MINIREVIEW ISJ 10: 84-93, 2013 ISSN 1824-307X MINIREVIEW Transcriptomic response to stress in marine bivalves Q Li, X Zhao, L Kong, H Yu College of Fisheries, Ocean University of China, Qingdao 266003, China Accepted September 25, 2013 Abstract Marine bivalves have a set of unique capabilities to adapt to the complicated conditions owing to their habitats, living habits and feeding ways. Meanwhile, marine bivalves can be the biosensors to monitor the quality of the intertidal zones or other habitats. It is interesting for every biologist to find out the mechanisms by which organisms adapt to environmental challenges and the factors limiting their adaptive capacities. The development of biotechnology over the past few decades has provided biologists with a vast repertoire of biosensors that allow testing mRNA expression in response to environmental factors. This minireview is focused on the transcriptomic responses to abiotic and biotic stressors in bivalves and the relative methods to provide new perspectives as well as improve applications for bivalve biomonitoring studies. Key Words: transcriptome; stress; bivalve Introduction Marine bivalves are an important component of the ecosystem and biodiversity (Dame, 2011), which have abundant species distributed worldwide from the intertidal zones to hydrothermal vents and cold seeps (Bettencourt et al., 2010; Boutet et al., 2011; Egas et al., 2012). Bivalve cultivation is one of the most important aquaculture industries globally (Forrest et al., 2009; Pawiro 2010). Furthermore, marine bivalves possess the unique adaptation to problematic surroundings. In light of the important status of marine bivalves in ecosystem and economy and their high adaptations, they are the valuable organisms to be investigated about their molecular mechanism responding to the variable environment. It is also meaningful to find relative gene expression index in marine bivalves to be the monitoring standard of the surroundings. Owing to no model organism in marine bivalves and the repetitive organization of the non-coding fraction in their genome, as well as their size, the development of the genome and transcriptome of them make a slow progress. Thanks to the technical advance, marine bivalves have acquired growing concerns and their genomic databases have enriched increasingly, such as MytiBase (http://mussel.cribi.unipd.it) for Mytilus galloprovincialis and DeepSeaVent ___________________________________________________________________________ Corresponding author: Qi Li College of Fisheries Ocean University of China Qingdao 266003, China E-mail: qili66@ouc.edu.cn (http://transcriptomics.biocant.pt:8080/deepSeaVent) for Bathymodiolus azoricus. Marine bivalves commonly inhabit variable and unstable conditions and most are the crisscross between anthropic zone and the nature. Complex ecological habitats bring about all kinds of stresses in the lives of bivalves and determine their distributions and abundance. These stresses are mostly from some reasons as follows. Firstly, intertidal zone that is one of the most important habitats for bivalves experiences large and sometimes rapid fluctuations caused by tidal fluctuations, rain and freshwater run-off (Shumway, 1977). Owing to this, intertidal bivalves are mostly exposed to multiple stressors including periodic hypoxia, hyposaline, temperature fluctuations and pollution (Ivanina et al., 2012). Meanwhile, in the recent past, anthropogenic inputs of contaminants, and combustion of fossil fuels and deposition of metals make the condition further complicated (Doney, 2010). Furthermore, some important cultured bivalve species, such as Pacific oyster and blue mussel, are subject to the “summer mortality” (Tremblay et al., 1998a; Tremblay et al., 1998b; Xiao et al., 2005; Samain et al., 2007; Lynch et al., 2012). Summer mortality has been reported to occur during the summer months in several countries (Myrand and Gaudreault, 1995; Cotter et al., 2010; Fleury and Huvet, 2012). This phenomenon is multifactorial, resulting from a complex interaction between organisms, environment and pathogens (Samain et al., 2008).   84 Secondly, climate changes will affect temperature extremes and averages, and hyposaline conditions in coastal areas due to extreme precipitation events and oceanic pH (Tomanek, 2012). Meanwhile, the changes of the environment can affect the distribution and abundance of the native species (Johnson et al., 2011), even lead to change the competitive interactions between invasive and native species (Lockwood et al., 2010; Lockwood et al., 2011). Lastly, it puts down to their living habits and feeding ways of themselves. Most bivalves are filter- feeding and their mobility is not great. They cannot escape the stresses by moving away quickly and have to adjust to the changing surroundings other than swimming organisms. It is more valuable