pellicle growth and crystal deposition in shell regeneration processes, while the organic shell framework of newborn prisms and nacre tablets was completely destroyed after HcKuSPI RNA interference (RNAi). Therefore, HcKuSPI secreted by the mantle may effectively neutralize excess proteases and bacterial proteases in the EPF during bacterial infection and could prevent matrix protein extracellular degradation by suppressing protease proteolytic activity, thereby ensuring a smooth shell biomineralization. In addition, GST-HcKuSPI was also crucial for crystal morphology regulation. These results have important implications for our understanding of the potential roles of SPIs during shell biomineralization.

Expression of bone morphogenetic protein 10 and its role in biomineralization in Hyriopsis cumingii.

Shells and pearls are the products of biomineralization of shellfish after ingesting external mineral ions. Bone morphogenetic proteins (BMPs) play a role in a variety of biological function, and the genes that encode them, are considered important shell-forming genes in mollusks and are associated with shell and pearl formation, embryonic development, and other functions, but bone morphogenetic protein 10 (BMP10) is poorly understood in Hyriopsis cumingii. In this study, we cloned Hc-BMP10 and obtained a 2477 bp full-length sequence encoding 460 amino acids with a conserved TGF-ß structural domain. During the embryonic developmental stages, the cleavage stage had the highest expression of Hc-BMP10, followed by juvenile clams; the expression in the mantle gradually decreased with increasing mussel age. A strong signal was detected on epidermal cells on the mantle edge by in situ hybridization. In both the shell notching and inserting operations of the pearl fragment assay, we found that the expression of Hc-BMP10 increased after the above treatments. RNA interference assays showed that the silencing of Hc-BMP10 resulted in a change in the morphology of the prismatic layer and nacreous layer, with the prismatic layer less closely aligned and the disordered aragonite flakes in the nacreous layer. These findings indicate that Hc-BMP10 is involved in the growth and development of H. cumingii, as well as the formation of shells and pearls. Therefore, this study provides some reference for selecting superior species for growth and pearl breeding of H. cumingii at a molecular level and further investigation of the molecular mechanism for biomineralization of Hc-BMP10.

Identification and Characterization of C-Mos in Pearl Mussel Hyriopsis cumingii and Its Role in Gonadal Development.

C-Mos, a proto-oncogene, regulates oocyte maturation by activating the classical MAPK pathway in cells. To examine the function of C-Mos in Hyriopsis cumingii, C-Mos was identified in this study. The full-length cDNA of C-Mos was 2213 bp, including 144 bp in the 5' UTR, 923 bp in 3' the UTR, and 1146 bp in the open reading frame (ORF) region. During early gonad development, the expression of C-Mos from 4 to 6 months of age in H. cumingii was significantly higher than that in other months, with the highest expression in 6-month-old H. cumingii, suggesting that C-Mos may be involved in early gonadal development in H. cumingii. Clear hybridization signals were found by in situ hybridization in the oocytes, oocyte nucleus and oogonium, and a small number of hybridization signals were found in the follicular wall of the male gonads. In addition, the C-Mos RNA interference (RNAi) assay results showed that the knockdown of C-Mos caused a down-regulation of ERK and P90rsk. In summary, these results indicate that C-Mos has a crucial part to play in gonadal development in H. cumingii.

Presence of immune factors in freshwater mussel (Hyriopsis cumingii) entails autologous serum an essential component in the culture of mantle cells.

