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Anthropogenic pressures in estuaries may impair not only the fitness of organisms and the evolutionary processes necessary to the survival, but also the management of exploited marine resources. In order to assess these impacts, phenotypic and genotypic data pertaining to young-of-the-year (YOY) sole ( Solea solea ) were screened in differentially contaminated North East Atlantic estuaries ranging from Portugal (Mondego) to the English Channel (Seine). YOY sole inhabiting each estuary was found being phenotypically distinct from each other; each of them being impacted by pollution when comparing observed phenotypic data with published literature. According to the strong pollution in the Seine estuary, the corresponding YOY sole sample showed significantly lower body mass, size and condition factor compared to other samples. Using fifteen genetic markers, the Mondego estuary demonstrated low, but significant levels of genetic differentiation with most of the samples. Using neutrality tests, two loci were found to be putatively under directional selection ( Soso7 and Soso23 ), but only locus Soso7 demonstrated significant positive heterozygosity–fitness correlation (HFC) for body size and body mass, suggesting a possible ongoing adaptive process. The candidate locus MT associated to a detoxifying metallothionein gene did not show significant genetic differentiation, but ( i ) significant deficit in heterozygote in the Seine estuary as already reported in other coastal samples impacted by metallic contaminants, and ( ii ) significant positive HFC with body mass were found. Selective rather than demographic processes better explain an observed genetic structure in YOY sole and are predominantly associated with the heavily contaminated Seine estuary whose nursery function and capacity are degraded.

International audience; Estuaries are important areas highly susceptible to anthropogenic degradations like pollution. Estuarine species have thus to cope with many types of constraints depending on the estuaries' characteristics. The European flounder Platichthys flesus is considered as a sentinel species for the monitoring of estuarine water quality. In this study, juvenile flounders (0+ group) were sampled from three contrasted Channel estuaries, i.e. the Seine, the Canche and the Tamar, and we characterized their liver proteomes by using a two-dimensional electrophoresis based proteomic approach. We showed that 27 protein spots differentially accumulated between the 3 populations. Six of these proteins were identified by MALDI TOF-TOF mass spectrometry. Flounders from the Seine and from the Tamar, two highly polluted estuaries, displayed common differences, i.e. an increase of the energetic- and the glutathione-metabolism. The most accumulated protein in the Seine's samples (6.7-fold) was a Vitelline Membrane Outer layer protein 1 homolog, suggesting oogenesis deregulation in these juvenile (sexually immature) flounders. Future works applying this kind of proteomic approach on flounders experimentally exposed to conditions that mimic environmental constraints will help to better understand the significance of these environmental proteomic signatures.

In this study, new DNA markers were explored for the flounder Platichthys flesus. cDNA and genomic sequences of the genes encoding the glyceraldehyde-3-phosphate-deshydrogenase (GAPDH), the cytosolic creatine kinase (CK), the prostaglandin D synthase (PGDS) and the betaine homocysteine methyltransferase (BHMT) were characterized. The tumour suppressor p53 gene structure was already described. A PCR–SSCP (Single Strand Conformation Polymorphism) analysis was finally conducted to study the genetic polymorphism of different populations of flounders collected along the French Atlantic coast. Four highly contaminated French estuaries (Seine, Vilaine, Loire and Gironde) were sampled and compared to a reference estuary (Ster) to explore possible selective effect of the environment on specific allelic frequencies. Our results showed that two loci p53 and PGDS, could be potential markers of chemical stress: p53A allele frequency increased in contaminated systems compared to the reference system. In the Vilaine estuary, PGDS polymorphism could be related to pesticide stress.

Genetic and physiological responses of flounder Platichthys flesus populations to chem- ical stress were investigated in 3 highly contaminated French estuaries (the Vilaine, Loire and Gironde) and compared to those observed in the Ster estuary, considered as a reference site because of its reletively low level of contamination. In the Vilaine, Loire and Gironde populations, multi-loci heterozygosity (allozymes: PGM, GPI-2, MPI, IDHP, AAT-1, AAT-2) was lower than in the reference site. The mean growth rate was measured by back-calculation from otoliths, and the mean condition factor corresponded to the somatic weight:fish length 3 ratio. The fish from contaminated estuaries displayed lower values for both parameters than those from the Ster. Among the alleles, PGM 85, AAT-1 95, and to a lesser extent MPI 90, MPI 95 and IDHP 90, could be considered as 'resistant' to contamination. In contaminated estuaries, these alleles were carried by individuals that displayed good fitness, measured as high DNA integrity (low coefficient of variation of DNA content in blood cells) by flow cytometry. The frequencies of these alleles were also more elevated compared to fish from the reference site. Moreover, the comparative analysis of the relationships between genotypic and phenotypic responses of flounder populations to pollution in the 4 studied estuaries highlighted some specificity in relation to contamination typology.

