Cadmium Highlights Common and Specific Responses of Two Freshwater Sentinel Species, Dreissena polymorpha and Dreissena rostriformis bugensis.

Zebra mussel (ZM), Dreissena polymorpha, commonly used as a sentinel species in freshwater biomonitoring, is now in competition for habitat with quagga mussel (QM), Dreissena rostriformis bugensis. This raises the question of the quagga mussel's use in environmental survey. To better characterise QM response to stress compared with ZM, both species were exposed to cadmium (100 µg·L-1), a classic pollutant, for 7 days under controlled conditions. The gill proteomes were analysed using two-dimensional electrophoresis coupled with mass spectrometry. For ZM, 81 out of 88 proteoforms of variable abundance were identified using mass spectrometry, and for QM, 105 out of 134. Interestingly, the proteomic response amplitude varied drastically, with 5.6% of proteoforms of variable abundance (DAPs) in ZM versus 9.4% in QM. QM also exhibited greater cadmium accumulation. Only 12 common DAPs were observed. Several short proteoforms were detected, suggesting proteolysis. Functional analysis is consistent with the pleiotropic effects of the toxic metal ion cadmium, with alterations in sulphur and glutathione metabolisms, cellular calcium signalling, cytoskeletal dynamics, energy production, chaperone activation, and membrane events with numerous proteins involved in trafficking and endocytosis/exocytosis processes. Beyond common responses, the sister species display distinct reactions, with cellular response to stress being the main category involved in ZM as opposed to calcium and cytoskeleton alterations in QM. Moreover, QM exhibited greater evidence of proteolysis and cell death. Overall, these results suggest that QM has a weaker stress response capacity than ZM.

Dreissena polymorpha responses under thermal and hypoxic stress: New insights in the tolerance of this freshwater sentinel species.

Dreissena polymorpha is a sentinel freshwater mussel providing key functional ecosystemic services like nutrient recycling and suspended matter filtration. Global warming and especially extreme events imply rapid fluctuations of environmental parameters that sessile organisms could not escape. The increase occurrence of heat waves and the subsequent expansion of hypoxic areas could challenge the survival of mussels. This study provided a deeper knowledge of energy management and cellular function during thermal (+15 °C) or hypoxic (30% of dissolved oxygen saturation) stress for 7 days. A potential metabolic rate depression was highlighted in D. polymorpha under hypoxia through a decline in the mitochondrial activity and a constant AMP content over time. A contrasted pattern of response was observed in thermal-stressed mussels between 24 h and 7 days of exposure. A global increase of metabolic activity was noticed in mussels after 24 h while a return to control level was noticed at the end of the experiment. Although D. polymorpha is considered as a temperature tolerant species, a significant increase of ADP:ATP ratio, related to a decrease of mitochondrial activity and density, suggested an overwhelming of organisms. This study pointed to the importance of considering time of exposure to natural factor variations in tolerance window of organisms in a long-term changing environment. The apparent short-term tolerance of D. polymorpha could hide much more deleterious consequences, i.e. mortality, if abiotic stresses persist, as suggested by climate change models.

Nanotherapeutics Plus Immunotherapy in Oncology: Who Brings What to the Table?

While the number of oncology-related nanotherapeutics and immunotherapies is constantly increasing, cancer patients still suffer from a lack of efficacy and treatment resistance. Among the investigated strategies, patient selection and combinations appear to be of great hope. This review will focus on combining nanotherapeutics and immunotherapies together, how they can dually optimize each other to face such limits, bringing us into a new field called nano-immunotherapy. While looking at current clinical trials, we will expose how passive immunotherapies, such as antibodies and ADCs, can boost nanoparticle tumor uptake and tumor cell internalization. Conversely, we will study how immunotherapies can benefit from nanotherapeutics which can optimize their lipophilicity, permeability, and distribution (e.g., greater tumor uptake, BBB crossing, etc.), tumor, tumor microenvironment, and immune system targeting properties.

Interest of a multispecies approach in active biomonitoring: Application in the Meuse watershed.

