The complete mitochondrial genome of soft coral, Eleutherobia rubra (Brundin, 1896) (Cnidaria; Anthozoa; Malacalcyonacea; Alcyoniidae).

The mitogenome of a soft coral, Eleutherobia rubra (Brundin, 1896), was completely sequenced for the first time. The total mitogenome length of E. rubra is 18,724 bp with 14 protein-coding genes, two ribosomal RNA genes, one transfer RNA gene (tRNA-Met), and one non-coding region (NCR). The gene order is also consistent with other Alcyoniidae species. The base composition is 30.1% A, 16.7% C, 19.5% G, and 33.7% T, with a G-C content of 36.2%. This is the first record of the complete mitogenome sequence of the genus Eleutherobia.

Transcriptome assemblies of two deep-sea octocorals Calyptrophora lyra and Chrysogorgia stellata from West Pacific seamount, Godin Guyot.

This is the first report of the transcriptome assemblies of the deep-sea octocorals Calyptrophora lyra and Chrysogorgia stellata, which were collected in a survey of the West Pacific seamounts area. We sequenced the transcriptomes of C. lyra and C. stellata using the Illumina NovaSeq 6000 System. De novo assembly and analysis of the coding regions predicted 193,796 unigenes from the total 116,441,796 reads of C. lyra and 235,513 unigenes from the total 122,031,866 reads of C. stellata. Our data are a valuable resource with which to understand the ecological and biological characteristics of the West Pacific deep-sea corals. The data will also contribute to the study of deep-sea environments as extreme and limited habitats and provide direction for future research and further insight into the organismal responses of deep-sea corals to environmental changes.

Comparative Genome and Evolution Analyses of an Endangered Stony Coral Species Dendrophyllia cribrosa Near Dokdo Islands in the East Sea.

Stony corals often harbor intracellular photosynthetic dinoflagellate algae that receive dissolved inorganic nutrients. However, Dendrophyllia cribrosa is a nonsymbiotic stony coral distributed in the western Pacific. We assembled a chromosome-level D. cribrosa genome using PacBio and Hi-C technologies. The final assembly was 625 Mb, distributed on 14 chromosomes, and contained 30,493 protein-coding genes. The Benchmarking Universal Single-Copy Orthologs analysis revealed a percentage of 96.8 of the metazoan genome. A comparative phylogenetic analysis revealed that D. cribrosa, which lacks symbionts, evolved to acquire cellular energy by expanding genes related to acyl-CoA metabolism and carbohydrate transporters. This species also has expanded immune-related genes involved in the receptor protein tyrosine kinase signaling pathway. In addition, we observed a specific expansion of calcification genes, such as coral acid-rich proteins and carbonic anhydrase, in D. cribrosa. This high-quality reference genome and comparative analysis provides insights into the ecology and evolution of nonsymbiotic stony corals.

Chromosome-Level Genome Assembly of the Butter Clam Saxidomus purpuratus.

Herein, we provide the first whole-genome sequence of the purple butter clam (Saxidomus purpuratus), an economically important bivalve shellfish. Specifically, we sequenced and de novo assembled the genome of Sa. purpuratus based on PromethION long reads and Hi-C data. The 978-Mb genome of Sa. purpuratus comprises 19 chromosomes with 36,591 predicted protein-coding genes. The N50 length of Sa. purpuratus genome is 52 Mb, showing the highest continuous assembly among bivalve genomes. The Benchmarking by Universal Single-Copy Orthologs assessment indicated that 95.07% of complete metazoan universal single-copy orthologs (n = 954) were present in the assembly. Approximately 51% of Sa. purpuratus genome comprises repetitive sequences. Based on the high-quality Sa. purpuratus genome, we resolved half of the immune-associated genes, namely, scavenger receptor (SR) proteins, which are collinear to those in the closely related Cyclina sinensis genome. This finding suggested a high degree of conservation among immune-associated genes. Twenty-two (19%) SR proteins are tandemly duplicated in Sa. purpuratus genome, suggesting putative convergence evolution. Overall, Sa. purpuratus genome provides a new resource for the discovery of economically important traits and immune-response genes.

Transcriptional response of the azooxanthellate octocoral Scleronephthya gracillimum to seawater acidification and thermal stress.