International audience; Platichthys flesus is often used as a sentinel species to monitor the estuarine water quality. In this study, we carried out an experimental contamination of fish using a PAHs/PCBs mixture, which was designed to mimic the concentrations found in the Seine estuary (C1) and 10 times these concentrations (C2). We used a proteomic approach to understand the molecular mechanisms implied in the response of P. flesus to these xenobiotics. We showed that 54 proteins were differentially accumulated in one or several conditions, which 34 displayed accumulation factors higher than two. 18 of these proteins were identified by MALDI TOF-TOF mass spectrometry. The results indicated the deregulation of oxidative stress- and glutathione metabolism-(GST, GPx) proteins as well as of several proteins belonging to the betaine demethylation pathway and the methionine cycle (BHMT, SHMT, SAHH), suggesting a role for these different pathways in the P. flesus response to chemical contamination.

We sampled and analyzed European flounder (Platichthys flesus) from two highly contaminated estuaries (Seine and Loire, France) and one moderately contaminated estuary (reference site: Ster, France). Significant and convergent modifications of the allelic frequencies for the loci phosphoglucomutase (PGM), glucose phosphate isomerase 2 (GPI-2), mannose phosphate isomerase (MPI), and aspartate aminotransferase (AAT-2) were evident for fish in the contaminated sites versus fish from the reference site. Back-calculation from otoliths showed that the average growth rate of fish between the first and the second winter was greater at the reference site (approximately 150 mm/year) than at the contaminated sites (approximately 100 mm/year). Flounder from the reference site also had a higher condition factor (somatic wt/(fish length)3) compared to fish from the two contaminated sites. However, the observed pattern of growth rate and condition factor might be biased by particular environmental conditions other than contaminants and must be confirmed by more extensive study. Flow cytometry analysis of fish blood revealed a significant difference in the frequency of abnormal profiles for fish from the Seine (20%) versus from the Ster (3%). We interpret this result as a marked genotoxic effect of contaminants on fish in the Seine system. Some genotypes, such as PGM 85/85, appeared to be linked to the measured components of fitness, particularly to DNA integrity. Thus, these genotypes might be considered to be more tolerant to pollutants. The frequency of the PGM 85 allele was clearly elevated in flounder from the more contaminated sites, compared to flounder from the reference site.

Pour decrire la cinetique de l'ovogenese et la strategie de ponte chez la limande, Limanda limanda (L., 1758) les ovaires de 74 femelles capturees en baie de Douarnenez et en baie de Seine en 1990 et 1991 ont fait l'objet de prelevements. Une echelle de developpement ovocytaire en six stades a ete utilisee pour classer les ovocytes. L'apparition d'alveoles corticaux a la peripherie du cytoplasme est le premier signe de la vitellogenese. Ils sont visibles dans les ovocytes dont le diametre est de 120 μ m, taille limite a utiliser lors d'etudes de la fecondite. Chez les femelles a ponte precoce un hiatus existe avant les pontes entre les ovocytes previtellogeniques et vitellogeniques. Ceci permet de classer la limande parmi les especes a fecondite determinee. Chez ces femelles l'intensite de l'atresie est faible et leur fecondite annuelle est proche de la fecondite potentielle. Par contre chez les femelles a ponte tardive tous les ovocytes vitellogeniques ne parviendront pas a maturite et ceux non encore emis a la fin du printemps degenereront.

International audience; The genetic structure of European flounder Platichthys flesus populations was analysed in the southern part of the species' distribution area, from England to Portugal; samples were collected in 5 estuaries which were chosen because of their latitudinal cline, the level of contaminants and the possibility of hypoxia: the Tamar in England; the Canche, Seine and Vilaine in France; and the Mondego in Portugal. Analysing the variability of a mitochondrial marker (cytochrome c oxidase subunit 1), we observed (1) a moderately higher nucleotide diversity in the Tamar and Canche, reflecting the probable existence of a major refugium in this sector during the last glacial maximum, and (2) a reduced genetic differentiation over the whole data set. The use of 8 microsatellites underlined the maintenance of genetic diversity in the peripheral population of the Mondego, located near the southern limit of the species' range; furthermore, significant pairwise differentiations of populations including Mondego underlined the particular genetic status of this Portuguese population. The polymorphism of a candidate gene involved in energetic metabolism, AMP-desaminase isoform 1, was investigated focusing on exon 1; we found the highest heterozygosities in the more polluted populations (Seine, Tamar) and marked differentiations compared to the other group of populations (Mondego, Vilaine, Canche). The pattern of variability observed for the AMP-desaminase suggests that in chronically contaminated flounder populations, cocktails of pollutants act as selective agents directly or indirectly on this locus.

