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To improve the utilization of by-products of the Hyriopsis cumingii, this study investigated the effect of Hyriopsis cumingii polysaccharide (HCP) on the proliferation and migration of normal cells, as well as its reparative effects on ultraviolet radiation-B (UVB)-damaged cells. First, HCP was isolated and purified using DEAE Cellulose-52 column chromatography, Sephadex G-100 column chromatography and nanofiltration. Then, structural analysis was conducted using high-performance liquid chromatography, high-performance gel permeation chromatography, and infrared spectroscopy. Following this, cell proliferation experiments, cell scratch tests, UVB damage experiments, and protein expression analyses were employed to explore the anti-UVB damage effects and corresponding mechanisms of polysaccharide components. The results indicated the three substances obtained by isolation and purification were all homogeneous polysaccharide components. Among them, HCP-2 played a major role in promoting cell proliferation in HCP and facilitated the migration of HaCaT cells. The recovery of UVB-damaged cell vitality was raised by the increasing concentration of HCP-2. Furthermore, the reparative effect was more substantial than the protective effect at the same sample concentration. It was also observed that HCP-2 up-regulated the expression of the anti-apoptotic protein bcl-2 while inhibited the expression of apoptosis-related proteins casp9, p-Akt, and p-p38, indicating that HCP-2 could repair HaCaT cells by regulating apoptosis of cells damaged by UVB. In conclusion, HCP had the ability to inhibit cell apoptosis which provide a theoretical basis for its future application in the development of food and health products. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) play an important biological role in pearl biomineralization in pearl mussels. In this study, based on the genome data of the triangular sail mussel (Hyriopsis cumingii), the genome-wide identification and bioinformatic analysis of BMP gene family were performed, and the expression pattern of the BMP genes was investigated by the insertion experiments. The results showed that a total of 12 BMP gene family members (BMP2a/2b, BMP3, BMP5a/5b, BMP7a/7b/7c, BMP9, BMP10a/10b, and BMP11) were identified, which were unevenly distributed on chromosome 3/14/18, encoding 169-- 583 amino acids, with molecular weights ranging from 19.32 to 65.99 kDa. BMP2a, BMP7b, and BMP10a were distributed, respectively, in the cytoplasm, endoplasmic reticulum and mitochondria, others were distributed in the nucleus. qRT-PCR results showed the significant tissue specificity in BMPs gene expression. The HcBMPs were differentially expressed in the mantle and visceral mass, and the expression level was higher in the visceral mass. The expressing trends of HcBMPs were not consistent between the mantle and visceral mass insertion, suggesting that HcBMPs may perform different functions. We also found that insertion surgery in the mantle and visceral mass significantly alters the expression profiling of the BMP gene family. Insertion of the mantle induced the biomineralization function of BMP2a, BMP7a, and BMP7b, while BMP3 and BMP10b played opposite roles in visceral mass insertion. Visceral mass insertion could suppress BMP9 expression at 5 d and BMP5b expression at 90 d after insertion This work lays the foundation and data support for the preliminary elucidation of regulatory role and mechanism of HcBMPs in the pearl-cultivating process of mantle and visceral mass. [ABSTRACT FROM AUTHOR]

For thousands of years, pearl and nacre powders have been important traditional Chinese medicines known for their skin whitening effects. To prepare the enzymatic hydrolysates of Hyriopsis cumingii nacre powder (NP-HCH), complex enzymatic hydrolysis by pineapple protease and of neutral protease was carried out after the powder was pre-treated with a high-temperature and high-pressure method. The peptides were identified using LC-MS/MS and picked out through molecular docking and molecular dynamics simulations. Subsequently, the tyrosinase inhibitory and antioxidant properties of novel tyrosinase inhibitory peptides were investigated in vitro. In addition, the enzymatic activity of tyrosinase in B16F10 cells as well as melanin content and antioxidant enzyme levels were also examined. The results showed that a tyosinase inhibitory peptide (Tyr-Pro-Asn-Pro-Tyr, YPNPY) with an efficient IC50 value of 0.545 ± 0.028 mM was identified. The in vitro interaction results showed that YPNPY is a reversible competitive inhibitor of tyrosinase, suggesting that it binds to the free enzyme. The B16F10 cell whitening test revealed that YPNPY can reduce the melanin content of B16F10 cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that YPNPY could be widely used as a tyrosinase inhibitor in whitening foods and drugs. [ABSTRACT FROM AUTHOR]

To improve the utilization of by-products of the Hyriopsis cumingii, this study investigated the effect of Hyriopsis cumingii polysaccharide (HCP) on the proliferation and migration of normal cells, as well as its reparative effects on ultraviolet radiation-B (UVB)-damaged cells. First, HCP was isolated and purified using DEAE Cellulose-52 column chromatography, Sephadex G-100 column chromatography and nanofiltration. Then, structural analysis was conducted using high-performance liquid chromatography, high-performance gel permeation chromatography, and infrared spectroscopy. Following this, cell proliferation experiments, cell scratch tests, UVB damage experiments, and protein expression analyses were employed to explore the anti-UVB damage effects and corresponding mechanisms of polysaccharide components. The results indicated the three substances obtained by isolation and purification were all homogeneous polysaccharide components. Among them, HCP-2 played a major role in promoting cell proliferation in HCP and facilitated the migration of HaCaT cells. The recovery of UVB-damaged cell vitality was raised by the increasing concentration of HCP-2. Furthermore, the reparative effect was more substantial than the protective effect at the same sample concentration. It was also observed that HCP-2 up-regulated the expression of the anti-apoptotic protein bcl-2 while inhibited the expression of apoptosis-related proteins casp9, p-Akt, and p-p38, indicating that HCP-2 could repair HaCaT cells by regulating apoptosis of cells damaged by UVB. In conclusion, HCP had the ability to inhibit cell apoptosis which provide a theoretical basis for its future application in the development of food and health products.

The triangle-sail mussel (Hyriopsis cumingii) is the most important freshwater pearl-bearing mussel in China. Nucleated pearls from the mantle of H. cumingii are emerging pearl cultivation products. Compared to traditional non-nucleated and nucleated pearls from visceral mass, nucleated pearls from the mantle show significant cultivation advantages, such as high pearl quality and short nurturing cycles. These nucleated pearls are widely known as "freshwater Akoya" in the pearl market because they resemble Japanese saltwater Akoya pearls. However, techniques for cultivating nucleated pearls from the mantle of H. cumingii are still immature and lack a targeted quality evaluation system. Therefore, this study was conducted to identify growth traits related to the quality of nucleated pearls from the mantle of H. cumingii. Two-year-old H. cumingii were implanted with 10 nuclei, 5 mm each in size per mussel. These host mussels were cultured for 19 months to form commercial pearls. Correlation and path analyses of the key quality traits of pearls (pearl number/pearl formation percentage, maximum diameter, minimum diameter, roundness, nacre thickness) and the growth traits of host mussels (shell length, shell width, shell height, body weight, shell weight, soft tissue weight) and nuclei (maximum diameter, minimum diameter, roundness) were performed. The results showed that the pearl formation rate varied from 50% to 100%, with 43.33% of mussels exhibiting a pearl formation rate of 70% and only 3.33% of mussels exhibiting a pearl formation rate of 50% or 100%. The average pearl formation rate was (70.00±10.82)%. The variation in pearl size (minimum diameter) was 4.72‒9.19 mm with an average of (6.91±0.67) mm. The variation in pearl roundness (diameter variation) was 0‒45.60% with an average of (6.74±7.78)%. The highest percentage of pearls were round, followed by near-round pearls, and then by positive round pearls at 21.90%. The nacre thickness was thin in 1.43% of pearls and extra thick in 98.57%. Pearl quantity and roundness were not correlated with all quantitative traits of pearls, host mussels, or nuclei (P > 0.05). Pearl size and nacre thickness were highly significantly and positively correlated with shell width, body weight, and shell weight (P < 0.01) and were significantly and positively correlated with shell height and soft tissue weight of the host mussels (P < 0.05). Among pearl quantitative traits, path analysis of pearl size and nacre thickness showed (highly) significant correlations with host mussels and nuclei. Stepwise regression analysis was performed with the pearl minimum diameter (y1) and nacre thickness (y2) as dependent variables and the shell length (x1), shell width (x2), shell height (x3), body weight (x4), shell weight (x5), and soft tissue weight (x6) of host mussels, along with the maximum diameter (x7), minimum diameter (x8), and roundness (x9) of the nuclei, as independent variables. The regression equations were y1 = 3.464 + 0.078x2 − 0.055x9 (R2 = 0.75; P < 0.01) and y2 = −1.243 + 0.077x2 − 0.055x9 (R2 = 0.75; P < 0.01). The direct effect of shell width on pearl size was the highest (0.69). The direct effect of nucleus roundness on pearl size was −0.23. The indirect effect of shell width on pearl size through nucleus roundness (0.05) was significantly greater than that of nucleus roundness on shell width (−0.02). The direct and indirect effects of shell width and nucleus roundness on nacre thickness were similar to those on pearl size. The direct determinant coefficient of shell width on pearl size (0.50) was significantly higher than that of nucleus roundness (0.08) and that of shell width via nucleus roundness (−0.03). The direct and indirect determinant coefficients of nucleus thickness by shell width and nucleus roundness in pearl-bearing mussels showed the same trend. These findings suggest that the shell width of host mussels is a key trait for controlling pearl size and pearl nacre thickness in nucleated pearls from the mantle of H. cumingii. These results will contribute to the development of China´s pearl industry.