to decipher the mechanisms of their unique capacities to adjust to the variable environment. There are kinds of reasons to induce the responses of the marine bivalves, and the factors involved in the stresses have multiple modes of interaction. Responses to the environmental stresses on the transcriptomic level are complicated and hard to explain by single gene or pathway. Actually, even against the single stress, there are a lot of genes participated and the complex networks between genes and pathways. Here we provide an overview about the transcriptomic responses to abiotic and biotic stressors in marine bivalves and the development of the relative technological methods to provide promising perspectives for a better comprehension of the mechanisms. Abiotic stress responses Temperature Temperature has been shown to be one of the most important determinants of survival, growth and reproduction (Helmuth et al., 2006; Menge et al., 2008). Understanding the underlying mechanisms by temperature driving organismal responses and physiological performance is becoming increasingly imperative as climate change alters habitat temperature (Somero, 2010). Meanwhile, sessile inhabitants of marine intertidal environments commonly face heat stress which is an important incentive in “summer mortality” (Tremblay et al., 1998a; Soletchnik et al., 2005). The largest changes in gene expression in response to heat-stress were among genes that encoded the molecular chaperone heat shock protein 70 (Hsp70) and his family (Lang et al., 2009; Lockwood et al., 2010; Chapman et al., 2011). The increased expression of molecular chaperones is a primary component of the cellular stress response and a key indicator of environment stress (Lockwood et al., 2010). Hsps, especially Hsp70 and Hsp90, can protect cells and organisms from thermal damages (Zhao and Jones, 2012) and are well known as molecular chaperones that help in the refolding of misfolded proteins and assist in their elimination if they become irreversibly damaged (Zhao and Jones, 2012). Peroxinectin is up- regulated during exposure to elevated temperature, which is reported in oysters (Lang et al., 2009; Chapman et al., 2011). Peroxinectin may perform adhesive and defensive functions (Lang et al., 2009), owing to higher temperature negatively impacting resistance to bacteria (Chapman et al., 2011). Transcriptomic responses to heat stress are, in part, characterized by the up-regulation at 32 oC of genes involved in proteolysis, which was specific to ubiquitin-mediated proteolysis in Mytilus trossulus (Lockwood et al., 2010). Proteolysis is the directed degradation of proteins and in the context of environmental stress serves to degrade and remove permanently denatured proteins, an indicator of severe cellular stress (Dahlhoff, 2004; Kültz, 2005). Heat shock can also affect the genes related to growth and reproduction. The relative genes such as suppressor of cytokine signaling-2, collagen, were up-regulated during heat stress (Lang et al., 2009). Salinity In intertidal zones, the salinity of seawater can strongly vary from nearly fresh water to highly saline (Meng et al., 2013). Some bivalves, such as the Pacific oysters, are able to survive in a range of salinity from 10‰ to 35‰ (Pauley et al., 1988). For mussels, the salinity may determine the outcome of competition between native and invasive mussels (Lockwood et al., 2011). During hypo-osmotic shock, all reported changes in gene expression mainly focus on osmoregulation and osmotic stress signaling (Lockwood et al., 2011; Zhao et al., 2012; Meng et al., 2013). Osmoregulation influences the up- regulation of genes encoding ion channel (Lockwood et al., 2011; Zhao et al., 2012; Meng et al., 2013) and free amino acid (FAA) metabolic key enzyme (Meng et al., 2013), as well as the down- regulation of the ion and amino acid transporter genes (Lockwood et al., 2011). Salt stress signal transduction pathways including calcium signaling cascade and phosphorylation regulation were observed to be up-regulated (Zhao et al., 2012; Meng et al., 2013). Furthermore, FAA metabolic pathways (including those for glycine, alanine, beta- alanine, arginine, proline, and taurine) were activated, altering the osmotic status in the oysters (Meng et al., 2013). In the study of mussels, ornithine decarboxylase, serving as the key regulatory enzyme in polyamine synthesis was highlighted, owing to different expression between two mussels, which regulated cell growth and cell viability and whose disruption can cause cancer or apoptosis (Lockwood et al., 2011). Moreover, many other types of metabolism, including immune responses and apoptosis, were shown to be enriched in the KEGG pathway analysis. Metals and chemicals Most species of marine bivalves have a long history as sentinel organisms for monitoring the status of marine ecosystems (Dondero et al., 