The stress responses to increased seawater temperature and marine acidification were investigated using a microarray to reveal transcriptional changes in S. gracillimum. For the study, corals were exposed to different stress experiments; high temperature only (26 °C, 28 °C and 30 °C), low-pH only (pH 7.5, pH 7.0 and pH 6.5) and dual stress experiments (28 °C + pH 7.8, 28 °C + pH 7.5 and 28 °C + pH 7.0), mortality and morphological changes in 24 h exposure experiments were investigated. The survival rates of each experimental group were observed. The gene expression changes in single and dual stress exposed coals were measured and the differentially expressed genes were classified with gene ontology analysis. The top three enriched gene ontology terms of DEGs in response to dual stress were metal ion binding (23.4%), extracellular region (17.2%), and calcium ion binding (12.8%). The gene showing the greatest increase in expression as a response to the dual stress was hemagglutinin/amebocyte aggregation factor, followed by interferon-inducible GTPase 5 and the gene showing the greatest decrease as a response to the dual stress was Fas-associating death domain-containing protein, followed by oxidase 2. These results represented the transcriptomic study focused on the stress responses of the temperate asymbiotic soft coral exposed to single and dual stresses. The combined effect of thermal and acidification stress on corals triggered the negative regulation of ion binding and extracellular matrix coding genes and these genes might serve as a basis for research into coral-specific adaptations to stress responses and global climate change.

Fluoxetine in the environment may interfere with the neurotransmission or endocrine systems of aquatic animals.

Antidepressants are extensively used to treat the symptoms of depression in humans, and the environmentally discharged drugs potentially threaten aquatic organisms. In this study, the acute toxic effects of fluoxetine (FLX) were investigated in two aquatic organisms, the freshwater polyp (Hydra magnipapillata) and Javanese medaka (Oryzias javanicus). The median lethal concentration (LC50) of FLX in H. magnipapillata was 3.678, 3.082, and 2.901 mg/L after 24, 48, and 72 h, respectively. Morphological observations of the FLX-exposed H. magnipapillata showed that 1.5 mg/L FLX induced the contraction of the tentacles and body column. The LC50 of FLX in O. javanicus was 2.046, 1.936, 1.532, and 1.237 mg/L after 24, 48, 72, and 96 h, respectively. Observation of the behavior of the FLX-exposed fish showed that FLX reduced their swimming performance at a minimum concentration of 10 µg/L. The half-maximal effective concentration (EC50) of FLX for swimming behavior in O. javanicus was 0.135, 0.108, and 0.011 mg/L after 12, 24, and 96 h, respectively. Transcriptomic analyses indicated that FLX affects various physiological and metabolic processes in both species. FLX exposure induced oxidative stress, reproductive deficiency, abnormal pattern formation, DNA damage, and neurotransmission disturbance in H. magnipapillata, whereas it adversely affected O. javanicus by inducing oxidative stress, DNA damage, endoplasmic reticulum stress, and mRNA instability. Neurotransmission-based behavioral changes and endocrine disruption were strongly suspected in the FLX-exposed fish. These results suggest that FLX affects the behavior and metabolic regulation of aquatic organisms.

First transcriptome assembly of a newly discovered vent mussel, Gigantidas vrijenhoeki, at Onnuri Vent Field on the northern Central Indian Ridge.

This is the first report of a transcriptome assembly of a newly discovered hydrothermal vent mussel, Gigantidas vrijenhoeki (Bivalvia: Mytilidae), on the Central Indian Ridge. Gigantidas vrijenhoeki was identified from material collected at the newly discovered Onnuri Vent Field (OVF) on the Central Indian Ridge in 2018, and was reported as a new species, distinct from another dominant hydrothermal vent mussel, Bathymodiolus marisindicus, in 2020. We sequenced the transcriptome of G. vrijenhoeki using the Illumina HiSeq X System. De novo assembly and analysis of the coding regions predicted 25,405 genes, 84.76% of which was annotated by public databases. The transcriptome of G. vrijenhoeki will be a valuable resource in studying the ecological and biological characteristics of this new species, which is distinct from other deep-sea mussels. These data should also support the investigation of the relationship between the environmental conditions of hydrothermal vents and the unique distribution of G. vrijenhoeki in the OVF of the Central Indian Ridge.

Transcriptomic profiling of Hydra magnipapillata after exposure to naproxen.