Hyriopsis cumingii is a major freshwater pearl mussel in the world. In order to investigate the functional roles of the growth- and mineralization-related genes involved in the pearl formation process after the inserting nucleus into the mantle tissue of H. cumingii, we conducted a transcriptome analysis of data from different time periods after the pearl-nucleus insertion. We screened a total of 1,898 growthrelated unigenes and 716 mineralization-related unigenes, including 12 growthrelated differential genes and eight mineralization-related differential genes that showed sustained differential expression throughout the pearl formation. The results of the gene expression patterns among samples at different time points showed that the Mcon group and theM05d group clustered together, theM50d group and the M90d group clustered together, while the M20d group clustered separately. The overall density distribution of differential gene analysis at different periods after pearlnucleus insertion of H. cumingii, cluster analysis, differential gene analysis, differential gene differential, and co-expression analysis together revealed that growth and mineralization-related genes have the same expression pattern and jointly regulate pearl formation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that the same functional cluster and pathway were together present on the enrichment results of both growth- and mineralization-related genes, suggesting a synergistic effect between growth and mineralization. We found that genes such as bone morphogenetic proteins (BMPs) and calmodulin (CALM) are jointly involved in growth and mineralization processes. The study indicate that growth genes cooperate with mineralization genes to play biological roles in the early process of pearl formation in H. cumingii. [ABSTRACT FROM AUTHOR]

In the classic molecular model of nacreous layer formation, unusual acidic matrix proteins rich in aspartic acid (Asp) residues are essential for nacre nucleation due to their great affinity for binding calcium. However, the acidic matrix proteins discovered in the nacreous layer so far have been weakly acidic with a high proportion of glutamate. In the present study, several silk-like matrix proteins, including the novel matrix protein HcN57, were identified in the ethylenediaminetetraacetic acid-soluble extracts of the nacreous layer of Hyriopsis cumingii. HcN57 is a highly repetitive protein that consists of a high proportion of alanine (Ala, 34.4%), glycine (Gly, 22.5%), and serine (Ser, 11.4%). It forms poly Ala blocks, GlynX repeats, an Ala-Gly repeat, and a Ser-Ala-rich region, exhibiting significant similarity to silk proteins found in spider species. The expression of HcN57 was specifically located in the dorsal epithelial cells of the mantle pallium and mantle center. Notably, expression of HcN57 was relatively high during nacreous layer regeneration and pearl nacre deposition, suggesting HcN57 is a silk matrix protein in the nacreous layer. Importantly, HcN57 also contains a certain content of Asp residues, making it an unusual acidic matrix protein present in the nacreous layer. These Asp residues are mainly distributed in three large hydrophilic acidic regions, which showed inhibitory activity against aragonite deposition and morphological regulation of calcite in vitro. Moreover, HcN57-dsRNA injection resulted in failure of nacre nucleation in vivo. Taken together, our results show that HcN57 is a bifunctional silk protein with poly Ala blocks and Gly-rich regions that serve as space fillers within the chitinous framework to prevent crystallization at unnecessary nucleation sites and Asp-rich regions that create a calcium ion supersaturated microenvironment for nucleation in the center of nacre tablets. These observations contribute to a better understanding of the mechanism by which silk proteins regulate framework construction and nacre nucleation during nacreous layer formation. [ABSTRACT FROM AUTHOR]

In triangle sail mussel (Hyriopsis cumingii), shell biomineralization is a complicated process that involves multiple gene products. Shell matrix proteins are involved in the formation of the organic framework and play an important role in the regulation of calcium carbonate deposition. In this study, A new shell matrix protein gene Hc-transgelin was identified in H. cumingii. The full-length cDNA of Hc-transgelin was 1200 bp, including a 501 bp open reading frame, which encoded 166 amino acids. Hc-transgelin is rich in lysine, it accounts for 11.40% of the protein. The predicted transgelin protein contained a conserved calmodulin homologous domain. A tissuespecific expression assay indicated that Hc-transgelin exhibited significantly highest expression in the mantle. Furthermore, Hc-transgelin in situ hybridization detected positive signals at the edge of the mantle outer fold, where nacre and prismatic layer biomineralization occur. An RNA interference assay showed that the shape of aragonite flakes in nacre changed and their growth was inhibited, and cracks appeared in the prismatic layer organic sheath when the expression of Hc-transgelin was suppressed. In a shell repair assay, a higher expression of Hc-transgelin appeared from day 12 to day 25 when the nacre accumulated quickly. These findings indicate that Hc-transgelin may be involved in the formation of aragonite flakes in the nacre and play a role in the formation of the organic sheath in the prismatic layer of the shell. This study provides new insights into the role of the Hctransgelin gene and also contributes to the molecular understanding of mollusk shell formation. [ABSTRACT FROM AUTHOR]

In order to screen out the feeding ratio of high‐quality algae strains in Hyriopsis cumingii, this experiment investigated the effects of different ratio of mixed feeding of two algae strains (Scenedesmus dimorphus and Cyclotella sp.) on the growth, bead production performance, trace elements, and amino acid composition of the newborn shell of H. cumingii. The results showed that the mixed feeding of two types of microalgae with different densities had significant effects on the growth performance, bead production quality, trace elements, and amino acid composition of the newly formed shell of H. cumingii (p < 0.05); the best body weight gain rate and shell growth rate were obtained by feeding S. dimorphus (30 × 105 cell/mL) and Cyclopella sp.(8 × 105 cell/mL); the pearl weight gain rate, amino acid ratio, Ca2+ and Mn2+ content were best when feeding Cyclotella sp. (16 × 105 cell/mL), but there was no significant difference compared with S. dimorphus (30 × 105 cell/mL) and Cyclopella sp.(8 × 105 cell/mL). In summary, the optimal feeding amount for the intensive breeding process of H. cumingii is S. dimorphus (30 × 105 cell/mL) and Cyclopella sp. (8 × 105 cell/mL). [ABSTRACT FROM AUTHOR]

Star3 is located in sex hormone synthesis signaling pathway associated with gonadal development. In this study, star3 was first cloned from the gonad of Hyriopsis cumingii. The cDNA of star3 in H. cumingii was 2072 bp in length, concluding an open reading frame (ORF) of 1419 bp, encoding 472 amino acids with a conserved START structural domain. Tissue distribution analysis showed star3 highest expressed mainly in gonad, which had higher transcription levels in testis than that in ovary. Significant hybridization signals were detected on the follicle wall cell of male gonad. Gonad expression patterns revealed star3 highest expressed at two months old during juvenile stages. After RNA interference with star3, expression levels of cyp17a and 17β-hsd11 was repressed. Furthermore, contents of pregnenolone, testosterone, and estradiol in gonad were reduced significantly. These results suggest star3 may be involved in sex hormone synthesis and play an important role in early gonadal development of H. cumingii.

