Hyriopsis Cumingii Research Articles

Thermal Oxidation Degradation Characteristics and Kinetic Analysis of Waste Shell Powders for Efficient Utilization and Environmental Protection

ABSTRACT The thermal oxidation degradation and kinetic behavior of four waste shell powders: Hyriopsis cumingii, abalone, scallop, and clam were investigated. The thermal oxidation degradation process was elucidated using thermogravimetric analysis, X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and the Coats-Redfern method. This process typically occurs in four stages, with the decomposition of CaCO₃ (Stage III) being pivotal. Notably, clam and scallop powders exhibited the highest CaO yields of approximately 55.1% and 55.0%, respectively. Although abalone powder had a lower yield of 50.2%, it demonstrated the lowest activation energy of 118.08 kJ/mol in Stage III, suggesting potential energy and cost savings during CaO conversion. High-temperature calcination altered the chemical composition and microstructure of the shell powders, resulting in rough and porous CaO surfaces conducive to heavy metal ion and pollutant adsorption. The research provides a theoretical framework and experimental validation for the sustainable utilization of calcined shell powders, fostering efficient shell resource management and environmental protection.

Cloning and Expression Analysis of TGF-β Type I Receptor Gene in Hyriopsis cumingii.

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.

Genome-wide association analysis reveals genetic architecture of growth and inner shell color traits in freshwater pearl mussel Hyriopsis cumingii (Lea 1852)

Triangle sail mussel (Hyriopsis cumingii, Lea 1852) is the most important freshwater pearl mussel in China. Growth traits of host mussels and inner shell color traits of donor mussels are critical determinants for pearl size and color. In this study, we performed GWAS analysis to explore the genetic architecture of growth and inner shell color traits. Using genotype by sequencing (GBS) technique, we obtained 80,562 SNPs from a total dataset of 463 mussels, based on these data, we found weak genetic relationship (−0.1–0.4) and rapid LD decay rate among the population. Moreover, GWAS analysis identified 12 growth-related significant SNPs and 29 inner shell color related significant SNPs. Five of the 12 SNPs linked to growth traits were validated to be credible SNPs in a new population. Cyclin, SNF, PMP22, CPED1, F-box and APC/C genes were found to be significantly associated with growth traits, which may influence the growth and development of H. cumingii by regulating cell mitosis, mantle epithelial tissue activity, and Ca2+ absorption and transport capacity. Notably, redness a* showed an obvious aggregation peak in chromosome 17, indicating that this region may be closely related to the inner shell color. Furthermore, three causal genes in chromosome 17, HSP78, MAS and PKS are inferred to be the key genes on color formation of purple inner shell. These results provide some new insights into the genetic basis and physiological regulation mechanism of growth and inner shell color traits in H. cumingii. However, detailed functional identification and regulation mechanism analysis merits more investigation.

In Vitro In Silico Screening Strategy and Mechanism of Novel Tyrosinase Inhibitory Peptides from Nacre of Hyriopsis cumingii.

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.

Identification and antibacterial activity of a novel phage-type lysozyme from the freshwater mussel Hyriopsis cumingii

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, Glu20 and Asp29, 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 Ni2+, 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.

Molecular characterization and transcriptional regulation between PPAR and CPT1 in freshwater bivalve Hyriopsis cumingii

Peroxisome proliferator activated receptors (PPARs) exert their roles in lipid metabolism and adaptive immunity by transactivating carnitine palmitoyltransferase 1 (CPT1). However, it remains unclear whether the PPAR-CPT1 signaling pathway exists in mollusks that only carry out innate immunity. This study cloned and characterized PPAR and CPT1 genes from Hyriopsis cumingii for the first time, designated as HcPPARs and HcCPT1s, respectively. The bioinformatics analysis revealed conservative molecular characteristics of these genes across species. Real-time quantitative PCR results indicated that higher expression levels of HcPPARs and HcCPT1s in the blood, mantle, and intestine suggested their potential involvement in lipid metabolism and innate immunity of mollusks. Treatments with agonists and inhibitors demonstrated a correlation in the expression of HcPPARs and HcCPT1s. Dual luciferase reporter assay identified regions with high transcriptional activities on promoters of HcCPT1s and potential binding sites for HcPPARs through prediction and mutation sites. These results suggested that the PPAR-CPT1 signaling might exist in H. cumingii. This research provides a necessary foundation for exploring the role of the PPAR-CPT1 signaling in innate immunity, and offers new theoretical evidence for the molecular regulatory mechanism of mollusks and the treatment of metabolic disorders and inflammatory diseases.

