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.

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.

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.

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.

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.

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.

Exposure to 2,3,7,8-tetrachlorodibenzo-paradioxin (TCDD) hampers the host defense capability of a bivalve species, Tegillarca granosa.

Though increasing reports of deleterious impacts of dioxins and polychlorinated biphenyls (PCBs) on a variety of marine organisms have been described, their effects on the host defense capability of marine bivalve mollusks remain poorly understood. In the present study we used 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a representative of dioxins and PCBs to investigate its impacts on the host defense capability of the blood clam, Tegillarca granosa. After exposure of clams to a range (0, 0.01, 0.1, 1, and 10 µg/L) of TCDD for 96 h, the total count, cell type composition, and phagocytic rate of haemocytes were analyzed. In addition, alkaline phosphatase (ALP) activity, cell viability, and the extent of DNA damage of haemocytes were also investigated. Our results showed that exposure to relatively high TCDD concentrations led to significant reductions in the total count and phagocytic activity of haemocytes, which could be accounted by aggravated DNA damage and reduced cell viability. In addition, the percentage of red granulocyte was significantly decreased whereas that of basophil granulocyte was significantly increased upon high doses TCDD exposure (effective concentrations are 1 µg/L and 10 µg/L for red and basophil granulocytes, respectively). Moreover, clams exposed to TCDD had a significant higher activity of ALP, may also indicate an enhanced ability to eliminate pathogens through direct dephosphorylation process whereas a suppressed inflammatory response through indirect regulating of downstream molecular cascade reaction. These findings suggest that TCDD may hamper the host defense capability and therefore render bivalve mollusks more vulnerable to pathogen infections.

Exposure to Waterborne nTiO2 Reduces Fertilization Success and Increases Polyspermy in a Bivalve Mollusc: A Threat to Population Recruitment.

Fertilization success is crucial for the population recruitment of an organism. However, little is known about the threat of nanoparticles (NPs) to the fertilization of broadcast spawners. Therefore, the effects of nTiO2 on fertilization success, polyspermy rate, sperm velocity, gametic DNA damage, sperm-egg collision probability, gamete fusion, and oocyte ultrastructure were investigated in a broadcast spawning bivalve, Tegillarca granosa. The results obtained show that fertilization success significantly decreased, whereas polyspermy risk markedly increased upon nTiO2 exposure. In addition, nTiO2 exposure led to a significant reduction in sperm swimming velocity, which would subsequently constrain gamete collisions. In addition, nTiO2 exposure resulted in a significant decline in gamete fusion per collision along with aggravated DNA damage in gametes. Furthermore, ultrastructural analysis illustrated the attachment of nTiO2 to the oocyte surface, which subsequently resulted in microvillus disassociation and plasma membrane damage. In conclusion, the results obtained suggest a significant threat from NP pollution to the recruitment of broadcast spawning invertebrates.

Waterborne Cd2+ weakens the immune responses of blood clam through impacting Ca2+ signaling and Ca2+ related apoptosis pathways.

Exposure to heavy metals such as Cadmium (Cd) may exert detrimental impacts on the immune responses of marine bivalve species. However, the immunotoxicity of Cd on blood clams remains unknown to date. Furthermore, though Cd2+ is known to compete with calcium (Ca2+) ions for their binding sites in cells and inhibit Ca2+ influx, whether Cd2+ weakens the immune responses of marine bivalves through inducing intracellular Ca2+ disorders still remains unclear. Therefore, the immunotoxicity of Cd2+ at different waterborne Ca2+ concentrations on blood clam, Tegillarca granosa, were investigated in the present study. Results obtained demonstrated that the total number, phagocytic activity, and red granulocytes ratio of the haemocytes were all significantly reduced after 10 days exposure of individuals to 25 µg/L Cd2+. However, when the waterborne Ca2+ concentrations were elevated by 10% and 20% (approximately 370 and 410 mg/L, respectively), mitigation effects on the immune responses of individuals were detected. In addition, though the expressions of genes from the Ca2+ signaling and Ca2+-related apoptosis pathways were significantly altered by Cd2+ exposure, the expression patterns of these genes were similar to that of the control when the waterborne Ca2+ concentrations were elevated, suggesting a relieving effect of waterborne Ca2+ on Cd2+ induced toxicity to haemocytes. The results obtained in the present study revealed that waterborne Cd2+ may hamper the immune responses of T. granosa through influencing Ca2+ signaling and Ca2+-related apoptosis pathways, which can be partially mitigated by elevating the waterborne Ca2+ concentrations.