In shellfish culturing and breeding practice, the width of shell growth lines is an important index to measure the growth rate of shellfish, and anal angle radial rib is the part with the widest growth lines. In order to explore the feasibility of taking anal angle radial rib length as a typical growth trait, this research analyzed the correlation between anal angle radial rib length （X1） and other growth traits, such as shell length （X2）, shell height （X3）, total height （X4）, shell width （X5）, live body weight （Y1）, soft tissue weight （Y2） and shell weight （Y3） of Hyriopsis cumingii at different ages and conducted the path analysis of body size traits to body weight traits （live body weight, soft tissue weight and shell weight）. The results of correlation analysis showed that there was a significant positive correlation between anal angle radial rib length and body weight in the first to third instars of H. cumingii. The correlation coefficient between shell length and live body weight was the highest in the first and second instars of mussel, while the correlation coefficient between anal angle radiating rib length and live body weight was the highest in the third instar of mussel. In addition, the correlation coefficient between anal angle radiating rib length and live body weight, soft tissue weight and shell weight showed an increasing trend with the increase of age. The data of path analysis and multiple regression analysis displayed that the main factors affecting the live body weight of 1 to 3 year old were anal angle radial rib length, shell length and shell width, the main factors affecting the soft tissue weight of 3 year old were shell length and shell width, the main factors affecting the shell quality of 1 and 2 year old mussel were shell length and shell width, and the main factors affecting the shell quality of the third instar mussel were anal angle radiating rib length, shell length and shell width. In conclusion, anal angle radial rib length, together with shell length and shell width, can be used as a new typical growth trait to represent the growth rate of H. cumingii. [ABSTRACT FROM AUTHOR]

Cyclophilin A (CypA) serves as a principal receptor of Cyclosporin A (CsA) within cells, exhibiting considerable biodiversity and playing an important role in immune system. In this study, the full-length cDNA sequence (GenBank ID: MN121742) of CypA of Hyriopsis cumingii was obtained using PCR and RACE technology. The cDNA sequence spans 1237 bp, comprising a 57 bp 5' noncoding region (UTR), a 688 bp 3' noncoding region, and a 492 bp open reading frame, encoding a total of 164 amino acids. The theoretical isoelectric point of the CypA cDNA sequence is 8.9, and the relative molecular weight is 17.29 kD. A search for homologous sequences in GenBank revealed high similarities between CypA amino acid sequence of Hyriopsis cumingii and those of other shellfish species, including Hyriopsis schlegelii (99.39%), Cyclina sinensis (77.44%), Ruditapes philippinarum (78.05%), Crassostrea ariakensis (78.66%), and Solen grandis (75.05%). Utilizing SWISS-MODEL, it was predicted that CypA of Hyriopsis cumingii contains 8 p-pleated sheets and 2 a helixes, potentially forming the active central region of CypA. A multiple sequence comparative analysis was carried out by using the MEGA 5.1 Software, and a phylogenetic tree of the genetic system was constructed employing the neighbor-joining method. The analysis revealed that CypA of Hyriopsis cumingii clustered with CypA of shellfish from the same genus, particularly exhibiting a close genetic relationship with CypA of Hyriopsis schlegelii. The expressions levels of CypA in different tissues of Hyriopsis cumingii were assessed through qRT-PCR, indicating the highest expression in the gonad, followed by the liver, kidney and gill. Upon exposure to Aeromonas hydrophila, a bacterial infection, expression levels of CypA in gonad, liver, gill, and intestines exhibited an initial increase followed by a subsequent decrease within the first 72h of infection. Specifically, peak expression levels were observed at 6h in gonad and gill, at 12h in liver, and at 24h in intestines. This proves that Aeromonas hydrophila infection significantly induce the expressions of CypA in Hyriopsis cumingii. Expression level of CypA in gonad reaches the peak earlier than that in liver and intestines. Moreover, the relative expression times (14.91) of CypA in gonad at peak is significantly higher than those in liver (9.89), intestines (9.78) and gill (7.53). It is speculated that CypA plays an important role in defending against bacterial infections in gonad, which may not only serve reproductive functions, but also contribute significantly to immune defense. [ABSTRACT FROM AUTHOR]

Sex steroid hormones are closely related to biological reproduction, gamete emission and gonadal development. And Srd5a1 plays an important role in the synthesis of sex steroid hormones. In order to investigate the effect of the sex steroid hormone synthesis-related enzyme gene Srd5a1 on ovarian development in female Hyriopsis cumingii, Srd5a1 was obtained from the gonads of H. cumingii by RACE （Rapid-amplificiantion of cDNA ends）. The expression characteristics of Srd5a1 were investigated by realtime fluorescence quantification （qRT-PCR）, in situ hybridization and in vitro induction of 17β-estradiol （E2） and 17α-methyltestosterone （MT） at different concentrations for 24 days. The results showed that the full-length cDNA of Srd5a1 was 2 224 bp, including ORF 825 bp, 3′-UTR 953 bp, and 5′-UTR 446 bp, encoding 274 amino acids. The expression of Srd5a1 in the gonad was bimodal, with higher expression in the ovary than that in the testis; Srd5a1 had the highest expression level in the ovarian discharge period, which was extremely significantly higher than that in all other periods. In situ hybridization results showed that Srd5a1 detected positive signals in ovarian oocytes and follicles as well as spermatocytes of the testis. The expression of Srd5a1 was changed after 24 days of treatment with different concentrations of E2 and MT. In conclusion, Srd5a1 may play a role in the developmental maturation of the ovary oocyte and gamete emission in the female H. cumingii. This study is important for exploring the gonadal growth and development of H. cumingii, and also provides a theoretical reference value for the realization of monogamous breeding of H. cumingii. [ABSTRACT FROM AUTHOR]

Abstract In order to screen out the feeding ratio of high‐quality algae strains in Hyriopsis cumingii, this experiment investigated the effects of different ratio of mixed feeding of two algae strains (Scenedesmus dimorphus and Cyclotella sp.) on the growth, bead production performance, trace elements, and amino acid composition of the newborn shell of H. cumingii. The results showed that the mixed feeding of two types of microalgae with different densities had significant effects on the growth performance, bead production quality, trace elements, and amino acid composition of the newly formed shell of H. cumingii (p

For thousands of years, pearl and nacre powders have been important traditional Chinese medicines known for their skin whitening effects. To prepare the enzymatic hydrolysates of Hyriopsis cumingii nacre powder (NP-HCH), complex enzymatic hydrolysis by pineapple protease and of neutral protease was carried out after the powder was pre-treated with a high-temperature and high-pressure method. The peptides were identified using LC-MS/MS and picked out through molecular docking and molecular dynamics simulations. Subsequently, the tyrosinase inhibitory and antioxidant properties of novel tyrosinase inhibitory peptides were investigated in vitro. In addition, the enzymatic activity of tyrosinase in B16F10 cells as well as melanin content and antioxidant enzyme levels were also examined. The results showed that a tyosinase inhibitory peptide (Tyr-Pro-Asn-Pro-Tyr, YPNPY) with an efficient IC50 value of 0.545 ± 0.028 mM was identified. The in vitro interaction results showed that YPNPY is a reversible competitive inhibitor of tyrosinase, suggesting that it binds to the free enzyme. The B16F10 cell whitening test revealed that YPNPY can reduce the melanin content of B16F10 cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that YPNPY could be widely used as a tyrosinase inhibitor in whitening foods and drugs.

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. [ABSTRACT FROM AUTHOR]

As micro-particle aggregates, bioflocs may be potential baits for ingestion by triangle sail mussels and supplementary feeding raises more concerns. Besides, a plasmid carrying the green fluorescent protein (GFP) gene was transformed into floc-forming Bacillus subtilis (GFP-B. subtilis) to visualize its colonization during the bioflocs ingestion. To investigate the potential benefits of bioflocs on H. cumingii, Chlorella vulgaris diets were supplemented with nothing (control group, CG) or bioflocs accounting for the microalgae biomass at 25% (T1), 50% (T2), and 100% (T3) respectively. The community composition of bioflocs was analyzed and some species like phylum Proteobacteria, order Rhodobacterales and genus Paracoccus with potentials to improve water quality were found. Furthermore, supplementary bioflocs could reduce the concentrations of inorganic nitrogen in the aquacultural environment. Differences in growth traits across all groups were insignificant, but the digestion, metabolic and immune response of H. cumingii were promoted remarkably. Interestingly, some colonies of GFP-B. subtilis were detected in the intestines of H. cumingii from T1, suggesting H. cumingii could uptake bioflocs efficiently and intestinal colonization of B. subtilis might be accomplished. Besides, supplementary bioflocs with the highest ratio (T3) could improve the physiological status of organs and suppress the dominance of potential pathogens such as Aeromonas and Pseudomonas. It was assumed that B. subtilis might stimulate the healthy intestine development via intestinal colonization. Overall, B. subtilis-mediated bioflocs is beneficial to H. cumingii and more feeding strategies of bioflocs in H. cumingii farming are expected to be developed in future.

