Unveiling potential virulence determinants in Vibrio isolates from Anadara tuberculosa through whole genome analyses.

The genus Vibrio includes pathogenic bacteria able to cause disease in humans and aquatic organisms, leading to disease outbreaks and significant economic losses in the fishery industry. Despite much work on Vibrio in several marine organisms, no specific studies have been conducted on Anadara tuberculosa. This is a commercially important bivalve species, known as "piangua hembra," along Colombia's Pacific coast. Therefore, this study aimed to identify and characterize the genomes of Vibrio isolates obtained from A. tuberculosa. Bacterial isolates were obtained from 14 A. tuberculosa specimens collected from two locations along the Colombian Pacific coast, of which 17 strains were identified as Vibrio: V. parahaemolyticus (n = 12), V. alginolyticus (n = 3), V. fluvialis (n = 1), and V. natriegens (n = 1). Whole genome sequence of these isolates was done using Oxford Nanopore Technologies (ONT). The analysis revealed the presence of genes conferring resistance to ß-lactams, tetracyclines, chloramphenicol, and macrolides, indicating potential resistance to these antimicrobial agents. Genes associated with virulence were also found, suggesting the potential pathogenicity of these Vibrio isolates, as well as genes for Type III Secretion Systems (T3SS) and Type VI Secretion Systems (T6SS), which play crucial roles in delivering virulence factors and in interbacterial competition. This study represents the first genomic analysis of bacteria within A. tuberculosa, shedding light on Vibrio genetic factors and contributing to a comprehensive understanding of the pathogenic potential of these Vibrio isolates.IMPORTANCEThis study presents the first comprehensive report on the whole genome analysis of Vibrio isolates obtained from Anadara tuberculosa, a bivalve species of great significance for social and economic matters on the Pacific coast of Colombia. Research findings have significant implications for the field, as they provide crucial information on the genetic factors and possible pathogenicity of Vibrio isolates associated with A. tuberculosa. The identification of antimicrobial resistance genes and virulence factors within these isolates emphasizes the potential risks they pose to both human and animal health. Furthermore, the presence of genes associated with Type III and Type VI Secretion Systems suggests their critical role in virulence and interbacterial competition. Understanding the genetic factors that contribute to Vibrio bacterial virulence and survival strategies within their ecological niche is of utmost importance for the effective prevention and management of diseases in aquaculture practices.

Triclosan exposure induces immunotoxic impacts by disrupting the immunometabolism, detoxification, and cellular homeostasis in blood clam (Tegillarca granosa).

Omnipresent presence of triclosan (TCS) in aqueous environment puts a potential threat to organisms. However, it's poorly understood about its immunometabolic impacts of marine invertebrates. In present study, we use a representative bivalve blood clam (Tegillarca granosa) as a model, investigating the effects of TCS exposure at 20 and 200 µg/L for 28 days on immunometabolism, detoxification, and cellular homeostasis to explore feasible toxicity mechanisms. Results demonstrated that the clams exposed to TCS resulting in evident immunotoxic impacts on both cellular and humoral immune responses, through shifting metabolic pathways and substances, as well as suppressing the expressions of genes from the immune- and metabolism-related pathways. In addition, significant alterations in contents (or activity) of detoxification enzymes and the expression of key detoxification genes were detected in TCS-exposed clams. Moreover, exposure to TCS also disrupted cellular homeostasis of clams through increasing MDA contents and caspase activities, and promoting activation of the apoptosis-related genes. These findings suggested that TCS might induce immunotoxic impacts by disrupting the immunometabolism, detoxification, and cellular homeostasis.

The transcriptomic and biochemical responses of blood clams (Tegillarca granosa) to prolonged intermittent hypoxia.

The blood clam (Tegillarca granosa), a marine bivalve of ecological and economic significance, often encounters intermittent hypoxia in mudflats and aquatic environments. To study the response of blood clam foot to prolonged intermittent hypoxia, the clams were exposed to intermittent hypoxia conditions (0.5 mg/L dissolved oxygen, with a 12-h interval) for 31 days. Initially, transcriptomic analysis was performed, uncovering a total of 698 differentially expressed genes (DEGs), with 236 upregulated and 462 downregulated. These genes show enrichments in signaling pathways related to glucose metabolism, sugar synthesis and responses to oxidative stress. Furthermore, the activity of the enzyme glutathione peroxidase (GPx) and the levels of gpx1 mRNA showed gradual increases, reaching their peak on the 13th day of intermittent hypoxia exposure. This observation suggests an indirect protective role of GPx against oxidative stress. The results of this study make a significantly contribute to our broader comprehensive of the physiological, biochemical responses, and molecular reactions governing the organization of foot muscle tissue in marine bivalves exposed to prolonged intermittent hypoxic conditions.