International audience; An epidemiological survey was conducted in the Seine estuary and in two smaller and relatively preserved estuaries on the French Atlantic coast in order to estimate the occurrence of liver lesions in European flounder, Platichthys flesus, and also to seek putative risk factors for the recorded pathologies. Four hundred and seventy-eight fish of both sexes and of different size ranges were sampled in the three studied areas, 338 of which in the Seine estuary. All fish were examined for histopathological liver lesions, while DNA adducts and otoliths were analyzed on a subsample. Five categories of hepatic lesions were recorded with the following prevalence for the Seine estuary: 36.7 % inflammations, 8 % parasites (mainly encysted nematodes), 6.5 % foci of cellular alteration (FCA), 5.3 % foci of necrosis or regeneration (FNR), and 1.5 % tumors. Inflammation occurrence increased according to age, contrary to parasitic infestations and FCA which were more prevalent in young fish, notably those of < 1 year old (group 0). Tumors were only observed in females of more than two winters. Females exhibited a higher prevalence of tumors (3.0 %) and FCA (6.5 %) than males (0 and 2.6 %, respectively). Parasitic and infectious lesions and FNR were equally distributed in males and females. The prevalence of FNR was also shown to vary according to sampling season, with significantly more occurrences of liver necrosis in the fish collected in summer than in spring. Spatial differences were observed with a higher occurrence of encysted parasites in flounders from the upper Seine estuary, while inflammations predominated in flounders living downstream. Temporal trends were also noted, with an increased prevalence of parasitic infestations, inflammations, and FCA in the 2002-2003 period in comparison to the 1996-1997 one. The three flounder populations from the Seine estuary (Normandy), Ster estuary (Brittany), and Bay of Veys (Normandy) showed different spectra of hepatic lesions. Flounders from the Bay of Veys had relatively few liver lesions as compared to flounders from the two other estuaries. Flounders from the Ster estuary exhibited the highest prevalence of parasites (37.2 %) and inflammations (51.1 %). Finally, FCA and liver tumors occurred at very similar levels in both flounder populations from the Seine and the Ster estuaries. Group 0 flounders inhabiting the upper Seine estuary were more prone to parasitic and pre-neoplastic hepatic lesions and had higher levels of liver DNA adducts than the older ones living downstream. It was postulated that group 0 European flounders may serve as valuable bioindicators for assessing the quality of estuarine waters and the health status of euryhaline fish populations.

International audience; 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.

International audience; European flounder responses to the chemical stress were assessed by a comparative approach on four estuaries displaying contrasted patterns of contamination. The contamination typology of the estuaries was investigated by individual measurements of contaminants in fish. Molecular and physiological responses were studied by gene expression, genotoxicity, neurotoxicity and growth rate. Fishes in contaminated estuaries were characterized by high levels of bioaccumulated contaminants, slow energetic metabolism and reduced growth rate, in contrast to the fish responses in the reference site. A seasonal effect was highlighted for contaminated flounder populations, with high PCB levels, high genotoxicity and elevated detoxification rate in summer compared with winter.

International audience; The widespread use of pesticides results in a growing contamination of the aquatic environment. The effects of (1) a simple mixture of a glyphosate-based formulation and AMPA (Aminomethylphosphonic acid -- a primary metabolite of glyphosate) and of (2) a more complex mixture of herbicides (glyphosate/AMPA/mecoprop/acetochlor/2,4D) were explored on the molecular and physiological responses of the European flounder Platichthys flesus, considering a long-term and environmentally realistic contamination. Molecular responses were identified using suppression subtractive hybridization on liver samples: the level of gene transcription was significantly different between contaminated fishes vs control ones for 532 sequences, after a 62-day contamination. Among them, 222 sequences were identified by homology with data-based sequences; they encoded several metabolic pathways including: methionine and lipid metabolism, immunity, protein regulation, coagulation and energetic metabolism. Expression pattern of nine transcripts in the liver was confirmed by real-time PCR. The molecular study underlined that potential markers of liver injury were expressed for both mixtures, in particular betaine homocysteine methyl transferase and chemotaxin. Physiological responses were analysed considering blood parameters and condition factor; after the two months contamination period; no significant physiological difference was detected between contaminated and control fish.