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. [ABSTRACT FROM AUTHOR]

Abstract Background Protein kinase C (PKC) is a multifunctional serine and PKC can phosphorylate serine residues in the cytoplasmic domain of tyrosinase, thereby regulating the activity of tyrosinase. Activated PKC is bound to the melanosome membrane, and unactivated PKC is free in the cytoplasm of melanocytes. In this study, we study the role of PKC gene in the melanin synthesis pathway and its effect on the color of the nacre of H. cumingii. Results In this study, a HcPKC gene in H. cumingii was cloned and its effects on melanin synthesis and nacre color were studied. HcPKC was expressed in both purple and white mussels, and the level of mRNA expression was higher in the purple mussels than in white mussels. Strong and specific mRNA signals were detected in the dorsal epithelial cells of the mantle pallial layer, indicating that HcPKC may be involved in nacre formation. After SNP association with inner shell color related traits, according to the principle that 0.25

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 labelfree 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. [ABSTRACT FROM AUTHOR]

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Simple Summary: Vaterite has been widely used in the pharmaceutical industry because of its large specific surface area, high solubility, high biocompatibility, low specific gravity, and other beneficial characteristics. It is an internal factor that affects the quality of freshwater pearls. The mollusk shell comprises more than 95% calcium carbonate and less than 5% organic matrix. The organic matrix of the shell includes shell matrix proteins, polysaccharides, and a small number of lipids, among which shell matrix proteins play a crucial role in the formation of shells and pearls, according to previous studies. In our present study, we cloned a novel matrix protein, cysrichin, and our in vitro experiments confirmed that the cysrichin peptide induced vaterite crystals. Cysrichin has repeat amino acid sequences and a modular structure that may play a key role in establishing a structural framework and inducing crystal nucleation and growth. We analyzed cysrichin at both molecular and protein levels and confirmed that it was an important matrix protein; this finding provided some new clues to the mineralization of mussels. In this study, we cloned a novel matrix protein, cysrichin, with 16.03% homology and a similar protein structure to the coral biomineralized protein galaxin. Tissue expression analysis showed that cysrichin was mainly expressed in mantle and gill tissues. In situ hybridization indicated that cysrichin mRNA was detected in the entire epithelium region of mantle tissue. RNAi analysis and shell notching experiment confirmed that cysrichin participates in the prismatic layer and nacreous layer formation of the shell. An in vitro crystallization experiment showed that the cysrichin protein induced lotus-shaped and round-shaped crystals, which were identified as vaterite crystals. These results may provide new clues for understanding the formation of vaterite in freshwater shellfish. [ABSTRACT FROM AUTHOR]

Luster is the index to measure the maximum value of pearl. To study the relative contribution of Hyriopsis cumingii donor mussel and pearl mussel to the luster of nucleated pearls, white mussel (inner shell is white) is used as material, shell length and green shell are used as selection traits. According to different selection strategies, eight groups of H. cumingii populations were established. Each group was used as donor mussel and host mussel respectively for combined nuclear insertion. After 18 months of cultivation, it was found that: Among the different pearl breeding combinations, the pearl luster value of YAA pearl breeding combination from the selected population of group I was the greatest, which was 15.46% and 21.51% higher than that of YGG and YHH respectively. There was a significant difference in the luster of pearls cultivated by different groups of donor mussel. There was a very significant positive correlation between the inner shell luster of donor mussel and the luster of pearls (r=0.717-0.939). The regression equation between inner shell luster and pearl luster was established as follows: y=16.81+0.93x(R²=0.777).There was no significant correlation between the growth traits of donor mussel and the pearl luster. There were also significant differences in the luster of pearls cultivated by different groups of host mussels, but there was no significant correlation between the growth traits and luster of host mussels and the luster of pearls. The above results show that the breeding of H. cumingii with green shell color at the juvenile mussel stage has achieved the goal of improving pearl luster, which can be regarded as an indirect trait of breeding H. cumingii with high luster, the inner shell luster of donor mussel is significantly correlated with pearl luster, and the inner shell luster is better as the target trait. [ABSTRACT FROM AUTHOR]

Biomineralization is a widespread phenomenon in marine mollusks and is responsible for the production of shells and pearls. However, the regulatory mechanisms governing the adaptive immune responses in the mollusk mantle and visceral mass during mineralization remain unclear. In this work, we examined the mantle and visceral mass immune responses of Hyriopsis cumingii during pearl culture using high-throughput sequencing techniques. A mantle transcriptome database was established using transcriptome sequencing technology and reference to the major databases. Digital gene expression profiling was used to identify the differentially expressed genes of mantle and visceral mass at different insertion periods. Moreover, quantitative real-time PCR was used to verify the expression of five immune-related genes. Transcriptome sequencing results showed 257,457 unigenes were identified. Digital gene expression profiles showed 1389, 3572, 1888, and 2613 differentially expressed genes (DEGs) in the mantle and visceral mass at 5, 20, 50, and 90 d after insertion, respectively, with the highest number at 20 d and the lowest at 5 d after insertion (q < 0.05). A cluster analysis of the DEGs showed similar clustering and expression features in the mantle to the control group, and at 5, 50 and 90 d, after mantle insertion. The DEGs in the visceral mass showed similar clustering and expression features to the control group and at 5, 20 and 50 d after insertion. We also screened 22 immune-related DEGs in the mantle and visceral mass during the same pearl culture period, including serine/threonine-protein kinase NLK, C-type lectin, and galectin. The greatest number of DEGs was found 90 d after insertion. Compared with the mantle, more immune-related DEGs were down-regulated than up-regulated in the visceral mass during pearl culture, indicating that the immune regulatory mechanisms in the visceral mass and the mantle differ during pearl culture, and that the visceral mass is liable to higher infection and mortality rates. Quantitative real-time PCR results showed that the expression of five immune-related genes was consistent with DGE results. Our findings will further knowledge of the immune systems that are present in the mantle and visceral mass during pearl culture.

Mollusk shells are characterized by hierarchical aggregation of calcium carbonate and organic matrix, and matrix protein is considered as a key active ingredient to understand shell biomineralization. In this study, a total of 21 proteins, including a novel matrix protein Hcprismatin-14 were identified in the EDTA-soluble matrix of the prismatic layer of the mussel Hyriopsis cumingii by liquid chromatography tandem mass spectrometry (LC-MS/MS). The full length of Hcprismatin-14 cDNA was cloned from the mantle of H. cumingii. Hcprismatin-14 contains a high proportion of Gly, Tyr, Arg and Asp residues, their concentrated distribution forms three structurally characteristic regions: a Gly/Tyr-rich region, a WDD-repeat region and a C-terminal basic tail. Hcprismatin-14 expression was high in mantle edge tissue in a tissue-specific analysis, and during disordered crystal deposition in a saibo transplantation assay. Knocking down Hcprismatin-14 expression with double-stranded RNA induced subgrains deposition inhibition and lost contact with chitinous scaffold. In addition, the WDD-repeat region polypeptide was involved in morphological regulation of calcite and had dose-dependent inhibitory activity against aragonite deposition in vitro. Based on these results, Hcprismatin-14 appears to be a dual-function prismatic-layer matrix protein, responsible for both framework recognition and crystal deposition. These findings contribute to understanding the relationship between the modular structure of matrix protein and their regulation mechanism during shell biomineralization in mollusks.

