Genome-wide association study reveals the underlying regulatory mechanisms of red blood traits in Anadara granosa.

BACKGROUND: Anadara granosa, commonly known as the blood clam, exhibits the unusual characteristic of having red blood among invertebrates. There is significant individual variation in blood color intensity among blood clams; individuals with vibrant red blood are deemed healthier and exhibit stronger stress resistance. However, the molecular basis underlying these red blood traits (RBTs) remains poorly understood. RESULTS: In this study, we performed genome-wide association studies (GWAS) in a population of 300 A. granosa individuals, focusing on RBTs as measured by hemoglobin concentration (HC), total hemocyte count (THC), and heme concentration (HEME). Our analysis identified 18 single nucleotide polymorphisms (SNPs) correlated with RBTs, subsequently selected 117 candidate genes within a 100 kb flanking region of these SNPs, potentially involved in the RBTs of A. granosa. Moreover, we discovered two haplotype blocks specifically associated with THC and HEME. Further analysis revealed eight genes (Septin7, Hox5, Cbfa2t3, Avpr1b, Hhex, Eif2ak3, Glrk, and Rpl35a) that significantly influence RBTs. Notably, a heterozygous A/T mutation in the 3'UTR of Cbfa2t3 was found to promote blood cell proliferation. These genes suggest that the hematopoietic function plays a significant role in the variability of RBTs in A. granosa. CONCLUSIONS: Our findings reveal a conservation of the regulatory mechanisms of RBTs between blood clams and vertebrates. The results not only provide a scientific basis for selective breeding in blood clams, but also offer deeper insights into the evolutionary mechanisms of RBTs in invertebrates.

Isolation and functional identification of immune cells in hemolymph of blood clams Tegillarca granosa.

Blood clam Tegillarca granosa is a type of economically cultivated bivalve mollusk with red blood, and it primarily relies on hemocytes in its hemolymph for immune defense. However, there are currently no reports on the isolation and identification of immune cells in T. granosa, which hinders our understanding of their immune defense. In this study, we employed single-cell transcriptome sequencing (scRNA-seq) to visualize the molecular profile of hemocytes in T. granosa. Based on differential expression of immune genes and hemoglobin genes, hemocytes can be molecularly classified into immune cells and erythrocytes. In addition, we separated immune cells using density gradient centrifugation and demonstrated their stronger phagocytic capacity compared to erythrocytes, as well as higher levels of ROS and NO. In summary, our experiments involved the isolation and functional identification of immune cells in hemolymph of T. granosa. This study will provide valuable insights into the innate immune system of red-blood mollusks and further deepen the immunological research of mollusks.

NO production of granulocytes associated with antibacterial immune response in Tegillarca granosa.

Nitric oxide (NO) is a signaling molecule and immune effector produced by the nitric oxide synthases (NOS), which involved to various physiological processes of animals. In marine bivalves, hemocytes play important roles in antimicrobial innate immune response. Although hemocyte-derived NO has been detected in several bivalves, the immune function of hemocyte-derived NO is not well understood. Here, we investigated the antibacterial response of hemocyte-derived NO in the blood clam Tegillarca granosa. Two types of hemocytes including erythrocytes and granulocytes were isolated by Percoll density gradient centrifugation, their NO production and TgNOS expression level were analyzed. The results showed that NO was mainly produced in granulocytes and almost no detected in erythrocytes. The granulocytes showed significantly higher NO level and TgNOS expression level than the erythrocytes. And the TgNOS expression level was significantly increased in granulocytes after Vibro parahemolyticus challenge. In addition, the NO donor sodium nitroprusside (SNP) significantly increased the NO production of hemocytes to kill pathogenic bacteria. In summary, the results revealed that granulocytes-derived NO play vital roles in the antimicrobial immune response of the blood clam.

Genome-wide identification and characteristic analysis of ETS gene family in blood clam Tegillarca granosa.

