Anadara Broughtonii Research Articles

Long-read transcriptomics of Ostreid herpesvirus 1 uncovers a conserved expression strategy for the capsid maturation module and pinpoints a mechanism for evasion of the ADAR-based antiviral defence

Abstract Ostreid herpesvirus 1 (OsHV-1), a member of the family Malacoherpesviridae (order Herpesvirales), is a major pathogen of bivalves. However, the molecular details of the malacoherpesvirus infection cycle and its overall similarity to the replication of mammalian herpesviruses (family Orthoherpesviridae) remain obscure. Here, to gain insights into the OsHV-1 biology, we performed long read sequencing of infected blood clams, Anadara broughtonii, which yielded over one million OsHV-1 long reads. This data enabled the annotation of the viral genome with 78 gene units and 274 transcripts, of which 67 were polycistronic mRNAs, 35 ncRNAs and 20 natural antisense transcripts (NATs). Transcriptomics and proteomics data indicate preferential transcription and independent translation of the capsid scaffold protein as an OsHV-1 capsid maturation protease isoform. The conservation of this transcriptional architecture across Herpesvirales likely indicates its functional importance and ancient origin. Moreover, we traced RNA editing events using short read sequencing and supported the presence of inosine nucleotides in native OsHV-1 RNA, consistent with the activity of ADAR1. Our data suggests that, whereas RNA hyper-editing is concentrated in specific regions of the OsHV-1 genome, single nucleotide editing is more dispersed along the OsHV-1 transcripts. In conclusion, we reveal the existence of a conserved pan-Herpesvirales transcriptomic architecture of the capsid maturation module and uncover a transcription-based viral counter defence mechanism, which presumably facilitates the evasion of the host ADAR antiviral system.

Analysis of the Polyculture Model of the Bivalves Anadara broughtonii and Chlamys farreri in Suspension Cages in Shallow Seas

Analysis of the Polyculture Model of the Bivalves Anadara broughtonii and Chlamys farreri in Suspension Cages in Shallow Seas

Localization and Tissue Tropism of Ostreid Herpesvirus 1 in Blood Clam Anadara broughtonii.

OsHV-1 caused detrimental infections in a variety of bivalve species of major importance to aquaculture worldwide. Since 2012, there has been a notable increase in the frequency of mass mortality events of the blood clam associated with OsHV-1 infection. The pathological characteristics, tissue and cellular tropisms of OsHV-1 in A. broughtonii remain unknown. In this study, we sought to investigate the distribution of OsHV-1 in five different organs (mantle, hepatopancreas, gill, foot, and adductor muscle) of A. broughtonii by quantitative PCR, histopathology and in situ hybridization (ISH), to obtain insight into the progression of the viral infection. Our results indicated a continuous increase in viral loads with the progression of OsHV-1 infection, reaching a peak at 48 h or 72 h post-infection according to different tissues. Tissue damage and necrosis, as well as colocalized OsHV-1 ISH signals, were observed primarily in the connective tissues of various organs and gills. Additionally, minor tissue damage accompanied by relatively weak ISH signals was detected in the foot and adductor muscle, which were filled with muscle tissue. The predominant cell types labeled by ISH signals were infiltrated hemocytes, fibroblastic-like cells, and flat cells in the gill filaments. These results collectively illustrated the progressive alterations in pathological confusion and OsHV-1 distribution in A. broughtonii, which represent most of the possible responses of cells and tissues to the virus.

Detection of Cyclic Imines (CIs) Toxins in Whole Body of Shellfishes: First Monitoring Report of CIs in South Korea Shellfishes

Cyclic imines (CIs) produced by microalgal species that accumulate in the food chains of marine organisms are novel biotoxins that do not belong to the classic group of marine biotoxins. In the past, CIs were found only in limited areas; however, in recent years, rapid changes in marine ecosystems have led to widespread CIs and increased exposure to toxic risks. In this study, we analyzed seven CI toxins, GYM-A, SPX (13-desmethyl spirolide C, 13, 19-dideMe spirolide C, 20-methyl spirolide G), and PnTX-E, F, and G, using LC/MRM-MS. Shellfish samples were purchased from a domestic Korean fish market (67 samples in 2021 and 216 samples in 2022). The entire body of the shellfish was ground and extracted with 50% methanol, followed by lipophilic-specific SPE. Only GYM-A, PnTX-G, and 13-desmethyl spirolide C were detected in all analyzed samples. The maximum concentrations of GYM-A is maximum 179 ppt (ng/kg) in Crassostrea nippona (March 2022), PnTX-G is maximum 7 ppt in Anadara broughtonii (April 2022), 13-desmethyl SPX C is maximum 58 ppt in Crassostrea nippona (April 2022). The southern coast exhibited the highest frequency of detection of these toxins, which was attributed to elevated sea-surface temperatures, aligned with conducive conditions for toxin-producing phytoplankton. According to the monitoring results, there were no significant CI toxins in the shellfish; however, it is important to monitor CI toxin accumulation in shellfish because of their high risk of toxicity.