In this study, we investigated the impact of water temperature on pearl formation and the molecular mechanisms underlying this biomineralization process in the mussel Hyriopsis cumingii. In the laboratory experiment, recipient mussels received saibo grafts from donor mussels and then were cultivated at five temperatures (16, 20, 24, 28, and 32 °C) for 1 month. We found that speed of pearl formation varied with water temperature: there was no visible pearl granules at 16 and 20 °C, tawny pearl granules were collected at 24 °C and lustrous pearl granules were collected at 28 and 32 °C. The expressions of several nacreous layer matrix protein genes (hic22, hic24, HcTyr Hc-upsalin and Hcperlucin) at 28 °C and 32 °C were peaked on day 28, and the expressions of other nacreous layer matrix protein genes (hic52 and hic74) on day 28 were significant higher than day 14. In the natural environment, pearl deposition rate also increased as water temperature rose, and the optimum range for pearl growth in H. cumingii was between 28.3 and 30.3 °C. Expression of the prismatic layer-related matrix protein genes hic31 and both prismatic and nacreous layer-related matrix protein gene silkmapin was significantly high in winter, and expressions of the nacreous layer-related matrix protein genes (hic24, hic74, HcTyr, Hc-upsalin and Hcperlucin) were always higher during higher temperature seasons compared with winter. These results indicate that high water temperature can speed up pearl formation and further regulate matrix protein secretion to accelerate pearl nacre deposition. [ABSTRACT FROM AUTHOR]

The quality of pearls is related closely to the position where the nucleus is inserted. The visceral mass of freshwater pearl mussels has the physiological condition for the culture of pearls, but the appropriate position for inserting the nucleus has not been determined. In this study, the physiological responses and cultured pearl biology were used to explore the most suitable of five positions in the visceral mass for inserting the nuclei (I: the front end axe foot visceral mass, II: the central axe foot visceral group, III: near the gonad department, IV: near the stomach and V: near the kidney). Overall, kidney damage (urea nitrogen/BUN, urine creatinine/Cr, uric acid/UA) and calcium metabolism indicators were in decline, whereas liver damage indicators (aspartate aminotransferase/AST, alanine aminotransferase/ALT) rose. Enzyme activity index was determined by enzyme‐linked immunosorbent assay. These results indicated that the insertion caused metabolic disorders and immune inflammation. The axe foot visceral group was not significant that compared with the control group, so it was suitable for nucleation. The peak of granulocytes and hyalinocytes in the blood cells appeared on day 10 (thd) and exerted an early immune defence function. Furthermore, the granulosa synthesized by the epidermal cells of the pearl capsule was in the mature stage of normal nacre secretion, thus forming a thicker deposit of pearls. This study filled the gaps in knowledge of visceral mass cultivating pearls and provided a theoretical basis for artificial pearl breeding. [ABSTRACT FROM AUTHOR]

The light plays a pivotal role in influencing the growth and body color for aquaculture animals. The freshwater pearl mussel Hyriopsis cumingii , inhabiting benthic environments naturally, is predominantly cultivated by hanging methods. This aquaculture method altersmutiple environmental factors, with light being one of the maximum variationfactor. In order to comprehend the impact of light change on the growth and inner shell color of H. cumingii , a total of 14 full-sibling families were established and cultivated separately under natural light and dim light conditions for a period of 13 months in this study. The results revealed that under natural light conditions, juvenile mussel exhibited accelerated growth rates, while dim light conditions expedited purple pigmentation in the inner shell. Genetic analysis indicated general heritability range for the entire experimental population falls between 0.07 and 0.28. heritability estimates for all traits under natural light conditions (NL) ranged from 0.33 to 0.72, indicating consistently high heritability. However, heritability estimates for all traits under dim light conditions (DL) ranged from 0.05 to 0.16. The growth and inner shell color traits showed significant G × E interactions under different light conditions. The anal angle radial rib length (AARRL) trait exhibited a significantly higher correlation with body weight (BW) compared to shell height (SH) and total shell height (TSH). Furthermore, the growth and inner shell color traits of different families were compared and evaluated under the two environmental conditions in this study. Families 1 and 6 excelled in various traits under both conditions, making them promising candidates for genetic improvement breeding. In this study, pronounced influences of light exposure on the growth of pearl mussels were identified, emphasizing the need for tailored breeding strategies for both bottom seeding and suspended cultivation methods. • The growth of Hyriopsis cumingii is accelerated in natural light and the inner shell color is enhanced in dark light. • The growth and inner shell color traits showed significant G × E interactions under different light conditions. • The newly defined growth trait, Anal Angle Radial Rib Length (AARRL), may be for breeding fast-growth mussels in future. • The identified excellent families offer genetic resourcesfor further breeding. [ABSTRACT FROM AUTHOR]

Uroporphyrinogen III synthetase (UROS) is an important enzyme involved in the structural formation of porphyrin, which is responsible for color intensity. A UROS homolog was isolated and characterized from the pearl mussel, Hyriopsis cumingii (HcUros). The HcUros cDNA was 858 bp in length with an open reading frame that encoded a 285 amino acid protein, which contained a conserved HemD domain. HcUros exhibited widespread tissue distribution, displaying particular enrichment in the mantle and haemolymph of purple mussels, compared to white mussels. In situ hybridization of HcUros in mantle tissue demonstrated that it was specifically expressed in the dorsal epithelial cells of the mantle pallia. Additionally, HcUros expression was upregulated following implantation of the pearl sac to produce pearls. The data suggest that HcUros contributes to shell pigmentation, and nacre formation and coloring in H. cumingii.

MAPKAPK2 (MK2) is an important regulator of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, which is involved in a plethora of cellular processes concluding the development of gamete cells in meiosis and resisting pathogenic bacterial infestation. Hyriopsis cumingii is a significant mussel resource in China and a good material for pearl breeding. To explore the role of MK2 in H. cumingii, MK2 was identified and cloned, whose full-length cDNA was 1568 bp, including 87 bp in 5′ UTR, 398 bp in 3′ UTR, and 1083 bp in the open reading frame (ORF) region, encoding 360 amino acids. The expression of MK2 was the highest in the gills. Meanwhile, there was a significant difference in the gonads. After Aeromonas hydrophila and Lipopolysaccharide (LPS) infestation, the transcript level of the MK2 was upregulated in the gills. It indicated that MK2 might be involved in the innate immune response of H. cumingii after a pathogenic attack. After quantifying H. cumingii of different ages, it was found that the expression of MK2 was highest at 1 year old. In situ hybridization (ISH) results showed that the blue-purple hybridization signal was very significant in the oocytes and egg membranes of the female gonads of H. cumingii. The expression of MK2 increased gradually at the age of 1 to 5 months and showed a downward trend at the age of 5 to 8 months. It was suggested that MK2 might play an important role in the formation of primitive germ cells in H. cumingii. To sum up, MK2 might not only be involved in the immune response against pathogenic bacterial infection but also might play an important role in the development of the gonads in H. cumingii. [ABSTRACT FROM AUTHOR]

Copyright of South China Fisheries Science is the property of South China Fisheries Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Protein kinase C (PKC) is a multifunctional serine and PKC can phosphorylate serine residues in the cytoplasmic domain of tyrosinase, thereby regulating the activity of tyrosinase. Activated PKC is bound to the melanosome membrane, and unactivated PKC is free in the cytoplasm of melanocytes. In this study, we study the role of PKC gene in the melanin synthesis pathway and its effect on the color of the nacre of H. cumingii. Results: In this study, a HcPKC gene in H. cumingii was cloned and its effects on melanin synthesis and nacre color were studied. HcPKC was expressed in both purple and white mussels, and the level of mRNA expression was higher in the purple mussels than in white mussels. Strong and specific mRNA signals were detected in the dorsal epithelial cells of the mantle pallial layer, indicating that HcPKC may be involved in nacre formation. After SNP association with inner shell color related traits, according to the principle that 0.25 < PIC < 0.5 is medium polymorphism and PIC < 0.25 is low polymorphism, the A + 332G site on the HcPKC gene was a site of moderate polymorphism, and the other four sites were low polymorphism sex sites. There was strong linkage disequilibrium among the five loci. A haplotype was constructed and it was found that the frequency of T1 (AGGAA)in the white population was significantly higher than that in the purple population (P < 0.05). Conclusion: The study found that HcPKC of H. cumingii can be used as a candidate gene related to inner shell color, and some of the SNP sites can be used for molecular-assisted breeding in the spinnaker mussel, providing a reference for cultivating high-quality freshwater pearls. [ABSTRACT FROM AUTHOR]

