Genomics in Aquaculture Chapter title: Mollusc 1. Life history and biology Molluscs represent a phylum with approximately fifty thousand species that is marked by tremendous diversity from cockles, genus Cerastoderma, to giant squids, genus Architeuthis. Taxonomic classes include bivalves, scaphopods, gastropods, cephalopods, monoplacophorans, polyplacophorans, and aplacophorans (including solenogastres and caudofoveata groups). Figure 1. Mollusca phylum. Font: Tree of life web project, http://tolweb.org/Mollusca/2488. Despite this diversity, key characters of this group are an epithelial derived mantle that can secrete shell(s) or spicules, a foot that develops from body wall muscles, and a radula, a feeding structure that has is not present in bivalves. In this chapter we will focus on those molluscs that make up a significant component on the global aquaculture market. Since prehistory, ponds were used for algae culture and for farming and maintenance of aquatic animals (Farber, 1997). Oysters were yet cultured in Japan, Greece or Rome more than two thousand years ago (Pillay, 1997). Nowadays, molluscan aquaculture contributes a significant proportion of global aquaculture with 15 million tonnes produced in 2012 (Food And Agriculture Organization Of The United States and Food and Agriculture Organization of the United Nations 2014). Specifically, we be characterizing genomic resources and describing how these resources are increasing our understanding of fundamental physiological processes. In considering the role of genomics in molluscan aquaculture and how this field could improve aquaculture production of this group of organisms, it is important to consider the basic hatchery and production techniques. First one must consider the general life history. A majority of species that are farmed are broadcast spawners, meaning the gametes are commonly released into the water where external fertilization occurs. This is the case for most bivalves including mussels, scallops, oysters, and clam as well as for gastropods such as abalone. Cephalopods such as octopus are an emerging aquacultured group (Vidal et al. 2014) are quite different in this respect as internal fertilization is accomplished with a copulatory organ, often a modified arm referred to as a hectocotylus. While there are no known hermaphroditic cephalopod species (Pechenik 2010), many of the other cultured species are, including examples of protandric hermaphrodites (oysters, ie Crassostrea), simultaneous hermaphrodites (scallops, ie Argopecten), and rhythmical consecutive hermaphrodites (oysters ie Ostrea). Larvae that then develop from external fertilization are consequently free-living, and remain in the water on the order of days to weeks where they undergo a metamorphosis. The length of time is going to be dependent on the taxa as well as environmental conditions. During this larval period molluscs development from an embryo to a trochophore. A velum soon appears, which is a ciliated organ used for locomotion, gas exchange, and food collection. At this point the larva is considered a veliger. In gastropods, such abalone, larvae undergo a process known as torsion when a 180 degree twisting of the nervous system and digestive system. Once metamorphosis is complete the body plan is more similar to that of the adult form and the larvae will settle out of the water column where the might attach to a substrate (eg mussels and oysters) or have limited locomotory capabilities (eg abalone, clams, and scallops). The primary diet of these molluscs consists of plankton (bivalves) and macroalgae (gastropods). Figure 2. Mussel (Mytilus galloprovincialis) life cycle. Photographs of larvae courtesy of Antonio Figueras lab. Mollusc aquaculture is different from most finfish aquaculture in that most adults are commonly placed back into open water with intensive culture practices focusing on larval rearing. Often the culture process will start with the “conditioning” of broodstock with the feeding of nutrient rich microalgae usually grown on premises. It is worth pointing out that a significant portion of mollusc aquaculture is in fact algae culture. Once selected broodstock are reproductively mature, spawning and fertilization is initiated by either manipulating environmental conditions (ie temperature, food availability) or in some cases gametes are harvested and controlled crosses carried out. In some instances juveniles that have recently metamorphosed and settled, referred to as seed, are collected from the wild. As embryos develop larvae are grown up through metamorphosis until the juveniles are of sufficient size to move out of the hatchery to either a nursery setting of open water to take advantage of the natural food supply. Adult culture technology varies and includes rope culture (ie mussels), cages / bags (ie oysters), and “planting” in the sediment (ie clams). 2. Genomes: diversity, structure, organization The interest in bivalve genomics has emerged from the late 90s mainly due to the importance of molluscs in aquaculture and fisheries. Genomics focuses on the study of genes and their function. It has changed dramatically from single gene to whole transcriptome approaches. The next big step, the genome sequence is a very powerful tool as it contains the codified information of the biology of every organism. Genomic research is basic to solve specific problems in mollusc aquaculture, including disease control. They can identify RNAs or proteins expressed in response to stress or pathogens and compensate the usual absence of clinical manifestations. Additionally, these tools can help to find genetic markers, which show the relation between genes, environmental factors and physiology. All this new information will increase the success of molluscan aquaculture, facilitating the monitoring of production, activation state of pathogens or the immune status of the animals. First genomic studies in bivalves were conducted using homology cloning. But this approach was not successful because of the limited information on bivalve genes in databases. The next step in the genomic approach was to describe the differentially expressed immune-related genes in bivalves using BAC libraries, EST collections from cDNA or subtractive hybridization libraries (SSH). These methodologies have been used to find gene markers related to pathogen resistance, and have led to a great increase in the number of sequences in databases. These collections were also used to design microarrays. Anyway, the major advance in recent genomic research occurred with the development of next-generation sequencing technologies (NGS). In bivalves, the NGS and the microarray technologies have been applied to investigate different physiological processes (reviewed in Romero et al., 2012 and in Suárez-Ulloa et al., 2013), such as shell deposition and repair, biomineralization, environmental monitoring, reproduction, skewed sex ratios of offspring or adaptation to climatic and pollution factors. In the review by Saavedra and Bachère, 2006, there are some interesting characteristics about the bivalve genome: The DNA content of the haploid bivalve genome could range from 0.65 pg to 5.4 pg, in the middle of the range for the metazoans. Haploid chromosome numbers for bivalves would be between 10 and 23. These chromosomes are very homogeneous in size making difficult cariotyping, which has to be assisted by FISH to obtain accurate results. FISH has also revealed that bivalves are the only invertebrates that carry the vertebrate telomeric sequence. Repetitive long DNA sequences are common in bivalves and some repetitive DNA seem to be related to transposons. Some of these characteristics have been confirmed after the publication of the first bivalve genome, the Pacific oyster (Crassostrea gigas) genome (Zhang et al., 2012). Genomic advances happen in an exponential way. In the next incoming years the amount of available information will allow researchers huge progresses regarding molecular biology of molluscs. Ten years ago the information about mollusc in the genomic databases was about 25,000 nucleotide sequences, nowadays more than 3 million sequences are publicly available in genbank and 2359 SRA projects are accessible. Regarding bivalve nucleotide sequences genbank returned more than 720,000 results, and the SRA repository has 771 entries. That is the result of 10 years of research in mollusc genomics. There are a handful of publicly available draft genomes and numerous transcriptomes and proteomes from cultured molluscs. The California sea hare, Aplysia californica, is the first mollusc to be sequenced. Its genome sequence will be useful in the study of many scientific areas, but it will be best used in the study of the sea hare’s remarkable nervous system. The sea hare whole genome shotgun assembly was produced by the Broad Institute using the Arachne assembler. The Broad Institute has sequenced to 11x coverage resulting in a total genome assembly size of approximately 712 Mb. More information is available at the dedicated web page: https://www.broadinstitute.org/scientific-community/science/projects/mammals-models/vertebrates-invertebrates/aplysia/aplysia-genom/ and in the genome browser: http://128.227.123.35:8889/cgi-bin/hgGateway?org=Sea+hare&db=aplCal1&hgsid=326/. The next mollusc genome to be sequenced was the Lottia gigantea genome (Simakov et al., 2013). This gastropod was chosen because the species is an emerging model in evolution and development, ecology, and conservation. In fact its publication describe the comparison among other animal genomes to investigate the origin and diversification of bilaterians from a