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This study examined use of the oyster Crassostrea gigas as a bioindicator of experimental pollution caused by two concentrations of five pesticides (atrazine, isoproturon, alachlore, metolachlore, and diuron) used in agricultural and urban activities. The effect of these pesticides on the genetic structure of the marine bivalve was studied aspart of an environmental biomonitoring project. This research was performed on two natural estuarine populations sampled along the FrenchAtlantic coast as part of an ongoing monitoring program to survey the ecosystem of Brittany using two approaches: identifying the geneticmarkers based on the alleles and genotypes associated with pollutioneffects, and searching for a correlation between these markers and the sensitivity or tolerance of individuals under stress conditions. Results indicate a differential survival of individuals subjected to the various pollutants examined. The sensitivity of alleles and genotypes to environmental stress can be assessed based on the significant differences in allele and genotype frequencies observed between resistant and sensitive individuals when subjected to the pesticides. Thisgenetic study included examination of five enzyme systems (Ak, Pgi, Cap, Pgm, and Mdh) involved in physiologic processes. A total of six alleles and five genotypes at three loci (Ak, Pgi, and Pgm) were identified as being markers of resistance or sensitivity. It is hypothesized that these markers could be used as potential genetic markers in estuarine ecosystem monitoring. [ABSTRACT FROM AUTHOR]

Abstract: Localization of heat shock proteins (HSPs) and metallothioneins (MTs) was investigated in a marine bivalve (Crassostrea gigas) by immunohistochemical methods. Differential protein expression was demonstrated in digestive gland, gonad and gills, using a polyclonal antibody against C. gigas proteins. Application of this technique showed the cellular and tissue immunolabelling specificity of the two proteins. HSPs and MTs were localized in the epithelium of the digestive gland and gills in contact with the palleal compartment. For the first time, localization of MTs was observed in mature gametes of bivalve molluscs. Our results establish a basis for the use of immmunodetection techniques to study the tissue-specific localization of stress proteins in marine bivalves exposed to metal stress. [Copyright &y& Elsevier]

In recent years, numerous studies conducted on teleost fish have highlighted the contribution of transcriptomic studies in elucidating the physiological mechanisms underlying the molecular events of oogenesis and follicular atresia, enabling the identification of potential genes and molecular networks that participate in both the reproductive cycle and the process of follicular atresia. Atresia can affect the reproductive potential of females by reducing the healthy eggs that a female can spawn in both aquaculture and wild populations. The substantial diversity of reproductive strategies exhibited by teleost fish has contributed to the difficulty in identifying common genes between species, but a set of core genes has emerged as potential markers for atresia in relation to apoptosis/autophagy, lipid metabolism, oxidative metabolism and other physiological processes similar to those identified in other vertebrates, even mammals. We review the current status of the genes that have been identified in ovaries with atretic oocytes. Our primary goal is to review the current status regarding gene expression during gonadal development and follicular atresia. This information will enable us to understand the factors and expression patterns involved in the follicular atresia of teleost fish., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Colonization of deep-sea hydrothermal vents by most invertebrates was made efficient through their adaptation to a symbiotic lifestyle with chemosynthetic bacteria, the primary producers in these ecosystems. Anatomical adaptations such as the establishment of specialized cells or organs have been evidenced in numerous deep-sea invertebrates. However, very few studies detailed global inter-dependencies between host and symbionts in these ecosystems. In this study, we proposed to describe, using a proteo-transcriptomic approach, the effects of symbionts loss on the deep-sea mussel Bathymodiolus azoricus' molecular biology. We induced an in situ depletion of symbionts and compared the proteo-transcriptome of the gills of mussels in three conditions: symbiotic mussels (natural population), symbiont-depleted mussels and aposymbiotic mussels., Results: Global proteomic and transcriptomic results evidenced a global disruption of host machinery in aposymbiotic organisms. We observed that the total number of proteins identified decreased from 1118 in symbiotic mussels to 790 in partially depleted mussels and 761 in aposymbiotic mussels. Using microarrays we identified 4300 transcripts differentially expressed between symbiont-depleted and symbiotic mussels. Among these transcripts, 799 were found differentially expressed in aposymbiotic mussels and almost twice as many in symbiont-depleted mussels as compared to symbiotic mussels. Regarding apoptotic and immune system processes - known to be largely involved in symbiotic interactions - an overall up-regulation of associated proteins and transcripts was observed in symbiont-depleted mussels., Conclusion: Overall, our study showed a global impairment of host machinery and an activation of both the immune and apoptotic system following symbiont-depletion. One of the main assumptions is the involvement of symbiotic bacteria in the inhibition and regulation of immune and apoptotic systems. As such, symbiotic bacteria may increase their lifespan in gill cells while managing the defense of the holobiont against putative pathogens.