International audience; The impacts of the fungicide Opus® (epoxiconazole) on marine phytoplankton communities were assessed in a 12-day field experiment using in situ microcosms maintained underwater at 6 m depth. Three community analysis methods were compared for their sensitivity threshold in fungicide impact detection. When phytoplankton communities were exposed to 1 μg l−1 of epoxiconazole, no effects could be demonstrated using TTGE (Temporal Temperature Gradient gel Electrophoresis), flow cytometry or HPLC. When exposed to 10 μg l−1, TTGE fingerprints from PCR amplified 18S rDNA of communities exhibited significant differences compared with controls (ANOSIM, P = 0.028). Neither flow cytometry counts, nor HPLC pigment profiles allowed to show significant differences in microcosms exposed to 10 μg l−1 of epoxiconazole. When exposed to 100 μg l−1, all three methods allowed to detect significant differences in treated microcosms, as compared to controls. The TTGE analysis appears in this study as the most sensitive method for fungicide impact assessment on eukaryote microbial communities.

The first record of the non-indigenous Fusinus rostratus in northern Brittany (north-west France) is reported. Seven specimens of this predatory gastropod were found on a muddy maerl matrix at about 10 m depth in the inner part of the bay of Morlaix. This is the first record for the Atlantic coast of France as the species originates from the warmer waters of the Mediterranean, Portugal and northern Spain, and was previously unrecorded in the English Channel. Some biometric measures are given.

In order to explore the population genetic structure of pollack (Pollachius pollachius) along the European coast, of 282 fish sampled from four locations along the Atlantic French coast and from one location off southern Norway were genotyped at six microsatellite loci. The limited genetic differentiation among samples may be due to high levels of larval dispersal, through passive drift with oceanic currents. Alternatively, populations may have diverged too recently for significant genetic differentiation to have become evident. Furthermore, small sample sizes and the limited number of loci may have hampered the detection of genetic structure. Nevertheless, a weak but significant genetic differentiation was detected between samples originating from the western English Channel and the Bay of Biscay.

International audience; In order to investigate the relative importance of historical processes and life-history traits in shaping the present-day genetic structure of European anglerfishes, 382 Lophius piscatorius and 134 Lophius budegassa were sequenced on the 5' end of the mitochondrial control region. Both species showed a limited genetic structure and some evidence of a demographic expansion that probably occurred not at the end of the Last Glacial Maximum, but earlier in the Pleistocene. The main discrepancy between the two anglerfishes concerned the genetic structure between Atlantic and Mediterranean populations, with weak but significant differentiation observed only in L. budegassa. This genetic structure was congruent with the existence of a phylogeographical break previously reported in several marine species across the Almeria-Oran front. The contrast observed between both anglerfishes was supposed to be induced by a possible more ancient (re)colonisation of the Mediterranean Sea by L. budegassa. Finally, the limited genetic structure and lack of isolation by distance observed in both species suggested high larval dispersal capacities that probably overwhelm the influence of the bathymetric distribution range on migrations of adults and juveniles.

International audience; Effects of pesticide exposure on the European flounder, Platichthys flesus, were investigated using a suppression subtractive hybridization method (SSH) to identify up- and down-regulated genes after a 30-day exposure to herbicides (a cocktail of atrazine, diuron, and isoproturon, and a single herbicide, glyphosate). A total of 256 expressed gene sequences were identified as having the potential for being differentially expressed, of which 116 could be identified by homology with databased sequences. The metabolic functions with which they are associated include energy production, general metabolism, signaling, transport, immune system, and structure. Expression of 14 of these genes was analyzed in liver, muscle, and gills by reverse transcriptase-polymerase chain reaction (RT-PCR) under experimental conditions (0, 15, and 30 days of exposure) and under field conditions (sampling in two estuaries displaying different levels of pesticide contamination). This study provides a first basis for studying the response of fish to pesticide exposure and allows the characterization of new potential genetic markers of pesticide contamination in the field.

