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.

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.

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.

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.

Anti-inflammatory action of ark shell (Scapharca subcrenata) protein hydrolysate in LPS-stimulated RAW264.7 murine macrophages.

Potential anti-inflammatory effects of ark shell (Scapharca subcrenata) protein hydrolysates were investigated. Ark shell protein hydrolysates were prepared using Alcalase® and pepsin and were designated ASAH and ASPH, respectively. The nitric oxide (NO) inhibitory activity of ASAH and ASPH was determined in lipopolysaccharides (LPS)-stimulated RAW264.7 murine macrophages, and the results showed that ASAH inhibited better NO inhibitory activity than ASPH. ASAH suppressed inflammatory mediator, a prostaglandin E2, secretion of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6), and production of reactive oxygen species (ROS) dose dependently. It inhibited the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and simulated heme oxygenase-1 (HO-1) protein expression. However, the pharmacological approach revealed that pretreatment with zinc protoporphyrin ІX (ZnPP), an inhibitor of HO-1, reversed the anti-inflammatory effect of ASAH. Moreover, ASAH upregulated phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK1/2, and p38 MAPK. To find out the role of MAPKs phosphorylation, MAPKs inhibitors were used, and the results showed that ASAH-mediated HO-1 protein expression and Nrf2 nuclear translocation were abolished. Taken all together, this study revealed that ASAH has a potential anti-inflammatory activity through regulation of the MAPK-dependent HO-1/Nrf2 pathway. PRACTICAL APPLICATIONS: Food-derived marine bioactive peptides, due to their pivotal role in biological activities, are gaining much attention recently. However, the anti-inflammatory activities of ark shell protein hydrolysates still remain to be investigated. This study investigated that ASAH shows potential anti-inflammatory activities through regulation of the MAPK-dependent HO-1/Nrf2 pathway in RAW264.7 murine macrophages. These findings indicated that ASAH may be used as a dietary supplement, functional food, and medicinal drug for the management of inflammation and inflammation-associated diseases.

Expression of ABCA3 transporter gene in Tegillarca granosa and its association with cadmium accumulation.

Exposure to cadmium (Cd), a heavy metal, can cause strong and toxic side effects. Cd can enter the body of organisms in several ways, leading to various pathological reactions in the body. Tegillarca granosa is a kind of bivalve shellfish favored by people in the coastal areas of China. Bivalve shellfish can easily absorb heavy metal pollutants from water bodies while filter feeding. T. granosa is considered a hyper-accumulator of Cd, and the TgABCA3 gene is highly expressed in individuals with a high content of Cd-exposed blood clam. However, it is unclear whether TgABCA3 is involved in Cd ion transport in blood clam and the molecular mechanism for the mechanism of the Cd-induced responses for maintaining cell homeostasis. In this study, the complete cDNA of the TgABCA3 gene was analyzed to provide insights into the roles of TgABCA3 in resistance against Cd in blood clam. The complete sequence of TgABCA3 showed high identity to that of TgABCA3 from other bivalves and contained some classical motifs of ATP-binding cassette transport proteins. TgABCA3 expression in different tissues was measured using real-time quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. The tissue-specific expression showed that TgABCA3 expression was highest in the gill tissue. The TgABCA3 expression in the gill tissue was silenced using the RNA interference technique. After TgABCA3 silencing, the TgABCA3 expression decreased, the Cd content increased, the oxygen consumption and ammonia excretion rates increased, and the ingestion rate decreased. These results showing that the extents of Cd accumulation and resulting toxic effects are related to expression levels and activity of TgABCA3 indicate that TgABCA3 has a protective function against Cd in the clam. This increase in Cd accumulation results in serious damage to the body, leading to the enhancement of its physiological metabolism. Therefore, the findings of the study demonstrated that TgABCA3 can participate in the transport of Cd ions in the blood clam through active transport and play a vital role in Cd detoxification.

Molecular cloning, characterization, and tissue distribution of c-Myc from blood clam Tegillarca granosa and its role in cadmium-induced stress response.