The relationship between the deep-sea mussel Bathymodiolus azoricus and its thiotrophic (SOX) and methanotrophic (MOX) symbionts has been ecologically and functionally well studied. Endosymbiosis is common in deep-sea hydrothermal vent fauna, yet little is known about the molecular mechanisms underlying the regulation of interactions between host and symbionts. In this study we focused on a group of pattern recognition receptors (PRR), called PGRPs that are able to recognize the peptidoglycan of bacterial cell wall. We first characterised the different PGRPs isoforms in B. azoricus gills and identified five paralogs. Among them two displayed a signal peptide. Then, specific probes designed for each paralog were used to perform real-time PCR quantification in gills of individuals showing various bacterial content as a result of in situ experimental procedures. Overall we found a decrease of PGRPs expression when symbionts amount decreases, suggesting an implication of PGRPs in the regulation of symbionts in B. azoricus gills. We therefore hypothesize that secreted proteins could act as cooperation signals to induce colonisation of symbiotic tissue while non-secreted proteins may regulate the density of endosymbionts within the gill tissue., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Temperature, perhaps more than any other environmental factor, is likely to influence the evolution of all organisms. It is also a very interesting factor to understand how genomes are shaped by selection over evolutionary timescales, as it potentially affects the whole genome. Among thermophilic prokaryotes, temperature affects both codon usage and protein composition to increase the stability of the transcriptional/translational machinery, and the resulting proteins need to be functional at high temperatures. Among eukaryotes less is known about genome evolution, and the tube-dwelling worms of the family Alvinellidae represent an excellent opportunity to test hypotheses about the emergence of thermophily in ectothermic metazoans. The Alvinellidae are a group of worms that experience varying thermal regimes, presumably having evolved into these niches over evolutionary times. Here we analyzed 423 putative orthologous loci derived from 6 alvinellid species including the thermophilic Alvinella pompejana and Paralvinella sulfincola. This comparative approach allowed us to assess amino acid composition, codon usage, divergence, direction of residue changes and the strength of selection along the alvinellid phylogeny, and to design a new eukaryotic thermophilic criterion based on significant differences in the residue composition of proteins. Contrary to expectations, the alvinellid ancestor of all present-day species seems to have been thermophilic, a trait subsequently maintained by purifying selection in lineages that still inhabit higher temperature environments. In contrast, lineages currently living in colder habitats likely evolved under selective relaxation, with some degree of positive selection for low-temperature adaptation at the protein level., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Quahog Parasite Unknown (QPX) is a fatal protistan parasite that causes severe losses in the hard clam (Mercenaria mercenaria) fisheries along the northeastern coast of the US. Field and laboratory studies of QPX disease have demonstrated a major role for water temperature and M. mercenaria genetic origin in disease development. Infections are more likely to occur at cold temperatures, with clam stocks originating from southern states being more susceptible than clams from northern origin where disease is enzootic. Even though the influence of temperature on QPX infection have been examined in susceptible and resistant M. mercenaria at physiological and cellular scales, the underlying molecular mechanisms associated with host-pathogen interactions remain largely unknown. This study was carried out to explore the molecular changes in M. mercenaria in response to temperature and QPX infection on the transcriptomic level, and also to compare molecular responses between susceptible and resistant clam stocks. A M. mercenaria oligoarray (15 K Agilent) platform was produced based on our previously generated transcriptomic data and was used to compare gene expression profiles in naive and QPX-infected susceptible (Florida stock) and resistant (Massachusetts) clams maintained at temperatures favoring disease development (13 °C) or clam healing (21 °C). In addition, transcriptomic changes reflecting focal (the site of infection, mantle) and systemic (circulating