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

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

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.

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 2917 bp 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.

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

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

Correction: Li et al. Identification and Functional Analysis of the Cell Proliferation Regulator, Insulin-like Growth Factor 1 (IGF1) in Freshwater Pearl Mussel (Hyriopsis cumingii). Biology 2022, 11, 1369.

In the original publication [...].

Transcriptome analysis of growth and shell color between two genetic variants of Corbicula fluminea with different shell colors.

To clarify the molecular mechanism of the black and yellow shell coloration, we performed a transcriptome analysis of whole tissue of Corbicula fluminea in Hongze Lake (Jiangsu Province, China). After assembly, 335,247 unigenes were obtained, and 136,804 unigenes were functionally identified using public databases (NR, GO, KEGG, eggnog, and Swissprot). 1567 differentially expressed genes (DEGs) were detected through pairwise comparisons, of which 941 DEGs were up-regulated and 626 were down-regulated in the black-shelled clam. We compared the DEGs between two clams and identified some coloration-related genes. Notably, the black-shelled clam was larger than the yellow-shelled. We speculated that higher digestion and anabolic ability of black-shelled clam might lead to this phenomenon. In contrast, the yellow-shelled clam appeared to be more sensitive to environmental stress. The metabolic energy of the yellow-shelled clam was depleted to maintain or recover from stress, and provide less energy for growth. In summary, our finding provides a theoretical basis for the molecular mechanism of pigmentation and the difference of somatotype in bivalve, as well as promotes the future breeding of more elite varieties.

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

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

Analysis of protein kinase C (HcPKC) gene expression and single-nucleotide polymorphisms related to inner shell color traits in Hyriopsis cumingii.

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.

Identification and Functional Analysis of the Cell Proliferation Regulator, Insulin-like Growth Factor 1 (IGF1) in Freshwater Pearl Mussel (Hyriopsis cumingii).

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.

Analysis of the genetic diversity and population structure of Monochasma savatieri Franch. ex Maxim using novel EST-SSR markers.

BACKGROUND: Monochasma savatieri Franch. ex Maxim is a medicinally valuable herb. However, the collection and protection of the wild germplasm resources of M. savatieri are still insufficient, and their genetic diversity and population structure have been poorly studied. RESULTS: We collected and examined 46 M. savatieri individuals from Fujian, Hunan, Jiangxi, and Zhejiang provinces for genetic diversity and population structure, using 33 newly developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. Applying these markers, we detected a total of 208 alleles, with an average of 6.303 alleles per locus. The polymorphic information content varied from 0.138 to 0.884 (average: 0.668), indicating a high level of polymorphism. At the population level, there was a low degree of genetic diversity among populations (I = 0.535, He = 0.342), with Zhejiang individuals showing the highest genetic diversity among the four populations (Fst = 0.497), which indicated little gene flow within the M. savatieri populations (Nm = 0.253). Mantel test analysis revealed a significant positive correlation between geographical and genetic distance among populations (R2 = 0.3304, p < 0.05), and structure and principal coordinate analyses supported classification of populations into three clusters, which was consistent with the findings of cluster analysis. CONCLUSIONS: As a rare medicinal plants, the protection of M. savatieri does not look optimistic, and accordingly, protective efforts should be beefed up on the natural wild populations. This study provided novel tools and insights for designing effective collection and conservation strategies for M. savatieri.

A proteomic approach reveals biomineralization and immune response for mantle to pearl sac in the freshwater pearl mussel (Hyriopsis cumingii).

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-ß (TGF-ß) 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.

Gene identification and functional analysis of a D-type cyclin (CCND2) in freshwater pearl mussel (Hyriopsis cumingii).

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.

Identification of a coproporphyrinogen-III oxidase gene and its correlation with nacre color in Hyriopsis cumingii.

Pearl color is an important factor influencing pearl value, and is affected by the nacre color of the shell in Hyriopsis cumingii. Coproporphyrinogen-III oxidase (CPOX) is a key enzyme in porphyrin synthesis, and porphyrins are involved in color formation in different organisms, including in the nacre color of mussels. In this study, a CPOX gene (HcCPOX) was identified from H. cumingii, and its amino acid sequence was found to contain a coprogen-oxidase domain. HcCPOX mRNA was expressed widely in the tissues of white and purple mussels, and the highest expression was found in the gill, followed by the fringe mantle. The expression of HcCPOX in all tissues of purple mussels (except in the middle mantle) was higher than that of white mussels. Strong hybridization signals for HcCPOX were observed in the dorsal epithelial cells of the outer fold of the mantle. The activity of CPOX in the gill, fringe mantle, and foot of purple mussels was significantly higher than that in white mussels. Moreover, the expression of HcCPOX and CPOX activity were decreased in RNA interference experiments. The findings indicate that HcCPOX might contributes to nacre color formation in H. cumingii by being involved in porphyrin synthesis.