The extensive use in humans and animals of nonsteroidal anti-inflammatory drugs (NSAIDs) increases their possible impact on aquatic organisms. In the present study, we investigated acute toxicity, morphological responses, and potential physiological and metabolic impacts of naproxen exposure on Hydra magnipapillata. The median lethal concentrations (LC50) of naproxen in H. magnipapillata were 51.999 mg/L, 44.935 mg/L, and 42.500 mg/L after exposure for 24, 48, and 72 h, respectively. Morphological observation of the exposed Hydra showed that 40 mg/L naproxen stimulated the contraction of body column and tentacles after 24 h. A KEGG pathway analysis of the genes differentially expressed in the Hydra after exposure to naproxen for 6, 24, or 48 h demonstrated various cellular and metabolic effects, including protein processing in the endoplasmic reticulum, Wnt signaling, and tryptophan metabolism. These results suggest that exposure to naproxen affects the genetic material, inflammatory processes, and metabolic processes of aquatic organisms.

The genome of the giant Nomura's jellyfish sheds light on the early evolution of active predation.

BACKGROUND: Unique among cnidarians, jellyfish have remarkable morphological and biochemical innovations that allow them to actively hunt in the water column and were some of the first animals to become free-swimming. The class Scyphozoa, or true jellyfish, are characterized by a predominant medusa life-stage consisting of a bell and venomous tentacles used for hunting and defense, as well as using pulsed jet propulsion for mobility. Here, we present the genome of the giant Nomura's jellyfish (Nemopilema nomurai) to understand the genetic basis of these key innovations. RESULTS: We sequenced the genome and transcriptomes of the bell and tentacles of the giant Nomura's jellyfish as well as transcriptomes across tissues and developmental stages of the Sanderia malayensis jellyfish. Analyses of the Nemopilema and other cnidarian genomes revealed adaptations associated with swimming, marked by codon bias in muscle contraction and expansion of neurotransmitter genes, along with expanded Myosin type II family and venom domains, possibly contributing to jellyfish mobility and active predation. We also identified gene family expansions of Wnt and posterior Hox genes and discovered the important role of retinoic acid signaling in this ancient lineage of metazoans, which together may be related to the unique jellyfish body plan (medusa formation). CONCLUSIONS: Taken together, the Nemopilema jellyfish genome and transcriptomes genetically confirm their unique morphological and physiological traits, which may have contributed to the success of jellyfish as early multi-cellular predators.

The Draft Genome of an Octocoral, Dendronephthya gigantea.

Coral reefs composed of stony corals are threatened by global marine environmental changes. However, soft coral communities of octocorallian species, appear more resilient. The genomes of several cnidarians species have been published, including from stony corals, sea anemones, and hydra. To fill the phylogenetic gap for octocoral species of cnidarians, we sequenced the octocoral, Dendronephthya gigantea, a nonsymbiotic soft coral, commonly known as the carnation coral. The D. gigantea genome size is ∼276 Mb. A high-quality genome assembly was constructed from PacBio long reads (29.85 Gb with 108× coverage) and Illumina short paired-end reads (35.54 Gb with 128× coverage) resulting in the highest N50 value (1.4 Mb) reported thus far among cnidarian genomes. About 12% of the genome is repetitive elements and contained 28,879 predicted protein-coding genes. This gene set is composed of 94% complete BUSCO ortholog benchmark genes, which is the second highest value among the cnidarians, indicating high quality. Based on molecular phylogenetic analysis, octocoral and hexacoral divergence times were estimated at 544 MYA. There is a clear difference in Hox gene composition between these species: unlike hexacorals, the Antp superclass Evx gene was absent in D. gigantea. Here, we present the first genome assembly of a nonsymbiotic octocoral, D. gigantea to aid in the comparative genomic analysis of cnidarians, including stony and soft corals, both symbiotic and nonsymbiotic. The D. gigantea genome may also provide clues to mechanisms of differential coping between the soft and stony corals in response to scenarios of global warming.

A simple and rapid method for toxicity evaluation of zinc oxide nanoparticle (ZnO NPs) in benthic animal Hydra magnipapillata.

Toxicity evaluation is necessary to investigate the possible risk of chemical or pollutants newly produced such as nanoparticles in the environments. The assessment should apply a method that is effective to determine the toxic concentration and the exposure time of the pollutants in an animal model. This study described three main stages including determining the median lethal concentrations (LC50) with Probit program and detecting toxic effects of ZnO NPs in morphology and regeneration observed by the changes in morphology of Hydra magnipapillata (H. magnipapillata). We also provide a strategy for culturing hydra in laboratory condition to use the animal for the experiment. The exposure to ZnO NPs led to the abnormality in regeneration such as formation of extraordinary number of tentacles and bifurcated tips in tentacles and the toxic effects in morphology appeared the clubbing tentacle, slender body, and retracting body column and tentacles by the exposure time. The method described here is simple and useful to evaluate the toxic effects of ZnO NPs using morphological characters in H. magnipapillata and could suggest the concentration and the exposure time for further investigations on cellular and molecular responses of the animal after exposure to other nanoparticles. •A simple method to evaluate the toxic effects of ZnO NPs using morphological characters of H. magnipapillata and other hydra species.•A rapid method to evaluate the toxic effects of ZnO NPs and other nanoparticles in H. magnipapillata.