BACKGROUND: ETS transcription factors, known as the E26 transformation-specific factors, assume a critical role in the regulation of various vital biological processes in animals, including cell differentiation, the cell cycle, and cell apoptosis. However, their characterization in mollusks is currently lacking. RESULTS: The current study focused on a comprehensive analysis of the ETS genes in blood clam Tegillarca granosa and other mollusk genomes. Our phylogenetic analysis revealed the absence of the SPI and ETV subfamilies in mollusks compared to humans. Additionally, several ETS genes in mollusks were found to lack the PNT domain, potentially resulting in a diminished ability of ETS proteins to bind target genes. Interestingly, the bivalve ETS1 genes exhibited significantly high expression levels during the multicellular proliferation stage and in gill tissues. Furthermore, qRT-PCR results showed that Tg-ETS-14 (ETS1) is upregulated in the high total hemocyte counts (THC) population of T. granosa, suggesting it plays a significant role in stimulating hemocyte proliferation. CONCLUSION: Our study significantly contributes to the comprehension of the evolutionary aspects concerning the ETS gene family, while also providing valuable insights into its role in fostering hemocyte proliferation across mollusks.

Comprehensive analysis on the regulation of differentially expressed of mRNA and ncRNA in different ovarian stages of ark shell Scapharca broughtonii.

BACKGROUND: Ovarian development is an important prerequisite and basis for animal reproduction. In many vertebrates, it is regulated by multiple genes and influenced by sex steroid hormones and environmental factors. However, relative information is limited in shellfish. To explore the biological functions and molecular mechanisms of mRNA and non-coding RNA that regulate ovarian development in Scapharca broughtonii, we performed whole transcriptome sequencing analysis on ovaries at three developmental stages. Furthermore, the biological processes involved in the differential expression of mRNA and ncRNA were analyzed. RESULTS: A total of 11,342 mRNAs, 6897 lncRNAs, 135 circRNAs, and 275 miRNAs were differentially expressed. By mapping the differentially expressed RNAs from the three developmental stages of Venn diagram, multiple groups of shared mRNAs and lncRNAs were found to be associated with ovarian development, with some mRNA and ncRNA functions associated with steroid hormone. In addition, we constructed and visualized the lncRNA/circRNA-miRNA-mRNA network based on ceRNA targeting relationships. CONCLUSIONS: These findings may facilitate our further understanding the mRNA and ncRNAs roles in the regulation of shellfish reproduction.

Immune responses of hemocytes in the blood clam Tegillarca granosa in response to in vivo Vibrio harveyi infection.

Aquaculture of the blood clam Tegillarca granosa accounts for approximately 50% of Arcidae (ark shell) production in China. Vibrio infection severely threatens the sustainability of the clam aquaculture industry. Exposure to Vibrio induces an immune response in blood clams. However, the underlying mechanism remains poorly understood. In this study, immune responses of hemocytes in blood clams were detected after Vibrio infection; the immersion method was used in vivo to mimic the clam's natural infection process. After 24 h of exposure to Vibrio infection, the Vibrio load in hemolymph fluid in both the treatment Ⅰ (25,033.33 ± 19,563.11 CFU/mL) and treatment Ⅱ (122,163.33 ± 194,409.49 CFU/mL) groups were significantly higher, than that in the control group (13.67 ± 37.73 CFU/mL) (P < 0.05). Correspondingly, the production of intracellular reactive oxygen species was approximately 1.40 (treatment Ⅰ) and 2.12 (treatment Ⅱ) fold higher than that in the control group (P < 0.05), and the induced DNA damage showed a similar trend (P < 0.05). Vibrio infection also significantly increased lysozyme content, adenosine triphosphate content, and peroxidase isozyme activity, in both the serum and hemocyte lysates (P < 0.05). The expression of immune-associated genes (ABCA3, c-Myc, Caspase 3, and HSP70) was upregulated under infection conditions. The phagocytic activity was approximately 1.99 (treatment Ⅰ) and 2.57 (treatment Ⅱ) fold that in control clams (P < 0.05). In addition, the total hemocyte count and red granulocyte percentage both significantly decreased by approximately 75-90% after Vibrio infection. These results provided novel insights into the mechanism of hemocyte immunity in T. granosa against Vibrio infection, which may aid in the future prevention and control of Vibrio infection in vivo.

Two common nanoparticles exert immunostimulatory and protective effects in Tegillarca granosa against Vibrio parahaemolyticus.