Mussel cell culture is a challenging problem and serum serves a crucial biological role in cell culture as an autologous supply and an immunizing agent. In this study, the biology (calcium ions, total protein, pH, and osmotic pressure) of fetal bovine serum (FBS) and Hyriopsis cumingii serum (HCS) was investigated, and the development of Hyriopsis cumingii (H. cumingii) mantle cells in HCS and FBS systems was examined. The results showed that total protein, calcium ions, and osmotic pressure varied significantly (p<0.05). The activity of mantle cells was superior in the HCS culture system to that in the FBS culture system. The label-free technique was used to distinguish the two serum proteins to investigate the supportive effect of autologous serum on cell culture. These were examined for 109 unique proteins and 35 particular HCS proteins. Most differentially expressed proteins (DEPs) were involved in immune response, cell differentiation, and calcium ion binding. Furthermore, immune factors such as HSP, CALR, APOB, C3 were identified with significant differences. HSP was significantly more present in HCS than in FBS as an endogenous protective protein that regulates immune system function, cell differentiation, transport, and activity regulation. Parallel reaction monitoring (PRM) analysis was carried out to validate the expression levels of 19 DEPs, indicating high reliability of the proteomic results. This study reveals the important role of immune factors in mussel cell culture, providing a theoretical basis for explaining the applicability of autologous serum in cell culture. It is also helpful in improving the cell culture conditions of mussels.

Low-complexity domain-containing protein (LCDP), induced accumulation of calcium carbonate individual crystals to irregular polycrystals in the triangle sail mussel Hyriopsis cumingii.

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12-21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO3) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality.

Biochemical and cellular responses of the freshwater mussel, Hyriopsis bialata, to the herbicide atrazine.

The present study aimed to evaluate biochemical and cellular responses of the freshwater mussel, Hyriopsis bialata, to the herbicide atrazine (ATZ). The mussels were exposed to environmentally-relevant concentrations of ATZ (0, 0.02 and 0.2 mg/L) and a high concentration (2 mg/L) for 0, 7, 14, 21 and 28 days. Tissues comprising male and female gonads, digestive glands and gills were collected and assessed for ethoxyresorufin-O-deethylase (EROD) activity, glutathione S-transferase (GST) activity, multixenobiotic resistance mechanism (MXR), histopathological responses, DNA fragmentation and bioaccumulation of ATZ and its transformation derivatives, desethylatrazine (DEA) and desisopropylatrazine (DIA). Additionally, circulating estradiol levels were determined. It appeared that ATZ did not cause significant changes in activities of EROD, GST and MXR. There were no apparent ATZ-mediated histopathological effects in the tissues, with the exception of the male gonads exhibiting aberrant aggregation of germ cells in the ATZ-treated mussels. Contrarily, ATZ caused significant DNA fragmentation in all tissues of the treated animals in dose- and time-dependent manners. In general, the circulating estradiol levels were higher in the females than in the males. However, ATZ-treated animals did not show significant alterations in the hormonal levels, as compared with those of the untreated animals. Herein, we showed for the first time differentially spatiotemporal distribution patterns of bioaccumulation of ATZ, DEA and DIA, with ATZ and DEA detectable in the gonads of both sexes, DEA and DIA in the digestive glands and only DEA in the gills. The differential distribution patterns of bioaccumulation of ATZ and its derivatives among the tissues point to different pathways and tissue capacity in transforming ATZ into its transformation products. Taken together, the freshwater mussel H. bialata was resistant to ATZ likely due to their effective detoxification. However, using DNA damage as a potential biomarker, H. bialata is a promising candidate for biomonitoring aquatic toxicity.

Hic12, a novel acidic matrix protein promotes the transformation of calcite into vaterite in Hyriopsis cumingii.

Shell acidic matrix proteins are widely considered to be essential for shell formation given their low affinity and high loading for calcium ion. In the present study, a novel matrix protein, hic12, was isolated from the mantle of Hyriopsis cumingii. High expression in tissue and positive signals with in situ hybridization were detected in the mantle center and mantle pallium, indicating that hic12 mainly participated in the biomineralization of the shell nacreous layer. The expression pattern of hic12 in the pearl sac during early pearl formation indicated that it was involved in pearl biomineralization. Moreover, the recombinant protein, rGST-Hic12, was successfully expressed and purified. The addition of rGST-Hic12 could accelerate the calcium carbonate deposition rate, change the morphology of crystals, and promote the conversion of calcite to vaterite. These results may provide new insights into the molecular mechanisms of aragonite mollusk shell formation.