2006; Hines et al., 2007; Milan et al., 2011; Varotto et al., 2013). Heavy metals including Cu2+, Cd2+, Hg2+, Ni2+, participate in the biological cycles of different groups of organisms (Zapata et al., 2009), affecting their distribution and abundance (Kudo et al., 1996). In previous studies, copper gives rise to the differentially expressed genes involved in the respiratory chain and stress response, development   85 and differentiation, cytoskeleton in Chilean scallop (Zapata et al., 2009). Metallothionein is the most well-known antioxidant that protects against metal toxicity, and was verified in marine bivalves (Venier et al., 2006; Dondero et al., 2011; Varotto et al., 2013). Moreover, Nickle modulated proliferation, growth and apoptosis as the same as copper, and highly regulated the genes encoding lipid metabolism (Dondero et al., 2011). Organic contaminants elicited defensin C and the defender against cell death 1 gene (whose defective function causes apoptotic death) that were under-represented in mussels other than from heavy metals (Venier et al., 2006). Similarly, in mussels, Chlorpyrifos decreased in acetylcholinesterase activity in the gills markedly, and up-regulated the chitinase activity, which play a role in digestion and participate in the innate immune response (Dondero et al., 2011). In addition, studies focused on the responses of Mytilus edulis to benzo[α]pyrene found that organic substance mostly disrupt the cellular redox status (Brown et al., 2006). Notably, gene expression levels primarily depend on the functional specificity of cells composing different organs and tissues (Venier et al., 2006). So that, to different tissues, the transcriptional responses to the pollution are specific. Hypoxia Oxygen deficiency is a common stressor in estuarine and coastal environments. Intertidal molluscs are among the animal champions of hypoxia tolerance owing to a suite of metabolic adaptions that allows them to survive prolonged periods without oxygen. They adapt to the fluctuation of oxygen by changing energy management and resource utilizations. In Crassostrea gigas, which exposed to hypoxia, an overall transcriptome study indicated many genes coding for enzymes involved in antioxidant defense and reactive oxygen species detoxification for the cellular redox balance except for genes related to stress exhibited over-expressed (Sussarellu et al., 2010). Meanwhile, some literatures were to characterize the some genes in bivalves and to define their potential regulation in the hypoxic response. Hypoxia-inducible factor-1 (HF-1), as the key regulator of oxygen homeostasis in aerobic organisms under hypoxia, had been verified to play a critical role in reactive oxygen species (ROS) production of hemocytes in C. gigas (Choi et al., 2013). And likewise, AMP-activated protein kinase α (AMPKα) was showed to participate in the metabolic response during hypoxia in the smooth muscle of C. gigas (Guévélou et al., 2013). However, Crassostrea virginica has both a better tissue aerobic capacity to compensate for reduced oxygen availability and a lower sensitivity to hypoxia than C. gigas, with a compensatory increase in activities of citrate synthase and cytochrome c oxidase after 2 weeks of hypoxia (Ivanina et al., 2011). Other abiotic stresses The different conditions of different vertical locations in intertidal zone depend on the tides. The tidal fluctuations changed many environmental factors such as temperature and food availability of the surroundings. Researchers caught this natural phenomenon and showed that low intertidal mussels altered their physiology very little with respect to the tide cycle, and mid-intertidal and high intertidal mussels reduced the gene expressions involved in metabolic processes (Place et al., 2012). Especially, in high intertidal zones, pathways associated with protein rescue, cellular repair and protein degradation and oxidative stress were activated (Place et al., 2012). Biotic stresses Biotic stress mainly refers to the stress that occurs as a result of damage to plants and animals by other living organisms such as bacteria, viruses, parasites and microalgae. Marine bivalves harbor an abundant and diverse microflora on their surface or inside their tissues. With evolution, marine bivalves have developed effective systems for maintaining their homeostasis and for controlling potentially harmful and pathogenic microorganisms and microalgae. The current literature shows that bivalves eliminate or limit the development of the microorganisms through different innate immune response in combination with other cellular mechanism, such as the apoptotic pathway by transcriptomic analysis (Wang et al., 2010; De Lorgeril et al., 2011; Morga et al., 2011; Brulle et al., 2012; Moreira et al., 2012a; Moreira et al., 2012b). In flat oyster (Ostrea edulis), Fas-ligand that was involved in