Rapid on-site authentication of three cockle species using real-time loop-mediated isothermal amplification (LAMP) assay

Cockles, crucial for commercial fishing and aquaculture, emerge as highly marketable shellfish. However, the morphological similarities among various cockle species are frequently exploited for shellfish fraud. In this study, we developed LAMP assays (end-point LAMP and real-time LAMP) to detect three commercially important cockle species: Tegillarca granosa, Anadara broughtonii, and Anadara kagoshimensis. The primers were designed based on mitochondrial DNA and used to develop LAMP assays. Exclusivity was assessed using 17 shellfish species, and no cross-reactivity was confirmed. The estimate of the detection limit was determined to be 0.0001 ng. The established assays were further refined to create an on-site real-time LAMP assay by combining fast DNA isolation with a real-time fluorometer for field application. The effectiveness of the on-site real-time LAMP assay was successfully verified using 38 commercial samples, with analysis accuracy confirmed using a DNA extraction kit. Consequently, the on-site real-time LAMP assay can complete the whole procedure, including DNA extraction, real-time LAMP reaction, and data analysis within a 45 min timeframe, maintaining the accuracy of detecting three cockle species from processed cockle samples in the field. Our method proves valuable for detecting shellfish fraud by identifying cockle species using samples of both raw and processed products in the field.

Deep learning-based phenotype classification of three ark shells: Anadara kagoshimensis, Tegillarca granosa, and Anadara broughtonii

The rapid and accurate classification of aquatic products is crucial for ensuring food safety, production efficiency, and economic benefits. However, traditional manual methods for classifying ark shell species based on phenotype are time-consuming and inefficient, especially during peak seasons when the demand is high and labor is scarce. This study aimed to develop a deep learning model for the automated identification and classification of commercially important three ark shells (Tegillarca granosa, Anadara broughtonii, and Anadara kagoshimensis) from images. The ark shells were collected and identified using a polymerase chain reaction method developed in a previous study, and a total of 1,400 images were categorized into three species. Three convolutional neural network (CNN) models, Visual Geometry Group Network (VGGnet), Inception-Residual Network (ResNet), and SqueezeNet, were then applied to two different classification sets, Set-1 (four bivalve species) and Set-2 (three ark shell species). Our results showed that SqueezeNet demonstrated the highest accuracy during the training phase for both classification sets, whereas Inception-ResNet exhibited superior accuracy during the validation phase. Similar results were obtained after developing a third classification set (Set-3) to classify six categories by combining Set-1 and Set-2. Overall, the developed CNN-based classification model exhibited a performance comparable or superior to that presented in previous studies and can provide a theoretical basis for bivalve classification, thereby contributing to improved food safety, production efficiency, and economic benefits in the aquatic products industry.

Temporal variation in the concentrations and profiles of paralytic shellfish toxins and tetrodotoxin in scallop (Mizuhopecten yessoensis) and bloody clam (Anadara broughtonii) collected from the coast of Miyagi Prefecture, Japan

For food safety, the concentrations and profiles of paralytic shellfish toxins (PSTs) and tetrodotoxin were examined in economically important scallops and bloody clams collected from the coast of the Miyagi Prefecture, Japan. PSTs were the major toxins in both species. The tetrodotoxin concentration in scallops increased in summer, although the highest value (18.7 μg/kg) was lower than the European Food Safety Authority guideline threshold (44 μg/kg). This confirmed the safety for tetrodotoxin in this area.