Cloning of a HcCreb gene and analysis of its effects on nacre color and melanin synthesis in Hyriopsis cumingii.

Creb (Cyclic AMP response element binding protein) is a nuclear regulatory factor that regulates transcription through autophosphorylation. In melanocytes, cAMP's corresponding elements bind to the Creb protein to autophosphorylation and activate MITF (Microphthalmia-associated transcription factor). MITF stimulates Tyrosine(tyr) to induce melanocytes to differentiate into eumelanin and pheomelanin. In this study, a HcCreb gene in Hyriopsis cumingii was cloned and its effects on melanin synthesis and nacre color were studied. HcCreb was expressed in both purple and white mussels, and there was a significant difference in expression between adductor muscle (p<0.01) and mantle tissue (p<0.05). Other tissues did not show significant differences (except for gill tissue), and in general, the level of mRNA expression was higher in purple mussels than in white mussels. In both white and purple mussels expression levels in gill tissue was the highest, followed by the mantle. Strong and specific mRNA signals were detected in the dorsal epithelial cells of the mantle pallial layer, indicating that HcCreb may be involved in nacre formation. After arbutin treatment, the expression of HcCreb decreased significantly. By further testing the changes in mantle melanin content it was found that the melanin content after arbutin treatment decreased significantly compared to the control group (p<0.05). It is speculated that the HcCreb gene plays a role in the process of melanin synthesis and nacre color formation in H. cumingii.

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

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

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

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

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

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

Integrated transcriptome analysis of immunological responses in the pearl sac of the triangle sail mussel (Hyriopsis cumingii) after mantle implantation.

For pearl culture of bivalve Hyriopsis cumingii, implantation of the sabio may cause nucleus discharge and increased host death rates. We performed a transcriptome analysis of the pearl sac of H. cumingii for 30 days after mantle implantation; 293863 unigenes were obtained, and 27176 unigenes were identified using nr, nt, KO, Swiss-Prot, Pfam, GO, and KOG databases. We detected 4878 differentially expressed genes (DEGs) through pairwise comparisons. We speculated that the physical condition of the recipient mussels returned to normal in about one month; the period was divided into six vital phases (0, 2 h-6 h, 12 h-24 h, 48 h to 7 days, 14 days and 30 days) on the basis of the overall similarities in DEGs. We compared the DEGs between time points and identified key immune-related genes. Our findings provide information on the immunological reactions induced by implantation in pearl mussels.

Integrative cytological analysis of the effects of Ca2+ and vitamin D3 on extracellular Ca2+ flux and intracellular Ca2+ reserves in the mantle of the pearl oyster (Hyriopsis cumingii Lea).

To examine Ca2+ absorption and transportation in the freshwater pearl oyster, Hyriopsis cumingii Lea, we studied the effects of different levels of either extracellular Ca2+ or 1,25(OH)2D3 on extracellular Ca2+ flux and intracellular Ca2+ concentrations in mantle cells using the non-invasive micro-test technique and laser scanning confocal microscopy. The inner and outer mantle (IM and OM) cells from mussels were cultured and then treated with different concentrations of Ca2+ and 1,25(OH)2D3. Extracellular Ca2+ flux and intracellular Ca2+ reserves were analyzed. The results showed that both extracellular Ca2+ and 1,25(OH)2D3 had significant effects on Ca2+ flux and reserves in mantle cells, especially in IM cells (P < .05). The increase in extracellular Ca2+ concentrations resulted in the conversion of extracellular Ca2+ flux into influx with an increase in flow rate (P < .05). The calcium ion fluorescence intensity of OM cells was higher than that of IM cells (P < .05). 1,25(OH)2D3 addition also significantly increased the influx rate of extracellular Ca2+, especially in IM cells, which were more sensitive to 1,25(OH)2D3 addition and had significantly higher Ca2+ influx rates than did OM cells (P < .05). Fluorescence intensities of intracellular Ca2+ first increased and then decreased with increasing 1,25(OH)2D3 levels. The study showed that IM cells play an important role in absorbing Ca2+ from the environment, while OM cells mainly function in the temporary storage and transportation of Ca2+ in the body. The current results suggested that high levels of extracellular Ca2+ (1.25 mM) or 1,25(OH)2D3 (over 100 IU/L) were favorable for Ca2+ uptake and maintenance in the body.

Identification of tumor necrosis factor receptor-associated factor 6 in the pearl mussel Hyriopsis cumingii and its involvement in innate immunity and pearl sac formation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.