the immune response against the parasite Bonamia ostreae was observed up- regulated. Meanwhile, according to the previous results, Fas-ligand is also associated in the apoptosis pathway with the inhibitors of apoptosis proteins (Morga et al., 2011). Furthermore, apoptosis as autophagy can be triggered by reactive oxygen species (ROS), and up-regulated genes are related to respiratory chain and particularly in ROS production (Wang et al., 2010; De Lorgeril et al., 2011). Above all, the apoptosis pathway is the most important response to the biotic stresses. In addition to the apoptosis pathway, other immune-related genes were reported, such as ferritin and lysozyme, several immune pathways and processes including the toll-like signaling pathway and the complement cascade (Wang et al., 2010; De Lorgeril et al., 2011; Moreira et al., 2012). Except for the immune response to the microorganisms, some bivalves exposed to Vibrio spp. (Gestal et al., 2007; Brulle et al., 2012; Moreira et al., 2012b), parasite including Bonamia (Martín- Gómez et al., 2012) and Perkinsus marinus (Tanguy et al., 2004) were elicited that such cytotoxic response led to the rearrangement of the cytoskeleton. Cytoskeleton is important in phagocytosis, and all phagocytosis processes are driven by rearrangement of the actin cytoskeleton (May et al., 2001). Besides the microorganisms, dinoflagellates and other microalgae can produce a wide spectrum of toxic molecules. During seasonal harmful algae blooms (HABs), many filter-feeding bivalves can   86 accumulate phycotoxins at extremely high levels, thus representing a serious threat to human health. Until now, a few researches about the transcriptome of marine bivalve induced by harmful algae have been reported. Manfrin et al., 2010 studied the molecular mechanism that M. galloprovincialis exposed to okadaic acid (OA) which is a lipophilic toxin. Its results indicated that the effects of OA mostly concentrated in genes related to stress response, apoptosis and cell structure function (Manfrin et al., 2010). These variations were also observed in the study that evaluated C. gigas hemocytes responses to purified PbTx-2 in vitro (Mello et al., 2012). In both researches, there were no genes associated to immune or antioxidant, which needs more experiments to be tested and verified. Interactive stresses Previous researches tended to control a single variable to discuss the transcriptomic responses and acquire the master genes, because the single variable is easier to control under the laboratory conditions. However, the natural circumstances consist of multifactor and are multivariable other than the single variable. In addition, the effect of two stresses do not coincide with the effect of the mix including the two stresses (Dondero et al., 2011). Several studies focused on the transcriptomic responses to the environmental stresses owing to “summer mortality” (Chaney et al., 2011; Fleury et al., 2012) and interactions with environmental variables that induced changes in gene expression profiles and affected the fitness of organisms (Chapman et al., 2009; Chapman et al., 2011; Philipp et al., 2012). Beyond that, the hydrothermal vent mussel (B. azoricus) itself thrives in a condition with the darkness, extreme cold, high pressure and rich in methane and sulfides (Egas et al., 2012) which is full of multi-stresses. Summer mortality causes a serious influence to the aquaculture of bivalves, particularly oysters. Environmental stress and pathogens are known to interact and lead to summer mortality outbreaks. Differentially expressed genes associated with “immune response” biological process were significant up-regulated (Chaney et al., 2011; Fleury et al., 2012). Moreover, genes of oysters associated with cell death and autopahgy would suggest that at least some proportion of genes is symptomatic that underwent “summer mortality” (Chaney et al., 2011). In addition to abiotic stressors, pathogen is an important factor in inducing “summer mortality”. Vibrio splendidus is associated with summer mortality of juvenile oysters (C. gigas) and make juvenile oysters reduce stress-response capacities (Lacoste et al., 2001). Similarly, the effects of vibrio may impair adult oyster immune defenses and cellular and immune functions that characterize the oyster capability to survive V. splendidus infections (De Lorgeril et al., 2011). Ostreid herpesvirus 1 (OsHV-1) infections have been reported around the world and are associated with high mortalities of C. gigas in summer (Segarra et al., 2010; Dégremont 2011). OsHV-1, same as V. splendidus, induced the oysters with significant changes in the expression of immune related genes (Renault et al., 2011). The interesting studies by Chapman et al. (2009, 2011) examined the transcriptomic responses of oysters to environmental stresses and land-use impacts, providing an