genomic perspective. The size of its genome is 348Mb, and the repetitive content represents the 21% of the assembled genome. There were predicted approximately 23,800 genes. The browser of this genome is available in http://genome.jgi.doe.gov/Lotgi1/Lotgi1.home.html/. With respect to mollusc that constitute a significant economic role in global aquaculture production, the Pacific oyster (Crassostrea gigas) has numerous genomic resources including a draft genome. One of the first integrative resources for this species was the GigasDatabase (Fleury et al. 2009) where publically available transcriptomic data such as expressed sequence tags (ESTs) were assemble into contigs and annotated. This resource is still available at the time of writing this chapter (http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.html). This resource greatly increased the rate of scientific discovery in the field of mollusc genomics. In mussel, the annotated ESTs obtained from different origins were gathered in a platform for expression studies, marker validation and genetic linkage analysis, the MytiBase (http://mussel.cribi.unipd.it) (Venier et al., 2009). There is also an available database for Bathymodiolus azoricus (Bettencourt et al., 2010), the DeepSeaVent database (http://transcriptomics.biocant.pt:8080/deepSeaVent). A draft genome was published in 2012 for Crassostrea gigas (Zhang et al 2012; Fang et al 2012). This effort used a short-read sequencing and fosmid pooling strategy that resulted in an assembly with 559 Mb and a scaffold N50 size of 401 kb (Zhang et al 2012). A total of 11969 scaffolds were assembled with more than 90% of the assembly covered by the longest 1670 scaffolds (Zhang et al). A total of 28,027 genes were predicted in this effort with 49 RNA-seq libraries from different developmental stages and different adult organs and 59 libraries sequenced from oysters subjected to various stressors (Zhang et al. 2012). Other general features described with the C. gigas genome include that 36% of the genome was repetitive sequence as well as evidence for active transposable elements. These oyster genome datasets were subsequently incorporated into the EnsemblMetazoa (http://metazoa.ensembl.org/) where the assembly is continually annotated. Only 7658 of the scaffolds have been used in the Ensembl platform, corresponding to 26,101 coding genes. Data can be accessed in numerous manners via the EnsemblMetazoa including via FTP (http://metazoa.ensembl.org/info/website/ftp/index.html) or using Biomar (http://metazoa.ensembl.org/biomart/martview). EnzemblMetazoa provides novel genome features compared to the source data including coordinate information for non-coding RNA and sequence repeats. A pearl oyster (Pinctada fuctata) draft genome has also been sequenced with represents approximately 1150 Mb and 23,257 complete gene models (Takeuchi et al. 2012). Over 800,000 scaffolds were assembled 629 at least 100,000 bp in length. The draft genome assembly and predicted gene products are available at http://marinegenomics.oist.jp/genomes/downloads?project_id=20. The octopus (Octopus bimaculoides) genome has recently been released (Albertin et al., 2015). With more than 300,000 scaffolds and a scaffold N50-length of 470 kb, the genome assembly captures more than 97% of expressed protein-coding genes and 83% of the estimated 2.7 Gb genome size. There are 33,638 protein-coding genes predicted. The unassembled fraction is dominated by high-copy repetitive sequences. Nearly 45% of the assembled genome is composed of repetitive elements. A browser of this genome assembly is available at http://octopus.metazome.net/. The Mytilus galloprovincialis genome is still in progress. The sequencing strategy was similar to this of C. gigas, fosmid pools and short-read sequencing of a wild female sampled in the Ría de Vigo. The actual assembly comprises 22,279 scaffolds and the estimated the genome size is 1.42Gb (personal communication). Species Resource Acc. nº Tissue Reference Crassostrea gigas Transcriptome Hemocytes Gueguen et al. (2003) Transcriptome PRJNA149121; GSM667902; GSM667901 Hemocytes Rosa et al., 2012 Transcriptome PRJNA167099 Gill Zhao et al. (2012) Transcriptome PRJNA198468 Whole body section Jouaux et al., 2013 BAC library Sperm Cunningham et al. (2006) Genome PRJNA70283 Adductor muscle, gill, mantle, gonad Zhang et al. (2012) Epigenome Gill Gavery and Roberts (2010) Proteome Gill Zhang et al. (2014b) Crassostrea virginica Transcriptome PRJNA82611 Hemocytes, gill, digestive gland, adductor muscle, mantle Zhang et al. (2014a) Transcriptome PRJNA248114 Whole juvenile McDowell et al. (2014) Transcriptome PRJNA227565 Gill, adductor muscle, mantle Eierman and Hare (2014) BAC Library Sperm Cunningham et al. (2006) Genome (in progress) Gómez-Chiarri et al., 2015 Mytilus edulis Transcriptome PRJEB2700 Digestive gland, foot, adductor muscle Philipp et al. (2012) Transcriptome PRNJNA182066 Hemocytes Tanguy et al. (2013) Transcriptome PRJNA252953 Larvae Bassim et al. (2014) Transcriptome PRJEB4516 Mantle Freer et al. (2014) Mytilus galloprovincialis Transcriptome PRJNA80091 Hemocytes Rosani et al. (2011) Transcriptome PRJNA230138 Hemocytes, gill, muscle, mantle Moreira et al., 2015 Genome (in progress) PRJNA178783 Mantle Nguyen et al. (2014) Genome (in progress) Mantle Murgarella et al., 2015 Proteome Hemolymph Oliveri et al. (2014) Patinopecten yessoensis Transcriptome PRJNA79873 Larvae; adult adductor muscle, digestive gland, gonad Hou et al. (2011) Transcriptome PRJNA186890 Gill, digestive gland Meng et al. (2013) Genome (in progress) PRJNA253231; PRJNA259405 Metagenome PRJNA242688 Pecten maximus Transcriptomes PRJNA222492 Hemocytes Pauletto et al. (2014) Pinctada fucata Transcriptome PRJDA63487 Pearl sac, mantle Kinoshita et al. (2011) Genome (draft) PRJDB2628 Sperm Takeuchi et al. (2012) Pinctata maxima Transcriptome PRJNA187136 Jones et al. (2011) Transcriptome PRJNA114555 Mantle Gardner et al. (2011) Ruditapes philipinarum Transcriptome SRX100159 Hemocytes Moreira et al., 2012 Transcriptome PRJNA234093; PRJNA234077; PRJNA2329 Octopus bimaculoides Genome PRJNA270931, PRJNA285380 Albertin et al., 2015 Table 1. Summary of genomic studies in molluscs with economic importance in aquaculture. To date there is yet to be a complete mollusc genome (ie chromosome organization) though there are other mollusc species with significant resources that allow for comparative analysis. These include the draft genome of the owl limpet (Lottia gigantea) (Simakov et al. 2013) and California sea hare (Aplysia californica) (https://www.broadinstitute.org/science/projects/mammals-models/vertebrates-invertebrates/aplysia/aplysia-genome-sequencing-project), both gastropods. Regarding Mytilus and Crassostrea genus, there are some studies about their karyotypes showing that 4 different species of mussel have 14 chromosome pairs (Pérez-García et al., 2014), meanwhile 5 different oyster species have 10 pairs (Wang et al., 2004). These works could help to solve the genomic architecture for these species. Figure 3. Mytilus karyotypes. Font: Pérez-García et al., 2014. With the ever increasing advances in DNA sequencing technology it is certain there will be a number of other draft genomes completed (undoubtedly before the publication of this chapter). For example, new genomic resources are expected for Crassostrea virginica(Gómez-Chiarri et al. 2015), Mytilus galloprovincialis(Maria et al. 2014)) and Patinopecten yessoensis (PRJNA253231; PRJNA259405), as showed in table 1. In this table it is also described the recent genomic research regarding the most valuable molluscs in terms of aquaculture interest: oysters, mussels, clams, scallops and octopus. As highlighted by Gómez-Chiarri et al. and Murgarella, et al. there are several challenges to proper and complete assembly of these genomes including the highly polymorphic nature and high percentage of repetitive elements. These features are likely associated with the life history characteristics of marine bivalves, a predominantly cultured mollusc. Several strategies have been tried to avoid these challenges. From the biological point of view, the development of highly inbred lines for these species is a strategy that could be of help. In the C. gigas genome, the use of inbred individuals reduced the polymorphism rate in a 44%. From the bioinformatic point of view long reads sequencing or fosmid libraries and novel assembly algorithms for highly polymorphic genomes are the proposed solutions. But even with this support the issue of polymorphisms and repetitions persist and are difficult to solve. It is also of interest in the mollusc research the genome sequencing of their pathogens. These efforts can be of help in aquaculture, especially for larval survival and adult fitness. Information regarding the genomes of bacterial (ie Vibrio, PRJNA256191, Schreier and Schott, 2014) and protozoan (ie Perkinsus, PRJNA46451 and PRJNA237117) pathogens is increasing each year. Viruses are still a challenge in this field, even so, the genome of the Ostreid herpesvirus-1 is available (PRJNA14552, Davison et al., 2005). Pathogen genomes would allow the identification of molecular variants and mechanisms of virulence, resulting in an increase of the performance in hatcheries and nurseries through specific management tools. Proteomic studies in marine molluscs are limited to the study of functions such as shell formation, reproduction or development (Diz et al., 2012, Diz et al., 2013, Huan et al., 2012, Marie et al., 2012, Marie et al., 2011); and focused on the response to environmental stress as it will be further explained in subsequent sections. 