The spatial distribution and physico-chemical characteristic of microplastics in the sediment and cockle (Anadara granosa) from the coastal waters of East Java, Indonesia, and the health hazards associated with cockle consumption.

This study evaluated microplastic (MP) abundances and physico-chemical characteristics in sediments and Anadara granosa along the East Java coast and their health implications. Fibers (74 %) dominated sediment MPs at south coast, while fragments (49-61 %) dominated north coast. Fiber (43-52 %) is the predominant MP in cockle tissues in all locations. Most MP in sediments (31-47 %) and cockle tissues (41-49 %) is black. The majority of microplastics (100-1500 µm) are found in sediment (73-90 %), and cockles (77-79 %). Very weak correlations found between the amount of MP and the length of the cockle shell. However, Spearman correlation shows that as the amount of MP in sediment increases, so does the amount of MP in cockle tissue. Each year, individuals of varying ages consume an average of 20,800 to 156,000 MP items. Cockles contain plasticizer components and microplastic polymers which are classified from II to V regarding of hazard levels, with V being the most hazardous.

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.

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.

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.

Organic enrichment reduces sediment bacterial and archaeal diversity, composition, and functional profile independent of bioturbator activity.

Eutrophication is a worldwide issue that can disrupt ecosystem processes in sediments. Studies have shown that macrofauna influences sediment processes by engineering environments that constrain microbial communities. Here, we explored the effect of different sizes of the Sydney cockle (Anadara trapezia), on bacterial and archaeal communities in natural and experimentally enriched sediments. A mesocosm experiment was conducted with two enrichment conditions (natural or enriched) and 5 cockle treatments (small, medium, large, mixed sizes and a control). This study was unable to detect A. trapezia effects on microbial communities irrespective of body size. However, a substantial decrease of bacterial richness, diversity, and structural and functional shifts, were seen with organic enrichment of sediments. Archaea were similarly changed although the magnitude of effect was less than for bacteria. Overall, we found evidence to suggest that A. trapezia had limited capacity to affect sediment microbial communities and mitigate the effects of organic enrichment.

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.

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.

Assessment of toxic metal pollution in Yueqing Bay and the extent of metal-induced oxidative stress in Tegillarca granosa raised in this water.

Yueqing Bay is an important economic shellfish culture zone in Zhejiang Province, China. However, increased pollution in the water caused by toxic metals has led to the bioaccumulation of toxic metals in cockles such as Tegillarca granosa, and the consequence of toxic metal-associated toxicity in these animals. This study aimed to assess the concentration of toxic metals in the water and sediment in four different sites (Baisha, Qingjiang, Nanyue, and Wengyang) within Yueqing Bay and to evaluate the extent of metal bioaccumulation in T. granosa raised in the aquaculture farms located within the four sites, as well as the changes in biomarkers in T. granosa in response to the metals. The assessment was carried out at two different times of the year, January and July. The water and sediment samples taken from the aquaculture farms in Baisha (S1), Qingjiang (S2) and Nanyue (S3) were found to have a comprehensive toxic metal pollution index (Pc) <1, indicating that these farms were not polluted. However, the water and sediment samples taken from the aquaculture farm in Wengyang (S4) had a Pc between 1 and 2, indicating mild toxic metal pollution. The edible risk assessments (HQ) of T. granosa in all four farms were <1, and therefore, these cockles could be considered safe for human consumption. The toxic metal enrichment in T. granosa exhibited a strong correlation with the toxic metal content in the sediment. In all four farms, CAT and SOD activity levels in the visceral mass of T. granosa were higher than those found in the foot, and a significantly higher level of CAT activity was detected in July compared with January. Similarly, MDA and H2O2 contents in the visceral mass were also higher in July than in January. Tegillarca granosa individuals taken from S4 and S3 farms exhibited significantly higher levels of metallothionein (MT) mRNA and MDA compared with individuals from S1 and S2 farms. Furthermore, the levels of MDA and MT mRNA showed significant positive correlations with Cd, Cr, Hg, and Cu. Elevation of lipid peroxidation in these cockles coincided with increasing levels of endogenous antioxidants. The visceral mass of T. granosa and its MDA level could be used as a tissue indicator and a biochemical marker, respectively, for detecting toxic metal pollution. MT mRNA might also be used as a molecular marker of toxic metal pollution. The integrated biomarker response version 2 (IBRv2) values of the four aquaculture farms in Yueqing Bay showed the order S4 > S3 > S2 > S1, indicating that S4 had the most serious metal-induced stress. Furthermore, the IBRv2 values correlated with the Nemerow composite index (Pc) for all the cockles examined. Thus, as far as the contamination of aquaculture farms in Yueqing Bay by toxic metals is concerned, the aquaculture farm in Wengyang (S4) was mildly contaminated by toxic metals. However, the contamination was relatively low, presenting a low risk for the local population of T. granosa.