extension of an earlier assessment of the relative gene expression patterns. Response to environmental stressors, genes encoding electron transport chain are important discriminators for the levels of metals, organic pollutants and nutrients. In addition, a suite of genes involved in the regulation of cell volume and growth, energy metabolism and stresses can also be the indicators of the environmental quality (Chapman et al., 2009, 2011). Through environmental cluster analysis, the environmental pH and the temperature were by far the leading environmental factors governing gene expression patterns with minor contributions of salinity and dissolved oxygen (Chapman et al., 2011). It is noteworthy that there is a strong negative correlation, suggesting genes that are up- regulated by higher temperatures are also up- regulated by lower pH and vice versa (Chapman et al., 2011). The hydrothermal vent mussel survival in such extreme conditions requires unique anatomical and physiological adaptions. It has been reported that they rely on unique capabilities to detect and respond to micro associated molecular patterns such as lipopolysaccharides (LPS), lipoteichoic acids, lipoproteins, peptidoglycan (PGN) and (1→3) β-D-glucans (Bettencourt et al., 2010). Under controlled hyperbaric pressure, genes of B. azoricus relative to heavy metal contaminants and oxidative stress differentially expressed, and the occurrence of glycosylation was changing with the elevanted hyperbaric pressure (Bettencourt et al., 2011). Furthermore, B. azoricus survives in reducing environments rich in methane and sulfides, owing to symbiotic association with methylotrophic or methanotrophic and thiotrophic bacteria (Egas et al., 2012). Enzymes involved in sulfur and methane oxidation have been found, but the molecular pathways underlying sulfur and methane oxidation within the hydrothermal vent mussel had no sufficient evidence (Egas et al., 2012). At “sea-level” condition, B. azoricus can be used a model organism to explore more information. The main studies focused on the transcriptomic response to stress in marine bivalves are summarized in Table 1. The transcriptomic approach Suppression subtractive hybridization (SSH) SSH is a PCR-based technique that allows the identification of genes that differentially expressed between two conditions (Diatchenko et al., 1996). Since this technology came to being in 1984 (Lamar et al., 1984), it has experienced several improvements to be more accurate and easier. Until 1996, SSH application has been maturation (Diatchenko et al., 1996; Diatchenko et al., 1999). This application is a powerful tool for the study of differential gene expression and the identification of   87 Table 1 Main studies related to transcriptomic response to stress in marine bivalve Study Stress Bivalve species Strategy Tanguy et al., 2004 Perkinsus marinus Crassostrea virginica and Crassostrea gigas SSH and real-time PCR on heamocytes Brown et al., 2006 benzo[a]pyrene Mytilus edulis SSH and macroarray on digestive glands of control and experimentally contaminated mussels Dondero et al., 2006 copper pollution gradient M. edulis Microarray and real-time PCR on digestive gland of experimental contaminated mussels Venier et al., 2006 metals and chemicals Mytilus galloprovincialis Microarray and real-time PCR on gills, digestive gland, muscles and mantle of naturally and experimentally contaminated mussels Gestal et al., 2007 mix of dead bacteria Ruditapes decussatus SSH on heamocytes of oysters exposed to the mix of dead bacteria Masson et al., 2007 atrazine M. edulis monitoring the gene of Dnak-type molecular chaperone Chapman et al., 2009 land-use influences C. virginica microarray on gills and hepatopancreas of oysters lived in 11 creeks along the Atlantic coast of the southeastern USA Green et al., 2009 hypoxia Saccostrea glomerata SSH on haemocytes, monitoring the expression of genes encoding anti-oxidant enzymes Lang et al., 2009 heat stress C. gigas Microarray and real-time PCR on gills of oysters exposed to high temperature Prado-Alvarez et al., 2009 Perkinsus olseni R. decussatus SSH on heamocytes exposed to perkinsus olseni in different time point Wang et al., 2009 Vibrio alginolyticus Pinctada fucata monitoring the expression of HSP 70 in the heamocytes of oysters responding to bacterial challenge Zapata et al., 2009 copper pollution gradient Argopecten purpuratus SSH and real-time PCR on post-larvae of scallop Bettencourt et al., 2010 conditions of the hydrothermal vent field Bathymodiolus azoricus RNA-seq on gills Dondero et al., 2010 nickel and chlorpyrifos M. galloprovincialis Microarry and real-time PCR on digestive gland of mussels exposed to nickel, chlorpyrifos and the mix (nickel and chlorpyrifos) Lockwood et al., 2010 osmotic stress M. galloprovincialis and Mytilus trossulus Microarray on gills of two species mussels