Acute toxic effects of zinc oxide nanoparticles on Hydra magnipapillata.

Zinc oxide nanoparticles (ZnO NPs) are increasingly used in various products as coating and additive materials for household goods, personal-care products, and drug delivery systems. Because of their broad applications, the potential risks to nontarget organisms associated with their input into aquatic environments have generated much concern. We investigated the acute toxicity, morphological responses, and potential impact on physiology and metabolism in polyps exposed to spherical ZnO NPs of either 20 nm (ZnO NP20) or 100 nm (ZnO NP100). The median lethal concentrations (LC50) of ZnO NP20 were 55.3, 8.7, and 7.0 µg/mL after exposure for 48, 72, and 96 h, respectively; and those of ZnO NP100 were 262.0, 14.9, and 9.9 µg/mL, respectively. The morphological responses of the hydra polyps to a range of ZnO NP concentrations suggest that ZnO NPs may negatively affect neurotransmission in Hydra. ZnO NPs may also induce abnormal regeneration in the polyps by affecting the expression of several genes related to the Wnt signaling pathway. The presence of ZnO NP20 in the hydra tissue was confirmed with electron microscopy. A Gene Ontology analysis of the genes differentially expressed in hydra polyps after exposure to ZnO NP20 for 12 or 24 h revealed changes in various processes, including cellular and metabolic process, stress response, developmental process, and signaling. A KEGG pathway analysis of hydra polyps after exposure of ZnO NP20 or ZnO NP100 for 12 or 24 h demonstrated various changes, including in the DNA replication and repair, endocytosis, lysosomes, Wnt signaling, and natural killer-cell-mediated cytotoxicity pathways, suggesting the mechanisms that maintain cellular homeostasis in response to ZnO NPs. Progesterone-mediated oocyte maturation was also affected by the ZnO NPs nanoparticles, suggesting that they are potential endocrine disruptors. This study should increase our concern regarding the dispersal of ZnO NPs in aquatic environments.

Detection of Metallothionein in Javanese Medaka (Oryzias javanicus), Using a scFv-Immobilized Protein Chip.

Environmental pollution by various industrial chemicals and biological agents poses serious risks to human health. Especially, marine contamination by potentially toxic elements (PTEs) has become a global concern in recent years. Many efforts have been undertaken to monitor the PTE contamination of the aquatic environment. However, there are few approaches available to assess the PTE exposure of aquatic organisms. In this research, we developed a strategy to evaluate the heavy metal exposure of marine organisms, by measuring the expression levels of metallothionein protein derived from Oryzias javanicus (OjaMT). OjaMT is a biomarker of heavy metal exposure because the expression level increases upon heavy metal exposure. The developed assay is based on a real-time, label-free surface plasmon resonance (SPR) measurement. Anti-OjaMT antibody and anti-OjaMT single-chain fragment of variable region (scFv) were used as detection probes. Two types of SPR sensor chips were fabricated, by immobilizing antibody or Cys3-tagged scFv (scFv-Cys3) in a controlled orientation and were tested for in situ label-free OjaMT detection. Compared to the antibody-presenting sensor chips, the scFv-presenting sensor chips showed improved performance, displaying enhanced sensitivity and enabling semi-quantitative detection. The portable SPR system combined with scFv-immobilized sensor chips is expected to provide an excellent point-of-care testing system that can monitor target biomarkers in real time.

Geographical variations in bacterial communities associated with soft coral Scleronephthya gracillimum.