hemocytes) responses were also assessed using the oligoarray platform. Results revealed significant regulation of multiple biological pathways by temperature and QPX infection, mainly associated with immune recognition, microbial killing, protein synthesis, oxidative protection and metabolism. Alterations were widely systemic with most changes in gene expression revealed in hemocytes, highlighting the role of circulating hemocytes as the first line of defense against pathogenic stress. A large number of complement-related recognition molecules with fibrinogen or C1q domains were shown to be specially induced following QPX challenge, and the expression of these molecules was significantly higher in resistant clams as compared to susceptible ones. These highly variable immune proteins may be potent candidate molecular markers for future study of M. mercenaria resistance against QPX. Beyond the specific case of clam response to QPX, this study also provides insights into the primitive complement-like system in the hard clam., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

It is commonly known that the nature of the diet has diverse consequences on larval performance and longevity, however it is still unclear which genes have critical impacts on bivalve development and which pathways are of particular importance in their vulnerability or resistance. First we show that a diet deficient in essential fatty acid (EFA) produces higher larval mortality rates, a reduced shell growth, and lower postlarval performance, all of which are positively correlated with a decline in arachidonic and eicosapentaenoic acids levels, two EFAs known as eicosanoid precursors. Eicosanoids affect the cell inflammatory reactions and are synthesized from long-chain EFAs. Second, we show for the first time that a deficiency in eicosanoid precursors is associated with a network of 29 genes. Their differential regulation can lead to slower growth and higher mortality of Mytilus edulis larvae. Some of these genes are specific to bivalves and others are implicated at the same time in lipid metabolism and defense. Several genes are expressed only during pre-metamorphosis where they are essential for muscle or neurone development and biomineralization, but only in stress-induced larvae. Finally, we discuss how our networks of differentially expressed genes might dynamically alter the development of marine bivalves, especially under dietary influence., (Copyright © 2015. Published by Elsevier Inc.)

Our understanding of disease processes and host-pathogen interactions in model species has benefited greatly from the application of medium and high-throughput genomic, metagenomic, epigenomic, transcriptomic, and proteomic analyses. The rate at which new, low-cost, high-throughput -omic technologies are being developed has also led to an expansion in the number of studies aimed at gaining a better understanding of disease processes in bivalves. This review provides a catalogue of the genetic and -omic tools available for bivalve species and examples of how -omics has contributed to the advancement of marine bivalve disease research, with a special focus in the areas of immunity, bivalve-pathogen interactions, mechanisms of disease resistance and pathogen virulence, and disease diagnosis. The analysis of bivalve genomes and transcriptomes has revealed that many immune and stress-related gene families are expanded in the bivalve taxa examined thus far. In addition, the analysis of proteomes confirms that responses to infection are influenced by epigenetic, post-transcriptional, and post-translational modifications. The few studies performed in bivalves show that epigenetic modifications are non-random, suggesting a role for epigenetics in regulating the interactions between bivalves and their environments. Despite the progress -omic tools have enabled in the field of marine bivalve disease processes, there is much more work to be done. To date, only three bivalve genomes have been sequenced completely, with assembly status at different levels of completion. Transcriptome datasets are relatively easy and inexpensive to generate, but their interpretation will benefit greatly from high quality genome assemblies and improved data analysis pipelines. Finally, metagenomic, epigenomic, proteomic, and metabolomic studies focused on bivalve disease processes are currently limited but their expansion should be facilitated as more transcriptome datasets and complete genome sequences become available for marine bivalve species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Brown Ring Disease (BRD) is a bacterial infection affecting the economically-important clam Ruditapes philippinarum. The disease is caused by a bacterium, Vibrio tapetis, that colonizes the edge of the mantle, altering the biomineralization process and normal shell growth. Altered organic shell matrices accumulate on the inner face of the shell leading to the formation of the typical brown ring in the extrapallial space (between the mantle and the shell). Even though structural and functional changes have been described in solid (mantle) and fluid (hemolymph and extrapallial fluids) tissues from infected clams, the underlying molecular alterations and responses remain largely unknown. This study was designed to gather information on clam molecular responses to the disease and to compare focal responses at the site of the infection (mantle and extrapallial fluid) with systemic (hemolymph) responses. To do so, we designed and produced a Manila clam expression oligoarray (15K Agilent) using transcriptomic data available in public databases and used this platform to comparatively assess transcriptomic changes in mantle, hemolymph and extrapallial fluid of infected clams. Results showed significant regulation in diseased clams of molecules involved in pathogen recognition (e.g. lectins, C1q domain-containing proteins) and killing (defensin), apoptosis regulation (death-associated protein, bcl-2) and in biomineralization (shell matrix proteins, perlucin, galaxin, chitin- and calcium-binding proteins). While most changes in response to the disease were tissue-specific, systemic alterations included co-regulation in all 3 tested tissues of molecules involved in microbe recognition and killing (complement-related factors, defensin). These results provide a first glance at molecular alterations and responses caused by BRD and identify targets for future functional investigations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The abalone production in Chile has increased considerably in recent years with no sign of tapering off. Open and semi-closed circuits in the marine water zones in the north and south of Chile are the preferred areas of culture. Coastal ecosystems are subjected to a wide variety of contaminants that generate stress that affects populations via their impacts to individuals at both physiological and genetic levels. This work investigated the genomic and cellular response of post-weaning juvenile Haliotis rufescens abalone under hatchery conditions, fed with probiotic diets, and subsequently challenged with Vibrio parahaemolyticus. The expression patterns of 16 selected genes associated with different metabolic pathways were analyzed using Real-Time PCR. Gene expression was then compared to immunological response parameters in the abalone and quantification of V. parahaemolyticus during the experimental period. Both transcriptomic and immunological analyses indicated significant alteration of physiological processes in H. rufescens correlated to exposure to the pathogenic bacteria, as well as to probiotic nutrition., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Alvinella pompejana (Polychaeta, Alvinellidae) is one of the most thermotolerant marine eukaryotes known to date. It inhabits chimney walls of deep-sea hydrothermal vents along the East Pacific Rise (EPR) and is exposed to various challenging conditions (e.g. high temperature, hypoxia and the presence of sulphides, heavy metals and radiations), which increase the production of dangerous reactive oxygen species (ROS). Two different allelic forms of a manganese-superoxide dismutase involved in ROS detoxification, ApMnSOD1 and ApMnSOD2, and differing only by two substitutions (M110L and A138G) were identified in an A. pompejana cDNA library. RFLP screening of 60 individuals from different localities along the EPR showed that ApMnSOD2 was rare (2 %) and only found in the heterozygous state. Dynamic light scattering measurements and residual enzymatic activity experiments showed that the most frequent form (ApMnSOD1) was the most resistant to temperature. Their half-lives were similarly long at 65 °C (>110 min) but exhibited a twofold difference at 80 °C (20.8 vs 9.8 min). Those properties are likely to be explained by the occurrence of an additional sulphur-containing hydrogen bond involving the M110 residue and the effect of the A138 residue on the backbone entropy. Our results confirm the thermophily of A. pompejana and suggest that this locus is a good model to study how the extreme thermal heterogeneity of the vent conditions may help to maintain old rare variants in those populations.