Cadmium (Cd) pollution threatens the cultivation of the blood clam Tegillarca granosa (T. granosa) in coastal regions of the East China Sea. The molecular mechanisms regulating Cd stress response and detoxification in blood clams are largely unclear. In the present study, the full-length T. granosa c-Myc (Tgc-Myc) cDNA was cloned for the first time. The 3063-bp cDNA consisted of a 129-bp 5' untranslated region (UTR), a 1746-bp 3' UTR, and a 1188-bp open reading frame encoding a predicted protein of 395 amino acid residues. The predicted protein had a calculated molecular weight of 44.9 kDa and an estimated isoelectric point of 6.82. The predicted protein contained an N-terminal transactivation domain and a C-terminal basic helix-loop-helix leucine zipper domain, which are conserved functional domains of c-Myc proteins. Tgc-Myc showed broad tissue distribution in blood clams, with the highest expression detected in the gill and hepatopancreas. Exposure to Cd, a major heavy metal pollutant in coastal regions of the East China Sea, induced Tgc-Myc expression in gill tissues. Tgc-Myc knockdown led to reduced expression of a variety of stress response/detoxification genes in blood clams cultivated in Cd-contaminated seawater. Tgc-Myc knockdown also led to decreased expression of IGF1R, a proto-oncogene that promotes cell proliferation. These findings indicated that Tgc-Myc regulates Cd-induced stress response and detoxification in blood clams. The upregulation of Tgc-Myc may serve as an approach to generate strains with an enhanced detoxification response and consequently a low heavy metal buildup.

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.

Modulatory function of calmodulin on phagocytosis and potential regulation mechanisms in the blood clam Tegillarca granosa.

Unlike vertebrate species, invertebrates lack antigen-antibody mediated immune response and mainly rely on haemocyte phagocytosis to fight against pathogen infection. Recently, studies conducted in model vertebrates demonstrated that the multifunctional protein calmodulin (CaM) plays an important role in regulating immune responses. However, the intrinsic relation between CaM and phagocytosis process remains poorly understood in invertebrate species such as bivalve mollusks. Therefore, in the present study, the immunomodulatory function of CaM on haemocyte phagocytosis was verified in the blood clam, Tegillarca granosa, using the CaM-specific inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Results obtained show that CaM inhibition significantly suppressed the phagocytic activity of haemocytes. In addition, CaM inhibition constrained intracellular Ca2+ elevation, hampered actin cytoskeleton assembly, suppressed calcineurin (CaN) activity, and disrupted NF-κB activation in haemocytes upon LPS induction. Furthermore, expression of seven selected genes from the actin cytoskeleton regulation- and immune-related pathways were significantly downregulated whereas those of CaM and CaN from the Ca2+-signaling pathway were significantly upregulated by in vitro incubation of haemocytes with W-7. For the first time, the present study demonstrated that CaM play an important role in phagocytosis modulation in bivalve species. In addition, the intracellular Ca2+ and downstream Ca2+-signaling-, actin cytoskeleton regulation-, and immune-related pathways offer candidate routes through which CaM modulates phagocytosis.

Microplastics in beach sediments and cockles (Anadara antiquata) along the Tanzanian coastline.

Little is known about the prevalence of microplastics (MPs) in East Africa. In the present study, sediments were sampled at 18 sites along the Tanzanian coast that exhibit different levels of anthropogenic activity and were extracted using floatation methodology. Cockles (Anadara antiquata) were collected only from eight sites and MPs were extracted following NaOH digestion. MPs were most abundant at Mtoni Kijichi Creek (MKC, 2972 ± 238 particles kg-1 dry sediment), an industrial port in Dar es Salaam, and significantly higher than all other sites where the abundance range was 15-214 particles kg-1 dry sediment (p < 0.05, one-way ANOVA). Fragments and fibers were found at all sites. Polypropylene and polyethylene were identified polymers. MPs were found in cockles from all sampled sites with both frequencies of occurrence and MPs per individual subject to site-specific variation. This study provides a baseline of MP data in a previously uninvestigated area.

Acetylcholine suppresses phagocytosis via binding to muscarinic- and nicotinic-acetylcholine receptors and subsequently interfering Ca2+- and NFκB-signaling pathways in blood clam.