Environmental impacts can alter relationships between a coral and its symbiotic microbial community. Furthermore, changes in the microbial community associated with increased seawater temperatures can cause opportunistic infections, coral disease and death. Interactions between soft corals and their associated microbes are not well understood. The species Scleronephthya gracillimum is distributed in tropical to temperate zones in coral assemblages along the Kuroshio Current region. In this study we collected S. gracillimum from various sites at different latitudes, and compared composition of their bacterial communities using Next Generation Sequencing. Coral samples from six geographically distinct areas (two sites each in Taiwan, Japan, and Korea) had considerable variation in their associated bacterial communities and diversity. Endozoicimonaceae was the dominant group in corals from Korea and Japan, whereas Mycoplasma was dominant in corals from Taiwan corals. Interestingly, the latter corals had lower relative abundance of Endozoicimonaceae, but greater diversity. These biogeographic differences in bacterial composition may have been due to varying environmental conditions among study locations, or because of host responses to prevailing environmental conditions. This study provided a baseline for future studies of soft coral microbiomes, and assessment of functions of host metabolites and soft coral holobionts.

Transcriptional changes caused by bisphenol A in Oryzias javanicus, a fish species highly adaptable to environmental salinity.

The Javanese medaka, Oryzias javanicus, is a fish highly adaptable to various environmental salinities. Here, we investigated the effects of the environmental pollutant bisphenol A (BPA; an endocrine disrupting chemical) on gene expression levels in this species acclimated to different salinities. Using cDNA microarrays, we detected the induction of differential expression of genes by BPA, and compared the transcriptional changes caused by chemical exposure at different salinities. There were marked transcriptional changes induced by BPA between treatments. While 533 genes were induced by a factor of more than two when O. javanicus was exposed to BPA in seawater, only 215 genes were induced in freshwater. Among those genes, only 78 were shared and changed significantly their expression in both seawater and freshwater. Those genes were mainly involved in cellular processes and signaling pathway. We then categorized by functional group genes specifically induced by BPA exposure in seawater or freshwater. Gene expression changes were further confirmed in O. javanicus exposed to various concentrations of BPA, using quantitative real-time reverse transcription PCR based on primer sets for 28 selected genes.

Transcript response of soft coral (Scleronephthya gracillimum) on exposure to polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are the most persistent organic pollutants in worldwide aquatic environments. The extensive isolation of genes responsive to PAH pollution in soft coral (Scleronephthya gracillimum) is described herein. Soft coral colonies were exposed to 100 µg/L of a standard mixture of PAHs. Gene candidates with transcript levels that changed in response to PAH exposure were identified by differential display polymerase chain reaction (DD-PCR). There were 37 types of candidate genes identified, of which 20 were upregulated in expression and 17 were downregulated. The functions of the genes identified included oxidative stress response, ribosomal structure maintenance, molecular chaperone activity, protein kinase activation and tumorigenesis, defense mechanisms, transcription, and other biological responses. mRNA quantification was carried out using real-time quantitative PCR in eight selected genes: cytosolic malate dehydrogenase, protein disulfide isomerase, ribosomal protein L6, ral guanine nucleotide dissociation stimulator-like 1, poly(ADP-ribose) polymerase 4, peptidylglycine α-hydroxylating monooxygenase, a disintegrin and metalloproteinase (ADAM) metallopeptidase protein, and eukaryotic initiation factor 4 gamma 3. Changes in transcript levels were consistent with DD-PCR results. The gene candidates isolated in this study were differentially expressed and therefore have potential as molecular biomarkers for understanding coral responses to environmental stressors.

Molecular toxicity identification evaluation (mTIE) approach predicts chemical exposure in Daphnia magna.

Daphnia magna is a bioindicator organism accepted by several international water quality regulatory agencies. Current approaches for assessment of water quality rely on acute and chronic toxicity that provide no insight into the cause of toxicity. Recently, molecular approaches, such as genome wide gene expression responses, are enabling an alternative mechanism based approach to toxicity assessment. While these genomic methods are providing important mechanistic insight into toxicity, statistically robust prediction systems that allow the identification of chemical contaminants from the molecular response to exposure are needed. Here we apply advanced machine learning approaches to develop predictive models of contaminant exposure using a D. magna gene expression data set for 36 chemical exposures. We demonstrate here that we can discriminate between chemicals belonging to different chemical classes including endocrine disruptors and inorganic and organic chemicals based on gene expression. We also show that predictive models based on indices of whole pathway transcriptional activity can achieve comparable results while facilitating biological interpretability.

Crucial role of TSC-22 in preventing the proteasomal degradation of p53 in cervical cancer.