The potential compensatory mechanism between pmbA and tldD governing the virulence of Aeromonas veronii and its implications on the immune response in freshwater bivalve (Hyriopsis cumingii)

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 LD50 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.

Enhancing Bioactivity through the Transfer of the 2-(Hydroxymethoxy)Vinyl Moiety: Application in the Modification of Tyrosol and Hinokitiol.

Utilizing Density Functional Theory (DFT) calculations at the B3LYP/QZVP level and incorporating the Conductor-like Polarizable Continuum Model (C-PCM) for solvation, the thermodynamic and chemical activity properties of 21-(hydroxymethoxy)henicosadecaenal, identified in cultured freshwater pearls from the mollusk Hyriopsis cumingii, have been elucidated. The study demonstrates that this compound releases formaldehyde, a potent antimicrobial agent, through dehydrogenation and deprotonation processes in both hydrophilic and lipophilic environments. Moreover, this polyenal exhibits strong anti-reductant properties, effectively scavenging free radicals. These critical properties classify the pearl-derived ingredient as a natural multi-functional compound, serving as a coloring, antiradical, and antimicrobial agent. The 2-(hydroxymethoxy)vinyl (HMV) moiety responsible for the formaldehyde release can be transferred to other compounds, thereby enhancing their biological activity. For instance, tyrosol (4-(2-hydroxyethyl)phenol) can be modified by substituting the less active 2-hydroxyethyl group with the active HMV one, and hinokitiol (4-isopropylotropolone) can be functionalized by attaching this moiety to the tropolone ring. A new type of meso-carrier, structurally modeled on pearls, with active substances loaded both in the layers and the mineral part, has been proposed.

HcN57, A Novel Unusual Acidic Silk-Like Matrix Protein from Hyriopsis cumingii, Participates in Framework Construction and Nacre Nucleation During Nacreous Layer Formation.

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.

Efficient U(VI) removal by Ti3C2Tx nanosheets modified with sulfidated nano zero-valent iron: Batch experiments, mechanism, and biotoxicity assessment

The MXenes, a new class of two-dimensional layered materials, have found extensive applications in water treatment for its excellent thermal stability, electrical conductivity, and excellent adsorption ability. Sulfidized nano zero-valent iron (S-nZVI) is a good reducing agent, however, the practical application of S-nZVI is currently restricted due to the tendency of nano materials to agglomerate. Herein, MXenes use as a support and in situ loading S-nZVI on it to prepare a new material (S-nZVI/Ti3C2Tx), and applied it to U(VI) removal in water treatment. The microscopic characterization proves that S-nZVI on Ti3C2Tx has good dispersion and effectively alleviates agglomeration. Batch experiments shown that S-nZVI/Ti3C2Tx has a very good effect on U(VI) removal, and the maximum adsorption capacity reaches 674.4 mg/g under the aerobic condition at pH=6.0. The pseudo-second-order kinetic model and the Langmuir isotherm model were found to be more appropriate for describing the adsorption behavior. This indicates that the removal process is a single molecular layer chemisorption. Moreover, the S-nZVI/Ti3C2Tx maintained a removal efficiency of over 85 % for U(VI) even after being reused five times, demonstrating its excellent reusability. It is worth noting that the material can remove 79.8% of 50 mg/L of U(VI) in simulated seawater, indicating that S-nZVI/Ti3C2Tx possessed an excellent uranium extraction performance from seawater. Experimental results and XPS analysis showed that U(VI) was removed by adsorption, reduction and co-precipitation. Moreover, S-nZVI/Ti3C2Tx was a low toxicity material to hyriopsis cumingii. Therefore, S-nZVI/Ti3C2Tx was expected to be a candidate as adsorbent with great potential in removal of uranium from wastewater and seawater.