A biomonitoring approach based on a single model species cannot be representative of the contaminations impacts on the ecosystem overall. As part of the Interreg DIADeM program ("Development of an integrated approach for the diagnosis of the water quality of the River Meuse"), a study was conducted to establish the proof of concept that the use of a multispecies active biomonitoring approach improves diagnostic of aquatic systems. The complementarity of the biomarker responses was tested in four model species belonging to various ecological compartments: the bryophyte Fontinalis antipyretica, the bivalve Dreissena polymorpha, the amphipod Gammarus fossarum and the fish Gasterosteus aculeatus. The species have been caged upstream and downstream from five wastewater treatment plants (WWTPs) in the Meuse watershed. After the exposure, a battery of biomarkers was measured and results were compiled in an Integrated Biomarker Response (IBR) for each species. A multispecies IBR value was then proposed to assess the quality of the receiving environment upstream the WWTPs. The effluent toxicity was variable according to the caged species and the WWTP. However, the calculated IBR were high for all species and upstream sites, suggesting that the water quality was already downgraded upstream the WWTP. This contamination of the receiving environment was confirmed by the multispecies IBR which has allowed to rank the rivers from the less to the most contaminated. This study has demonstrated the interest of the IBR in the assessment of biological impacts of a point-source contamination (WWTP effluent) but also of the receiving environment, thanks to the use of independent references. Moreover, this study has highlighted the complementarity between the different species and has emphasized the interest of this multispecies approach to consider the variability of the species exposition pathway and sensibility as well as the mechanism of contaminants toxicity in the final diagnosis.

Cutibacterium acnes and Staphylococcus epidermidis: the unmissable modulators of skin inflammatory response.

Acne is a multifactorial inflammatory dermatose that affects all age categories from teenagers to adults, resulting in important psychological impacts. Multiple hypotheses currently attempt to decrypt the physiopathology of this disease, and four main actors were identified as highly implicated in it: hyperkeratinization of the pilosebaceous follicle, hyperseborrheae, host factors (innate immunity) and skin microbiota. In this letter, we present results illustrating the impact of skin microbiota on inflammatory skin response, and how far the proper balance between each bacterial community, especially C. acnes and S. epidermidis, is crucial to maintain an appropriate inflammatory response on the skin. The data presented in this study demonstrate that within the skin microbiota, an imbalance between Cutibacterium acnes and Staphylococcus epidermidis, is able to induce the activation of inflammation-related markers such as IL-1ra, IL-6, IL-8, G-CSF and the molecules C5/C5a, soluble CD14 MIP-3beta, Serpin E1, VCAM-1 and beta-defensin-2. Moreover, S. epidermidis appears to have a more important role than C. acnes on the induction of inflammation-related markers, particularly on IL-6. This work is the basis of future in vitro studies to further understand acne physiopathology, inspiring the development of future innovative therapies based on skin microbiota modulation.

SK Channels Modulation Accelerates Equilibrium Recovery in Unilateral Vestibular Neurectomized Rats.

We have previously reported in a feline model of acute peripheral vestibulopathy (APV) that the sudden, unilateral, and irreversible loss of vestibular inputs induces selective overexpression of small conductance calcium-activated potassium (SK) channels in the brain stem vestibular nuclei. Pharmacological blockade of these ion channels by the selective antagonist apamin significantly alleviated the evoked vestibular syndrome and accelerated vestibular compensation. In this follow-up study, we aimed at testing, using a behavioral approach, whether the antivertigo (AV) effect resulting from the antagonization of SK channels was species-dependent or whether it could be reproduced in a rodent APV model, whether other SK channel antagonists reproduced similar functional effects on the vestibular syndrome expression, and whether administration of SK agonist could also alter the vestibular syndrome. We also compared the AV effects of apamin and acetyl-DL-leucine, a reference AV compound used in human clinic. We demonstrate that the AV effect of apamin is also found in a rodent model of APV. Other SK antagonists also produce a trend of AV effect when administrated during the acute phase of the vertigo syndrome. Conversely, the vertigo syndrome is worsened upon administration of SK channel agonist. It is noteworthy that the AV effect of apamin is superior to that of acetyl-DL-leucine. Taken together, these data reinforce SK channels as a pharmacological target for modulating the manifestation of the vertigo syndrome during APV.

Effect of reproduction cycle stage on energy metabolism responses in a sentinel species (Dreissena polymorpha) exposed to cadmium: What consequences for biomonitoring?