There are many studies revealed that metal-based nanoparticles (NPs) possess excellent bactericidal effect on multitudinous bacteria and fungi. However, the control effect of NPs as antimicrobial agents to against Vibrio parahaemolyticus infection remain in poorly understood for blood clam, Tegillarca granosa. In order to evaluate the effect, the changes in six physiological parameters and the immune-related genes expression of clams exposed to V. parahaemolyticus alone or along with NPs (nZnO or nCuO) were investigated in present study. Results showed that both tested NPs exerted prominent redemptive or mitigative effect in an inverse dose-dependent way on physiological indexes of clam, especially in the total counts, phagocytosis and the cell viability of haemocytes, as well as the concentration and activity of lysozymes, when co-exposed with Vibrio. Gene expression analysis showed NPs at a concentration of 0.1 mg/L generally mitigated the downregulation of immune-related genes after clam exposure to V. parahaemolyticus. The combination of 0.1 mg/mL nZnO and nCuO additives has been shown to significantly enhance the humoral immunity of blood clam, suggesting its potential as a protective measure against V. parahaemolyticus infection in T. granosa.

A C-type lectin with a single carbohydrate-recognition domain (CRD) containing unique QPN/WDD motifs from Tegillarca granosa is involved in the innate immune defense.

C-type lectins (CTLs), a superfamily of Ca2+-dependent carbohydrate-recognition proteins, serve as pattern recognition receptors (PRRs) in the immune response of many species. However, little is currently known about the CTLs of the commercially and ecologically important bivalve species, blood clam (Tegillarca granosa). In this study, a CTL (designated as TgCTL-1) with a single carbohydrate-recognition domain (CRD) containing unique QPN/WDD motifs was identified in the blood clam through transcriptome and whole-genome searching. Multiple alignment and phylogenetic analysis strongly suggested that TgCTL-1 was a new member of the CTL superfamily. Expression analysis demonstrated that TgCTL-1 was highly expressed in the hemocytes and visceral mass of the clam under normal condition. In addition, the expression of TgCTL-1 was shown to be significantly up-regulated upon pathogen challenge. Moreover, the recombinant TgCTL-1 (rTgCTL-1) displayed agglutinating and binding activities against both the gram-positive and gram-negative bacteria tested in a Ca2+-dependent manner. Furthermore, it was found that the in vitro phagocytic activity of hemocytes was significantly enhanced by rTgCTL-1. In general, our results showed that TgCTL-1 was an inducible acute-phase secretory protein, playing crucial roles in recognizing, agglutinating, and binding to pathogenic bacteria as well as modulating phagocytic activity of hemocytes in the innate immune defense of blood clam.

First report of Perkinsus olseni infections in blood cockles Tegillarca granosa on the south coast of Korea.

The protozoan parasite Perkinsus olseni has become a focus of attention since it has been responsible for mass mortalities and economic losses in a wide range of bivalve hosts globally. The P. olseni host range along the south coast of Korea may extend beyond what was previously understood, and blood cockles in the Family Arcidae are also suggested to be potential hosts of P. olseni. In the present study, we applied histology and molecular techniques to identify Perkinsus sp. infections in the blood cockles Tegillarca granosa, which have been commercially exploited on the south coast of Korea for several decades. Histology and molecular techniques, including genus-specific immunofluorescence assay, species-specific fluorescence in situ hybridization, and phylogeny based on the ribosomal DNA internal transcribed spacer region revealed that T. granosa is infected by P. olseni, although the prevalence was low (0.5%). Histology revealed massive hemocyte infiltrations in the mantle, gill, and digestive gland connective tissues, indicating that the infection exerts negative impacts on the host cockles.

Absorption of iron from Tegillarca granosa using an in vitro simulated digestion and Caco-2/HepG2 co-culture system.