Effects of hemoglobin extracted from Tegillarca granosa on the gut microbiota in iron deficiency anemia mice.

Iron deficiency anemia (IDA) is a serious threat to the health of humans around the world. Tegillarca granosa (T. granosa) is considered as an excellent source of iron due to its abundant iron-binding protein hemoglobin. This study aimed to investigate the effects of hemoglobin from T. granosa on the gut microbiota and iron bioavailability in IDA mice. Compared to normal mice, IDA mice showed reduced microbiota diversity and altered relative abundance (reduced Muribaculaceae and increased Bacteroides). After 4 weeks of administration, hemoglobin restored the dysbiosis of the intestinal microbiota induced by IDA and decreased the Firmicutes/Bacteroidota ratio and the abundance of Proteobacteria. Analysis of the hemoglobin regeneration efficiency of mice treated with hemoglobin confirmed that hemoglobin exhibited high iron bioavailability, particularly at low-dose administration, suggesting that a small amount of hemoglobin from T. granosa markedly elevated the blood hemoglobin level in mice. These findings suggested that IDA could be alleviated by administration of hemoglobin with excellent iron bioavailability, and its therapeutic mechanism may be partially attributed to the regulation of the intestinal microbiota composition and relative abundance. These results indicated that T. granosa hemoglobin may be a promising iron supplement to treat IDA and promote the utilization of aquatic-derived proteins.

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.

Identificaiton and characterization of a novel hemoglobin gene (Tgr-HbIII) from blood clam Tegillarca granosa.

Hemoglobin (Hb) is the major protein component of red blood cells (hemocytes) of the blood clam Tegillarca granosa. Three T. granosa hemoglobin genes have been mentioned in the literature, designated Tgr-HbI, Tgr-HbIIA and Tgr-HbIIB. Previously, our group identified another novel gene, Tgr-HbIII, in the Hb cluster of the chromosome-level genome but the issue of whether this Hb gene expresses functional protein remains unclear. In the current study, phylogenetic analysis revealed that Tgr-HbIII resembles an ancient Hb gene. Sequence alignment and three-dimensional structural modeling results showed that Tgr-HbIII does not bind heme due to the completely different structure at amino acid position 96-100 and replacement of the N100 residue in known Tgr-Hbs with Q100, what causes loss of a single hydrogen bond linking heme with the globin fold. Interface prediction data suggest that Tgr-HbIII forms a homodimer (ΔG = -5.6 kcal/mol) with a similar conformation to the Tgr-HbI homodimer (ΔG = -3.5 kcal/mol). In adult T. granosa, mRNA expression of Tgr-HbIII was lower than that of Tgr-HbIIA and Tgr-HbIIB (up to 100 × ), but comparable to that of Tgr-HbI. Notably, protein expression of Tgr-HbIII was extremely low. Single-cell RNA sequencing analysis of Hb expression showed that all adult hemocytes expressed Tgr-HbI, Tgr-HbIIA and Tgr-HbIIB, while only 43 % (3872 of 8978) expressed Tgr-HbIII. Based on the collective data, we speculate that Tgr-HbIII carried oxygen prior to mutation of N100 to Q100 and subsequently evolved into a known functional remnant of Hb with an adequate mRNA/low protein expression profile. The current study provides a foundation for further research on the origin, evolution and function of molluscan Hbs.

Assessment of mercury and lead contamination using the bivalve Anadara tuberculosa (Arcidae) in an estuary of the Colombian Pacific.