Simple Summary: Hyriopsis cumingii (H. cumingii, mussels) is an important freshwater pearl shellfish species, and its mantle is the most important tissue during shell and pearl mineralization. However, the low proliferative capacity of mantle cells during in vitro culture hinders the establishment of stable cell lines. In this study, we identified and explored the function of IGF1. The results show that IGF1 has a typical IIGF structural domain, exerts biological functions through the PI3K/AKT signaling pathway, and that the recombinant protein significantly promotes the proliferation of mantle cells. Our findings will help to further extend the culture time of mantle cells as well as to understand the growth regulation mechanism of mussels. Insulin-like growth factor 1 (IGF1) plays an important regulatory role in the regulation of growth, differentiation, and anabolism in a variety of cells. In this study, the full-length cDNA of the IGF1 gene was cloned from Hyriopsis cumingii, named HcIGF1. The expression level of HcIGF1 in six tissues (adductor muscle, foot, hepatopancreas, gill, mantle, and gonad) was determined. In addition, the localization of HcIGF1 in the mantle was analyzed by in situ hybridization, and finally the function of HcIGF1 was explored by RNA interference and prokaryotic expression. The results showed that the amino acid sequence contained a typical IIGF structural domain. The phylogenetic tree showed that HcIGF1 clustered with other marine bivalve sequences. Quantitative real-time PCR and in situ hybridization analysis showed that HcIGF1 was expressed in all tissues. The highest expression was in the foot and the lowest was in the mantle. In the mantle tissue, the hybridization signal was mainly concentrated in the outer mantle. After RNA interference, the expression of IGF1 was found to be significantly decreased (p < 0.05), and its related genes IGF1R, AKT1, and cyclin D2 were downregulated, while MAPK1 were upregulated. The recombinant HcIGF1 protein was purified and its growth-promoting effect was investigated. The results showed that the recombinant HcIGF1 protein could significantly promote the proliferative activity of the mantle cells of mussels, with the best proliferative effect at 12.5 μg/mL. The results of this study provide a new method to solve the problem of weak proliferation of shellfish cells in vitro and lay the foundation for further understanding of the growth regulation mechanism of H. cumingii, as well as a better understanding of the physiological function of IGF1 in mollusks. [ABSTRACT FROM AUTHOR]

The TGF-β signaling pathway plays an important role in wound healing and immune response. In this study, a TGF-β type I receptor (TGF-βRI) homolog was cloned and characterized from freshwater mussel Hyriopsis cumingii . The full-length cDNA of the TGF-β RI gene was 2017 bp, with a 1554 bp open reading frame (ORF), and encoded 517 amino acids. The predictive analysis further identified distinct regions within the TGF-βRI protein: a signal peptide, a membrane outer region, a transmembrane region, and an intracellular region. Real-time quantitative PCR results showed that the TGF-β RI gene was expressed in all tissues of healthy mussels. The transcripts of TGF-β RI in hemocytes and hepatopancreas were significantly up-regulated at different periods after stimulation with Aeromonas hydrophila and peptidoglycan (PGN) ( P < 0.05). The mRNA expression of TGF-β RI progressively increased from day 1 to day 10 after trauma ( P < 0.05), and it returned to the initial level by day 15. The expression levels of TGF-β , Smad5 , MMP1/19 , and TIMP1/2 , but not Smad3/4 , were significantly up-regulated at different time points after trauma. However, the expression levels of TGF-β , MMP1/19 , and TIMP2 were decreased after treatment with the inhibitor SB431542. Furthermore, the recombinant TGF-βRI proteins were expressed in vitro and existed in the form of inclusion bodies. Western blotting results showed that TGF-βRI proteins were expressed constitutively in various tissues of mussels, and their expression was up-regulated after trauma, which was consistent with the mRNA expression trend. These results indicate that TGF-β RI is involved in the process of wound repair and immune response.

A cDNA encoding a phage-type lysozyme, designated as HcPLYZ, was successfully cloned from Hyriopsis cumingii. The full-length cDNA sequence of HcPLYZ was determined to be 896 base pairs in length. Analysis revealed the absence of a signal peptide at its N-terminus, and identified two highly conserved phage-type lysozyme activity sites, Glu 20 and Asp 29 , within the deduced amino acid sequence of HcPLYZ. The results of the cloning and sequencing symbiotic bacteria in tissues were consistent with those obtained using tissue cDNA as the template, suggesting that HcPLYZ may originate a symbiotic bacterium. The expression levels of HcPLYZ mRNA exhibited significant variations across different tissues. Successful expression was induced using IPTG, and the native recombinant protein was subsequently purified through affinity chromatography employing Ni 2+ , and the optimal pH and temperature of which were determined to be 5.5 and 50 °C, respectively. Following exposure to Aeromonas hydrophila, there was a significant increase in the levels of HcPLYZ mRNA in the hemocytes, hepatopancreas, and gills. HcPLYZ was demonstrated the inhibition activity of 55% and 83% against Micrococcus lysodeikticus under pH 5.5 and 50 °C conditions, respectively. These results suggested that HcPLYZ possessed antibacterial activity against both A. hydrophila and M. lysodeikticus., (Copyright © 2024. Published by Elsevier Ltd.)

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.

The process of spermatogenesis is complex and controlled by many genes. In mammals, Testis-expressed gene 11 (Tex11) and meiosis expressed gene 1 (Meig1) are typical spermatogenesis-related genes. In this study, we obtained the full length cDNAs for Tex11 (3143bp) and Meig1 (1649bp) in Hyriopsis cumingii by cloning. Among them, Hc-Tex11 contains 930 amino acids and Hc-Meig1 contains 91 amino acids. The protein molecular masses (MW) of Hc-Tex11 and Hc-Meig1 were 105.63 kDa and 10.95 kDa, respectively. Protein secondary structure analysis showed that Hc-TEX11 protein has three TPR domains. The expression of Hc-Tex11 and Hc-Meig1 in different tissues showed higher levels in testes. At different ages, the expression of Hc-Tex11 and Hc-Meig1 was higher levels in 3-year-old male mussels. During spermatogenesis, the mRNA levels of Hc-Tex11, Hc-Meig1 gradually increased with the development of spermatogonia and reached a peak during sperm maturation. Hc-Tex11 and Hc-Meig1 mRNA signals were detected on spermatogonia and spermatocytes by in situ hybridization. In addition, RNA interference (RNAi) experiments of Hc-Tex11 caused a downregulated of Dmrt1, KinaseX, Tra-2 and Klhl10 genes and an up-regulated of ß-catenin gene. Based on the above experimental results, it can be speculated that Hc-Tex11 and Hc-Meig1 are important in the development of the male gonadal and spermatogenesis in H. cumingii, which can provide important clues to better comprehend the molecular mechanism of Tex11 and Meig1 in regulating spermatogenesis of bivalves. [ABSTRACT FROM AUTHOR]

In the process of production of freshwater pearl, implanted mantle pieces undergo a series of complex physiological and biochemical processes to form pearl sac, which produce pearl. This is a very important site of occurrence due to immune-induced biomineralization, while its molecular regulatory mechanism is still unclear. Here, we use proteomics to identify differentially expressed proteins (DEPs) of the mantle and pearl sac and examine the biomineralization and immune response of the pearl sac formation process in Hyriopsis cumingii. Using iTRAQ technology and bioinformatics analysis, we obtained DEP profiles between the mantle and pearl sac. A total of 1871 proteins were identified. Of these, 74 DEPs were found between the pearl sac and outer mantle, 112 DEPs between the pearl sac and inner mantle, and 124 DEPs between the outer and inner mantles. Bioinformatics analysis revealed that the screened biomineralization-related DEPs were mainly enriched in signaling pathways associated with calcium signaling, regulation of the actin cytoskeleton and protein processing in the endoplasmic reticulum, while the immune-related DEPs were mainly enriched in the Notch, Hippo, nuclear factor kappa-B (NF-κB), and transforming growth factor-β (TGF-β) signaling pathways. In addition, the expression of six biomineralization-related and four immune-related proteins were verified at the transcriptional level using quantitative real-time PCR. Our findings contribute to furthering the understanding of the mechanisms of pearl formation and immune response, and have long-term implications for future studies on the production of high-quality freshwater pearls and development of the freshwater pearl industry. • We used comparative proteomics to analyze immune- and biomineralization-associated DEPs and analyzed the possible joint role of both in the process of pearl sac formation. • We subdivided the mantle into outer mantle and inner mantle and compared them with each other with the pearl sac. • Our data will contribute to the understanding of the molecular mechanisms involved in pearl formation and will have implications for future research on the production of freshwater pearls. [ABSTRACT FROM AUTHOR]