Genotype by environment interactions for growth and inner shell color disparity in Hyriopsis cumingii under different light conditions

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.

Zero-waste approach to fluoride removal by contact precipitation using Hyriopsis cumingii shells with value-added products

We propose a zero-waste approach using Hyriopsis cumingii shells for fluoride (F) removal via contact precipitation with value-added products. H3PO4 was used as the precipitant for F removal. The F concentration could be reduced from 9.5 mg/L to 0.09–0.91 mg/L for over 900 BV with running-backwashing cycle. The precipitates formed during contact precipitation were easily stripped from the shell matrix by periodic backwashing, thus supporting the continuous dissolution of calcium (Ca) from the shell particles. X-ray diffraction (XRD)-Rietveld analysis showed that the backwashing residue was fluorapatite (FAP) with a small amount of hydroxy substitution. Therefore, FAP can be used as a raw material in phosphate fertilizer manufacturing. Economic analysis and sustainability evaluation showed that the contact precipitation process with H. cumingii shells as the substrate not only reduces chemical costs but also generates value-added products; thus, this process is environmentally friendly compared to traditional methods of F removal. The proposed approach for F elimination, waste shell utilization, and F and Ca resource recovery in the contact precipitation process represents a novel sustainable water treatment technology.

The mechanism underlying increased myosin solubility in pearl mussels (Hyriopsis cumingii) by epigallocatechin gallate at low NaCl level

The poor water solubility of myosin hinders the full utilization of its functions and limits its application in the food industry. The aim of this study was to investigate the effect of epigallocatechin gallate (EGCG) on the solubility of myosin in pearl mussel (Hyriopsis cumingii) muscle at low NaCl level and the underlying action mechanism. The results showed that myosin solubility increased with the increase of EGCG concentration at 0.2 M NaCl level. The polyphenol binding equivalent and thermodynamic characterization analysis implied that EGCG was mainly bound to myosin via hydrogen bonding and van der Waals force to form an EGCG-myosin complex, and compared with 0.5 M NaCl, the binding affinity of EGCG and myosin decreased at low NaCl level. Structural analysis suggested that EGCG caused myosin endogenous fluorescence quenching, a wavelength redshift and a β-sheet content increase, exposing the aromatic amino acid residues inside myosin molecules to the surface in a polar environment. Scanning electron microscopy observation and particle size result indicated that the introduction of EGCG reduced the particle size of myosin, which was more distinct at 0.2 M NaCl level. Therefore, EGCG ordered and stabilized the myosin structure at low NaCl level by forming a complex with myosin, effectively improving the myosin solubility.

Ultrasonic treatment enhanced the binding capacity of volatile aldehydes and pearl mussel (Hyriopsis cumingii) muscle: Investigation of underlying mechanisms

This study was aim to investigate the influencing mechanism of ultrasonic treatment on the interaction between volatile aldehydes and myosin. The results showed that when the mass concentration ratio of myosin to heptanal/hexanal was 1:0.3, ultrasonic treatment could enhance the binding capacity of myosin to heptanal/hexanal, especially the binding of myosin to hexanal. The entropy and enthalpy values of their interaction were negative, indicating that the interaction was mainly driven by hydrogen bond and van der Waals force. After ultrasonic treatment, the fluorescence wavelength of myosin-heptanal/hexanal complex was redshifted, the α-helix content was increased, while its roughness values, particle size and the polydispersity index were decreased. These demonstrated that ultrasonic treatment was conducive to myosin binding to heptanal/hexanal, thereby restraining the release of volatile flavor compounds from myosin, which could provide new insights for the regulation of volatile flavor compounds.