The synergic impacts of TiO2 nanoparticles and 17ß-estradiol (E2) on the immune responses, E2 accumulation, and expression of immune-related genes of the blood clam, Tegillarca granosa.

The extensive use of TiO2 nanoparticles (nTiO2) in industrial products has led to their release into the marine environment, thereby posing a potential risk to marine organisms. However, in addition to affecting marine organisms through its inherent properties, nTiO2 can also act as a vehicle for other toxic pollutants due to their strong adsorption ability through the "Trojan horse" effect. Due to their potential hazard, the endocrine disrupting chemicals (EDCs) such as 17ß-estradiol (E2), have been considered as one of the most serious anthropogenic threats to biodiversity and ecosystem health. However, there is still a lack of knowledge regarding the possible synergistic effects of nTiO2 and endocrine disrupting chemicals (EDCs) on marine organisms to date. Therefore, the combined effects of nTiO2 and 17ß-estradiol (E2) on the immune responses of the blood clam, Tegillarca granosa, were investigated in this study. After 10 days of treatment, the total number, phagocytic activity, red granulocytes ratio, and the phagocytosis of hemocytes were significantly reduced in almost all treatment groups. Furthermore, expressions of genes from NFκß and Toll-like receptor signaling pathways were significantly altered after exposure to nTiO2 and/or E2, indicating a reduced sensitivity to pathogen challenges. In addition, compared to exposure to E2 alone, co-exposure to E2 and nTiO2 led to a significant increase in the content of alkali-labile phosphate (ALP) in hemolymph, suggesting an enhanced E2 bioconcentration in the presence of nTiO2. In general, the present study demonstrated that nTiO2 enhanced the immunotoxicity of E2 to the blood clam, which may be due to the increased E2 uptake in the presence of nTiO2.

Laboratory simulation reveals significant impacts of ocean acidification on microbial community composition and host-pathogen interactions between the blood clam and Vibrio harveyi.

It has been suggested that climate change may promote the outbreaks of diseases in the sea through altering the host susceptibility, the pathogen virulence, and the host-pathogen interaction. However, the impacts of ocean acidification (OA) on the pathogen components of bacterial community and the host-pathogen interaction of marine bivalves are still poorly understood. Therefore, 16S rRNA high-throughput sequencing and host-pathogen interaction analysis between blood clam (Tegillarca granosa) and Vibrio harveyi were conducted in the present study to gain a better understanding of the ecological impacts of ocean acidification. The results obtained revealed a significant impact of ocean acidification on the composition of microbial community at laboratory scale. Notably, the abundance of Vibrio, a major group of pathogens to many marine organisms, was significantly increased under ocean acidification condition. In addition, the survival rate and haemolytic activity of V. harveyi were significantly higher in the presence of haemolymph of OA treated T. granosa, indicating a compromised immunity of the clam and enhanced virulence of V. harveyi under future ocean acidification scenarios. Conclusively, the results obtained in this study suggest that future ocean acidification may increase the risk of Vibrio pathogen infection for marine bivalve species, such as blood clams.

Immunotoxicity of nanoparticle nTiO2 to a commercial marine bivalve species, Tegillarca granosa.

The increasing production and extensive application of nanoparticles (NPs) inevitably leads to increased release of NPs into the marine environment and therefore poses a potential threat to marine organisms, especially the sessile benthic bivalves. However, the impacts of NPs on the immunity of commercial and ecological important bivalve species, Tegillarca granosa, still remain unknown to date. In addition, the molecular mechanism of the immunotoxicity of NPs still remains unclear in marine invertebrates. Therefore, the immunotoxicity of nTiO2 exposure to T. granosa at environmental realistic concentrations was investigated in the present study. Results obtained showed that the total number, phagocytic activity, and red granulocytes ratio of the haemocytes were significantly reduced after 30 days nTiO2 exposures at the concentrations of 10 and 100 µg/L. Furthermore, the expressions of genes encoding Pattern Recognition Receptors (PPRs) and downstream immune-related molecules were significantly down-regulated by nTiO2 exposures, indicating a reduced sensitivity to pathogen challenges. In conclusion, evident immunotoxicity of nTiO2 to T. granosa at environmental realistic concentrations was detected by the present study. In addition, the gene expression analysis suggests that the PRRs (both TLRs and RIG1 investigated) may be the molecules for NPs recognition in marine invertebrates.