BACKGROUND: Iron-deficiency anemia is one severe micronutrient malnutrition and has captured worldwide attention. This study evaluated the in vitro iron absorption of two iron-binding proteins (hemoglobin and ferritin) from Tegillarca granosa. In addition, the protein structure-iron absorption relationship and the regulatory effect of hepcidin on cellular iron absorption were explored. RESULTS: Our findings revealed that both hemoglobin and ferritin extracted from T. granosa contained abundant iron-binding sites, as evidenced by stronger peaks in amide I and II regions compared with the two proteins from humans. Less ß-sheet (27.67%) structures were found in hemoglobin compared with ferritin (36.40%), probably contributing to its greater digestibility and more release of available iron. This was confirmed by the results of Caco-2/HepG2 cell culture system that showed iron absorption of hemoglobin was 26.10-39.31% higher than that of ferritin with an iron content of 50-150 µmol L-1 . This high iron absorption of hemoglobin (117.86-174.10 ng mg-1 ) could also be due to more hepcidin produced by HepG2 cells, thereby preventing ferroportin-mediated iron efflux from Caco-2 cells. In addition, the possible risk of oxidative stress was evaluated in cells post-iron exposure. In comparison with ferrous sulfate, a common iron supplement, Caco-2 cells treated with the iron-binding proteins had a 9.50-25.73% lower level of intracellular reactive oxygen species, indicating the safety of hemoglobin and ferritin. CONCLUSION: Collectively, the data of this research would be helpful for understanding the key features and potential of developing hemoglobin and ferritin from T. granosa as novel iron supplements. © 2022 Society of Chemical Industry.

A comparative analysis of mitochondrial ORFs provides new insights on expansion of mitochondrial genome size in Arcidae.

BACKGROUND: Arcidae, comprising about 260 species of ark shells, is an ecologically and economically important lineage of bivalve mollusks. Interestingly, mitochondrial genomes of several Arcidae species are 2-3 times larger than those of most bilaterians, and are among the largest bilaterian mitochondrial genomes reported to date. The large mitochondrial genome size is mainly due to expansion of unassigned regions (regions that are functionally unassigned). Previous work on unassigned regions of Arcidae mtDNA genomes has focused on nucleotide-level analyses to observe sequence characteristics, however the origin of expansion remains unclear. RESULTS: We assembled six new mitogenomes and sequenced six transcriptomes of Scapharca broughtonii to identify conserved functional ORFs that are transcribed in unassigned regions. Sixteen lineage-specific ORFs with different copy numbers were identified from seven Arcidae species, and 11 of 16 ORFs were expressed and likely biologically active. Unassigned regions of 32 Arcidae mitogenomes were compared to verify the presence of these novel mitochondrial ORFs and their distribution. Strikingly, multiple structural analyses and functional prediction suggested that these additional mtDNA-encoded proteins have potential functional significance. In addition, our results also revealed that the ORFs have a strong connection to the expansion of Arcidae mitochondrial genomes and their large-scale duplication play an important role in multiple expansion events. We discussed the possible origin of ORFs and hypothesized that these ORFs may originate from duplication of mitochondrial genes. CONCLUSIONS: The presence of lineage-specific mitochondrial ORFs with transcriptional activity and potential functional significance supports novel features for Arcidae mitochondrial genomes. Given our observation and analyses, these ORFs may be products of mitochondrial gene duplication. These findings shed light on the origin and function of novel mitochondrial genes in bivalves and provide new insights into evolution of mitochondrial genome size in metazoans.

Genomic Insights into the Origin and Evolution of Molluscan Red-Bloodedness in the Blood Clam Tegillarca granosa.

Blood clams differ from their molluscan kins by exhibiting a unique red-blood (RB) phenotype; however, the genetic basis and biochemical machinery subserving this evolutionary innovation remain unclear. As a fundamental step toward resolving this mystery, we presented the first chromosome-level genome and comprehensive transcriptomes of the blood clam Tegillarca granosa for an integrated genomic, evolutionary, and functional analyses of clam RB phenotype. We identified blood clam-specific and expanded gene families, as well as gene pathways that are of RB relevant. Clam-specific RB-related hemoglobins (Hbs) showed close phylogenetic relationships with myoglobins (Mbs) of blood clam and other molluscs without the RB phenotype, indicating that clam-specific Hbs were likely evolutionarily derived from the Mb lineage. Strikingly, similar to vertebrate Hbs, blood clam Hbs were present in a form of gene cluster. Despite the convergent evolution of Hb clusters in blood clam and vertebrates, their Hb clusters may have originated from a single ancestral Mb-like gene as evidenced by gene phylogeny and synteny analysis. A full suite of enzyme-encoding genes for heme synthesis was identified in blood clam, with prominent expression in hemolymph and resembling those in vertebrates, suggesting a convergence of both RB-related Hb and heme functions in vertebrates and blood clam. RNA interference experiments confirmed the functional roles of Hbs and key enzyme of heme synthesis in the maintenance of clam RB phenotype. The high-quality genome assembly and comprehensive transcriptomes presented herein serve new genomic resources for the super-diverse phylum Mollusca, and provide deep insights into the origin and evolution of invertebrate RB.