Metal trace elements such as cadmium (Cd) are commonly present in ecosystems and could lead to impairment of mitochondrial functions and energy imbalance in aquatic organisms including molluscs. Combined exposure to increasing temperatures and Cd could enhance such an impact on animals. Seasonal fluctuations, such as temperature, and the corresponding reproduction cycle can affect biomarker responses. However, the reproduction cycle stage is rarely taken into account in ecotoxicological studies. Thus, this work aimed at understanding energy metabolism responses in a sentinel species, Dreissena polymorpha. Mussels were collected during the rest and the reproduction periods and were exposed to 10 µg.L-1 of cadmium (Cd) at two temperatures (in situ temperature and in situ temperature + 5°C) during 7 days. Energy metabolism was monitored by measuring reserves and energy nucleotides charge and by assessing aerobic and anaerobic metabolism markers, and upstream regulation pathways. Markers related to OXPHOS activity revealed seasonal variations under laboratory conditions. Conversely, adenylate nucleotides, glycogen, lipid and transcript levels of AMP-activated protein kinase, citrate synthase, ATP synthase and cytochrome b encoding genes remained steady after the acclimation period. No evident effect of Cd on energy metabolism markers was noticed for both exposures although the transcript level of succinate dehydrogenase and citrate synthase encoding genes decreased with Cd during the rest period. Cellular stress, revealed by lipid peroxidation and catalase mRNA levels, only occurred in Cd and warming co-exposed mussels during the reproduction period. These results suggest that contaminant impact might differ according to the reproduction cycle stage. The effect of confounding factors on biomarker variations should be further investigated to have a deeper knowledge of metabolism responses under laboratory conditions.

Corrigendum: Quantitative Evaluation of a New Posturo-Locomotor Phenotype in a Rodent Model of Acute Unilateral Vestibulopathy.

[This corrects the article DOI: 10.3389/fneur.2020.00505.].

Quantitative Evaluation of a New Posturo-Locomotor Phenotype in a Rodent Model of Acute Unilateral Vestibulopathy.

Vestibular pathologies are difficult to diagnose. Existing devices make it possible to quantify and follow the evolution of posturo-locomotor symptoms following vestibular loss in static conditions. However, today, there are no diagnostic tools allowing the quantitative and spontaneous analysis of these symptoms in dynamic situations. With this in mind, we used an open-field video tracking test aiming at identifying specific posturo-locomotor markers in a rodent model of vestibular pathology. Using Ethovision XT 14 software (Noldus), we identified and quantified several behavioral parameters typical of unilateral vestibular lesions in a rat model of vestibular pathology. The unilateral vestibular neurectomy (UVN) rat model reproduces the symptoms of acute unilateral peripheral vestibulopathy in humans. Our data show deficits in locomotion velocity, distance traveled and animal mobility in the first day after the injury. We also highlighted alterations in several parameters, such as head and body acceleration, locomotor pattern, and position of the body, as well as "circling" behavior after vestibular loss. Here, we provide an enriched posturo-locomotor phenotype specific to full and irreversible unilateral vestibular loss. This test helps to strengthen the quantitative evaluation of vestibular disorders in unilateral vestibular lesion rat model. It may also be useful for testing pharmacological compounds promoting the restoration of balance. Transfer of these novel evaluation parameters to human pathology may improve the diagnosis of acute unilateral vestibulopathies and could better follow the evolution of the symptoms upon pharmacological and physical rehabilitation.

Seasonal monitoring of cellular energy metabolism in a sentinel species, Dreissena polymorpha (bivalve): Effect of global change?

Aquatic organisms such as bivalves are particularly sensitive to seasonal fluctuations associated with climate changes. Energy metabolism management is also closely related to environmental fluctuations. Changes in both biotic and abiotic conditions, such as the reproduction status and temperature respectively, may affect the organism energy status. A bivalve sentinel species, Dreissena polymorpha was sampled along its one-year reproduction cycle in situ (2018-2019) to study natural modulations on several markers of energy metabolism regarding seasonal variations in situ. A panel of different processes involved in energy metabolism was monitored through different functions such as energy balance regulation, mitochondrial density, and aerobic/anaerobic metabolism. The typical schema expected was observed in a major part of measured responses. However, the monitored population of D. polymorpha showed signs of metabolism disturbances caused by an external stressor from April 2019. Targeting a major part of energy metabolism functions, a global analysis of responses suggested a putative impact on the mitochondrial respiratory chain due to potential pollution. This study highlighted also the particular relevance of in situ monitoring to investigate the impacts of environmental change on sentinel species.