Buenaventura Bay is considered the main estuary on the Pacific coast of Colombia, because of port and industrial activities. Mining and agriculture are major sources of contamination in the estuary. This study used the bivalve Anadara tuberculosa to evaluate the presence of mercury and lead in the Dagua River estuary (Colombia), one of the main tributaries coming into the bay. Five samplings for sediments and six for tissues of A. tuberculosa were carried out between 2016 and 2017. Additionally, a series of bioassays were conducted to determine its ability to bioaccumulate metals. A mean mercury concentration of 0.57 ± 0.74 mg*kg-1 and lead concentration of 0.87 ± 0.68 mg*kg-1 in sediment were found. Bioassays allowed us to conclude that at low mercury exposure concentrations (0.25 mg*kg-1), lesions are generated in animal tissue, mainly in sexual cells. The high concentrations of mercury found in organisms exceed the maximum permitted concentration established in Colombia. In addition, the contamination factors for mercury and lead were considered very high. This research aims at contributing to the use of Anadara tuberculosa, as prospective bioindicator for pollution biomonitoring in mangrove coastal ecosystems.

Comparative metabarcoding analysis of phytoplankton community composition and diversity in aquaculture water and the stomach contents of Tegillarca granosa during months of growth.

Filter-feeder bivalves and phytoplankton are interdependent. Their interaction plays important role in estuarine and coastal ecosystem. The correlation between bivalve feeding and phytoplankton is highly species specificity and environment dependent. In the background of miniature and nondiatom trend of phytoplankton in coastal seawaters, how bivalve respond and how the response play roles in the phytoplankton community are poorly known. In the present study, by applying DNA metabarcoding approach based on plastid 23S rDNA, this question was addressed by comparing the phytoplankton composition in the seston and the stomach content of blood clam Tegillarca granosa sampled during the growth period from March to November 2020 in an experimental farm on tidal flat in Xiangshan Bay, East China Sea. The result showed that, a total of seven phyla, 55 genera and 73 species of phytoplankton were identified for all samples. Chlorophyta, Bacillariophyta, and Cyanobacteria were found to be three dominant phyla both in the stomach contents and seston. High diversity of pico-sized phytoplankton, which was easy overlooked by microscopy, was revealed both in seston and stomach contents. This result indicated that the clam was able to feed on the pico-sized algae. At the genus level, the most abundant genera were the pico-sized green alga Ostreococcus (6.12 %-67.88 %) in seston and Picochlorum (4.07 %-35.33 %) in the stomach contents. In addition, microalgae of high nutritional value showed trend of higher proportion in stomach contents than that in seston, especially in July and September when significant growth of T. granosa was observed during this period (the body size increased 155 %). Biodiversity of phytoplankton in the seston was totally higher than that in stomach content, however, the changes among the months showed respective trend. Especially in July when the biodiversity was the lowest in seston, that in the stomach content showed the highest. The results indicated that blood clam farming might influence the phytoplankton composition, including those of pico-sized level, although the particular species in seston were mainly correlating with the dominant environmental factors such as temperature, salinity, pH respectively. These results extend the understanding of roles that bivalve aquaculture may play in the changing of coastal phytoplankton community.

Characterization of a novel polysaccharide from Arca subcrenata and its immunoregulatory activities in vitro and in vivo.

Arca subcrenata is an economical edible shellfish. A novel water-soluble α-D-glucan (ASPG-1) with a molecular weight of 2.56 × 106 Da was purified and characterized from A. subcrenata. Its structure was characterized as a repeating unit consisting of α-D-Glcp, (1 → 6)-α-D-Glcp and (1 → 4,6)-α-D-Glcp. ASPG-1 exerted potent immunoregulatory activity by promoting the viability of splenic lymphocytes. Moreover, it enhanced pinocytic capacity, and promoted the secretion of NO and cytokines in RAW264.7 cells. The immunomodulatory mechanism of ASPG-1 involved the activation of the TLR4-MAPK/Akt-NF-κB signaling pathway. ASPG-1 inhibited tumor growth in 4T1 breast cancer mice and its combination with doxorubicin increased antitumor efficacy. The ASPG-1 combination with DOX-treated group (64.8%) showed an improved tumor inhibition rate compared to that of the DOX-treated group (53.3%). The antitumor mechanism of ASPG-1 may involve an enhancement of the immune response of mice to tumors. These results indicated that ASPG-1 could be developed as a potential adjuvant in tumor immunotherapy.

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.

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.