Effects of spawning stress on the immune capacity of blood cockle Tegillarca granosa occurring on the south coast of Korea.

Spawning in marine bivalves is a great energy-demanding process, and it often results in lethal and sublethal stresses during the post-spawning period, including depressed immune capacity. The blood cockle Tegillarca granosa (Linnaeus, 1758) distributes widely in silty-mud tidal flats on the south coast of Korea, and they spawn in late summer. To understand the impacts of spawning on immune parameters, we analyzed the total hemocyte count (THC), hemocyte mortality, phagocytosis capacity, and reactive oxygen species (ROS) production of T. granosa in pre-, and post-spawning condition using a flow cytometer. Histology indicated that the blood cockles occurring on the south coast of Korea ripe and ready to spawn in July, and they spawned in August and September. The THC in the blood cockle hemolymph declined from pre-spawning (1.2 × 108 cell mL-1) to post-spawning (0.9 × 108 cell mL-1), possibly due to the spawning stress and the massive infiltration of hemocytes in the gonad to phagocytose and resorb the residual gametes during the post-spawning period. The hemocyte mortality increased linearly from August (4.1%) to November (9.1%), as the histology revealed that the blood cockle completed spawning, and they resorbed the relict gametes. The granulocyte phagocytosis capacity declined dramatically from July (12.7%) to September (6.0%), when the cockles were engaged in active spawning. The flow cytometry revealed that the production of reactive oxygen species (ROS) from the granulocytes and the erythrocytes type II increased linearly from August (0.8-0.9 × 105 A U.) to December (2.1-2.8 × 105 A U.), which may cause stresses at a cellular level during this period. As the data indicated, spawning is a stressful activity inducing depressed immunological capacities in the blood cockles.

Genome-wide identification and immune response analysis of serine protease inhibitor genes in the blood clam Tegillarca granosa.

Serine protease inhibitors (SPIs) are the main regulators of serine protease activities. In this study, we present a genome-wide identification of SPI genes in T. granosa（TgSPI genes）and their expression characteristics in respond to Vibrio stress. A total of 102 TgSPI genes belonging to eight families, including Serpin, TIL (trypsin inhibitor like cysteine rich domain), Kunitz, Kazal, I84, Pacifastin, WAP (whey acidic protein) and A2M (Alpha-2-macroglobulin) were identified, while no genes belonging to Bowman-Birk, amfpi and Antistasin families were identified. The Kazal family has the most TgSPI genes with 38, and 11 TgSPI genes belong to the mollusc-specific I84 family. The TgSPI genes were found to be randomly distributed on 17 chromosomes with 12 tandem duplicate gene pairs. Expression profiles showed that most TgSPI genes were mainly expressed in immune-related tissues such as hepatopancreas, gill and mantle. In the hepatopancreas, most of TgSPI genes were sensitive to Vibrio stress, 28 and 29 TgSPI genes were up-regulated and down-regulated, respectively. Some up-regulated genes with signal peptides, such as the TgSPIs of I84 family, may act as a mechanism to directly prevent Vibrio from invasion. Six Kazal-type TgSPIs (TgSPI29, 45, 49, 50, 51 and 52) were intracellular proteins and their expression was down-regulated in hemocytes after Vibrio stress. This may have boosted protease activity in hemocytes to the point that more hemoglobin derived peptides were produced and secreted into the hemolymph to exert their anti-Vibrio effects. These findings may provide valuable information for further clarifying the roles of SPIs in the immune defense and will benefit future exploration of the immune function of SPIs in molluscs.

Genome-wide identification and characteristic analysis of PGRP gene family in Tegillarca granosa reveals distinct immune response of the invasive pathogen.