3. Functional and applied aspects Much of what we have learned with regard to genome activity (gene and protein expression) in aquacultured molluscs has centered around fundamental physiological processes that are critical for improving aquaculture production. In this section we will address key aspects of biology with first reviewing how genomic approaches have improved our fundamental understanding, followed by direct comparison of the genomic underpinnings of beneficial traits. Note that here we will be focusing primarily on the expressed part of the genome and not as much on genetic approaches, those that target DNA variation and manipulation. a) Growth and Nutrition Like any agriculture product, growth is an important factor that considered in terms of reaching market size with minimum expenditure of resources. There are numerous aspects with regard to factors contributing to growth with functional genomic studies provide important insight insight into underlying mechanisms. In bivalves, particular Crassostrea gigas, growth heterosis is observed whereby hybrids often grow faster than respective parental lines (Pace et al. 2006). In one of the earliest high-throughput sequencing efforts in bivalves, (Hedgecock et al. 2007) used massively parallel signature sequencing (MPSS) to examine gene expression patterns in two partially inbred and two hybrid larval populations. Previous work had shown that the hybrid oysters have higher feeding rates and feeding efficiencies than inbred oysters (Bayne et al. 1999, Pace et al. 2006) but nothing was known about underlying transcriptomic differences. (Hedgecock et al. 2007) found that approximately 1.5% genes control the phenotypes associated with oyster growth heterosis, and that protein metabolism was involved based gene annotation results. Later work (Meyer and Manahan 2010) also found protein metabolism drives growth variation in a study where using the same genetic families as (Hedgecock et al. 2007), examined 188 candidate genes. In this study, the majority of candidate genes were ribosomal proteins and the authors indicate that given the high metabolic cost of protein synthesis and degradation, inefficiencies in this process could certainly impact energy available for growth (Meyer and Manahan 2010). b) immune function and health According to the FAO, the future of the molluscan production needs to be planned as an integral program that includes aspects such as the creation of a special research plan on molluscan pathologies. The high mortality of bivalves in the larval stage and in juveniles and adults are the main problem in mollusc cultures. Mortalities cause dramatic economic losses to the aquaculture industry. Nevertheless, the knowledge of the immune response in these species is still limited and the fight against the pathologies is based in preventive strategies and the removal of diseased individuals. The molecular basis of the immune response in bivalves has been of great interest in the last decade and many studies have been published about bivalve genomics, reviewd in Saavedra and Bachère, 2006 and Romero et al., 2012. Vertebrate immune system is characterized by an innate unspecific response and acquired response, with memory after a previous contact with the pathogen. Bivalves lack the acquired response in a narrow sense, but in addition of physical barriers such as the shell and the mucus, they have a potent and efficient cellular and humoral innate immune system. Defense cells in bivalve mollusks are the hemocytes. These cells are traditionally classified depending on their morphologic and functional characteristics in granulocytes and hyalinocytes. Hemocytes trigger their defense mechanisms after pathogen recognition and eliminate these cells or foreign particles by phagocyting and destroying them by lysosomal enzymes, Reactive oxigen species (ROS) synthesis or nitric oxide (NO) production. Figure 4. Hyalinocyte and granulocyte of M. galloprovincialis. Font: Carballal et al., 1997. Humoral immunity is mediated by the proteins found in the hemolymph, the hemocytes or both. These proteins are the focus of the omics studies in mollusc immunology. Traditionally, the research in molluscs pathology was mainly based on functional an observational studies such as phagocytosis, ROS/NO production or mortality and histology assays. Then, studies focused in a molecular point of view, and characterization of specific immune-related molecules were performed with techniques such as homology cloning, BAC libraries or Expressed Sequence Tags (EST) collections from Suppression Subtractive Hybridization (SSH). These methods were especially useful in organisms where genomic data were scarce or not available. They facilitated the identification of new genes through the study of differential gene expression after the interaction between the host immune system and different pathogens. In molluscs, the first published library in an immune framework was constructed in C. virginica (Jenny et al., 2002). After that similar studies were performed in oyster, clam or mussel to identify genes related to pathogen infections (ie Vibrio sp and Perkinsus sp) or focused on differential susceptibility to disease (reviewed in Romero et al., 2012). The SSH technology and the information available in the MytiBase led to the detection of a considerable number of antimicrobial peptides (AMPs) in different mollusc species. AMPs are small amphipathic proteins highly conserved thoughout evolution. Their antimicrobial function relies principally on their integration on the pathogen’s membrane, unstabilizing it and generating pores which lyse the cells, but AMPs can also act intracellularly avoiding DNA, protein or cell wall synthesis. AMPs were originally classified in four main groups: defensins, mytilins, myticins and mytimycins (Mitta et al., 2000). Their importance in bivalve immune response lead to a large number of recent publications, such as the various studies carried on myticin C to explain its high diversity (Costa et al., 2009), its antiviral and immunoregulatory properties (Balseiro et al., 2011) and its genomic organization, molecular diversification, and evolution (Vera et al., 2011). There is also a recent study on the variability of the AMPs (Rosani et al., 2011). Even in a group such as AMPs, very studied in the last 15 years, there have been recent discoveries, like the characterization of big defensins and mytimacins (Gerdol et al., 2012); myticusins (Liao et al., 2013) and mytichitins (Qin et al., 2014). More recently, the NGSs technology were the boost that the genomics research in mollusc immunology needed to enrich the databases and explore new tools, such as microarrays and RNAseq to study host pathogen interactions. The European project ReProSeed (http://www.reproseed.eu/), finished in 2013, made an important effort to enrich the databases in sequences of several species of cultured bivalves: oyster (Crassostrea gigas), mussels (Mytilus galloprovincialis and Mytilus edulis), clams (Ruditapes decussatus) and scallop (Pecten maximus). This techniques lead to the identification and characterization of relevant immune-related genes in molluscs. Several immune processes such as apoptosis, the Toll-like signalling pathway and the complement cascade were described for Ruditapes philippinarum, together with a large number of other immune related genes (Moreira et al., 2012). Valuable information about the apoptotic process in Mytilus galloprovincialis was obtained from the MytiBase database by Romero et al. (2011). They were able to identify for the first time the most important molecules involved in apoptosis, as a result 6 different caspase genes were characterized. Transcriptomic studies lead to many publications with the microarrays technology. Microarrays were principally used to study the response to several pathogens of protozoan, bacterial and viral origin wich infect a wide variety of marine molluscs. Perkinsus spp. belongs to a family of protozoan parasites that are associated with mass mortalities. It is the causal agent of the dermo disease. This parasite was used as a challenge in several microarray studies in Crassostrea virginica (Wang et al., 2010), Ruditapes decusattus (Leite et al., 2013) and Ruditapes philippinarum (Romero et al., 2015). All of them used different approaches to study this chronic condition: an end point infection, natural infection and a time course but all found that pathogen recognition (as Perkinsus is an intracellular pathogen these are important genes in the initial stages of the infection for the parasite to be internalized), antimicrobial activity, redox processes and, especially, apoptosis were important processes to fight the infection. Regarding bacterial infections, Vibrio spp. is a universal marine pathogen that has been studied in mollusc hosts such as Mytilus galloprovincialis (Venier et al., 2011), Ruditapes philippinarum (Moreira et al., 2014; Allam et al., 2014) and the disk abalone Haliotis discus discus, a species with small and recent representation in aquaculture (De Zoysa et al., 2011). Although the pathogen species was different and the infection timing differs among studies a general tendency can be followed to fight the acute infection: apoptosis, as in the case of Perkinsus infections, is an important process activated in the first hours after the infection. Recognition molecules interacting with bacterial PAMPs, genes related to signaling pathways and transcription factors are present in all the studied molluscs, as well as cytokines such as LITAF, MIF or IL-17. It is also observed oxidative burst-related genes. Chemotactic and phagocytic behavior