As members of arrestins family, β-arrestins are widely expressed in monocytes, macrophages, neutrophils and other immune cells. They can regulate the immune response of bodies through various ways. In the present study, a β-arrestin homolog named Hcβ-arrestin was cloned and identified from Hyriopsis cumingii. Predicted Hcβ-arrestin protein contained a conserved arrestin domain, which could be further divided into arrestin-N (39-192aa) and arrestin-C (211-365aa). Amino acid sequence alignment showed that it had the highest identity with Mytilus galloprovincialis and Mytilus edulis counterpart, which was 89.02% and 87.68%, respectively. Furthermore, real-time quantitative PCR analysis showed that the Hcβ-arrestin gene was widely expressed in the detected tissues and with the highest expression in hepatopancreas. The transcription of Hcβ-arrestin in hepatopancreas and gill of mussels was significantly up-regulated after stimulation with peptidoglycan, lipopolysaccharide (LPS) and polyinosinic polycytidylic acid. Knockdown of Hcβ-arrestin gene significantly increased the expression of some antibacterial effector genes, such as lysozyme, LPS-binding protein/bactericidal permeability increasing protein and theromacin in hepatopancreas and gills of LPS stimulated mussels, but only had little effect on TLR pathway genes. In addition, GST pull-down assay confirmed that Hcβ-arrestin can bind to HcTRAF6 protein in vitro. Dual luciferase reporter assay showed that the co-expression of HcTRAF6 and Hcβ-arrestin inhibited the activation of NF-κB reporter by HcTRAF6. These findings indicated that Hcβ-arrestins could interact with HcTRAF6 to negatively regulate the NF-κB pathway in H. cumingii. • Hcβ-arrestin was identified from Hyriopsis cumingii. • The transcription level of Hcβ-arrestin was significantly up-regulated after stimulation of PAMPs. • Knockdown of Hcβ-arrestin significantly increased the expression of antibacterial genes. • Hcβ-arrestin can bind to HcTRAF6 protein in vitro. • Co-expression of Hcβ-arrestin and HcTRAF6 inhibited the activation of NF-κB reporter by HcTRAF6. [ABSTRACT FROM AUTHOR]

Background: Cyclin D (CCND) plays an important role in the cell cycle and is a rate-limiting factor that facilitates the G1/S transition. Methods: In this study, the full-length cDNA of Hc-CCND2 was isolated from freshwater pearl mussel (Hyriopsis cumingii; Hc) and amplified using the 3´/5´ RACE system. The Hc-CCND2 expression profiles were analysed by quantitative real-time PCR. Functional analysis of the Hc-CCND2 genes was examined by both RNA interference (RNAi) and overexpression in H. cumingii. Results: Hc-CCND2 protein sequences were 295 amino acids long, possessed D-type cyclin signature motifs and contained conserved cyclin box domains. Hc-CCND2 was expressed in all examined tissues (adductor, foot, visceral mass, gill, outer mantle, inner mantle and gonad), with the highest expression levels found in the gill (P < 0.05). During the different developmental periods of the embryo, the relative expression of Hc-CCND2 increased with embryonic development, peaking at the blastula stage and decreasing significantly in the gastrula stage. After knockdown of Hc-CCND2 by RNAi, a significant decrease in CDK6 expression levels was found, while the percentage of cells in the G0/G1 phase significantly increased. Overexpression of Hc-CCND2 in mantle cells led to increased proliferation of cultured cells (P < 0.05). Conclusions: Our results demonstrated that Hc-CCND2 may promote cell cycle progression in H. cumingii, and that overexpression of Hc-CCND2 promotes mantle cell proliferation. These findings may provide a novel approach for improving the slow proliferation rate of shellfish cells in in vitro cultures. [ABSTRACT FROM AUTHOR]

Aeromonas veronii is an opportunistic pathogen that causes diseases in aquatic animals, but its key virulence factors remain unclear. In order to evaluate the feasibility of potential compensatory mechanism to be a novel target for developing live attenuated vaccine, virulence-related phenotypes and the immune response of H. cumingii were measured. Then two single-deletion mutants ΔPmbA, ΔTldD and one double-deletion mutant ΔP&T were constructed by homologous recombination. The hemolytic activity of double-deletion mutant ΔP&T showed dramatically lower than wide-type GL2 (P < 0.05), and the two genes could significantly regulate the proteolytic activity on skimmed milk (P < 0.05), except for casein (P > 0.05). All mutants exhibited attenuated capacities to form biofilms (P < 0.05), and pmbA mRNA was upregulated dramatically (P < 0.05) under exogenous threats modeled by osmotic stress, in which tldD mRNA was overlapped. Besides, the absence of one gene resulted in significantly higher (P < 0.05) mRNA level of another gene. ftsH mRNA was restrained dramatically (P < 0.05) in all mutants, and aerA mRNA was suppressed remarkably (P < 0.05) in ΔP&T. Challenge tests showed that the pathogenicity of ΔP&T on Hyriopsis cumingii was further reduced compared with the two single-deletion mutants, and its LD 50 was 6.31-fold than that of GL2. In addition, enzymatic activities in the hemolymph and hepatopancreas of ΔP&T group were significantly lower (P < 0.05) than those in GL2 group at different time points, and their peaks in hepatopancreas were found almost at 12 h post challenge, which were supported by the qPCR results of HcIL-17 and TR. Besides, histopathological examination displayed that the degree of tissue damage in ΔP&T group was alleviated remarkably compared with GL2 group. Finally, bacterial loads in the hemolymph and hepatopancreas of ΔP&T group were lower significantly (P < 0.05) than other groups. The above results demonstrate that the compensatory mechanism between the genes pmbA and tldD is a novel target to attenuate A. veronii , and the double-deletion mutant ΔP&T is helpful for the development of live attenuated vaccine. • Absence of pmbA or tldD leads to the transcriptional compensation of the other. • pmbA and tldD , the upstreams of aerA , regulate A. veronii hemolysis. • pmbA and tldD are in response to exogenous threats on A. veronii. • Deletion of pmbA and tldD weakens A. veronii infection to H. cumingii. • Double-deletion mutant ΔP&T is potential live attenuated vaccine. [ABSTRACT FROM AUTHOR]

Hyriopsis cumingii has attracted attention because of its pearl production performance and water purification capacity. Realizing sustainable industrialized culture of H. cumingii or applying it to bivalve biomanipulation for controlling water eutrophication needs urgent studies about the selection of suitable algae and the effects of different microalgae on mussel physiology. To contrast molecular and biochemical effects of high-quality microalgal diets (Chlorella vulgaris, Navicula pelliculosa, and Cyclotella sp.) with toxic Microcystis aeruginosa on metabolism and immune physiology of H. cumingii, levels of related enzymes and genes were analyzed during the 28-day exposure period. Results showed that the Cyclotella sp. diet could significantly (p < 0.05) maintain higher levels of metabolic enzymes (glutamic oxaloacetate transaminase (GOT), glutamic pyruvate transaminase (GPT), pyruvate kinase (PK), and hexokinase (HK)) and genes (CPT1 and LDLR). C. vulgaris and N. pelliculosa treatments significantly (p < 0.05) reduced activities of these metabolic parameters. The M. aeruginosa treatment significantly (p < 0.05) enhanced levels of immune enzymes (alkaline phosphatase (AKP), superoxide dismutase (SOD), and catalase (CAT)) and genes (HcIL-17 and IAP) on day 1 or 7, and there was a significant (p < 0.05) reduction on day 28. Results suggested that Cyclotella sp. was the suitable algae for H. cumingii, followed by C. vulgaris and N. pelliculosa, and toxic algae caused metabolic disorders, immune injury, and poor physiological status. The study has practical significance in the sustainable cultivation of H. cumingii and provides a theoretical basis for bivalve biomanipulation in eutrophic water.