Physiological and genetic responses of flounder Platichthys flesus populations were investigated along the French Atlantic Coast in one moderately contaminated estuary (Ster) and three contaminated estuaries (Seine, Loire and Gironde). The focus of this study was to explore the relationship between stress resistance and energetic trade-offs, in order to detect possible differential physiological capacities or performances between individuals carrying particular alleles or genotypes (allozyme data) characterised as "tolerant" or "sensitive". A general reduction of the relative fecundity, the growth rate and the condition factor was highlighted in contaminated fish populations, suggesting that survival in such polluted systems implies energetic costs for fish thus reducing the energy available for particular functions. A lower observed heterozygosity was also detected in contaminated populations with respect to the Ster, suggesting a general decrease in genetic variability in response to chemical stress (with an exception for the Seine estuary). This study confirmed the previously detected relationship between PGM 85, AAT1 95 alleles and reduced DNA damage in contaminated fish [Marchand, J., Tanguy, A., Laroche, J., Quiniou, L., Moraga, D., 2003. Responses of European flounder Platichthys flesus populations to contamination in different estuaries along the Atlantic coast of France. Mar. Ecol. Prog. Ser. 260, 273-284] and furthermore suggested that, reduced fecundity and condition factor associated to the individuals carrying the previous alleles, were also reflecting the cost of resistance to stress in polluted populations. The cost of tolerance to stress as well as the high gene flow from neighbouring populations less exposed to contamination may explain the apparently moderate increase of the suspected "tolerant" alleles in contaminated flounder populations.

Estuaries are important areas highly susceptible to anthropogenic degradations such as pollution. Estuarine species have to cope not only with environmental constraints inherent to estuarine habitats, but also with the presence of contaminants, the occurrence of hypoxic events or with waters warming. The European flounder Platichthys flesus is considered as a sentinel species in estuarine water quality monitoring. This study investigates the molecular mechanisms allowing the European flounder to cope with these different stress factors, both in environmental and experimental conditions, in order to eventually identify potential biomarkers of the response to contamination. A proteomic approach using 2-dimensional electrophoresis followed by MALDI TOF-TOF mass spectrometry allowed us to identify differentially expressed proteins in flounder livers, and then to better understand the mechanisms and a pathway implied in the response of flounder to environmental constraints. These observations were completed by targeted markers analyses by qPCR and enzymatic activity measurements. First, farm flounders were experimentally contaminated with two concentrations of PAH/PCB cocktails (concentrations found in the Seine and ten times this concentration). After 29 days of contamination, energetic metabolism was deregulated in contaminated flounder livers. Proteins involved in anti-oxidative defenses and detoxification were also accumulated. We suggest that BHMT could be implied in a pathway leading to the production of GSH allowing detoxification and anti-oxidative defense. Then, Flounders were fished in contrasted estuaries along the French Atlantic coast. The liver proteomic patterns of Flounders from the Seine (France) and the Tamar (UK), two estuaries displaying different contamination patterns, and from the Canche (France), were compared to characterize the proteins differentially expressed between these sites. Proteome profiles reflected the contamination patterns in each estuary., Les estuaires sont des zones de transition entre les eaux fluviales et les océans soumis à de fortes contraintes d’origine anthropique, telles que la pollution chimique. Pourtant, les estuaires sont d’une importance écologique primordiale, et sont notamment des zones de nourricerie ou de reproduction pour de nombreuses espèces. Le Flet européen Platichthys flesus est un modèle souvent utilisé comme espèce sentinelle dans les estuaires. Le but de ces travaux est d’explorer les mécanismes de réponse du Flet européen à la contamination chimique, afin éventuellement d’identifier de nouveaux biomarqueurs. Il a donc été choisi de mener cette étude par une approche de protéomique par électrophorèse en deux dimensions. Les protéines différentiellement accumulées en réponse à la pollution ont été identifiées par MALDI TOF-TOF. Ces études par protéomique ont été complétées par des mesures ciblées de transcription de gènes. Tout d’abord, des flets juvéniles issus d’un élevage ont été contaminés expérimentalement avec deux doses d’un cocktail de HAP et PCB (concentration retrouvée dans la Seine, et 10 fois celle-ci). Après 29 jours de contamination, le métabolisme énergétique paraît dérégulé. Des protéines impliquées dans les défenses anti-oxydantes et la détoxification sont accumulées dans le foie des poissons contaminés. Les résultats nous ont conduit à poser l’hypothèse de l’implication de la BHMT dans un cycle aboutissant à la production de GSH, et qui pourrait donc participer à la détoxification ou aux défenses anti-oxydantes. Ensuite, les profils protéiques de foies de poissons issus de 3 estuaires de la Manche ont été comparés. Les profils protéiques des foies de poissons de la Seine et de la Tamar, 2 estuaires présentant des profils de contamination différents, se sont révélés différents des profils protéiques des foies des poissons de la Canche. Des différences moins marquées entre les profils protéiques des foies de poissons de la Seine et de la Tamar pourraient refléter les différences entre les contaminants présents dans ces estuaires.