of hemocytes is also important in the critical phase of the infection. In the phase of overcoming the infection a predominant down-regulation of gene expression, as well as activation of processes related to wound healing such as biomineralization are observed. Only one study was performed to date to study the interaction between viruses and molluscs, Ostreid herpes virus type 1 (OsHV-1) in Pacific oyster Crassostrea gigas (Jouaux et al., 2013). As well as against Perkinsus and Vibrio infections apoptosis plays an important role to overcome the infection. Immune system has a more limited activity to fight viral infections but cell signaling in the host and its ability to regulate the OsHV-1 genome activity seems to be the key to survival. Indeed, another study with resistant and susceptible C. gigas to summer mortality (a phenomenon resulting from viral and bacterial infection of individuals weakened by abiotic stress and reproduction) (Fleury & Huvet, 2012) shows that the regulation of the NF–κB signaling pathway is key to determine the susceptibility or resistance to summer mortality. These genes are possible candidates to allow marker-aided selection to improve oyster and probably other molluscs culture. RNA-seq studies in molluscs are still scarce but several are available or in progress. The first SOLiD RNA sequencing work was performed in Crassostrea gigas (Gavery & Roberts, 2012) and it demonstrated that this technology was able to generate novel information and identify differentially expressed genes, which is very useful in non-model species. In the next years other approaches in M. chilensis, Octopus vulgaris, Pecten maximus, and Mytilus galloprovincialis were released. These studies covered different areas like SNP identification, yielding 20,306 polymorphisms associated to immune-related genes in M. chilensis (Núñez-Acuña & Gallardo-Escárate, 2013); or tissue characterization in M. galloprovincialis (Moreira et al., 2015) showing the great importance of the immune system in all the tissues and finding new functions like the hematopoietic, antifungal and sensorial functions of mantle. Of course, it was studied the interaction with PAMPs and pathogens. There was generated new information about coccidiosis in octopus, which revealed genes involved in NF-kB and TLR pathways and in the complement cascade (Castellanos-Martínez et al., 2014), there were identified at least four TLRs in scallop hemocyte transcriptome (Pauletto et al., 2014), and new virulence factors in Perkinsus (Pales et al., 2014). c) Reproduction As indicated above the aquaculture production cycle starts with the reproductive maturation of broodstock which will lay the foundation for larval production and subsequent adult harvest. From functional genomics perspective, there has been deserved attention towards understanding processes associated with reproductive maturation and spawning. Having a better understanding reproductive biology has the potential and promise to increase efficiency and output. While there have been numerous efforts targeting candidate gene function during reproduction, one example of a global approach to examining gametogenesis in an aquaculture relevant species was Dhielly and colleagues (2012) (Dheilly et al. 2012) study using a microarray platform in Crassostrea gigas. This oligonucleotide microarray was composed of 31,918 expressed sequence tags (ESTs) (Dheilly et al. 2011). In comparing males and females Dhielly et al identified 77 genes that were sex specific, which could have practical application in identifying broodstock. For example, there are several aquacultured mollusc where sexual dimorphism is lacking. Gene or possibly protein expression could be used to distinguish sexes as well as developmental status. More recently there have been numerous studies that examine reproductive genes other molluscs ( ie (de Sousa et al. 2014, Teaniniuraitemoana et al. 2014, 2015, Valenzuela-Muñoz et al. 2014). Taken together we now have a more complete understanding gametogenesis in commercially important. Primordial germ cells are the cells which development into gametes, which go onto to create a new individual through sexual reproduction. The establishment of the germline varies across taxa with a general difference of either being specified via inherited factors (preformation) or by inductive signals (epigenesis) (see (Extavour and Akam 2003) for review). In several molluscs it is not clear (or varies) on the origin of the germline. Using a vasa-like gene as a marker in Crasssostrea gigas, (Fabioux et al. 2004) concluded that in C. gigas that the germline is specified by maternal cytoplasmic determinants (preformation) through the 4d cell lineage and larvae possess putative PGCs. Also using vasa gene expression (Kranz et al. 2010) inferred that in the Abalone (Haliotis asinina) that PGCs are not determined completely by maternal determinants, but also by inductive signals. The germ cell eventually become sexually differentiated and enter gametogenesis. As indicated in the above section, sexual reproduction can be quite complex in this group different temporal degrees of hermaphroditism and sex change. A recent study by (Zhang et al. 2014) used transcriptome sequencing to identify genes in sex-determining pathways. These gonia then go through oogenesis and spermatogenesis and ripen into mature ova and spermatozoa. Gametogenesis in many marine invertebrates, is a substantial energy demanding process as these organisms have high fecundity. Fecundity and subsequent larval survival is key concern in aquaculture as this serves as the base of production. As indicated the lability of sex in molluscs is complex (and fascinating) and functional genomic approaches are actively being used in the better understand this. As with many aspects of physiology this includes attention to how large scale environmental change might influence reproductive biology. The first stage of gametogenesis (Stage 0) is the initial differentiation of primordial germ cells where the sex cannot be determined. The next stage (Stage 1) is when germ cells undergo mitosis and a large number of gonia are produced. Stage 2 gonads contain maturing cells that depend on energy from surrounding tissues. Stage 3 gonads are considered fully mature. Using a microarray platform to study the gonadal maturation process in males (spermatogenesis) and females (oogenesis) (Dheilly et al. 2012) identified a suite of genes indicative of the respective processes. In mature male gonads, there was a number of genes involved in ubiquitination and and proteasomal degradation of ubiquitinated proteins. In females, researchers found evidence of elevated cell cycle activity in maturing gonads and interestingly found Methyl-CpG binding domain protein 2 to be highly expressed in mature female gonads. Methyl-CpG binding domain protein 2 is involved in binding methylated DNA and thus presumably involved in gene regulatory activity. (Dheilly et al. 2012) suggested that the high expression of methyl-CpG binding domain protein indicated a epigenetic transfer of information. This process could have important implication for aquaculture production as it would indicate parental factors (ie maternal RNA) has direct impact on larval development, thus broodstock physiology is important to consider. Maternal factors are considered important for fertilization and embryonic genome activation (Li et al. 2010). The maternal contribution is also receiving attention in the aquaculture production of teleost fish (ie (Sullivan et al. 2015)). (Dheilly et al. 2012) also found a number of genes associated with early embryological development (ie Forkhead box Q2, Frizzled) Focusing on another important aquaculture species, the European clam (Ruditapes decussatus), (de Sousa et al. 2015) used a microarray to identify potential biomarkers of oocyte quality, also using D-larval yield as a metric. From this work it was determined that chaperones molecules are main determinants of good quality oocytes and genes involved in stress response, including apoptosis, are associated with poor oocyte quality. Proteomic approaches are increasingly becoming important tools to ass gamete and xx quality, particularly for species with abundant transcriptomic resources necessary for annotation. Using a 2-DE proteomic approach (Corporeau et al. 2012) identified factors indicative of oocyte quality. Specifically oocyte quality was determined by D-larva yields. In low quality oocytes, there was a higher expression of 10 proteins, including five vitellogenins proteins some of which were breakdown products. It was suggested that cleavage products could be an indication of resorbtion and or oocyte aging. In high quality oocytes, again vitellogenin was identified as well as molecular chaperones and protein involved in cell cycle control. In an analogous study that set out to identify proteins associated with mature spermatozoa (Kingtong et al. 2013) used 2-DE proteomics to compare mature sperm with germ / less mature cells. Here researchers identified 31 differentially expressed proteins with mature spermatozoa having increased expression of proteins associated with flagellum, energy production, and acrosome reaction. Researchers proposed that the suite of proteins identified would be good biomarkers for spermatozoa in Pacific oysters. This use of biomarkers would be particularly useful in this species as the Pacific oyster is one organism where gonad stripping (dissection) is commonly used for direct, controlled breeding. 