The novel matrix protein hic7 of hyriopsis cumingii participates in the formation of the shell and pearl.

Shell matrix proteins have important roles in the biomineralization of shells. In this study, we isolated and identified a novel shell matrix protein gene, hic7, from the mussel Hyriopsis cumingii. The cDNA of hic7 was 459 bp long, including a 240-bp open reading frame. It encoded a 79 amino acid-long protein, with amino acids 1-19 constituting the signal peptide. The resulting hic7 is rich in cysteine (16.5%). After removing the signal peptide, the molecular weight was 8.85 kDa and the theoretical isoelectric point was 6.34, indicating that hic7 is a weakly acidic shell matrix protein. Hic7 is mainly expressed in the mantle tissue of H. cumingii. In situ hybridization showed hic7 signals at the edge and dorsal region of the mantle outer fold, indicating that it is related to the formation of the prismatic and nacreous layer of the shell. RNA interference indicated that when hic7 was inhibited by 80%, the crystal morphology of the prism and nacre layers of the shell were irregular and disordered. In addition, the expression of hic7 during the early development of the pearl sac indicated that it has an important role in the transformation of calcium carbonate crystals from a disordered to an orderly deposition pattern. These results suggest that matrix protein hic7 take part in constructing the framework of crystal nucleation and regulating the calcium carbonate crystal morphology of the nacreous and prismatic layers of shells and pearls.

The mantle exosome proteins of Hyriopsis cumingii participate in shell and nacre color formation.

Pearl color is closely related to the nacre color of shell in Hyriopsis cumingii, and pearl color has a huge impact on its price. The nacre is an important part of the shell, and studies have suggested that mantle exosomes participated in shell formation. Exosomes contain many different proteins that are involved in different biological processes. In this study, exosomes were extracted from mantles of mussels with different nacre color. TMT quantitative proteome sequencing analysis was performed on purple and white mussel mantle exosomes, and 4861 proteins were obtained. Based on the standard of (|log2 (Fold change)| ≥ 1.2 or ≤ 0.83 and p-value ≤ 0.05), a total of 758 differentially expressed proteins were found. Some proteins involved in shell and nacre color formation were predicted with the proteins annotate, GO classification system. Moreover, 14 differentially expressed proteins (including eight up-regulated proteins and six down-regulated proteins) were validated using parallel reaction monitoring (PRM) assays. Overall, this information will be useful to improve our understanding of the molecular mechanisms of shell and nacre color formation in H. cumingii.

Bioaccumulation, Metabolism, and Biomarker Responses in Hyriopsis cumingii Exposed to 4-Mono-Chlorinated Dibenzothiophene.

Polychlorinated dibenzothiophenes (PCDTs) are sulfur analogues of polychlorinated dibenzofurans with prevalent occurrence in aquatic environments and potential ecological risks. However, data on the behavior and toxicity of PCDTs in aquatic organisms remain scarce. In the present study, the bioaccumulation, metabolism, and oxidative damage of 4-mono-chlorinated dibenzothiophene (4-mono-CDT) in freshwater mussel (Hyriopsis cumingii) were investigated after exposure to 4-mono-CDT in semistatic water. The uptake rates, depuration rates, half-lives, and bioconcentration factors of 4-mono-CDT in hepatopancreas, gill, and muscle tissues ranged from 0.492 to 1.652 L d-1 g-1 dry weight, from 0.117 to 0.308 d-1 , from 2.250 to 5.924 d, and from 2.903 to 8.045 × 103 L kg-1 dry weight, respectively. A dechlorinated metabolite (dibenzothiophene) was detected in hepatopancreas tissue, indicating that dechlorination was the main metabolic pathway of 4-mono-CDT. As the exposure time increased, the activities of superoxide dismutase, catalase, and glutathione peroxidase were induced or inhibited in the different experimental groups. The malondialdehyde content increased with increasing 4-mono-CDT dose and exposure time. A higher concentration of 4-mono-CDT corresponded to a greater integrated biomarker response in each tissue and greater oxidative damage. The antioxidant enzymes in hepatopancreas were more sensitive to 4-mono-CDT than those in gill. The results provide useful information on the behavior and ecotoxicity of PCDTs in freshwater mussels. Environ Toxicol Chem 2021;40:1873-1882. © 2021 SETAC.