Sussarellu et al., 2010 hypoxia C. gigas Microarray and real-time PCR on the digestive gland Wang et al., 2010 Perkinsus marinus C. virginica Microarray and real-time PCR on gills Bettencourt et al., 2011 hydrostatic pressure B. azoricus real-time PCR on selected genes of gills and mantles Boutet et al., 2011 environmental factors of the hydrothermal vent field B. azoricus compare differentially expressed genes in gills of two groups, one is rich in methanotrophic bacteria and the other is rich in thiotrophic bacteria using SSH and microarray Chaney et al., 2011 summer mortality C. gigas microarray on heamocytes of survival and mortal oysters Chapman et al., 2011 environmental factors C. virginica microarray on gills and hepatopancreas of oysters lived in 11 creeks along the Atlantic coast of the southeastern USA De Lorgeril et al., 2011 Vibrio spp. C. gigas DGE analysis on heamocytes of oysters infected by virulent and avirulent strains Fu et al., 2011 osmotic stress and bacterial challenge Crassostrea hongkongensis monitoring the expression of HSP 90 in the heamocytes of oysters under stresses Ivanina et al., 2011 cadmium and hypoxia C. virginica real-time PCR on selected genes of hepatopancreas Lockwood et al., 2011 heat stress M. galloprovincialis and M. trossulus Microarray on gills of two species mussels Milan et al., 2011 temperature and salinity Ruditapes philippinarum RNA-seq and microarry on individuals of clams exposed to quick changes of temperature of salinity   88 Brulle et al., 2012 Vibrio tapetis R. philippinarum SSH and real-time PCR on heamocytes exposure to V.tapetis and V.splendidus Egas et al., 2012 endosymbionts and free-living deep-sea bacteria B. azoricus RNA-seq on gills Fleury and Huvet, 2012 summer mortality C. gigas Microarray on muscle, gills, gonad of natural mussels in different time points Martín-Gómez et al., 2012 Bonamia spp Ostrea edulis SSH and real-time PCR on heamocytes exposed to Bonamia spp Mello et al., 2012 brevetoxin C. gigas real-time PCR on selected genes following heamocytes exposure to brevetoxin Moreira et al., 2012a V. alginolyticus R. philippinarum and R. decussatus real-time PCR on selected genes following heamocytes exposure to vibrio Moreira et al., 2012b Vibrio anguillarum R. philippinarum RNA-seq on heamocytes of clams exposed to alive and heat-inactivated vibrio Morga et al., 2012 Bonamia spp O. edulis SSH and real-time PCR on heamocytes Place et al., 2012 Pacific tides Mytilus californianus Microarray on gills, muscle and mantle of mussels inhabiting different vertical locations Wang et al., 2012 V. alginolyticus Pinctada martensii SSH and real-time PCR on heamocytes Zhao et al., 2012 osmotic stress C. gigas RNA-seq on gills of oysters exposed to different salinity gradient seawater Choi et al., 2013 hypoxia C. gigas monitoring the effects of hypoxia-inducible factor-alpha on respiratory burst activity Guevelou et al., 2013 hypoxia C. gigas monitoring the expression of AMP-activated protein kinase α Meng et al., 2013 osmotic stress C. gigas RNA-seq on gills of oysters exposed to different salinity gradient seawater Varotto et al., 2013 combined metal salts M. galloprovincialis Microarray and real-time PCR on gills genes involved in specific biological functions, especially in organisms where genomic data are not available (Zhang et al., 2001). In molluscs, most studies aim to identify the molecular basis of the most common pathologies reviewed in Romero et al., 2012. Some studies focused on several genes by SSH-cDNA libraries, such as Hsp90, ubiquitin gene response to osmotic stress and bacterial challenge and two catalase homologs response to bacterial infection and oxidative stress in Crassostrea hongkongensis (Fu et al., 2011; Zhang et al., 2011), a Dnak-type molecular chaperone exposed to atrazine in blue mussels (Masson et al., 2007), and Hsp70 in the haemocytes of pearl oyster responding to bacterial challenge (Wang et al., 2009). Some studies investigated genes of the same categories, and mostly aimed at immune-related genes (Xu et al., 2009; Martín-Gómez et al., 2012; Wang et al., 2012; Gestal et al., 2007) and anti-oxidant genes (Green et al., 2009). With the increase of the EST databases in marine bivalves, studies containing more coverage of transcripts were undertaken, involved transciptome in response to parasites (Tanguy et al., 2004; Prado-Alvarez et al., 2009; Morga et al., 2011), virus (Brulle et al., 2012), and heavy metals (Zapata et al., 2009). The SSH technology is a quick and effective method to distinguish the genes differentially expressed by high specificity and sensitivity. It can isolate dozens of, even hundreds of genes differently expressed and is easy to operation. However, this technology bases on the hybridization, so that the genes that have no or little restriction enzyme cutting site cannot be isolated by the SSH. Microarrays