The p53 tumor suppressor function can be compromised in many tumors by the cellular antagonist HDM2 and human papillomavirus oncogene E6 that induce p53 degradation. Restoration of p53 activity has strong therapeutic potential. Here, we identified TSC-22 as a novel p53-interacting protein and show its novel function as a positive regulator of p53. We found that TSC-22 level was significantly down-regulated in cervical cancer tissues. Moreover, over-expression of TSC-22 was sufficient to inhibit cell proliferation, promote cellular apoptosis in cervical cancer cells and suppress growth of xenograft tumors in mice. Expression of also TSC-22 enhanced the protein level of p53 by protecting it from poly-ubiquitination. When bound to the motif between amino acids 100 and 200 of p53, TSC-22 inhibited the HDM2- and E6-mediated p53 poly-ubiquitination and degradation. Consequently, ectopic over-expression of TSC-22 activated the function of p53, followed by increased expression of p21(Waf1/Cip1) and PUMA in human cervical cancer cell lines. Interestingly, TSC-22 did not affect the interaction between p53 and HDM2. Knock-down of TSC-22 by small interfering RNA clearly enhanced the poly-ubiquitination of p53, leading to the degradation of p53. These results suggest that TSC-22 acts as a tumor suppressor by safeguarding p53 from poly-ubiquitination mediated-degradation.

Toxaphene affects the levels of mRNA transcripts that encode antioxidant enzymes in Hydra.

We evaluated toxaphene-induced acute toxicity in Hydra magnipapillata. The median lethal concentrations of the animals (LC(50)) were determined to be 34.5 mg/L, 25.0 mg/L and 12.0 mg/L after exposure to toxaphene for 24 h, 48 h and 72 h, respectively. Morphological responses of hydra polyps to a range of toxaphene concentrations suggested that toxaphene negatively affects the nervous system of H. magnipapillata. We used real-time quantitative PCR of RNA extracted from polyps exposed to two concentrations of toxaphene (0.3 mg/L and 3 mg/L) for 24 h to evaluate the differential regulation of levels of transcripts that encode six antioxidant enzymes (CAT, G6PD, GPx, GR, GST and SOD), two proteins involved in detoxification and molecular stress responses (CYP1A and UB), and two proteins involved in neurotransmission and nerve cell differentiation (AChE and Hym-355). Of the genes involved in antioxidant responses, the most striking changes were observed for transcripts that encode GPx, G6PD, SOD, CAT and GST, with no evident change in levels of transcripts encoding GR. Levels of UB and CYP1A transcripts increased in a dose-dependent manner following exposure to toxaphene. Given that toxaphene-induced neurotoxicity was not reflected in the level of AChE transcripts and only slight accumulation of Hym-355 transcript was observed only at the higher of the two doses of toxaphene tested, there remains a need to identify transcriptional biomarkers for toxaphene-mediated neurotoxicity in H. magnipapillata. Transcripts that respond to toxaphene exposure could be valuable biomarkers for stress levels in H. magnipapillata and may be useful for monitoring the pollution of aquatic environments.

Differentially displayed genes with oxygen depletion stress and transcriptional responses in the marine mussel, Mytilus galloprovincialis.

Hypoxic events affecting aquatic environments have been reported worldwide and the hypoxia caused by eutrophication is considered one of the serious threats to coastal marine ecosystems. To investigate the molecular-level responses of marine organisms exposed to oxygen depletion stress and to explore the differentially expressed genes induced or repressed by hypoxia, differential display polymerase chain reaction (DD-PCR) was used with mRNAs from the marine mussel, Mytilus galloprovincialis, under oxygen depletion and normal oxygen conditions. In total, 107 cDNA clones were differentially expressed under hypoxic conditions relative to the control mussel group. The differentially expressed genes were analyzed to determine the effects of hypoxia. They were classified into five functional categories: information storage and processing, cellular processes and signaling, metabolism, predicted general function only, and function unknown. The differentially expressed genes were predominantly associated with cellular processing and signaling, and they were particularly related to the signal transduction mechanism, posttranslational modification, and chaperone functions. The observed differences in the DD-PCR of 10 genes (encoding elongation factor 1 alpha, heat shock protein 90, calcium/calmodulin-dependent protein kinase II, GTPase-activating protein, 18S ribosomal RNA, cytochrome oxidase subunit 1, ATP synthase, chitinase, phosphoglycerate/bisphosphoglycerate mutase family protein, and the nicotinic acetylcholine receptor) were confirmed by quantitative RT-PCR and their transcriptional changes in the mussels exposed to hypoxic conditions for 24-72 h were investigated. These results identify biomarker genes for hypoxic stress and provide molecular-level information about the effects of oxygen depletion on marine bivalves.