The peptidoglycan recognition proteins (PGRPs) are conserved innate immune molecular in invertebrates and vertebrates, which play important roles in immune system by recognize the peptidoglycans of bacterial cell walls. Although PGRPs have been extensively characterized in insects, a systematic analysis of PGRPs in bivalves is lacking. In the present study, the phylogenic relationships, gene structures and expression profiles of PGRPs in marine bivalves were analyzed. The results indicated that the most PGRPs of bivalves were predicted to degrade the peptidoglycans and prevent excessive immunostimulation of bacteria. In addition, the results of the present study showed that the protein diversity of PGRPs in most marine bivalves was mainly generated by the alternative splicing of genes, however the alternative splicing of PGRP gene family was absent in Tegillarca granosa. The differences of PGRPs might be related to the genetic and environmental differences of marine bivalves. Spatiotemporal expression profiling in T. granosa suggested that PGRPs play important roles in the immune response of invasive pathogens. The present study describes a comprehensive view of PGRPs in the blood clam T. granosa and provides a foundation for functional characterization of this gene family in innate immune of marine bivalves.

Prediction and characterization of a novel hemoglobin-derived mutant peptide (mTgHbP7) from Tegillarca granosa.

The hemoglobin (Hb) is identified in Tegillarca granosa and its derived peptides have been proved to possess antibacterial activity against gram-positive and gram-negative bacteria. In this study, we identified a series of novel antimicrobial peptides (AMPs) and artificially mutated AMPs derived from subunits of T. granosa Hbs, among which, a mutant T. granosa hemoglobin peptide (mTgHbP) mTgHbP7, was proved to possess predominant antibacterial activity against three bacteria strains (Vibrio alginolyticus, V. parahaemolyticus and Escherichia coli). Besides, mTgHbP7 was predicted to form α-helical structure, which was known to be an important feature of bactericidal AMPs. Furthermore, upon contact with HEK293 cell line, we confirmed that mTgHbP7 had no cytotoxicity to mammalian cell even at a high concentration of 160 µM. Therefore, the findings reported here provide a rationalization for antimicrobial peptide prediction and optimization from mollusk hemoglobin, which will be useful for future development of antimicrobial agents.

Characterization of a novel activating protein-1 (AP-1) gene and the association of its single nucleotide polymorphisms with vibrio resistance in Tegillarca granosa.

The blood clam Tegillarca granosa is a commercial marine bivalve of economic value, accounting for approximately 50% of clam production in China. In recent years, the yield of blood clams has been threatened by bacterial infections caused by marine Vibrio species that thrive under a rising sea temperature. The transcription factor activating protein-1 (AP-1) is emerging as an important player in the innate immunity of marine bivalves against viral or bacterial infections. In this study, the full-length cDNA of a novel T. granosa AP-1 (TgAP-1) was cloned for the first time. The 1591-bp cDNA encoded a protein of 292 amino acid residues with a calculated molecular weight of 32.8 kDa. The TgAP-1 protein contained an N-terminal Jun domain and a C-terminal basic region leucine zipper domain typically found in Jun proteins (a subfamily of AP-1 proteins). TgAP-1 was ubiquitously expressed in T. granosa, with the highest expression detected in the gill and foot, followed by the mantle, hemolymph, and hepatopancreas. Exposure to Vibrio harveyi induced TgAP-1 expression in gill tissues and the expression levels of TgAP-1 of resistant blood clams were always lower than that of control population whether Vibro infection or not. A total of 18 single nucleotide polymorphisms (SNPs) of TgAP-1 were detected in T. granosa. SNP-typing and haplotyping of resistant and susceptible populations revealed that six SNPs (AG type of TgSNP-1, GA type of TgSNP-2, TG type of TgSNP-4, CT type of TgSNP-7, AG type of TgSNP-11, and GA type of TgSNP-12) and four haplotypes (fHap2, fHap3, fHap6, and fHap7) were significantly associated with V. harveyi resistance. Risk assessment showed that fHap2 (CG) and fHap7 (GA) were associated with an increased resistance, while fHap3 (CT) and fHap6 (AG) were associated with an increased susceptibility. The results from this study supported a potential role of TgAp-1 in the anti-Vibro immunity of T. granosa. The discovery of the genetic molecular markers and haplotypes related to Vibrio resistance can provide guidance for selective breeding of T. granosa in the future.

Identification, characterization, and antimicrobial activity of a novel big defensin discovered in a commercial bivalve mollusc, Tegillarca granosa.