Energy allocation in two dreissenid species under metal stress.

Measurements of biological responses on living organisms are essential in aquatic biomonitoring. In freshwaters, Dreissena polymorpha is an invasive bivalve commonly used in ecotoxicological studies and considered as a model organism. However, D. polymorpha abundances are declining while another species colonizes most of the freshwaters: Dreissena rostriformis bugensis. This species has already been studied in ecophysiology but there is still a lack of data concerning its responses to stressors before its use as a bioindicator of environmental pollution. This study aims to compare the responses of the two species exposed to metal stress. Responses at different levels of biological organization were targeted with measurement of sub-cellular and individual biomarkers following an exposure of up to 7 days to cadmium at 10 µg.L-1. At the individual level, the scope for growth (SFG) was measured. It corresponds to the energy allocated to growth and reproduction. D. polymorpha exhibits variations in biomarker measurements as well as in the SFG in presence of Cd. D. r. bugensis shows no variation in its responses at the different targeted levels. According to the present results, energy metabolism seems to have an essential role for these species when facing a metal stress. Different energy allocation strategies were evidenced between the two species, although the link with biochemical biomarkers is more evident for D. polymorpha than for D. r. bugensis.

Daily use of extracorporeal CO2 removal in a critical care unit: indications and results.

BACKGROUND: While outcome improvement with extracorporeal CO2 removal (ECCO2R) is not demonstrated, a strong pathophysiological rational supports its use in the setting of acute respiratory distress syndrome (ARDS) and COPD exacerbation. We aimed to describe our single-center experience of ECCO2R indications and outcome. METHODS: Patients treated with ECCO2R in our medial ICU, from March 2014 to November 2017, were retrospectively enrolled. Primary end point was evolution of ventilator settings during the two first days following ECCO2R start. RESULTS: Thirty-three patients received ECCO2R. Seventeen were managed with Hemolung®, 10 with Prismalung®, 4 with ILA®, and 2 with Cardiohelp®. Indications for ECCO2R were mild or moderate ARDS (n = 16), COPD exacerbation (n = 11), or uncontrolled hypercapnia due to other causes (n = 6). Four patients were not intubated at the time of ECCO2R start. Median duration of ECCO2R treatment was 7 days [5-10]. In ARDS patients, between baseline and day 2, median tidal volume and driving pressure decreased from 5.3 [4.4-5.9] mL/kg and 10 [8-15] to 3.8 [3.3-4.1] mL/kg and 9 [8-11], respectively. Prone positioning was performed in 10 of the 16 patients, without serious adverse event. In COPD patients, between baseline and day 2, median ventilation minute and PaCO2 decreased significantly from respectively 7.6 [6.6-8.7] L/min and 9.4 [8.4-10.1] kPa to 5.8 [4.9-6.2] L/min and 6 [5.3-6.8] kPa. Four out of 11 COPD patients were extubated while on ECCO2R. Device thrombosis occurred in 5 patients (15%). Hemolysis was documented in 16 patients (48%). One patient died of intracranial hemorrhage, while on ECCO2R. Twenty-four patients were discharged from ICU alive. Twenty-eight day mortality was 31% in ARDS, 9% in COPD patients, and 50% in other causes of refractory hypercapnic respiratory failure. CONCLUSION: ECCO2R was useful to apply ultra-protective ventilation among ARDS patients and improved PaCO2, pH, and minute ventilation in COPD patients.

Field biomonitoring using the zebra mussel Dreissena polymorpha and the quagga mussel Dreissena bugensis following immunotoxic reponses. Is there a need to separate the two species?