Teosin, a novel basic shell matrix protein from Hyriopsis cumingii induces calcium carbonate polycrystal formation.

In this study, a novel matrix protein (teosin) was isolated from Hyriopsis cumingii. Gene expression analysis showed that teosin is mainly expressed in the mantle and blood, and a hybridization signal was found in dorsal epithelial cells of the mantle pallial by in situ hybridization. Moreover, teosin expression during pearl formation indicated its participation in initial nacreous layer biomineralization, and suppressing teosin expression resulted in irregular crystal morphology and disordered arrangement in RNAi assay. In vitro crystallization assays indicated teosin could increase the size of calcite. By turning the sample stage about 15°, we got the high-resolution TEM images of the crystals' edges. This is a novel method to observe the crystal which is over 200 nm under TEM. In the control experiment group, the calcite show the character of long range order. The calcite induced by teosin were composed of nano-grains, and the polycrystal character were confirmed by EDS. These results suggested that teosin is involved in regulating crystal morphology regulation and inducing polycrystal formation during nacreous-layer formation.

Acute toxicity of lead in fresh water bivalves Lamellidens jenkinsianus obesa and Parreysia (Parreysia) corrugata with evaluation of sublethal effects on acetylcholinesterase and catalase activity, lipid peroxidation, and behavior.

The median lethal concentrations (LC50) of Pb at 96 h were 8.84, 192.14, and 3.22 mg L-1 for pre-reproductive and reproductive individuals of Lamellidens jenkinsianus obesa and reproductive individuals of Parreysia (Parreysia) corrugata, respectively. Thus, young L. j. obesa were much more sensitive to Pb than its adults, while P. (P.) corrugata was the most sensitive. However, all the three values were much higher than the Pb levels commonly found in natural waters, and indicative of the tolerance of these mollusks to acute, short-term Pb exposure. In contrary to these findings, acetylcholinesterase (AChE) and catalase (CAT) activities were affected and lipid peroxidation (LPO) elevated in young L. j. obesa and P. (P.) corrugata in 21-day sublethal toxicity tests at 26-68 ppb Pb concentrations, which might be considered environmentally realistic. Some behavioral patterns such as number of movement (Mov) and durations of foot mobilization together with siphon extensions (FSE) were reduced, and the durations of valves remaining completely closed without any extension of foot and siphons (VC) increased significantly at 25-69 ppb Pb as well. Thus, the study revealed significant interspecific differences as well as that between life stages of the same species, suggesting that apparently hardy species could be impacted by low Pb concentrations in their young stages. Further, a multi-biomarker approach involving biological effects, anti-oxidative enzyme activity and easy-to-measure behavioral elements could comprise a valuable tool in assessment of Pb-induced stress in freshwater bivalves.

Biochemical responses of freshwater mussel Unio tumidus to titanium oxide nanoparticles, Bisphenol A, and their combination.