4. Future directions It is evident that molluscan aquaculture will be faced with challenges associated with an increasing human population including both feeding said population as well as persisting in a changing environment. Functional genomic research will play an important role on both of these fronts. There is still a considerable amount fundamental biology that we do not fully understand regarding the ability of molluscs to adapt to changing environment. There have been a considerable number of studies that have examined acute impacts of temperature gradients as well as ocean acidification on molluscs (Wang et al., 2015). While generally these studies project a unfavorable impact on aquaculture, there are a limited number of studies that suggest some species will not be significantly impacted (maybe? REFS). In order to get a better idea of how commercially important species will respond to a changing environment, more attention should be paid to the capacity for an evolutionary response as opposed to a within generation response. From a practical perspective this refers to the possibility that on one hand an acute exposure to predicted future environmental conditions might result in significant mortality, while from an evolutionary perspective those individuals that survive could contribute to more robust broodstock. Functional genomic research efforts will play an central role in evaluating population responses, determining mechanisms, and providing information for modelling responses under different scenarios. Specifically this will include the linking genotypes and epigenotypes to phenotype (including interactive effects) and measuring the inherent genomic variation that occurs across generations. The latter is something that is not necessarily considered in the context of aquaculture production as the livestock counterparts (ie cattle, pigs, sheeps) have a considerably different life history strategies. Aquacultured molluscs can produce millions of offspring and whether an advantageous trait and/or a result of excessive gametogenesis, there evidence that genomic variation occurs at the nucleotide and transposable element level. Given the number of offspring produced at each spawning event and the propensity for variation, genomic tools that can quantify variation, selection, and mutations will be critical to understanding how to optimize aquaculture practices and predict yield. Although the genomes, transcriptomes and proteomes for marine molluscs are scarce compared to those for vertebrates, especially the model organisms, the new, low-cost, high-throughput sequencing technologies were able to encourage marine researchers to immerse in this new approach to better understand mollusc biology. As a result, many works have been recently published. This is an indicator of the high importance of these animals in diverse scientific fields like gene characterization (Saranya et al., 2012; Maldonado-Aguayo et al., 2014; Niu et al., 2014), genomics, transcriptomics and proteomics, toxicity and stress (Suárez-Ulloa et al., 2013; Menike et al., 2014), ontology (Bassim et al., 2014; Balseiro et al., 2013) or immunology. All this work in molluscs is of great interest for scientists and for the related industries. As a consequence of these years of research, the enrichment of the mollusc sequences in the databases and after the release of several genomes, five functional categories specific to molluscan biology, such as "Byssus Formation" and "Shell Formation", have been added to the Gene Ontology database (Kawashima et al., 2013). The resulting data will serve as a useful reference for future genomic analyses in related species. But this is only the beginning, and in the years to come new genomes, as well as new GO categories will further enrich the databases, as the researchers demand them. Currently, the knowledge about the molecular and cellular mechanisms involved in the physiological processes of interest in aquaculture (reproduction, growth, immunity) is rapidly increasing thanks to the genomic research. The next challenge is to find the specific functional roles of all these new genes and proteins, and to curate the possible issues in sequencing, mapping and assembly or annotation. The number of genomic studies is rising every year but, in contrast, the identification and characterization of proteins of interesting pathways is not accompanying these findings. A strong effort focused on the characterization of genes and proteins is needed, and will be facilitated, now that many large transcriptomic databases and some genomes are available. On the other hand, the 'big data' obtained by NGS tends to generate large numbers of errors, requiring monitoring for methodological biases and strategies for replication and validation (Goldman et al., 2014). Despite the recent progress omic tools have enabled in the field of mollusk biology, there is much more work to be done. To date, only a few mollusk genomes have been sequenced completely, but the inaccuracy in assembly is still a problem difficult to solve. The Pacific oyster genome assembly is still ongoing (Hedgecock, 2015) after linkage maps detected errors in the genome. As other researchers have pointed before, more genomes need to be sequenced. Model species studies have proved that comparisons between related species contain many times more inferential power than possible from analysis of one genome alone. With the information of all these fonts it is possible to identify quantitative trait locus (QTL). A QTL is a section of DNA that correlates with variation in a phenotype (the quantitative trait). QTLs are usually linked to genes that control that phenotype and they are extensively used in fish aquaculture. Marker-assisted selection has the potential to improve the efficiency of selection but this technology has not been used in mollusc aquaculture yet. Other ways to use genetic markers are the comparative studies of the divergence of quantitative traits and neutral molecular markers, known as QST–FST comparisons. This application can provide means for researchers to distinguish between natural selection and genetic drift as causes of population differentiation in complex polygenic traits (Leinonen et al., 2013). Methods based on the QST–FST approach could be used to analyze various types of omics data in new and revealing ways. In the near future, interesting possibilities for bivalve research are epigenetics or ribosome profiling. Epigenetics is an almost unexplored technique regarding mollusc biology. This branch of science only has been applied to bivalves in the case of the Pacific oyster (Crassostrea gigas) (Gavery and Roberts, 2014; Olson and Roberts, 2014). DNA methylation has regulatory functions in C. gigas, particularly in gene families that have inducible expression, including those involved in stress, environmental responses and male gamete tissue gene regulatory activity. Ribosome profiling (Ingolia et al., 2009) is a method that reflects the translation process of specific tissues and developmental stages or conditions. As a result, it was suggested to estimate the translation efficiency of genes as well as the interaction between translation initiation, elongation, and termination. Expression and sequencing tools alone cannot explain critical aspects of gene expression regulation. This technique fills the gap between gene expression and protein quantification. In combination with methods such as RNA immunoprecipitation, miRNA profiling or proteomics, it is possible to get a new point of view of post-transcriptional and translational gene regulation. In addition, these techniques also provide new insight into new regulatory elements, such as alternative open reading frames and translation regulation under different conditions. A constant effort in all the omics disciplines together will improve our knowledge in genomes, metagenomes, epigenomes, transcriptomes, proteomes, and metabolomes of many diverse mollusc species. The analysis of all this information will result in a better understanding of mollusc physiology, and eventually it can be translated into a better management of these commercially and ecologically important species. The future of all the expression and sequencing techniques is exciting and promising. These tools have never stopped their technical improvement and science is always finding new applications and updates to solve the infinite questions that the new discoveries set out. Concluding remarks Molluscs are an interesting group, not only in terms of aquaculture production but also because they have an important ecological role in the depuration of waters and as environmental sentinels. Their sedentary life and filter-feeding behavior leads to the bioaccumulation of pollutants, which makes them ideal species for research in immunology and toxicology (Wootton et al., 2003; Campos et al., 2012). Additionally, this group includes intermediate hosts for serious parasitic human diseases like schistosomiasis (Morgan et al., 2001). On the other hand, the presence of toxins of phytoplanktonic origin in molluscs, a direct effect of red tides, is the cause of several diseases in humans after consumption (Bricelj and Shumway, 1998) and they are known to alter the immune response in molluscs (Rijcke et al., 2015). The recent use of new sequencing technologies has drastically increased the number of mollusc genomic sequences in the databases. These sequences are the basis for understanding physiological processes and the immune response against diseases and for solving problems in the bivalve industry. Genomic information will help find specific answers to a number of questions: Can this information be used to discover specific genes linked to resistance, growth, fitness? Can these conditions be enhanced or manipulated by stimulating specific responses? Can the interesting individuals be selected, and will these characteristics be maintained and passed to descendants? Will this information open alternatives to selection? Which is the influence of the environmental factors? And finally, of critical importance, is this information a priority to the mollusc industry? Summarizing, the study of molluscs from the point of view of the molecular biology, genomics and proteomics covers not only the intensive aquaculture production, this research has also ecological and health interests. Additionally, the study of the larval stages at cellular and molecular levels, diet design and growth, as well as factors related to disease resistance, stress or pollution are aspects of bivalve physiology that can be studied using genomic techniques. REFERENCES Bayne, B.L., Hedgecock, D., McGoldrick, D., and Rees, R., 1999. Feeding behaviour and metabolic efficiency contribute to growth heterosis in Pacific oysters [Crassostrea gigas (Thunberg)]. Journal of experimental marine biology and ecology, 233 (1), 115–130. Corporeau, C., Vanderplancke, G., Boulais, M., Suquet, M., Quéré, C., Boudry, P., Huvet, A., and Madec, S., 2012. Proteomic identification of quality factors for oocytes in the Pacific oyster Crassostrea gigas. Journal of proteomics, 75 (18), 5554–5563. Dheilly, N.M., Lelong, C., Huvet, A., and Favrel, P., 2011. Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns. BMC genomics, 12, 468. Dheilly, N.M., Lelong, C., Huvet, A., Kellner, K., Dubos, M.-P., Riviere, G., Boudry, P., and Favrel, P., 2012. Gametogenesis in the Pacific oyster Crassostrea gigas: a microarrays-based analysis identifies sex and stage specific genes. PloS one, 7 (5), e36353. Extavour, C.G. and Akam, M., 2003. Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development , 130 (24), 5869–5884. Fabioux, C., Huvet, A., Lelong, C., Robert, R., Pouvreau, S., Daniel, J.Y., Minguant, C., and Le Pennec, M., 2004. Oyster vasa-like gene as a marker of the germline cell development in Crassostrea gigas. Biochemical and biophysical research communications, 320 (2), 592–598. Fleury, E., Huvet, A., Lelong, C., de Lorgeril, J., Boulo, V., Gueguen, Y., Bachere, E., Tanguy, A., Moraga, D., Fabioux, C., Lindeque, P., Shaw, J., Reinhardt, R., Prunet, R., Davey, G., Lapegue, S., Sauvage, C., Corporeau, C., Moal, J., Gavory, F., Wincker, P., Moreews, F., Klopp, C., Mathieu, M., Boudry, P., and Favrel, B., 2009. Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase. BMC genomics, 10, 341. Food And Agriculture Organization Of The United States and Food and Agriculture Organization of the United Nations, 2014. State of the World Fisheries and Aquaculture 2014. Fao. Gómez-Chiarri, M., Marta, G.-C., Warren, W.C., Ximing, G., and Dina, P., 2015. Developing tools for the study of molluscan immunity: The sequencing of the genome of the eastern oyster, Crassostrea virginica. Fish & shellfish immunology, 46 (1), 2–4. Hedgecock, D., Lin, J.-Z., DeCola, S., Haudenschild, C.D., Meyer, E., Manahan, D.T., and Bowen, B., 2007. Transcriptomic analysis of growth heterosis in larval Pacific oysters (Crassostrea gigas). Proceedings of the National Academy of Sciences of the United States of America, 104 (7), 2313–2318. Kingtong, S., Kellner, K., Bernay, B., Goux, D., Sourdaine, P., and Berthelin, C.H., 2013. Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas. Journal of proteomics, 82, 81–91. Kranz, A.M., Tollenaere, A., Norris, B.J., Degnan, B.M., and Degnan, S.M., 2010. Identifying the germline in an equally cleaving mollusc: Vasa and Nanos expression during embryonic and larval development of the vetigastropod Haliotis asinina. Journal of experimental zoology. Part B, Molecular and developmental evolution, 314 (4), 267–279. Li, L., Zheng, P., and Dean, J., 2010. Maternal control of early mouse development. Development , 137 (6), 859–870. Maria, M., André, C., Tyler, A., Beatriz, N., Antonio, F., David, P., and Carlos, C., 2014. Genomic characterization of the aquaculture resource Mytilus galloprovincialis. Frontiers in Marine Science, 1. Meyer, E. and Manahan, D.T., 2010. Gene expression profiling of genetically determined growth variation in bivalve larvae (Crassostrea gigas). The Journal of experimental biology, 213 (5), 749–758. Pace, D.A., Marsh, A.G., Leong, P.K., Green, A.J., Hedgecock, D., and Manahan, D.T., 2006. Physiological bases of genetically determined variation in growth of marine invertebrate larvae: A study of growth heterosis in the bivalve Crassostrea gigas. Journal of experimental marine biology and ecology, 335 (2), 188–209. Pechenik, J.A., 2010. Biology of the Invertebrates. McGraw-Hill Higher Education. Simakov, O., Marletaz, F., Cho, S.-J., Edsinger-Gonzales, E., Havlak, P., Hellsten, U., Kuo, D.-H., Larsson, T., Lv, J., Arendt, D., Savage, R., Osoegawa, K., de Jong, P., Grimwood, J., Chapman, J.A., Shapiro, H., Aerts, A., Otillar, R.P., Terry, A.Y., Boore, J.L., Grigoriev, I.V., Lindberg, D.R., Seaver, E.C., Weisblat, D.A., Putnam, N.H., and Rokhsar, D.S., 2013. Insights into bilaterian evolution from three spiralian genomes. Nature, 493 (7433), 526–531. De Sousa, J.T., Milan, M., Bargelloni, L., Pauletto, M., Matias, D., Joaquim, S., Matias, A.M., Quillien, V., Leitão, A., and Huvet, A., 2014. A microarray-based analysis of gametogenesis in two Portuguese populations of the European clam Ruditapes decussatus. PloS one, 9 (3), e92202. De Sousa, J.T., Milan, M., Pauletto, M., Bargelloni, L., Joaquim, S., Matias, D., Matias, A.M., Quillien, V., Leitão, A., and Huvet, A., 2015. A microarray-based analysis of oocyte quality in the European clam Ruditapes decussatus. Aquaculture , 446, 17–24. Sullivan, C.V., Chapman, R.W., Reading, B.J., and Anderson, P.E., 2015. Transcriptomics of mRNA and egg quality in farmed fish: Some recent developments and future directions. General and comparative endocrinology. Teaniniuraitemoana, V., Huvet, A., Levy, P., Gaertner-Mazouni, N., Gueguen, Y., and Le Moullac, G., 2015. Molecular signatures discriminating the male and the female sexual pathways in the pearl oyster Pinctada margaritifera. PloS one, 10 (3), e0122819. Teaniniuraitemoana, V., Huvet, A., Levy, P., Klopp, C., Lhuillier, E., Gaertner-Mazouni, N., Gueguen, Y., and Le Moullac, G., 2014. Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes. BMC genomics, 15, 491. Valenzuela-Muñoz, V., Bueno-Ibarra, M.A., and Escárate, C.G., 2014. Characterization of the transcriptomes of Haliotis rufescens reproductive tissues. Aquaculture research, 45 (6), 1026–1040. Vidal, E.A.G., Villanueva, R., Andrade, J.P., Gleadall, I.G., Iglesias, J., Koueta, N., Rosas, C., Segawa, S., Grasse, B., Franco-Santos, R.M., Albertin, C.B., Caamal-Monsreal, C., Chimal, M.E., Edsinger-Gonzales, E., Gallardo, P., Le Pabic, C., Pascual, C., Roumbedakis, K., and Wood, J., 2014. Cephalopod culture: current status of main biological models and research priorities. Advances in marine biology, 67, 1–98. Zhang, N., Xu, F., and Guo, X., 2014. Genomic analysis of the Pacific oyster (Crassostrea gigas) reveals possible conservation of vertebrate sex determination in a mollusc. G3 , 4 (11), 2207–2217. Albertin CB, Simakov O, Mitros T, Wang ZY, Pungor JR, Edsinger-Gonzales E, Brenner S, Ragsdale CW, Rokhsar DS. The octopus genome and the evolution of cephalopod neural and morphological novelties. Nature. 2015; 524: 220-224. Allam B1, Pales Espinosa E2, Tanguy A3, Jeffroy F4, Le Bris C4, Paillard C4. Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease. Fish Shellfish Immunol. 2014 Nov;41(1):2-11 Balseiro P, Falcó A, Romero A, Dios S, Martínez- López A, Figueras A, et al. Mytilus galloprovincialis myticin C: a chemotactic molecule with antiviral activity and immunoregulatory properties. PLoS One. 6: e23140, 2011. Bassim S, Genard B, Gauthier-Clerc S, Moraga D, Tremblay R. Ontogeny of bivalve immunity: assessing the potential of next-generation sequencing techniques. Reviews in Aquaculture 2014, 6: 1–21.Balseiro P1, Moreira R, Chamorro R, Figueras A, Novoa B. Immune responses during the larval stages of Mytilus galloprovincialis: metamorphosis alters immunocompetence, body shape and behavior. Fish Shellfish Immunol. 2013 Aug;35(2):438-47. Bassim, S., Tanguy, A., Genard, B., Moraga, D., Tremblay, R. Identification of Mytilus edulis genetic regulators during early development. Gene , 2014.551, 65–78. Bayne, B.L., Hedgecock, D., McGoldrick, D., and Rees, R., 1999. Feeding behaviour and metabolic efficiency contribute to growth heterosis in Pacific oysters [Crassostrea gigas (Thunberg)]. Journal of experimental marine biology and ecology, 233 (1), 115–130. Bettencourt R1, Pinheiro M, Egas C, Gomes P, Afonso M, Shank T, Santos RS. High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus. BMC Genomics. 2010 Oct 11;11:559. Campos A, Tedesco S, Vasconcelos V, Cristobal S. Proteomic research in bivalves: towards the identification of molecular markers of aquatic pollution. J Proteomics. 2012; 75:4346-4359. Corporeau, C., Vanderplancke, G., Boulais, M., Suquet, M., Quéré, C., Boudry, P., Huvet, A., and Madec, S., 2012. Proteomic identification of quality factors for oocytes in the Pacific oyster Crassostrea gigas. Journal of proteomics, 75 (18), 5554–5563. Castellanos-Martínez S, Arteta D, Catarino S, Gestal C. De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana. PLoS One. 2014 Oct 16;9(10):e107873. Costa MM, Dios S, Alonso-Gutierrez J, Romero A, Novoa B, Figueras A. Evidence of high individual diversity on myticin C in mussel (Mytilus galloprovincialis). Dev. Comp. Immunol. 33: 162-170, 2009. Cunningham, C., Hikima, J., Jenny, M.J., Chapman, R.W., Fang, G.