Though immunomodulation via cholinergic neurotransmitter acetylcholine (ACh), an important part of neuroendocrine-immune (NEI) regulatory network, has been well established in vertebrate species, the mechanisms remain poorly understood in invertebrates. In the present study, the immunomodulatory effect of ACh on haemocyte phagocytosis was investigated in an invertebrate bivalve species, Tegillarca granosa. Data obtained showed that in vitro ACh incubation suppressed phagocytic activity of haemocytes along with a significant elevation in intracellular Ca2+. In addition, the expressions of genes from Ca2+ signaling pathway were significantly induced whereas those from NF-κB signaling pathway were significantly down-regulated by ACh incubation. Furthermore, these adverse impacts of ACh were significantly relieved by the blocking of muscarinic acetylcholine receptors (mAChRs) or nicotinic acetylcholine receptors (nAChRs) using corresponding antagonists. Our study suggests that ACh suppresses phagocytosis via binding to both mAChRs and nAChRs, which disrupts intracellular Ca2+ homeostasis and subsequently interferes with downstream Ca2+ and NF-κB signaling pathways.

De novo transcriptome analysis of stressed blood clam (Anadara broughtonii) and identification of genes associated with hemoglobin.

BACKGROUND: Blood clam (Anadara broughtonii) is a commercially important marine bivalve characterised by the red blood. Recently, the clams have been subjected to severe resource recession. Multiple environmental stressors are indispensable for the recession. OBJECTIVE: We aimed to investigate the transcriptome information of blood clam under environmental stressors. METHODS: Paired-end Illumina HiSeq™ 2500 sequencing technology was employed for cDNA library construction and Illumina sequencing. Several public databases were introduced for gene annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathways analyses. The Open Reading Frame of annotated hemoglobin (Hb) was predicted and validated by DNAMAN 6.0 and NCBI BLASTx, respectively. RESULTS: A total of 242,919 transcripts were generated, 116,264 unigenes were subsequently assembled with an average length of 747 base pairs, and 33,776 unigenes were successfully annotated. Gene Ontology (GO) categories indicated that the terms of cellular processes, metabolic processes, cell, cell part, binding, and catalytic activity were dominant. KEGG pathway analyses suggested ribosome, oxytocin, focal adhesion, Ras, and PI3K-Akt were the largest signaling pathway groups, and many presented pathways (Ras, Rap1, and MAPK, etc.) were related to apoptosis, immune and stress response. In addition, a total of 19,306 potential simple sequence repeats (SSRs) were detected in 15,852 sequences. Six hemoglobin-related genes with complete conserved domain sequences were identified and 3 of them were predicted as HbI, HbIIα, and HbIIß. CONCLUSION: This study provides transcriptome responses to multiple environmental stressors in blood clams and would provide interesting hints for further studies.

Single and joint oxidative stress of cadmium and phenanthrene on the Bivalve Anadara subcrenata.

Single and joint oxidative stress of cadmium (Cd) and phenanthrene (Phe) on Anadara subcrenata were investigated under laboratory conditions with biomarkers such as reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (TAOC). The results indicated that the ROS level in Anadara subcrenata exposed to Cd and Phe significantly (P < 0.05) increased during the early exposure stage, and the TAOC level and SOD activity were induced as the response of the antioxidant defense system, and this followed a damage, recovery or acclimation event. The MDA content was negatively correlated with SOD activity. As the time went on, the ROS level decreased gradually and was close to that in the control after 9 days; the TAOC level decreased accordingly, but the recovery rate of the SOD activity was slower than that of the TAOC level. High concentrations of Cd or Phe had a stronger oxidative stress effect on Anadara subcrenata. Phe had a stronger effect on the SOD activity of Anadara subcrenata. The joint effects of Cd and Phe on the levels of ROS and TAOC, SOD activity and MDA content in Anadara subcrenata are dependent on their concentration combination, which were called the double-dose dependent effects and time-dependent effects, respectively. The binary mixture treatments of 477.33 µg/L Cd and 48.08 µg/L Phe had the highest oxidative stress on Anadara subcrenata.

Morphologic, cytometric and functional characterisation of Anadara kagoshimensis hemocytes.

Circulating hemocytes of ark clam, Anadara kagoshimensis, were investigated using light microscopy and flow cytometry. Hemolymph contained 3 morphotype of cells, amebocytes, erythrocytes and intermediate type cells, which formed two distinct subpopulations on flow cytometric dot plots. Large cells (intermediate morphotype and erythrocytes) amounted 85.6 ± 2.8% total cells in hemolymph. Erythrocytes were hemoglobin-containing cells with up to 40 granules presented in the cytoplasm. All hemocyte types, observed in the ark clam hemolymph demonstrated equal capacity to spontaneous production of reactive oxygen species.