Multiple interactions between different pollutants in the surface waters can cause unpredictable consequences. The aim of the study was to evaluate the combined effect of two widespread xenobiotics, titanium oxide nanoparticles (TiO2) and bisphenol A (BPA), on freshwater bivalve Unio tumidus. The specimens were exposed for 14 days to TiCl4 (Ti, 1.25 µM), TiO2 (1.25 µM), BPA (0.88 nM), or their combination (TiO2 + BPA). Every type of exposure resulted in a particular oxidative stress response: TiO2 had antioxidant effect, decreasing the generation of reactive oxygen species (ROS) and phenoloxidase (PhO) activity, and doubling reduced glutathione (GSH) concentration in the digestive gland; Ti caused oxidative changes by increasing levels of ROS, PhO and superoxide dismutase; BPA decreased the GSH level by a factor of two. In the co-exposure treatment, these indices as well as lysosomal membrane stability were not affected. All Ti-containing exposures caused elevated levels of metalated metallothionein (Zn,Cu-MT), its ratio to total metallothionein protein, and lactate/pyruvate ratio. Both BPA-containing exposures decreased caspase-3 activity. All exposures, and particularly co-exposure, up-regulated CYP450-dependent oxidation, lipid peroxidation and lipofuscin accumulation, lysosomal cathepsin D and its efflux, as well as alkali-labile phosphates in gonads and caused DNA instability (except for TiO2). To summarize, co-exposure to TiO2 + BPA produced an overlap of certain individual responses but strengthened the damage. Development of water purification technologies using TiO2 requires further studies of the biological effects of its mixtures. U. tumidus can serve as a sentinel organism in such studies.

The Mantle Exosome and MicroRNAs of Hyriopsis cumingii Involved in Nacre Color Formation.

The nacre color of shells has an effect on the pearl color in Hyriopsis cumingii and is an important indicator for its value. The nacre is part of the shell, and some studies have shown that exosomes of the mantle are involved in the formation of shells. Most of the RNA contained in exosomes are microRNAs (miRNAs); however, little information is available on the roles of exosomes and miRNAs on the formation of nacre color in mussels. In this study, exosomes of mantles were extracted from white and purple mussels. High-throughput Illumina sequencing was performed on the white and purple mussel mantle exosomes, and 7,665,167 and 10,994,115 reads were harvested. Using the standard of |log2(Fold change)| ≥ 2, and a p value ≤ 0.05, a total of 54 differentially expressed miRNAs were identified. The miRNAs that regulated the target genes (hcApo, HcTyr, HcTyp-1, HcMitf, HcSRCR1, and HcSRCR2) involved in shell color formation were predicted. Moreover, miR-15b negatively regulated hcApo, which plays important roles in the absorption and transport of ß-carotene in H. cumingii. These results improve our understanding of the molecular mechanisms of nacre color formation in H. cumingii.

miR-4504 is involved in nacre color formation in Hyriopsis cumingii.

Pearl color is affected by the nacre color of shells in Hyriopsis cumingii, and is the primary indicator of its value. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in many biological processes, including pigmentation. In this study, we used a luciferase reporter assay to identify that miR-4504 can interact with the 3'-untranslated region of the MITF gene in H. cumingii (HcMitf). After injecting mussels with the miR-4504 antagomir, the expression of miR-4504 was inhibited. Upon miR-4505 silencing, the expression of HcMitf and its downstream gene, HcTyr, were simultaneously increased. Tyrosinase activity and melanin content were also increased. The collective findings indicated that miR-4504 was involved in melanin synthesis in H. cumingii. These findings also improve our understanding of the molecular mechanisms of nacre color formation in H. cumingii.

First occurrence and composition assessment of microplastics in native mussels collected from coastal and offshore areas of the northern and central Adriatic Sea.