Molluscs, the second largest animal phylum on earth, primarily rely on cellular and humoral immune responses to fight against pathogen infection. Although antimicrobial peptides (AMPs) such as big defensin play crucial roles in the humoral immune response, it remains largely unknown in the ecological and economic important blood clam (Tegillarca granosa). In this study, a novel big defensin gene (TgBD) was identified in T. granosa through transcripts and whole genome searching. Bioinformatic analyses were conducted to explore the molecular characteristics of TgBD, and comparisons of TgBD with those reported in other molluscs were performed by multiple alignments and phylogenetic analysis. In addition, the expression patterns of TgBD in various tissues and upon bacterial challenge were investigated while the antimicrobial activity of synthetic N-terminal domain of TgBD was confirmed in vitro by radial diffusion experiment. Results obtained showed TgBD had an open reading frame (ORF) of 369 bp, encoding a prepropeptide containing a signal peptide and a propeptide. Similar to big defensins reported in other species, TgBD consists of a hydrophobic N-terminal domain containing ß1-α1-α2-ß2 folds and a cysteine-rich cationic C-terminal domain with three disulfide bonds between C1-C5, C2-C4, and C3-C6. Phylogenetic analysis showed that TgBD shared 76.80% similarity to its close relative ark shell (Scapharca broughtoni). In addition, TgBD expression was observed in all tissues investigated under normal conditions and was significantly induced by injection of Vibrio parahaemolyticus. Furthermore, synthetic N-terminal peptide of TgBD exhibited strong antimicrobial activity against Gram-positive bacteria tested. Our results indicated that TgBD is a constitutive and inducible acute phase AMP, which provides a universal and prompt protection for T. granosa.

A Novel Peptide Derived from Arca inflata Induces Apoptosis in Colorectal Cancer Cells through Mitochondria and the p38 MAPK Pathway.

Colorectal carcinoma (CRC) is one of the major causes of cancer-related incidence and deaths. Here, we identified a novel antitumor peptide, P6, with a molecular weight of 2794.8 Da from a marine Chinese medicine, Arca inflata Reeve. The full amino acid sequence and secondary structure of P6 were determined by tandem mass de novo sequencing and circular dichroism spectroscopy, respectively. P6 markedly inhibited cell proliferation and colony formation, and induced apoptosis in CRC cells. Mechanistically, transcriptomics analysis and a serial functional evaluation showed that P6 induced colon cancer cell apoptosis through the activation of the p38-MAPK signaling pathway. Moreover, it was demonstrated that P6 exhibited antitumor effects in a tumor xenograft model, and induced cell cycle arrest in CRC cells in a concentration-dependent mode. These findings provide the first line of indication that P6 could be a potential therapeutic agent for CRC treatment.

Proximate Composition, Antioxidant Properties, and Hepatoprotective Activity of Three Species of Shellfish of the Pacific Coast of Russia.

The objective of the present study was to investigate the proximate composition, antiradical properties and hepatoprotective activity of three species of shellfish, Corbicula japonica, Spisula sachalinensis, and Anadara broughtonii, from the coastal areas of Far East Russia. Biologically active peptides such as taurine (3.74 g/100 g protein) and ornithine (2.12 g/100 g protein) have been found in the tissues of A. broughtonii. C. japonica contains a high amount of ornithine (5.57 g/100 g protein) and taurine (0.85 g/100 g protein). The maximum DPPH and ABTS radical scavenging activity (36.0 µg ascorbic acid/g protein and 0.68 µmol/Trolox equiv/g protein, respectively) was determined for the tissue of C. japonica. The protein and peptide molecular weight distribution of the shellfish tissue water extracts was investigated using HPLC. It was found that the amount of low molecular weight proteins and peptides were significantly and positively correlated with radical scavenging activity (Pearson's correlation coefficient = 0.96), while the amount of high molecular weight proteins negatively correlated with radical scavenging activity (Pearson's correlation coefficient = -0.86). Hepatoprotective activity, measured by the survival rate of HepG2 hepatocytes after cotreatment with t-BHP, was detected for C. japonica. The highest protection (95.3 ± 2.4%) was achieved by the cold water extract of C. japonica at the concentration of 200 mg/mL. Moreover, oral administration of hot water extract of C. japonica to rats before the treatment with CCl4 exhibited a markedly protective effect by lowering serum levels of ALT and AST, inhibiting the changes in biochemical parameters of functional state of rat liver, including MDA, SOD, GSH and GST.