The transcriptomic responses of the ark shell, Anadara broughtonii, to sulfide and hypoxia exposure.

Sulfide and hypoxia threaten marine organisms in various ways. Anadara broughtonii, a commercial marine bivalve in China which has great potential exposure to sulfide and hypoxia, was selected to test the responses to these stresses. Digital gene expression profile (DGE) analysis was performed on the juveniles' gills after exposed to normal condition (CG group), hypoxia (LO group), and low/high concentration of sulfide (LS/HS group, administered in hypoxia), respectively, using RNA-seq technology. A total of over 30 million clean reads were filtered from each DGE library and over 90% of them were annotated successfully. In total, 774 significant differentially expressed genes (DEGs) were detected and assigned to Gene ontology (GO) classification and KEGG Pathway enrichment analysis. The results show that many of the upregulated DEGs are related to hemoglobin, immunology, and stress responding. In the stressed A. broughtonii, cytochrome P450 and phosphoenolpyruvate carboxykinase may stimulate the glycolysis process to reduce oxygen consumption; Aminoacyl-tRNA synthetases, heat shock protein and protein disulfide isomerase probably help to maintain the genome integrity; Baculoviral IAP repeat-containing protein 2/3, mitogen-activated protein kinase and tumor necrosis factor pathways were probably responsible for protein repair, proteolysis, apoptosis and immune responses to high concentration of sulfide. Combined challenges also induced alternative oxidase and sushi repeat-containing protein, which have indistinct but probably indispensable function in invertebrates. For the first time, comprehensive transcriptome information on A. broughtonii in response to sulfide and hypoxia were provided. Our research offers new insights into the molecular mechanism behind the resistance of shellfish to sulfide and hypoxia.

The health risk for seafood consumers under future ocean acidification (OA) scenarios: OA alters bioaccumulation of three pollutants in an edible bivalve species through affecting the in vivo metabolism.

The current knowledge about the effect of pCO2-driven ocean acidification on the bioaccumulation of pollutants in marine species is still scarce, as only limited types of pollutants have been investigated. Therefore, to obtain a better understanding of the effect of ocean acidification on the process of bioaccumulation and subsequent food safety, the accumulation of benzo[a]pyrene (B[a]P), chloramphenicol (CAP), and nitrofurazone (NFZ) in an edible bivalve species, Tegillarca granosa, under present and near-future ocean acidification scenarios was investigated in the present study. The health risks associated with consuming contaminated blood clams were also assessed using target hazard quotient (THQ), lifetime cancer risk (CR), or margin of exposure (MoE). To explain the alterations in bioaccumulation of these pollutants, the expressions of genes encoding corresponding key metabolic proteins were analyzed as well. The results obtained showed that ocean acidification exerted a significant effect on the accumulation of B[a]P, NFZ, and CAP in the clams. After four-week exposure to B[a]P, NFZ, or CAP contaminated seawater acidified with CO2 at pH 7.8 and 7.4, significantly greater amounts of B[a]P and lower amounts of NFZ and CAP were accumulated in the clams compared to that in the control. Although no non-carcinogenic risk of consuming B[a]P-contaminated blood clams was detected using the THQ values obtained, the CR values obtained indicated a high life-time risk in all groups. In addition, according to the MoE values obtained, the health risks in terms of consuming NFZ- and CAP-contaminated clams were significantly reduced under ocean acidification scenarios but still cannot be ignored, especially for children. The gene expression results showed that the ability of clams to eliminate B[a]P may be significantly constrained, whereas the ability to eliminate NFZ and CAP may be enhanced under ocean acidification scenarios, indicating that the changes in the accumulation of these pollutants may be due to the altered in vivo metabolism.

Allometric relationship in the bioaccumulation of radionuclides (134Cs &241Am) and delineation of contamination pathways (food and seawater) in bloody cockle Anadara senilis using radiotracer techniques.