-C., Saski, C., Lundqvist, M.L., Wing, R.A., Cupit, P.M., Gross, P.S., Warr, G.W., Tomkins, J.P., 2006. New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas). Mar. Biotechnol. 8, 521–533. Davison, A.J., Trus, B.L., Cheng, N., Steven, A.C., Watson, M.S., Cunningham, C., Deuff, R.-M.L., Renault, T., 2005. A novel class of herpesvirus with bivalve hosts. J. Gen. Virol. 86, 41–53. http://dx.doi.org/10.1099/vir.0.80382-0. De Sousa, J.T., Milan, M., Bargelloni, L., Pauletto, M., Matias, D., Joaquim, S., Matias, A.M., Quillien, V., Leitão, A., and Huvet, A., 2014. A microarray-based analysis of gametogenesis in two Portuguese populations of the European clam Ruditapes decussatus. PloS one, 9 (3), e92202. De Sousa, J.T., Milan, M., Pauletto, M., Bargelloni, L., Joaquim, S., Matias, D., Matias, A.M., Quillien, V., Leitão, A., and Huvet, A., 2015. A microarray-based analysis of oocyte quality in the European clam Ruditapes decussatus. Aquaculture , 446, 17–24. De Zoysa M, Nikapitiya C, Oh C, Lee Y, Whang I, Lee JS, Choi CY, Lee J. Microarray analysis of gene expression in disk abalone Haliotis discus discus after bacterial challenge. Fish Shellfish Immunol. 2011 Feb;30(2):661-73. Dheilly, N.M., Lelong, C., Huvet, A., and Favrel, P., 2011. Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and Dheilly, N.M., Lelong, C., Huvet, A., Kellner, K., Dubos, M.-P., Riviere, G., Boudry, P., and Favrel, P., 2012. Gametogenesis in the Pacific oyster Crassostrea gigas: a microarrays-based analysis identifies sex and stage specific genes. PloS one, 7 (5), e36353. Diz, A.P., Dudley, E., Cogswell, A., MacDonald, B.W., Kenchington, E.L.R., Zouros, E., Skibinski, D.O.F., 2013. Proteomic analysis of eggs from Mytilus edulis females differing in mitochondrial DNA transmission mode. Mol. Cell. Proteomics 12, 3068–3080. Diz, A.P., Dudley, E., Skibinski, D.O.F., 2012. Identification and characterization of highly expressed proteins in sperm cells of the marine mussel Mytilus edulis. Proteomics 12, 1949–1956. Eierman, L.E., Hare, M.P. Transcriptomic analysis of candidate osmoregulatory genes in the eastern oyster Crassostrea virginica. BMC Genomics , 2014.15, 503. Extavour, C.G. and Akam, M., 2003. Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development , 130 (24), 5869–5884. Fabioux, C., Huvet, A., Lelong, C., Robert, R., Pouvreau, S., Daniel, J.Y., Minguant, C., and Le Pennec, M., 2004. Oyster vasa-like gene as a marker of the germline cell development in Crassostrea gigas. Biochemical and biophysical research communications, 320 (2), 592–598. Farber JM. Ancient Hawaiian fishponds: can restoration succeed on Molokaʻi? Cornell University. First Edition. Neptune House Publications (California). 1997. Fleury E, Huvet A. Microarray analysis highlights immune response of pacific oysters as a determinant of resistance to summer mortality. Mar Biotechnol (NY). 2012 Apr;14(2):203-17. Fleury, E., Huvet, A., Lelong, C., de Lorgeril, J., Boulo, V., Gueguen, Y., Bachere, E., Tanguy, A., Moraga, D., Fabioux, C., Lindeque, P., Shaw, J., Reinhardt, R., Prunet, R., Davey, G., Lapegue, S., Sauvage, C., Corporeau, C., Moal, J., Gavory, F., Wincker, P., Moreews, F., Klopp, C., Mathieu, M., Boudry, P., and Favrel, B., 2009. Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase. BMC genomics, 10, 341. Freer, A., Bridgett, S., Jiang, J., Cusack, M. Biomineral proteins from Mytilus edulis mantle tissue transcriptome. Mar. Biotechnol. , 2014.16, 34–35. Food And Agriculture Organization Of The United States and Food and Agriculture Organization of the United Nations, 2014. State of the World Fisheries and Aquaculture 2014. Fao. Gardner, L., Mills, D., Wiegand, A., Leavesley, D., Elizur, A. Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima. BMC Genomics , 2011.12, 455. Gavery MR, Roberts SB. A context dependent role for DNA methylation in bivalves. Brief Funct Genomics. 2014; 13: 217-222. Gavery MR, Roberts SB. Characterizing short read sequencing for gene discovery and RNA-Seq analysis in Crassostrea gigas. Comp Biochem Physiol Part D Genomics Proteomics. 2012;7:94-9. Gavery, M.R., Roberts, S.B., 2010. DNA methylation patterns provide insight into epigenetic regulation in the Pacific oyster (Crassostrea gigas). BMC Genomics 11, 483. Gerdol M, De Moro G, Manfrin C, Venier P, Pallavicini A. Big defensins and mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis. Dev. Comp. Immunol. 36: 390-399, 2012. Goldman D, Domschke K. Making sense of deep sequencing. Int J Neuropsychopharmacol. 2014 17: 1717-1725. Gómez-Chiarri, M., Marta, G.-C., Warren, W.C., Ximing, G., and Dina, P., 2015. Developing tools for the study of molluscan immunity: The sequencing of the genome of the eastern oyster, Crassostrea virginica. Fish & shellfish immunology, 46 (1), 2–4. Gueguen, Y., Cadoret, J.-P., Flament, D., Barreau-Roumiguière, C., Girardot, A.-L., Garnier, J., Hoareau, A., Bachère, E., Escoubas, J.-M., 2003. Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas. Gene 303, 139–145. Hedgecock, D., Lin, J.-Z., DeCola, S., Haudenschild, C.D., Meyer, E., Manahan, D.T., and Bowen, B., 2007. Transcriptomic analysis of growth heterosis in larval Pacific oysters (Crassostrea gigas). Proceedings of the National Academy of Sciences of the United States of America, 104 (7), 2313–2318. Hedgecock D. SECOND-GENERATION LINKAGE MAPS REVEAL ERRORS IN THE ASSEMBLY OF THE PACIFIC OYSTER (Crassostrea gigas) GENOME AND FACTORS AFFECTING MAP LENGTHS AND MARKER ORDERS. ISGA XII abstract book, O36, p82. http://www.isga2015.com/isga-2015-Abstract-Book.pdf. Hou, R., Bao, Z., Wang, S., Su, H., Li, Y., Du, H., et al. Transcriptome sequencing and de novo analysis for Yesso scallop (Patinopecten yessoensis) using 454 GS FLX. PLoS One , 2011.6, e21560. Huan, P., Wang, H., Dong, B., Liu, B., 2012. Identification of differentially expressed proteins involved in the early larval development of the Pacific oyster Crassostrea gigas. J. Proteomics 75, 3855–3865. Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009; 324: 218-223. Jenny MJ, Ringwood AH, Lacy ER, Lewitus AJ, Kempton JW, Gross PS, et al. Potential indicators of stress response identified by expressed sequence tag analysis of hemocytes and embryos from the American oyster, Crassostrea virginica. Mar. Biotechnol. 4: 81- 93, 2002. Jones, D., Zenger, K., Jerry, D. In silico whole genome EST analysis reveals 2322 novel microsatellites for the silver-lipped pearl oyster, Pinctada maxima. Mar. Genomics, 2011. 4, 287–290. Jouaux A, Lafont M, Blin JL, Houssin M, Mathieu M, Lelong C. Physiological change under OsHV-1 contamination in Pacific oyster Crassostrea gigas through massive mortality events on fields. BMC Genomics. 2013 Aug 29;14:590. Kawashima T, Takeuchi T, Koyanagi R, Kinoshita S, Endo H, Endo K. Initiating the mollusk genomics annotation community: toward creating the complete curated gene-set of the Japanese Pearl Oyster, Pinctada fucata. Zoolog Sci. 2013; 30: 794-6. Kingtong, S., Kellner, K., Bernay, B., Goux, D., Sourdaine, P., and Berthelin, C.H., 2013. Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas. Journal of proteomics, 82, 81–91. Kinoshita, S., Wang, N., Inoue, H., Maeyama, K., Okamoto, K., Nagai, K., Kondo, H., Hirono, I., Asakawa, S. Deep sequencing of ESTs from nacreous and prismatic layer producing-tissues and a screen for novel shell formation related genes in the Pearl Oyster. , 2011.PLoS One 6, e21238. Kranz, A.M., Tollenaere, A., Norris, B.J., Degnan, B.M., and Degnan, S.M., 2010. Identifying the germline in an equally cleaving mollusc: Vasa and Nanos expression during embryonic and larval development of the vetigastropod Haliotis asinina. Journal of experimental zoology. Part B, Molecular and developmental evolution, 314 (4), 267–279. Leinonen T1, McCairns RJ, O'Hara RB, Merilä J. QST-FST comparisons: evolutionary and ecological insights from genomic heterogeneity. Nat Rev Genet. 2013 Mar;14(3):179-190. Li, L., Zheng, P., and Dean, J., 2010. Maternal control of early mouse development. Development , 137 (6), 859–870. Liao Z1, Wang XC, Liu HH, Fan MH, Sun JJ, Shen W.Molecular characterization of a novel antimicrobial peptide from Mytilus coruscus. Fish Shellfish Immunol. 2013;34(2):610-6. Maldonado-Aguayo W, Teneb J, Gallardo-Escárate C. A galectin with quadruple-domain from red abalone Haliotis rufescens involved in the immune innate response against to Vibrio anguillarum. Fish Shellfish Immunol. 2014; 40: 1-8. Maria, M., André, C., Tyler, A., Beatriz, N., Antonio, F., David, P., and Carlos, C., 2014. Genomic characterization of the aquaculture resource Mytilus galloprovincialis. Frontiers in Marine Science, 1. Marie, B., Joubert, C., Tayalé, A., Zanella-Cléon, I., Belliard, C., Piquemal, D., Cochennec-Laureau, N., Marin, F., Gueguen, Y., Montagnani, C., 2012. Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell. Proc. Natl. Acad. Sci. U.S.A. 109, 20986–20991. Marie, B., Trinkler, N., Zanella-Cleon, I., Guichard, N., Becchi, M., Paillard, C., Marin, F., 2011. Proteomic identification of novel proteins from the calcifying shell matrix of the Manila clam Venerupis philippinarum. Mar. Biotechnol. 13, 955–962. McDowell, I.C., Nikapitiya, C., Aguiar, D., Lane, C.E., Istrail, S., Gomez-Chiarri, M. Transcriptome of American oysters, Crassostrea virginica, in response to bacterial challenge: insights into potential mechanisms of disease resistance. PLoS One , 2014. 