Understanding the concept of diversity, abundance, and distribution of marine invertebrates through practicum students of the Biology Education, Pattimura University

The application of marine biology courses cannot be separated from field practicum activities which are integrated with the main scientific pattern of Pattimura University, namely build nobleh marine, meaning that the marine biology lecture process is a goal of developing human resources. One of the competencies of marine biology courses which emphasizes is that students must understand the life of various types of marine invertebrates that live in each coastal water ecosystem and must also master how to observe and research various types of marine invertebrate life found in each zone of coast (intertidal zone and subtidal). The results of the findings of various types of marine invertebrates were carried out by diversity analysis, the results were 2.0 <H'=2.363836 ≤ 3.0, meaning the level of diversity was medium. The relative density of invertebrates on various substrates includes sand 25%, density on sandy gravel substrates 9.9%, muddy substrates 11.4%, coral substrates 15%. The distribution patterns of various types of invertebrates can also be explained according to the results of analyzes including (1) clustered distribution patterns occur in Crustacea (Emita sp, Harpiosquilla raphidae, Ocypoda cursor), Gastropoda (Nerita polita, Conus sponalis), Bivalvia (Venerupis corrugate), (2) The distribution pattern is uniformly occurs in invertebrates of Bivalvia (Anadara broughtonii, Mytilus trossulus, Tridacna rosewateri, Pinctada radiate), (Diadema setosum, Holothuria scabra), Gasropoda (Strombus decorus). Proving the truth of the concept or theory of various types of invertebrates in biology courses the truth of the sea has been tested through field practicums in the coastal waters of Salahutu District, Ambon Island

Genome-wide SNP-based diversity analysis and phylogeographic inference in the ark shell (Anadara broughtonii)

The ark shell (Anadara broughtonii) is a marine bivalve inhabiting the West Pacific coasts. Its natural resources have declined considerably in recent years due to overexploitation. Understanding of the genetic diversity and population structure of A. broughtonii is important for conservation and species-specific management. The Specific-Locus Amplified Fragment Sequencing (SLAF-seq) method was used to screen the SNPs for exploring the genetic diversity and phylogeographic characterization in 40 Anadara broughtonii individuals, 10 each from 4 sample locations: China, Japan, Korea, and Russia. We generated 394,768 SLAFs and developed 452,144 polymorphic SNPs. The observed and expected heterozygosity (Ho and He) ranged from 0.1805 to 0.3216 and from 0.1504 to 0.2156, respectively. Relatively strong differentiation was detected between populations from China/Russia and those from Japan/Korea, possibly related to the barrier effects from the Shandong Peninsula and the Korean Peninsula. Phylogenetic and Discriminant analysis of principal components (DAPC) supported the 40 individuals were mainly classified into four genetically distinct groups according to their sampling locations. Overall, Japanese and Korean populations owned higher genetic diversity. However, the two native populations may probably be affected by human activities such as artificial culture and commercial transportation. In order to avoid the further admixture of the native population of A. broughtonii, more attention should be paid to the germplasm resources protection of this species. Our results demonstrated the capability and high resolution of SNPs derived from SLAF-seq to measure the population genetic diversity and phylogeographic structure of A. broughtonii.

Morphological Analysis of the Hemolymph Cell Composition in the Bivalve Mollusk <i>Anadara</i> <i>broughtonii</i> Schrenck, 1867 (Sea of Japan)

The hemolymph cells of the ark clam Anadara broughtonii were examined using light microscopy, flow cytometry and gradient centrifugation. All three methods of analysis made it possible to identify two main types of cells in the hemolymph of the ark clam Anadara broughtonii – large granular erythrocytes and small agra-nular amebocytes. Erythrocytes accounted for 95.6 ± 0.9% of the total number of hemolymph cells. Erythrocytes were hemoglobin-containing cells with a great number of granules in the cytoplasm, a low nuclear-cytoplasmic ratio (NCR) and a lower intensity of cellular respiration compared to amebocytes. Amebocytes are cells of predominantly irregular shape with a high number of cells that do not contain or contain no more than 10 granular inclusions in the cytoplasm. All types of hemocytes found in the ark clam hemolymph demonstrated the same ability to spontaneously produce reactive oxygen species. For the first time, it has been shown that red blood cells of the ark clam Anadara broughtonii are capable of phagocytosis. At the same time, amebocytes absorbed on the average two times more zymosan particles (10.3 ± 0.7 pcs.), compared to erythrocytes (5.3 ± 0.1 pcs.).

Antioxidant and cellular immune response to acute hypoxia stress in the ark shell (Anadara broughtonii)

In this work we studied antioxidant status and cellular immunity of ark shells (A. broughtonii) to determine the range of the adaptive potential to hypoxia. For this, the effects of 24 h and 72 h periods of exposure to dissolved oxygen concentration 1.5–1.9 mg O2 L−1 were assessed. Hypoxia was not associated with changes in hemocyte morphology as well as with shifts in hemolymph cellular composition. Intracellular reactive oxygen species (ROS) production by hemocytes decreased following the 24 h exposure period but recovered after 72 h of hypoxia. Hypoxia contributed more to reduced phagocytosis and mitochondrial membrane potential of hemocytes. In gills, the activity of catalase and superoxide dismutase was suppressed upon exposure to hypoxia but the expression levels of the appropriate genes were stable. Metabolic depression state appeared to be the main driver for reduced cellular immune status and the activity of antioxidant enzymes in ark shells. Overall, ark shells demonstrated stability of functional state upon exposure to hypoxia and stabilization of cellular immune parameters under prolonged incubation to low dissolved oxygen concentrations.