Benzo[a]pyrene exposure under future ocean acidification scenarios weakens the immune responses of blood clam, Tegillarca granosa.

Persistent organic pollutants (POPs) are known to converge into the ocean and accumulate in the sediment, posing great threats to marine organisms such as the sessile bottom burrowing bivalves. However, the immune toxicity of POPs, such as B[a]P, under future ocean acidification scenarios remains poorly understood to date. Therefore, in the present study, the impacts of B[a]P exposure on the immune responses of a bivalve species, Tegillarca granosa, under present and future ocean acidification scenarios were investigated. Results obtained revealed an increased immune toxicity of B[a]P under future ocean acidification scenarios in terms of reduced THC, altered haemocyte composition, and hampered phagocytosis, which may attribute to the synergetic effects of B[a]P and ocean acidification. In addition, the gene expressions of pathogen pattern recognition receptors (TLR1, TLR2, TLR4, TLR6), pathway mediators (TRAF6, TAK1, TAB2, IKKα and Myd88), and effectors (NF-ĸB) of the important immune related pathways were significantly down-regulated upon exposure to B[a]P under future ocean acidification scenarios. Results of the present study suggested an increased immune toxicity of B[a]P under future ocean acidification scenarios, which will significantly hamper the immune responses of T. granosa and subsequently render individuals more susceptible to pathogens challenges.

The first description of the male Euophrys atrata and E. bulbus from southern China (Araneae: Salticidae).

The males of Euophrys atrata Song & Chai,1992 and Euophrys bulbus Bao & Peng, 2002 are described and illustrated for the first time from southern China.

Hypoxia-mediated immunotoxicity in the blood clam Tegillarca granosa.

In marine ecosystems, dissolved oxygen (DO) is essential for maintaining intracellular energy balance during aerobic metabolism. Bivalve mollusks are frequently exposed to hypoxia environments due to tides, temperature changes, and anthropogenic activities. The blood clam, Tegillarca granosa, mainly inhabits intertidal mudflats and is more susceptible to low oxygen events. In this study, we investigated the effect of hypoxia on immune responses in clams, and showed that hypoxia exposure reduced total hemocyte counts (THC), hemoglobin concentrations, and intracellular reactive oxygen species (ROS) levels. Also, phagocytic and cell activities of hemocyte were significantly inhibited. Furthermore, immune-related gene expression was also down-regulated. In conclusion, hypoxia greatly affected immune functions in blood clams, and our research provided the foundation for further mechanistic studies on hypoxia tolerance in clams.

Acute sulfide exposure induces hemocyte toxicity and microbiota dysbiosis in blood clam Tegillarca granosa.

Sulfide are widely accumulated in aquatic environments under anaerobic conditions, which cause health problems of aquatic animals, yet their toxic effects to benthic bivalves are not well understood. We investigated the effects of sulfide on innate immunity of the blood clam Tegillarca granosa. Immunity-related indicators and hemolymph microbiota were investigated in the clams exposed to sulfide (via 10, 100 and 1000 µmol/L of Na2S) over a 7-day period. The results showed that cellular immune responses in T. granosa were affected by exposure to high sulfide concentration (1000 µmol/L Na2S), as indicated by total counts of hemocytes (THC), cell viability, ROS levels and phagocytic activities, suggesting that sulfide stress induces T. granosa more vulnerable to pathogen challenges. In addition, the Na2S-induced stress also reshaped the hemolymph microbial community structure of T. granosa that some original genera decreased, such as Lactobacillus, Desulfovibrio and Akkermansia; some genera increased, such as Vibrio and Pseudoalteromonas in sulfide stress group. Sulfide exposure promoted the proliferation of opportunistic pathogen and reduced the diversity of microbial community in the hemolymph of T. granosa. In summary, sulfide stress had marked hemocytotoxicity, reduced immune-cell activity and increased bacterial infections in the blood clam.

Integrated RNA-seq and RNAi Analysis of the Roles of the Hsp70 and SP Genes in Red-Shell Meretrix meretrix Tolerance to the Pathogen Vibrio parahaemolyticus.