Cyclin dependent kinase 6 (CDK6) is a serine/threonine kinase that plays important roles in cell cycle progression and differentiation. In this study, full-length cDNA of Hc-CDK6 was obtained from freshwater pearl mussels (Hyriopsis cumingii, Hc) with 3´,5´ rapid-amplification of cDNA ends (RACE). The Hc-CDK6 expression profiles were analyzed with quantitative real-time PCR and in situ hybridization. The function of the Hc-CDK6 gene was studied with both RNA interference (RNAi) and overexpression in H. cumingii. Hc-CDK6 was found to encode 331 amino acids and to have a CDK4/6-like serine/threonine kinase catalytic structural domain. In terms of the amino acid sequence, the protein Hc-CDK6 was most closely related to its homolog in Crassostrea gigas, with a similarity of 75.23%. Hc-CDK6 was expressed in all examined tissues (adductor, foot, visceral mass, gill, outer mantle, inner mantle and gonads), and the highest expression was observed in the gonads (P

In this study, we cloned a novel matrix protein, cysrichin, with 16.03% homology and a similar protein structure to the coral biomineralized protein galaxin. Tissue expression analysis showed that cysrichin was mainly expressed in mantle and gill tissues. In situ hybridization indicated that cysrichin mRNA was detected in the entire epithelium region of mantle tissue. RNAi analysis and shell notching experiment confirmed that cysrichin participates in the prismatic layer and nacreous layer formation of the shell. An in vitro crystallization experiment showed that the cysrichin protein induced lotus-shaped and round-shaped crystals, which were identified as vaterite crystals. These results may provide new clues for understanding the formation of vaterite in freshwater shellfish.

Nowadays, the cultivation mode of Hyriopsis cumingii urgently needs transformation and upgrading, and realizing sustainable aquaculture lacks the selection of suitable algae and the exploration of culture conditions. This experiment is to evaluate combined effects of ration levels (low ration, medium ration and high ration) and temperature (13.0 ± 0.2°C, 23.0 ± 0.2°C, and 33.0 ± 0.2°C) on immune responses of H. cumingii. Activities of immune enzymes (alkaline phosphatase, superoxide dismutase, catalase and lysozyme) and expression of immune‐related genes (interleukin‐17 and inhibitor of apoptosis protein) in mussels were analysed during 21 days exposure period. Results suggested that the diatom Navicula pelliculosa was suitable algae for H. cumingii, the optimal ration level and temperature of H. cumingii were the medium ration and 23℃. Semi‐starvation, excessive dietary, low‐ or high‐temperature stimulated immune activities, and when beyond the immune regulation range, most immune enzyme activities decreased. Acute stress also stimulated expression of HcIL‐17 and IAP, and long‐term stress of discomfort temperature and ration levels led to immune dysfunction and affected expression of these genes. The study has practical significance in realizing the sustainable aquaculture of H. cumingii and provides a broader perspective regarding the water ecosystem. [ABSTRACT FROM AUTHOR]

Color is an important trait of pearls which significantly affects their economic value. Golden seawater pearls with excellent luster are becoming popular among consumers, but golden freshwater pearls are rare. Hyriopsis cumingii is the most important freshwater mussel for pearl production in China and a selective breeding program has recently been developed to improve the yellowness and luster of the inner shell of H. cumingii for better production of golden freshwater pearls. As part of this breeding program, it has been necessary to evaluate the genetic parameters of the target traits and possible genotype-by-environment interactions. In this study, we investigated the effects of the environment on the genetic parameters of the inner shell color and luster of F1 golden strain H. cumingii, reared at two sites (Chongming and Wuyi). Inner shell color parameters and inner shell luster were recorded in 570 19-month-old individuals. Microsatellite-based DNA parentage analysis was used to assign the mussels to 19 maternal half-sib families, including 41 full-sib families. The results showed that the estimated heritability of the inner shell color parameters L*, a*, b* and dE were low, 0.13 ± 0.04, 0.14 ± 0.04, 0.12 ± 0.03 and 0.14 ± 0.05, respectively. The heritability of inner shell luster was medium (0.28 ± 0.07). The correlations between the genetic and phenotypic parameters for inner shell color and inner shell luster were insignificant at less than 0.8. Genetic correlations between the inner shell color parameters and inner shell luster in Chongming and Wuyi ranged from −0.07 ± 0.28 to −0.37 ± 0.24 and showed significant genotype-by-environment interaction. This study demonstrated that inner shell color parameters and inner shell luster could be improved by selective breeding. Environmental influences on genotype for inner shell color and inner shell luster should be taken into account in future selective breeding programs in golden strain H. cumingii.

Bone morphogenetic proteins (BMPs) play an important biological role in pearl biomineralization in pearl mussels. In this study, based on the genome data of the triangular sail mussel ( Hyriopsis cumingii ), the genome-wide identification and bioinformatic analysis of BMP gene family were performed, and the expression pattern of the BMP genes was investigated by the insertion experiments. The results showed that a total of 12 BMP gene family members ( BMP2a/2b , BMP3 , BMP5a/5b, BMP7a/7b/7c , BMP9 , BMP10a/10b , and BMP11 ) were identified, which were unevenly distributed on chromosome 3/14/18, encoding 169-583 amino acids, with molecular weights ranging from 19.32 to 65.99 kDa. BMP2a , BMP7b , and BMP10a were distributed, respectively, in the cytoplasm, endoplasmic reticulum and mitochondria, others were distributed in the nucleus. qRT-PCR results showed the significant tissue specificity in BMPs gene expression. The HcBMPs were differentially expressed in the mantle and visceral mass, and the expression level was higher in the visceral mass. The expressing trends of HcBMPs were not consistent between the mantle and visceral mass insertion, suggesting that HcBMPs may perform different functions. We also found that insertion surgery in the mantle and visceral mass significantly alters the expression profiling of the BMP gene family. Insertion of the mantle induced the biomineralization function of BMP2a , BMP7a , and BMP7b , while BMP3 and BMP10b played opposite roles in visceral mass insertion. Visceral mass insertion could suppress BMP9 expression at 5 d and BMP5b expression at 90 d after insertion This work lays the foundation and data support for the preliminary elucidation of regulatory role and mechanism of HcBMPs in the pearl-cultivating process of mantle and visceral mass., Competing Interests: SL was employed by Shanghai Mugao Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Liu, Yao, Sun, Chen, Yu, Xuan, Bian and Li.)

Wnt2 is a significant factor in the Wnt signaling pathway, which is associated with a variety of physiological activities, including inflammatory response, cell apoptosis, reproductive system development, and cell differentiation. Hyriopsis cumingii is the main pearl breeding mussel in China. However, the role of wnt2 in this species remains unclear. In this study, wnt2 from H. cumingii was cloned and identified. The full-length cDNA of wnt2 is 1524 bp, containing a 963 bp open reading frame (ORF), encoding 320 amino acid residues. The tissue distribution of H. cumingii indicated that wnt2 was predominantly highly expressed in the ovary and gill. And the expression profile after Aeromonas hydrophila or LPS injection indicated that wnt2 was up-regulated in gill, suggesting its role in the innate immune response. The expression of wnt2 was high at 4-month-old of early gonadal development and throughout ovarian development. In situ hybridization (ISH) showed significant hybridization signals on the gills and mature eggs of female gonads. In addition, miR-1988b-5p was found to negatively regulate wnt2 to affect the expression of key genes (frizzled-5 , ctnnb1 , and tcf7l) in the Wnt signaling pathway. Thus, these findings suggest a key role for wnt2 in immune regulation and gonadal development in H. cumingii. • wnt2 of Hyriopsis cumingii was identified and characterized. • The highest wnt2 expression was observed in ovary and gill. • wnt2 is up-regulated in gill following Aeromonas hydrophila /LPS exposure. • wnt2 is involved in immune regulation and gonadal development of H.cumingii. • MiR-1988b-5p affects Wnt signaling pathway genes expression by targeting wnt2. [ABSTRACT FROM AUTHOR]

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