Genetic and ecophysiological responses of oyster, Crassostrea gigas, populations to environmental stress were investigated in three highly contaminated French estuaries (the Gironde, Loire and Vilaine) and compared to a control, the Belon estuary. A strong response in both metallothionein CgMT4 mRNA expression, as determined by semi-quantitative RT-PCR, and amount of protein, as determined by ELISA tests, was demonstrated in estuarine populations subjected to differential contamination, with an inhibition in the area most highly contaminated with metals. In these same estuarine populations, we found polymorphisms of the metallothionein CgMT4 gene and three other genes (glutamine synthetase--GS, delta-9 desaturase--D9 and phosphoglucomutase--PGM) involved in stress response of C. gigas. We showed that genetic differentiation was observed for MT4 and PGM genes in the Gironde estuary which is highly contaminated with metals. A strong seasonal effect was observed. Phenotype-genotype coupling revealed that one particular MT4 allele and one PGM allele seemed to be associated with metal sensitivity expressed as lower detoxification efficiency and higher metal bioaccumulation. The MT4 gene is a good physiological and genetic marker of stress response and susceptibility., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The marine gastropod Concholepas concholepas, locally known as the "loco", is the main target species of the benthonic Chilean fisheries. Genetic and genomic tools are necessary to study the genome of this species in order to understand the molecular basis of its development, growth, and other key traits to improve the management strategies and to identify local adaptation to prevent loss of biodiversity. Here, we use pyrosequencing technologies to generate the first transcriptomic database from adult specimens of the loco. After trimming, a total of 140,756 Expressed Sequence Tag sequences were achieved. Clustering and assembly analysis identified 19,219 contigs and 105,435 singleton sequences. BlastN analysis showed a significant identity with Expressed Sequence Tags of different gastropod species available in public databases. Similarly, BlastX results showed that only 895 out of the total 124,654 had significant hits and may represent novel genes for marine gastropods. From this database, simple sequence repeat motifs were also identified and a total of 38 primer pairs were designed and tested to assess their potential as informative markers and to investigate their cross-species amplification in different related gastropod species. This dataset represents the first publicly available 454 data for a marine gastropod endemic to the southeastern Pacific coast, providing a valuable transcriptomic resource for future efforts of gene discovery and development of functional markers in other marine gastropods., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The deep-sea vent shrimp Rimicaris exoculata dominates the vagile megafauna at most vent sites along the Mid-Atlantic Ridge. This shrimp swarms around the hot end of the hydrothermal biotope where temperature can exceed its critical maximal temperature (33-38.5 ± 2°C). It may therefore be subjected to a thermal regime that is assumed to be stressful for animals. In this study, we used a global transcriptomic approach by constructing suppression subtractive hybridization cDNA libraries in order to identify specific up- and down-regulated genes in R. exoculata exposed to a severe heat stress (1h at 30°C). A total of 218 sequences representing potentially highly expressed genes in thermally stressed shrimp were obtained. Expression of 11 genes involved in various cell functions was quantified in control and heat shocked specimens using real-time PCR. Differential expression was observed for some specific genes such as mannose receptor C1, metalloprotease, histone H1, and hemocyanin with a strong up-regulation of several genes encoding heat shock proteins. These results suggest that R. exoculata is affected at both cellular and molecular levels by sustained exposure at 30°C. The sequenced ESTs presented here will provide an excellent basis for future thermal stress studies on deep-sea vent fauna., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Hydrothermal vent mussels belonging to the genus Bathymodiolus are distributed worldwide and dominate communities at shallow Atlantic hydrothermal sites. While organisms inhabiting coastal ecosystems are subjected to predictable oscillations of physical and chemical variables owing to tidal cycles, the vent mussels sustain pronounced temperature changes over short periods of time, correlated to the alternation of oxic/anoxic phases. In this context, we focused on the short-term adaptive response of mussels to temperature change at a molecular level. The mRNA expression of 23 genes involved in various cell functions of the vent mussel Bathymodiolus azoricus was followed after heat shocks for either 30 or 120 min, at 25 and 30 degrees C over a 48 h recovery period at 5 degrees C. Mussels were genotyped at 10 enzyme loci to explore a relationship between natural genetic variation, gene expression and temperature adaptation. Results indicate that the mussel response to increasing temperature is a depression in gene expression, such a response being genotypically correlated at least for the Pgm-1 locus. This suggests that an increase in temperature could be a signal triggering anaerobiosis for B. azoricus or this latter alternatively behaves more like a 'cold' stenotherm species, an attribute more related to its phylogenetic history, a cold seeps/wood fall origin.

The 70-kDa heat shock protein (Hsp) family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. We sequenced 2 genes encoding an Hsp70 and an Hsc70 in the Pacific oyster Crassostrea gigas. The Cghsc70 gene contained introns, whereas the Cghsp70 gene did not. Moreover, the corresponding amino acid sequences of the 2 genes presented all the characteristic motifs of the Hsp70 family. We also investigated the expression of Hsp70 in tissues of oysters experimentally exposed to metal. A recombinant Hsc72 was used as an antigen to produce a polyclonal antibody to quantify soluble Hsp70 by enzyme-linked immunosorbent assay in protein samples extracted from oysters. Our results showed that metals (copper and cadmium) induced a decrease in cytosolic Hsp70 level in gills and digestive gland of oysters experimentally exposed to metal. These data suggest that metals may inhibit stress protein synthesis.