In recent years, the occurrence of microplastics in the aquatic environment has gathered increasing scientific interest. Several studies have shown that the ingestion of microplastics may negatively influence the physiology of marine organisms having different feeding strategies, particularly in those species which cannot discriminate between food sources. Recent studies highlighted the potential for such particles to accumulate in the food web, posing risks to human health via the consumption of seafood. Furthermore, early findings also indicated the role of microplastics as vectors of chemical pollutants either used as additives during synthesis of the plastics or adsorbed directly from seawater, i.e., PAHs, PCB, and surfactants. Despite the importance of microplastics in adsorption and transport of hydrophobic pollutants, little is known about their distribution and accumulation in marine food webs, or their direct and indirect harmful effects. The Adriatic Sea represents a semi-enclosed basin with a low water recirculation rate and high anthropogenic pressures associated with unsustainable fishing and inputs of contaminants. The body burden, accumulation rates, polymer composition, and recurring morphotypes of microplastics in native blue mussels (M. galloprovincialis) were examined. Organisms collected offshore were compared to those collected in coastal areas. Microplastics were recovered from the soft tissues of all analyzed mussels. Coastal organisms showed a load of 1.06-1.33 fragments g-1 (wet weight) and 0.62-0.63 fibers g-1 (wet weight) while offshore organisms showed an accumulation of 0.65-0.66 fragments g-1 (wet weight) and 0.24-0.35 fibers g-1 (wet weight). The size class distribution revealed a marked prevalence of smaller particles (20 µm to 40 µm range) and the most recurring polymer type in analyzed organisms was PE followed by PP, PET, and equal amounts of PS, PLY, and PVC. A significant site-, time-, and oceanographic-related distribution trend was observed. Based on the findings presented here, there is a clear need to implement a seafood safety monitoring program to better understand actual human health-related risks.

A Kunitz proteinase inhibitor (HcKuPI) participated in antimicrobial process during pearl sac formation and induced the overgrowth of calcium carbonate in Hyriopsis cumingii.

Proteinase inhibitors with the ability to inhibit specific proteinases are usually closely connected with the immune system. Interestingly, proteinase inhibitors are also a common ingredient in the organic matrix of mollusk shells. However, the molecular mechanism that underlies the role of proteinase inhibitors in immune system and shell mineralization is poorly known. In this study, a Kunitz serine proteinase inhibitor (HcKuPI) was isolated from the mussel Hyriopsis cumingii. HcKuPI was specifically expressed in dorsal epithelial cells of the mantle pallium and HcKuPI dsRNA injection caused an irregular surface and disordered deposition on the aragonite tablets of the nacreous layer. These results indicated that HcKuPI plays a vital role in shell nacreous layer biomineralization. Moreover, the expression pattern of HcKuPI during LPS challenge and pearl formation indicated its involvement in the antimicrobial process during pearl sac formation and nacre tablets accumulation during pearl formation. In the in vitro calcium carbonate crystallization assay, the addition of GST-HcKuPI increased the precipitation rate of calcium carbonate and induced the crystal overgrowth of calcium carbonate. Taken together, these results indicate that HcKuPI is involved in antimicrobial process during pearl formation, and participates in calcium carbonate deposition acceleration and morphological regulation of the crystals during nacreous layer formation. These findings extend our knowledge of the role of proteinase inhibitors in immune system and shell biomineralization.

Cloning and characteristic of MMP1 gene from Hyriopsis cumingii and collagen hydrolytic activity of its recombinant protein.

Matrix metalloproteinases (MMPs) play an essential role in a variety of biological processes including wound healing, inflammation, cell invasion, angiogenesis and immune defense. In this study, a putative MMP1 cDNA was cloned and characterized from Hyriopsis cumingii (designated as HcMMP1). The cDNA was 1822 bp in length and encoded a putative protein of 510 amino acids, with a predicted molecular mass of 58.28 kDa and an isoelectric point (pI) of 9.27. HcMMP1 contained all prototype MMPs family signatures, such as signal peptide, prodomain, catalytic center, hinge region, and hemopexin like domain. Quantitative real time-PCR (qRT-PCR) revealed that in mussels HcMMP1 mRNA was expressed in all tissues tested, and the transcriptional expression levels were significantly up-regulated in hepatopancreas and hemocytes after Aeromonas hydrophila, peptidoglycan stimulations and in mantle after wounding. Moreover, the recombination HcMMP1 protein, successfully expressed in Escherichia coli, was purified by affinity chromatography with the concentration of final yield at 0.3 mg/mL. The recombinase had an essentially hydrolytic activity toward rat type I collagen, mouse II and IV collagen after renaturation.