The "Wanlihong" Meretrix meretrix (WLH-M) clam is a new variety of this species that has a red shell and stronger Vibrio tolerance than ordinary M. meretrix (ORI-M). To investigate the molecular mechanisms responsible for the WLH-M strain's tolerance to Vibrio, we challenged clams with Vibrio parahaemolyticus and then assessed physiological indexes and conducted transcriptome analysis and RNA interference experiments. The mortality, tissue bacterial load, and hemocyte reactive oxygen species level of ORI-M were significantly higher than those of WLH-M, whereas the content and activity of lysozyme were significantly lower. Gene Ontology functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that immune and metabolic pathways were enriched in Vibrio-challenged clams. The expressions of the heat shock protein 70 (Hsp70) and serine protease (SP) genes, which are involved in antibacterial immunity, were significantly upregulated in WLH-M but not in ORI-M, while the expression of the kynurenine 3-monooxygenase gene, a proinflammatory factor, was significantly downregulated in WLH-M. RNA interference experiments confirmed that Hsp70 and SP downregulation could result in increased mortality of WLH-M. Therefore, we speculate that Hsp70 and SP may be involved in the antibacterial immunity of WLH-M in vivo. Our data provided a valuable resource for further studies of the antibacterial mechanism of WLH-M and provided a foundation for the breeding of pathogen-resistant strains.

Impacts of four commonly used nanoparticles on the metabolism of a marine bivalve species, Tegillarca granosa.

The rapid development of nanotechnology boosts the massive production and utilization of various nanoparticles (NPs). However, the NPs escaped into environments form emergent pollutants, which pose a potential threat to marine organisms and ecosystems. Due to their sessile filter-feeding lifestyle, marine bivalves live in pollution-prone coastal areas are more susceptible to land-sourced pollutants such as NPs. However, the impacts of many NPs on the metabolism, one of the most critical physiological processes of an organism, still remain largely unknown in bivalve species. To fill up this knowledge gap, in this study the impacts of four commonly used NPs (nZnO, nFe2O3, nCuO, and multi-walled carbon tube (MWCNT)) on the filtration rate, oxygen consumption rate, ammonia excretion rate, and O:N ratio were investigated in the blood clam, Tegillarca granosa. In addition, the expressions of ten key metabolism-related genes upon exposure to these NPs were also analyzed. The results demonstrated that exposure of blood clams to the NPs resulted in a reduction in the food intake (indicated by declined filtration rate), a shift in the metabolism substance, and disruptions in key metabolism-related molecular pathways (i.e., glycolysis and tricarboxylic acid cycle), which may render blood clam in energy shortage and thus pose significant threat to the health of this important bivalve species.

Chemical and electrochemical conversion of magnetic nanoparticles to Prussian blue for label-free and refreshment-enhanced electrochemical biosensing of enrofloxacin.

Magnetic biosensor takes advantage of rapid and facile magnetic separation/collection of targets, however, generally relies on additional signal labels to generate signal in a tedious and high-cost way. Here, we proposed a chemical and electrochemical conversion (C-ECC) method to develop a label-free electrochemical magnetic biosensor to detect antibiotics enrofloxacin (ENR). The C-ECC method integrates the chemical decomposition of magnetic beads (MBs) to release ironic ions and the simultaneous electrochemical deposition of Prussian blue (PB) analogs through the reaction of ironic ions and co-existing K4Fe(CN)6. Unlike conventional method that relies on the physical magnetic property of MBs, the C-ECC method fully exploited the chemical/electrochemical properties of MBs to produce electrochemically active PB to generate signal, thus endowing MBs with dual roles in both sample treatment and signal generation. The incorporation of chemical and electrochemical conversion produced more PB with higher electroactivity when compared with sole chemical or electrochemical conversion. Moreover, an interesting electrochemical refreshment (ER) was designed to remove insulative species on the electrode surface to improve electroactivity of electrode and benefit amperometric detection significantly. Under optimized conditions, the C-ECC-based biosensor presented limit of detection (LOD) of 4.17 pg mL-1 for ENR, which is lower than most analogs, as well as satisfactory specificity. The biosensor also performed well in fish and chicken meat samples, with LODs lower than maximum residue limits of national standards. The C-ECC method may create a new way to design magnetic sensors and contribute to rapid, facile and sensitive detection in agriculture/food, clinic diagnosis and environmental monitoring.