9, e105097. Meng, X., Lui, M., Jiang, K., Wang, B., Tian, X., Sun, S., Luo, Z., Qiu, C., Wang, L. De novo characterization of Japanese Scallop, Mizuhopecten yessoensis transcriptome and analysis of its gene expression following cadmium exposure. PLoS One , 2013.8, e6485. Menike U, Lee Y, Oh C, Wickramaarachchi WD, Premachandra HK, Park SC, Lee J, De Zoysa M.Oligo-microarray analysis and identification of stress-immune response genes from manila clam (Ruditapes philippinarum) exposure to heat and cold stresses. Mol Biol Rep. 2014; 41: 6457-6473. Meyer, E. and Manahan, D.T., 2010. Gene expression profiling of genetically determined growth variation in bivalve larvae (Crassostrea gigas). The Journal of experimental biology, 213 (5), 749–758. Mitta G, Vandenbulcke F, Roch P. Original involvement of antimicrobial peptides in mussel innate immunity. FEBS Lett. 2000;486:185-190. Moreira R, Balseiro P, Planas JV, Fuste B, Beltran S, Novoa B, Figueras A.Transcriptomics of in vitro immune-stimulated hemocytes from the Manila clam Ruditapes philippinarum using high-throughput sequencing. PLoS One. 2012;7(4):e35009. Moreira R, Milan M, Balseiro P, Romero A, Babbucci M, Figueras A, Bargelloni L, Novoa B. Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray. BMC Genomics. 2014 Apr 7;15:267. Moreira R, Pereiro P, Canchaya C, Posada D, Figueras A, Novoa B. RNA-Seq in Mytilus galloprovincialis: comparative transcriptomics and expression profiles among different tissues. BMC genomics. 2015, 16: 728. Murgarella M, Corvelo A, Alioto T, Novoa B, Figueras A, Posada D and Canchaya CA. Genomic characterization of the aquaculture resource Mytilus galloprovincialis. Front. Mar. Sci. Conference Abstract: IMMR | International Meeting on Marine Research 2014. doi: 10.3389/conf.FMARS.2014.02.00113 Nguyen TT1, Hayes BJ, Guthridge K, Ab Rahim ES, Ingram BA Use of a microsatellite-based pedigree in estimation of heritabilities for economic traits in Australian blue mussel, Mytilus galloprovincialis. J Anim Breed Genet. 2011 Dec;128(6):482-90 Niu D, Xie S, Bai Z, Wang L, Jin K, Li J. Identification, expression, and responses to bacterial challenge of the cathepsin C gene from the razor clam Sinonovacula constricta. Dev Comp Immunol. 2014; 46: 241-245. Núñez-Acuña G, Gallardo-Escárate C.Identification of immune-related SNPs in the transcriptome of Mytilus chilensis through high-throughput sequencing. Fish Shellfish Immunol. 2013 Dec;35(6):1899-905. Oliveri, C., Peric, L., Sforzini, S., Banni, M., Viarengo, A., Cavaletto, M., Marsano, F. Biochemical and proteomic characterisation of haemolymph serum reveals the origin of the alkali-labile phosphate (ALP) in mussel (Mytilus galloprovincialis). Comp. Biochem. Physiol. D: Genomics Proteomics , 2014. 11, 29–36. Olson CE, Roberts SB. Genome-wide profiling of DNA methylation and gene expression in Crassostrea gigas male gametes. Front Physiol. 2014; 5: 224. Pace, D.A., Marsh, A.G., Leong, P.K., Green, A.J., Hedgecock, D., and Manahan, D.T., 2006. Physiological bases of genetically determined variation in growth of marine invertebrate larvae: A study of growth heterosis in the bivalve Crassostrea gigas. Journal of experimental marine biology and ecology, 335 (2), 188–209. Pales Espinosa E, Corre E, Allam B. Pallial mucus of the oyster Crassostrea virginica regulates the expression of putative virulence genes of its pathogen Perkinsus marinus. Int J Parasitol. 2014 Apr;44(5):305-17. Pauletto M, Milan M, Moreira R, Novoa B, Figueras A, Babbucci M, Patarnello T, Bargelloni L. Deep transcriptome sequencing of Pecten maximus hemocytes: a genomic resource for bivalve immunology. Fish Shellfish Immunol. 2014 Mar;37(1):154-65 Pechenik, J.A., 2010. Biology of the Invertebrates. McGraw-Hill Higher Education. Pérez-García C, Morán P, Pasantes JJ. Karyotypic diversification in Mytilus mussels (Bivalvia: Mytilidae) inferred from chromosomal mapping of rRNA and histone gene clusters. BMC Genet. 2014; 15:84. Philipp, E.E.R., Kraemer, L., Melzner, F., Poustka, A.J., Thieme, S., Findeisen, U., Schreiber, S., Rosenstiel, P. Massively parallel RNA sequencing identifies a complex immune gene repertoire in the lophotrochozoan Mytilus edulis. PLoS One , 2012.7, e33091. Pillay TVR. Economic and social dimensions of aquaculture management. Aquacult Econ Manag 1997; 1: 3-11. Qin CL1, Huang W1, Zhou SQ2, Wang XC1, Liu HH1, Fan MH1, Wang RX1, Gao P1, Liao Z3. Characterization of a novel antimicrobial peptide with chitin-biding domain from Mytilus coruscus. Fish Shellfish Immunol. 2014 Dec;41(2):362-70. Romero A, Estévez-Calvar N, Dios S, Figueras A, Novoa B.New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis. PLoS One. 2011 Feb 11;6(2):e17003 Romero A, Novoa B, Figueras A. Genomics, immune studies and diseases in bivalve aquaculture. Invertebr Surviv J. 2012; 9: 110‐121. Romero A1, Forn-Cuní G1, Moreira R1, Milan M2, Bargelloni L2, Figueras A1, Novoa B3 An immune-enriched oligo-microarray analysis of gene expression in Manila clam (Venerupis philippinarum) haemocytes after a Perkinsus olseni challenge. Fish Shellfish Immunol. 2015 Mar;43(1):275-86. Rosa, R.D., de Lorgeril, J., Tailliez, P., Bruno, R., Piquemal, D., Bachère, E., 2012. A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections. BMC Genomics 13, 252 Rosani U, Varotto L, Rossi A, Roch P, Novoa B, Figueras A, et al. Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis. PLoS ONE 6: e26680, 2011. Saavedra C, and Bachère E. Bivalve genomics. Aquaculture. 2006; 256: 1‐14. Saranya Revathy K, Umasuthan N, Lee Y, Choi CY, Whang I, Lee J.First molluscan theta-class Glutathione S-Transferase: identification, cloning, characterization and transcriptional analysis post immune challenges. Comp Biochem Physiol B Biochem Mol Biol. 2012; 162: 10-23. Schreier, H.J., Schott, E.J., 2014. Draft genome sequence of the shellfish bacterial pathogen Vibrio sp. strain B183. Genome Announc. 2. http://dx.doi.org/10.1128/genomeA.00914-14, e00914–14. Simakov O1, Marletaz F, Cho SJ, Edsinger-Gonzales E, Havlak P, Hellsten U, Kuo DH, Larsson T, Lv J, Arendt D, Savage R, Osoegawa K, de Jong P, Grimwood J, Chapman JA, Shapiro H, Aerts A, Otillar RP, Terry AY, Boore JL, Grigoriev IV, Lindberg DR, Seaver EC, Weisblat DA, Putnam NH, Rokhsar DS. Insights into bilaterian evolution from three spiralian genomes. Nature. 2013 Jan 24;493(7433):526-31. Suárez-Ulloa V, Fernández-Tajes J, Manfrin C, Gerdol M, Venier P, Eirín-López JM. Bivalve omics: state of the art and potential applications for the biomonitoring of harmful marine compounds. Mar Drugs. 2013; 11: 4370-4389. Sullivan, C.V., Chapman, R.W., Reading, B.J., and Anderson, P.E., 2015. Transcriptomics of mRNA and egg quality in farmed fish: Some recent developments and future directions. General and comparative endocrinology. Tanguy, M., McKenna, P., Gauthier-Clerc, S., Pellerin, J., Danger, J.-M., Siah, A Sequence analysis of a normalized cDNA library of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain. Results Immunol. ., 2013.3, 40–50 Teaniniuraitemoana, V., Huvet, A., Levy, P., Gaertner-Mazouni, N., Gueguen, Y., and Le Moullac, G., 2015. Molecular signatures discriminating the male and the female sexual pathways in the pearl oyster Pinctada margaritifera. PloS one, 10 (3), e0122819. Teaniniuraitemoana, V., Huvet, A., Levy, P., Klopp, C., Lhuillier, E., Gaertner-Mazouni, N., Gueguen, Y., and Le Moullac, G., 2014. Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes. BMC genomics, 15, 491. Valenzuela-Muñoz, V., Bueno-Ibarra, M.A., and Escárate, C.G., 2014. Characterization of the transcriptomes of Haliotis rufescens reproductive tissues. Aquaculture research, 45 (6), 1026–1040. Venier P, De Pittà C, Bernante F, Varotto L, De Nardi B, Bovo G, et al. MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences. BMC Genomics 9; 10- 72, 2009. Venier P, Varotto L, Rosani U, Millino C, Celegato B, Bernante F, Lanfranchi G, Novoa B, Roch P, Figueras A, Pallavicini A. Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis. BMC Genomics. 2011 Jan 26;12:69 Vera M, Martínez P, Poisa-Beiro L, Figueras A, Novoa B. Genomic organization, molecular diversification, and evolution of antimicrobial peptide myticin-C genes in the mussel (Mytilus galloprovincialis). PLoS ONE 6: e24041, 2011. Vidal, E.A.G., Villanueva, R., Andrade, J.P., Gleadall, I.G., Iglesias, J., Koueta, N., Rosas, C., Segawa, S., Grasse, B., Franco-Santos, R.M., Albertin, C.B., Caamal-Monsreal, C., Chimal, M.E., Edsinger-Gonzales, E., Gallardo, P., Le Pabic, C., Pascual, C., Roumbedakis, K., and Wood, J., 2014. Cephalopod culture: current status of main biological models and research priorities. Advances in marine biology, 67, 1–98. Wang S, Peatman E, Liu H, Bushek D, Ford SE, Kucuktas H, Quilang J, Li P, Wallace R, Wang Y, Guo X, Liu Z: Microarray analysis of gene expression in eastern oyster (Crassostrea virginica) reveals a novel combination of antimicrobial and oxidative stress host responses after dermo (Perkinsus marinus) challenge. Fish Shellfish Immunol 2010, 29:921–929. Wang Y, Xu Z, Guo X. Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca). Biol Bull. 2004; 206:46-54. Zhang, L., Li, L., Zhu, Y., Zhang, G., Guo, X., 2014a. Transcriptome analysis reveals a rich gene set related to innate immunity in the eastern oyster (Crassostrea virginica). Mar. Biotechnol. 16, 17–33. Zhang, N., Xu, F., and Guo, X., 2014. Genomic analysis of the Pacific oyster (Crassostrea gigas) reveals possible conservation of vertebrate sex determination in a mollusc. G3 , 4 (11), 2207–2217. Zhang, Y., Sun, J., Mu, H., Li, Jun., Hang, Y., Qian, P., Qiu, J., Yu, Z., 2014b. Proteomic basis of stress responses in the gills of the pacific oyster Crassostera gigas. J. Proteome Res. J Proteome Res. 2015 Jan 2;14(1):304-17. Zhao, X., Yu, H., Kong, L., Li, Q., 2012c. Transcriptomic responses to salinity stress in the pacific oyster Crassostrea gigas. PLoS One 7, e46244.