The uptake and depuration kinetics of 134Cs and 241Am were investigated in the bloody cockle Anadara senilis exposed via seawater and food in controlled conditions, using animals of different weight groups in order to assess how their bioaccumulation is affected by allometry and, hence, the individual's age. This study is one of the few experiments investigating bioaccumulation capacities of radionuclides in a West-African bivalve. Results showed that allometric relationships were mainly dependent on the exposure pathway considered. Significant relationships with body weight of bloody cockles were found during the uptake from dissolved phase for both radionuclides; they followed inverse power functions: smaller cockles concentrated both radionuclides more than larger ones. In contrast, radionuclide absorption and assimilation efficiencies from water and food, respectively, did not show any significant relationship with weight: only slight variation was observed between small and large organisms for the retention of 241Am accumulated from food. A bioaccumulation model was used to assess the contribution of each pathway of exposure (food vs. water) in organisms grouped in small and large individuals. We found that, regardless of the size, 134Cs was mainly bioaccumulated through the dietary pathway. In the case of 241Am, the relative contribution of each pathway is weight-dependent: major contribution of dissolved pathway in smaller organisms and the major dietary contribution in larger organisms.

Transcriptome analysis of sex-related genes in the blood clam Tegillarca granosa.

BACKGROUND: Blood clams (Tegillarca granosa) are one of the most commercial shellfish in China and South Asia with wide distribution in Indo-Pacific tropical to temperate estuaries. However, recent data indicate a decline in the germplasm of this species. Furthermore, the molecular mechanisms underpinning reproductive regulation remain unclear and information regarding genetic diversity is limited. Understanding the reproductive biology of shellfish is important in interpreting their embryology development, reproduction and population structure. Transcriptome sequencing (RNA-seq) rapidly obtains genetic sequence information from almost all transcripts of a particular tissue and currently represents the most prevalent and effective method for constructing genetic expression profiles. RESULTS: Non-reference RNA-seq, an Illumina HiSeq2500 Solexa system, and de novo assembly were used to construct a gonadal expression profile of the blood clam. A total of 63.75 Gb of clean data, with at least 89.46% of Quality30 (Q30), were generated which was then combined into 214,440 transcripts and 125,673 unigenes with a mean length of 1,122.63 and 781.30 base pairs (bp). In total, 27,325 genes were annotated by comparison with public databases. Of these, 2,140 and 2,070 differentially expressed genes (DEGs) were obtained (T05 T08 vs T01 T02 T04, T06 T07 vs T01 T02 T04; in which T01-T04 and T05-T08 represent biological replicates of individual female and male clams, respectively) and classified into two groups according to the evaluation of biological replicates. Then 35 DEGs and 5 sex-related unigenes, in other similar species, were investigated using qRT-PCR, the results of which were confirmed to data arising from RNA-seq. Among the DEGs, sex-related genes were identified, including forkhead box L2 (Foxl2), sex determining region Y-box (Sox), beta-catenin (ß-catenin), chromobox homolog (CBX) and Sex-lethal (Sxl). In addition, 6,283 simple sequence repeats (SSRs) and 614,710 single nucleotide polymorphisms (SNPs) were identified from the RNA-seq results. CONCLUSIONS: This study provided the first complete gonadal transcriptome data for the blood clam and allowed us to search many aspects of gene sequence information, not limited to gender. This data will improve our understanding of the transcriptomics and reproductive biology of the blood clam. Furthermore, molecular markers such as SSRs and SNPs will be useful in the analysis of genetic evolution, bulked segregant analysis (BSA) and genome-wide association studies (GWAS). Our transcriptome data will therefore provide important genetic information for the breeding and conservation of germplasm.

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.

Hemoglobins Likely Function as Peroxidase in Blood Clam Tegillarca granosa Hemocytes.

Hemoglobins are a group of respiratory proteins principally functioning in transport of oxygen and carbon dioxide in red blood cells of all vertebrates and some invertebrates. The blood clam T. granosa is one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity of T. granosa hemoglobins (Tg-Hbs) was characterized and the associated mechanism of action was deciphered via structural comparison with other known peroxidases. We detected that purified Tg-Hbs catalyzed the oxidation of phenolic compounds in the presence of exogenous H2O2. Tg-Hbs peroxidase activity reached the maximum at pH 5 and 35°C and was inhibited by Fe2+, Cu2+, SDS, urea, and sodium azide. Tg-Hbs shared few similarities in amino acid sequence and overall structural characteristics with known peroxidases. However, the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (HRP) and myeloperoxidase (MPO). This research represented the first systemic characterization of hemoglobin as a peroxidase.