Distribution and evolution of the serine/aspartate racemase family in invertebrates. II. Frequent and widespread parallel evolution of aspartate racemase.

Our previous studies showed that invertebrate animal serine racemase (SerR) and aspartate racemase (AspR) evolved from a common ancestral gene and are widely distributed. However, the overall molecular evolutionary background of these genes has remained unclear. In the present study, we have cloned, expressed and characterized five SerR and three AspR genes from six invertebrate species. The coexistence of SerR and AspR paralogs has been observed in some species, and the presence of both SerR and AspR is here confirmed in the flatworm Macrostomum lignano, the feather star Anneissia japonica, the ark shell Anadara broughtonii and the sea hare Aplysia californica. Comparison of the gene structures revealed the evolution of SerR and AspR. The ancestral species of metazoans probably had a single SerR gene, and the first gene duplication in the common ancestor species of the eumetazoans occurred after the divergence of porifera and eumetazoans, yielding two SerR genes. Most eumetazoans lost one of the two SerR genes, while the echinoderm A. japonica retained both genes. Furthermore, it is clear that invertebrate AspR genes arose through parallel evolution by duplication of the SerR gene followed by substitution of amino acid residues necessary for substrate recognition in multiple lineages.

Rapid On-Site Identification for Three Arcidae Species (Anadara kagoshimensis, Tegillarca granosa, and Anadara broughtonii) Using Ultrafast PCR Combined with Direct DNA Extraction

Granular ark (Tegillarca granosa), broughton’s ribbed ark (Anadara broughtonii), and half-crenate ark (Anadara kagoshimensis) are important fishery resources throughout Asia; granular ark exhibiting a higher economic value due to its rarity. However, due to the similar morphological characteristics of the three species, the less valuable species could be exploited for food fraud. In this study, we developed a rapid on-site identification method based on a microfluidic chip for the detection of the three ark shell species. We designed new species-specific primers, targeting the genes encoding mitochondrial cytochrome b or cytochrome c oxidase I, for the identification of the three ark shells and estimated their specificity against 17 species, which amplified only the target species. The sensitivity of each primer was 0.001 ng. In addition, this method was further improved to develop a direct ultrafast polymerase chain reaction (PCR) for on-site food monitoring, which would allow for completing the entire procedure (from sampling to obtaining the results) within 25 min without DNA extraction. Our direct, ultrafast PCR was successfully applied to differentiate the three species from 29 commercial products. Therefore, this assay could be used as a rapid and cost-effective approach for the on-site identification of ark shells in commercial food products.

The critical roles of mitochondrial alternative chains in juvenile ark shells (Anadara broughtonii) exposed to acute hypoxia with or without sulfide.

Reduced oxygen levels and increased sulfide concentrations have become a concern for marine animals. This study examines respiratory and energetic adaption to acute (0-96h) hypoxia (0.5mg/L dissolved oxygen) with or without sulfide (0.2mM, 1mM, 3mM) in the hypoxia-resistant and sulfide-tolerant ark shell, Anadara broughtonii. The different states of aerobic respiration, energy-balance, and activity of the mitochondrial sulfide oxidation chain (MSOC) under these conditions were evaluated. The results indicated that the anaerobic pathway was activated by hypoxia at 24h without sulfide, but was activated in the presence of sulfide at only 2h. Exposure to sulfide resulted in significant accumulation of ATP, probably due to the activated MSOC and lowered metabolism via suppression of Na+-K+ ATPase activity and protein synthesis. During hypoxia, both enzyme activity and mRNA levels of alternative oxidase (AOX) increased while the key enzymes in MSOC, sulfide: quinone oxidoreductase (SQR) and sulfur dioxygenase (SDO), were not altered. With additional sulfide, the enzyme activity and mRNA levels of AOX, SQR, SDO significantly increased. Classical aerobic respiration was significantly inhibited, and induction of alternative respiration was detected. The corresponding alternative electron transport chain (AETC) accepted the electrons originating from both the tricarboxylic acid cycle and MSOC during the challenge, indicating that the capacity of aerobic respiration and sulfide-oxidation under a reduction state might greatly depend on AETC. The synergistically induced alternative chains (AETC and MSOC) and anaerobic pathway suggested energy-balance between respiration and sulfide-oxidation, which might contribute to the endurance of ark shells to acute sulfide exposure.