Aeromonas veronii tolC modulates its virulence and the immune response of freshwater pearl mussels, Hyriopsis cumingii

Aeromonas veronii is an opportunistic pathogen that causes diseases in aquatic animals, but its key virulence factors remain unclear. We screened the gene tolC with significantly different expression levels in the two isolates, A. veronii GL2 (higher virulence) and A. veronii FO1 (lower virulence). Therefore, we constructed mutant strain ΔtolC and analyzed its immunological properties. ΔtolC exhibited the reduced ability of biofilms formation, inhibited envelope stress response mediated by several antibiotics except cefuroxime, implying the ability to evade host immunity might be restrained. Challenge tests showed that the LD50 of ΔtolC was 10.89-fold than that of GL2. Enzymatic activities of ΔtolC group were significantly lower and peak time was delayed to 12 h, as demonstrated by qRT-PCR results. Histopathological examination displayed that the degree of tissue damage in ΔtolC group was alleviated. The results show that tolC is an important virulence factor of A. veronii, which provides references for live-attenuated vaccine.

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.

The effects of different ratios of mixed feeding ofScenedesmus dimorphustoCyclotellasp. on the growth performance, pearl production ability, and biochemical components of the new shell tissue ofHyriopsis cumingii

AbstractIn order to screen out the feeding ratio of high‐quality algae strains inHyriopsis cumingii, this experiment investigated the effects of different ratio of mixed feeding of two algae strains (Scenedesmus dimorphusandCyclotellasp.) on the growth, bead production performance, trace elements, and amino acid composition of the newborn shell ofH. 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 ofH. cumingii(p &lt; 0.05); the best body weight gain rate and shell growth rate were obtained by feedingS. dimorphus(30 × 105cell/mL) andCyclopellasp.(8 × 105cell/mL); the pearl weight gain rate, amino acid ratio, Ca2+and Mn2+content were best when feedingCyclotellasp. (16 × 105cell/mL), but there was no significant difference compared withS. dimorphus(30 × 105cell/mL) andCyclopellasp.(8 × 105cell/mL). In summary, the optimal feeding amount for the intensive breeding process ofH. cumingiiisS. dimorphus(30 × 105cell/mL) andCyclopellasp. (8 × 105cell/mL).

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-β 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.

Study on the Role of Mitophagy Receptor PHB2 in Doubly Uniparental Inheritance of Hyriopsis cumingii.

In bivalves, the heterogeneity of mitochondrial DNA and its unique mode of transmission have been the focus of attention, which is called doubly uniparental inheritance (DUI). Prohibitin-2 (phb2) is a mitochondrial inner membrane protein that is a key mitophagy receptor for parental mitochondrial removal. Hyriopsis cumingii is a freshwater bivalve in China, the full-length cDNA of H. cumingii phb2 (named Hcphb2) is 2917bp and encodes a total of 300 amino acids, a highly conserved sequence. Hcphb2 was highly expressed in the ovary. In the gonadal tissues of 5- to 8-month-old female mussels, the expression level of Hcphb2 continued to significantly increase. After Hcphb2 siRNA interference in 6-month-old female mussels, the expression of M-COII, a marker gene on M-type mitochondria, showed a considerable increase (p < 0.05). In contrast, the expression of autophagosome formation and maturation-related genes, atg4b, atg5, atg12, and atg16l, in the ATG family genes was significantly decreased (p < 0.01). Subcellular localization showed that Hcphb2 appeared in spermatogonia, spermatocyte, spermatid, and sperm, and its location changes synchronize with the behavior of M-type mitochondria location changes in DUI species. And it was found that miR-184 negatively regulated Hcphb2. The above results suggest that the mitochondrial autophagy receptor gene Hcphb2 may be associated with the degradation of M-type mitochondria in the freshwater mussel. This process requires multiple genes to participate, of which Hcphb2 and autophagy genes are only some of those that may play a role.

Transcriptome and digital gene expression analysis reveal immune responses of mantle and visceral mass pearl culturing in Hyriopsis cumingii

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 &amp;lt; 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.