Microarrays are on the basis of abundant genes or gene segments with known sequences. The construction of the numerous libraries has led to a significant increase in the number of ESTs in databases, which contain genes that are modulated in response to environmental stresses and can be used to design probes in microarrays. Microarrays have various applications, including the analysis of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs) (Heller, 2002). In molluscs, microarrays were mostly used on the gene expression profiling of responses to environmental stresses. Lockwood et al. (2010, 2011) analyzed the effects of heat stress and hypo- osmotic stress between invasive and native mussels. The relative studies about gene expression profiles of the oysters exposed to hypoxia (Sussarellu et al., 2010), heat (Lang et al., 2009), and parasites (Wang et al., 2010) were performed. Moreover, microarrays were used to analyze the interactions of multifactor in oysters (Chapman et al., 2009; Manfrin et al., 2010; Chaney et al., 2011; Chapman et al., 2011; Dondero et al., 2011; Fleury et al., 2012). Microarrays were also   89 used to decipher the effects of the environmental factors on gene expression in the deep-sea mussels (Boutet et al., 2011). Owing to the limitation that microarrays have to base on the known sequences, the studies about the marine bivalves with limited gene databases have only concentrated on several species that are important under the commercial point of view. Meanwhile, the relevant researches are so few that the costing of microarrays in marine bivalves is relatively high. The next-generation sequencing technologies The occurrence of the next-generation sequencing technologies is a giant leap in genomic and transcriptomic research. They make large-scale sequencing possible by high-throughput and cost- efficiency (Marguerat et al., 2010). Although these powerful and rapidly evolving technologies have only been available for a couple of years, they are already making substantial contributions to our understanding of genome expression and regulation. There are three main commercially technologies, including Roche (454 Life Sciences), Illumina (Solexa Sequencing Technology), and Applied Biosystems (Life Technologies/APG). The approach to exploit dynamic transcriptomes by the next-generation sequencing technologies termed RNA-seq. The application of these technologies is a fast and efficient approach for gene discovery and enrichment of transcriptomes in non-model organisms. Currently, transcriptomes have been sequenced for various marine bivalves. In relation to the responses to environmental stresses in molluscs, the researches concentrated in oysters to discuss the relative genes and pathways against osmotic stresses and virus infections (De Lorgeril et al., 2011; Zhao et al., 2012; Menge et al., 2013). Furthermore, there were some literatures about the immune system of mussels (Philipp et al., 2012), and clams (Moreira et al., 2012). The next-generation technologies make the sequencing of the non-model organisms possible. With the development and popularization of high- thoughput sequencing technologies, the genomes and transcriptomes of more and more marine bivalves would be sequenced. Conclusion The ecological status of the marine bivalves is always an important advantage to monitor the environmental quality of the intertidal zones. Along with the fast development of technologies and analysis methods, the information of the molluscs genomes increased drastically. The genomic information is the basis for understanding how the mollusks respond to environmental stresses and solving important problems in the bivalve production such as the “summer mortality”. In this minireview, we summarized the recent studies about the transcriptomic response to stress in marine bivalve. Owing to the characteristic of bivalve’s genome and no model organism, the development of the molecular studies made a slow progress in a period. However, advanced technologies bring genome and transcriptome of bivalve new insights and progressive directions. Increasing researchers devote themselves to the mechanisms of bivalve adapted to the complex conditions at molecular level. Nevertheless, we face several problems. Due to the decreasing cost of the next-generation technologies, we need to increase the biological repeats to increase the accuracy of the data. To date, most experiments are based on the laboratory conditions. However, environmental factors interact with each other in the nature. The researches in the future should consider the combination of imitation and the nature. Eventually, transcript levels are only a proxy for protein expression, and cannot be identical completely with protein expression because of post-translational modifications or other reasons. 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