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

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

Two-sided effects of prolonged hypoxia and sulfide exposure on juvenile ark shells (Anadara broughtonii)

Oxygen deficit and sulfide have been restrictive factors in mariculture zones. However, the adaptive mechanism in aquatic lives is still unclear. The commercial ark shells Anadara broughtonii were selected to test the tolerance and adaptive responses to prolonged and intermittent hypoxia with or without exogenous sulfide (mild, moderate, high) by evaluating their behavior, mortality, oxidative level, antioxidant responses, and the MAPK-mediated apoptosis in gills. The results indicated that the clams were tolerant to hypoxia and sulfide exposure but vulnerable during reoxygenation from the challenges. Even so, sulfide had remarkable effect on attenuating the accumulation of reactive oxygen species (ROS) and lipid peroxides caused by reoxygenation from prolonged hypoxia. The increase of glutathione level was probably as an early and primary protective response to prevent the expected reperfusion injury from reoxygenation. The challenges suppressed the oxidative level with a dose-dependent effect of sulfide, with an exception when exposed to mild sulfide. Synchronously, biphasic effects of exogenous sulfide on apoptotic cascade, which was induced by mild sulfide while it was inhibited by higher sulfide, were also detected in gills. The induced or inhibited apoptosis by hypoxia and sulfide kept to a typical ROS-MAPK-CASPASE cascade, desiderating further investigation.

Effects of temperature on the respiratory metabolism, feeding and expression of three heat shock protein genes in Anadara broughtonii

Anadara broughtonii is one of the main commercially important species of shellfish in northern China. A. broughtonii lives in relatively stable subtidal zone where the clam could respond to environmental changes with minimum energy. Therefore, slight fluctuations in water environment may have a great impact on physiological processes such as feeding and metabolism. Large-scale mortality often occurs during the intermediate rearing processes, and high temperatures in summer are considered the leading cause of mortality. To understand the physiological and molecular response patterns of A. broughtonii at high temperatures and to estimate the appropriate metabolism temperature for A. broughtonii, the effects of temperature on the respiratory metabolism and food intake at different growth stages were studied. The response patterns of heat shock protein genes to heat stress and post-stress recovery were also explored. Results show that the temperature and growth stage of A. broughtonii were two important factors affecting metabolism and feeding. The optimum temperature for feeding and physiological activities in each shell-length group was 24 °C. The temperature at which the peak metabolic rate occurred in each shell-length group increased with the increase in shell length. With the increase in heat stress, the expression of three heat shock protein genes (Abhsp60, Abhsp70, and Abhsp90) in the tissues of two size groups of A. broughtonii increased significantly when temperature was above 24 °C and reached a peak at 30 °C. After heat shock at 30 °C for 2 h, the expression of the three heat shock protein genes rapidly increased, peaked at 2 h after the heat shock, and then gradually decreased to the level of the control group at 48 h after the heat shock. The three heat shock protein genes respond rapidly to heat stress and can be used as indicators to the cellular stress response in A. broughtonii under an environmental stress.

Antiradical Activity of Hydrolysates and Extracts from Mollusk A. broughtonii and Practical Application to the Stabilization of Lipids.

The antiradical properties of hydrolysates and hydrothermal extracts of bivalve mollusks (Anadara broughtonii) from the Far Eastern Region of Russia and their influence on lipid oxidation in mayonnaise were investigated. The radical binding activity of hydrolysates and extracts of A. broughtonii varies from 55% to 89%. The maximum radical-binding activity was observed for acid hydrolysates. The antiradical efficiency of acid hydrolysates is 35%–41% of the BHT (butylhydroxytoluene) index. The antiradical activity depends on the (method of) technological and biotechnological processing of raw materials. Acid and enzymatic hydrolysates and hydrothermal extracts of A. broughtonii in mayonnaise slow down the process of oxidation of lipids and hydrolysis of triglycerides. Acid hydrolysates reduce the speed of oxidation and hydrolysis of lipids in mayonnaise more efficiently than the enzymatic hydrolysates.

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

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. We aimed to investigate the transcriptome information of blood clam under environmental stressors. 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. 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 HbIIβ. This study provides transcriptome responses to multiple environmental stressors in blood clams and would provide interesting hints for further studies.

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.