The zebra mussel, Dreissena polymorpha constitutes an extensively used sentinel species for biomonitoring in European and North American freshwater systems. However, this invasive species is gradually replaced in freshwater ecosystem by Dreissena bugensis, a closely related dreissenid species that shares common morphological characteristics but possess some physiological differences. However, few are known about differences on more integrated physiological processes that are generally used as biomarkers in biological monitoring studies. Declining of zebra mussel populations raises the question of the sustainability of using one or both species indifferently to maintain the quality of environmental pollution monitoring data. In our study, we performed a field comparative study measuring immune-related markers and bioaccumulation of PCBs, PAHs and PBDEs in sympatrically occurring mussel populations from three sites of the St. Lawrence River. For tested organisms, species were identified using RFLP analysis. Measurement of bioaccumulated organic compounds indicated a higher accumulation of PCBs and PBDEs in D. bugensis soft tissues compared to D. polymorpha while no differences were noticed for PAHs. Results of hemocytic parameters highlighted that differences of hemocyte distributions were associated to modulations of phagocytic activities. Moreover, marked differences occurred in measurement of hemocytic oxidative activity, indicating divergences between the two species for ROS regulation strategies. This physiological characteristic may deeply influence species responses facing environmental or pollution related stress and induce bias if the two species are not differentiated in further biomarker or bioaccumulation measurement-based studies.

Differential sensitivity to cadmium of immunomarkers measured in hemocyte subpopulations of zebra mussel Dreissena polymorpha.

Increasing discharge of industrial wastes into the environment results in pollution transfer towards hydrosystems. These activities release heavy metals such as cadmium, known as persistent pollutant that is accumulated by molluscs and exercise immunotoxicological effects. Among molluscs, the zebra mussel, Dreissena polymorpha constitutes a suitable support for freshwater ecotoxicological studies. In molluscs, homeostasis maintain is ensured in part by hemocytes that are composed of several cell populations involved in multiple physiological processes such as cell-mediated immune response or metal metabolism. Thus, hemocytes constitute a target of concern to study adverse effects of heavy metals. The objectives of this work were to determine whether immune-related endpoints assessed were of different sensitivity to cadmium and whether hemocyte functionalities were differentially affected depending on hemocyte subpopulation considered. Hemocytes were exposed ex vivo to concentrations of cadmium ranging from 10-6 M to 10-3 M for 21h prior flow cytometric analysis of cellular markers. Measured parameters (viability, phagocytosis, oxidative activity, lysosomal content) decreased in a dose-dependent manner with sensitivity differences depending on endpoint and cell type considered. Our results indicated that phagocytosis related endpoints were the most sensitive studied mechanisms to cadmium compared to other markers with EC50 of 3.71±0.53×10-4M for phagocytic activity and 2.79±0.19×10-4M considering mean number of beads per phagocytic cell. Lysosomal content of granulocytes was less affected compared to other cell types, indicating lower sensitivity to cadmium. This suggests that granulocyte population is greatly involved in metal metabolism. Mitochondrial activity was reduced only in blast-like hemocytes that are considered to be cell precursors. Impairment of these cell functionalities may potentially compromise functions ensured by differentiated cells. We concluded that analysis of hemocyte activities should be performed at sub-population scale for more accurate results in ecotoxicological studies.

Functional features of hemocyte subpopulations of the invasive mollusk species Dreissena polymorpha.

Dreissena polymorpha is a mussel species that invaded many lotic and lentic inland waters in Western Europe and North America. Its positive or negative interactions with biotic and abiotic components of ecosystems are numerous, making this bivalve the subject of numerous studies in ecology, ecophysiology and ecotoxicology. In these contexts, the functional characterization of the zebra mussel hemocytes is of particular interest, as hemocytes are central cells involved in vital functions (immunity, growth, reproduction) of molluscan physiology. Dreissena polymorpha circulating hemocytes populations were characterized by a combination of structural and functional analysis. Assessments were performed during two contrasted physiological periods for mussels (gametogenesis and spawning). Three hemocyte types were identified as hyalinocytes and blast-like cells for agranular hemocytes and one granulocyte population. Flow cytometry analysis of hemocytes functionalities indicated that blast-like cells had low oxidative and mitochondrial activities and low lysosomal content. Hyalinocytes and granulocytes are fully equipped to perform innate immune response. Hyalinocytes exhibit higher oxidative activity than granulocytes. Such observation is not common since numerous studies show that granulocytes are usually cells that have the highest cellular activities. This result demonstrates the significant functional variability of hemocyte subpopulations. Moreover, our findings reveal that spawning period of Dreissena polymorpha was associated with an increase of hyalinocyte percentage in relation to low levels of biological activities in hemocytes. This reduction in hemocyte activity would reflect the important physiological changes associated with the spawning period of this invasive species known for its high reproductive potential.

Juvenile roach (Rutilus rutilus) increase their anaerobic metabolism in response to copper exposure in laboratory conditions.

This study aims to determine the potential impairment of cell energy synthesis processes (glycolysis and respiratory chain pathways) by copper in juvenile roach at different regulation levels by using a multi-marker approach. Juvenile roach were exposed to 0, 10, 50, and 100 µg/L of copper for 7 days in laboratory conditions. The glycolysis pathway was assessed by measuring the relative expression levels of 4 genes encoding glycolysis enzymes. The respiratory chain was studied by assessing the electron transport system and cytochrome c oxidase gene expression. Muscle mitochondria ultrastructure was studied, and antioxidant responses were measured. Furthermore, the main energy reserves-carbohydrates, lipids, and proteins-were measured, and cellular energy was evaluated by measuring ATP, ADP, AMP and IMP concentrations. This study revealed a disturbance of the cell energy metabolism due to copper exposure, with a significant decrease in adenylate energy charge in roach exposed to 10 µg/L of copper after 1 day. Moreover, ATP concentrations significantly decreased in roach exposed to 10 µg/L of copper after 1 day. This significant decrease persisted in roach exposed to 50 µg/L of copper after 7 days. AMP concentrations increased in all contaminated fish after 1 day of exposure. In parallel, the relative expression of 3 genes encoding for glycolysis enzymes increased in all contaminated fish after 1 day of copper exposure. Focusing on the respiratory chain, cytochrome c oxidase gene expression also increased in all contaminated fish at the two time-points. The activity of the electron transport system was not disturbed by copper, except in roach exposed to 100 µg/L of copper after 1 day. Copper induced a metabolic stress. Juvenile roach seemed to respond to the ensuing high energy demand by increasing their anaerobic metabolism, but the energy produced by the anaerobic metabolism is unable to compensate for the stress induced by copper after 7 days. This multi-marker approach allows us to reach a greater understanding of the effects of copper on the physiological responses of juvenile roach.

Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes.

Heavy metals are pollutants which affect all organisms. Since a small number of eukaryotes have been investigated with respect to metal resistance, we hypothesize that many genes that control this phenomenon remain to be identified. This was tested by screening soil eukaryotic metatranscriptomes which encompass RNA from organisms belonging to the main eukaryotic phyla. Soil-extracted polyadenylated mRNAs were converted into cDNAs and 35 of them were selected for their ability to rescue the metal (Cd or Zn) sensitive phenotype of yeast mutants. Few of the genes belonged to families known to confer metal resistance when overexpressed in yeast. Several of them were homologous to genes that had not been studied in the context of metal resistance. For instance, the BOLA ones, which conferred cross metal (Zn, Co, Cd, Mn) resistance may act by interfering with Fe homeostasis. Other genes, such as those encoding 110- to 130-amino-acid-long, cysteine-rich polypeptides, had no homologues in databases. This study confirms that functional metatranscriptomics represents a powerful approach to address basic biological processes in eukaryotes. The selected genes can be used to probe new pathways involved in metal homeostasis and to manipulate the resistance level of selected organisms.

Expression of candidate genes related to metabolism, immunity and cellular stress during massive mortality in the American oyster Crassostrea virginica larvae in relation to biochemical and physiological parameters.

Quantification of mRNA of genes related to metabolism, immunity and cellular stress was examined in relation to a massive mortality event during the culture of American oyster larvae, Crassostrea virginica which was probably, in regard to previous microbiological analysis, induced by Vibrio infection. To document molecular changes associated with the mortality event, mRNA levels were compared to biochemical and physiological data, previously described in a companion paper. Among the 18 genes studied, comparatively to the antibiotic control, 10 showed a lower relative gene expression when the massive mortality occurred. Six of them are presumed to be related to metabolism, corroborating the metabolic depression associated with the mortality event suggested by biochemical and physiological analyses. Relationships between the regulation of antioxidant enzyme activities, lipid peroxidation, and the mRNA abundance of genes linked to oxidative stress, cytoprotection, and immune response are also discussed. Finally, we observed an increase in the transcript abundance of two genes involved in apoptosis and cell regulation simultaneously with mortality, suggesting that these processes might be linked.

Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality.

Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.

Responses of Pacific oyster Crassostrea gigas populations to abiotic stress in environmentally contrasted estuaries along the Atlantic coast of France.

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.