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. / 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.

Le flet européen (Platichthys flesus) est un poisson catadrome dont l’aire de répartition dans le Nord-Est Atlantique s’étend du cercle polaire Arctique jusqu’au Portugal. Depuis quelques décennies, la limite Sud de son aire de distribution remonte le long des côtes Portugaises. Une étude préliminaire sur la biologie des populations de flet a identifié un métabolisme énergétique spécifique pour une population périphérique Sud au Portugal vs des populations situées dans le Golfe de Gascogne et en Manche. Dans la présente étude, nous avons combiné une approche en génétique de populations avec des challenges expérimentaux menés en situation de common garden, pour approfondir nos connaissances sur la différentiation génétique entre les populations de flets sur la façade Atlantique et sur le possible différentiel phénotypique qui pourrait en résulter, particulièrement en terme de bioénergétique et de traits d’histoire de vie. L’approche génétique considérant le polymorphisme de marqueurs de type microsatellites et de différents gènes candidats met en évidence une différentiation génétique très significative entre les populations de la Péninsule Ibérique et celles situées plus au Nord. Une pression de sélection différentielle s’exerce probablement sur un gène impliqué dans le métabolisme énergétique (GAPDH) et confirme la particularité génétique des populations Ibériques. Un challenge thermique et hypoxique conduit sur des poissons juvéniles et mettant en oeuvre une combinaison d’approches ciblées (activités enzymatiques sur des proxies du métabolisme énergétique en aérobiose vs anaérobiose, et de l’anabolisme) et sans a priori (protéomique par électrophorèse 2D) suggère une meilleure aptitude des populations centrales (Canche et Vilaine, France) et de la population périphérique (Lima, Portugal) à se maintenir respectivement en conditions froide vs chaude et hypoxique. Enfin, l’analyse de normes de réaction des traits de vie pour des stades embryo-larvaires exposés à un gradient thermique met en évidence une probable adaptation locale des populations de flet à leur environnement thermique. / The European flounder (Platichthys flesus) is a catadromous fish species distributed along the North-East Atlantic coasts, from the Arctic polar circle to the Iberian Peninsula. For several decades, the southern limit of its distribution area is shifting towards the North of the Portuguese coast. A preliminary study on the flounder population biology has detected a specific energetic metabolism for a southern peripheral population in Portugal compared to Northern populations located in the Bay of Biscay and in the Channel. In the present study, we have combined population genetics with experimental challenges in the laboratory to improve our knowledge on the genetic structure of flounder populations over Europe, and on the potentially resulting phenotypic differences among populations, mainly considering bioenergetics and life-history traits. The polymorphism of microsatellite markers and of several candidate genes highlighted a significant genetic differentiation between Iberian Peninsula populations and Northen populations. A thermal and hypoxia challenge has been conducted on flounder juveniles, considering (1) enzymatic activities linked to energetic metabolism (aerobiosis and anaerobiosis) and anabolism, and (2) a proteomic approach; this common garden experiment suggested a higher capacity of central populations (from Canche and Vilaine, France) and of one peripheral Southern population (from Lima, Portugal) to cope with respectively cold condition, warm and hypoxia condition. Finally, the analysis of thermal reaction norms in fish early life history traits, considering embryo-larval stages submitted to a thermal gradient, underlined a possible local adaptation of the flounder populations to their thermal environments.

Our understanding of turbot and flounder ecology in the Baltic Sea is insufficient for sound management decisions. This thesis aims to fill some gaps in current knowledge by providing information of the ecology of turbot and flounder within their juvenile habitat, and to relate these findings to issues assumed relevant for recruitment variation. Main focus is on turbot due to its relatively low abundance and high variability in recruitment. The distribution of both species was studied on different scales, as was environmental effects on food consumption in 0-group turbot. The 0-group turbot display a relatively restricted spatial distribution compared to flounder. This is possibly due to a more specialized diet, which may make them more vulnerable to habitat degradation, especially eutrophication as a strong negative correlation was found with the organic content in the sediment. The species show high temporal and spatial overlap when settling in July-September, with peak abundances in August, and at depths <1 m. Both species display sedentary behavior within the nursery ground. Compared to flounder, turbot was more mobile potentially due to its restricted diet calling for extended searches. For turbot, feeding conditions appear to vary between size groups, which potentially could cause variations in survival between years through size-selective mortality. But, the predation may be low in central Baltic Sea as the abundance of the main predator, brown shrimp are comparatively low during flatfish occupancy. A significant positive relationship was found between the recruitment of turbot and flounder, which suggests that no inter-specific interactions during the juvenile stage affect recruitment. This co-variation also suggests that the recruitment of the species is determined by the same phenomena, potentially by large scale abiotic factors during the egg- and larval stage. For turbot, additional variability is potentially generated during the juvenile stage due to its relatively restricted food and habitat requirements. The specific habitat demands of turbot revealed in this thesis may be used to protect and restore essentially nursery grounds. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 3: Submitted. Paper 4: Submitted.

The reproductive success for pelagic spawning Baltic flounders is strongly linked to the hydrodynamics in the spawning areas. Egg survival is dependent upon the ability to achieve neutral buoyancy at a depth interval where temperature and oxygen concentrations are favourable for egg development. The main focus of this thesis was to compare egg survival probabilities of pelagic eggs from Baltic flounder in the Bornholm, Gdansk and Gotland basins, prior to and after the saline water inflow in December 2014. The results showed greatly enhanced survival probabilities in Bornholm basin 2015 (p<0.01), as egg survival increased from 47% in 2014 to 100% the following year. In Gdansk basin the situation was similar, and survival probability increased from 13% to 100% (p<0.01). In Gotland basin no difference in survival probability was identified, although the dominant cause of mortality shifted from sedimentation, i.e. due to low salinity conditions in 2014, to oxygen deficiency in 2015 (p<0,01). / BONUS INSPIRE-project, the joint Baltic Sea research and development programme (Art 185), funded jointly by the European Union’s Seventh Programme for research, technological development and demonstration and the Swedish Research Council Formas

Effects of man made pollutants on an ecosystem are initiated at the cellular level where a prime determinant for survival of an organism is its ability to metabolise and excrete toxic chemicals or their metabolites, thereby preventing cellular toxicity or damage to germ cell DNA. Cytochrome P450 (CYP) enzymes are responsible (in concert with the remainder of the Ah battery enzymes) for the metabolism of numerous xenobiotics and endogenous compounds, including the metabolic activation of most environmental toxic chemicals and carcinogens. Genetic polymorphisms which affect performance of these enzymatic detoxification systems may alter tolerance to pollutants and thus survival in polluted environments. Alterations in the susceptibility of individuals and the development of resistant populations has arisen by forced selection of populations with variant genes, resulting in increased detoxification capacity. There is evidence for such scenarios of variations in activities of pollutant biotransforming enzymes of fish contributing to survival in polluted estuarine environments and several chemically resistant populations have been identified in the USA and Europe. In fish it has been demonstrated that CYP1A enzyme activity is required to activate some carcinogenic xenobiotics to a metabolic state in which they can form DNA adducts. The mechanism of reduced CYP1A expression in highly contaminated populations may therefore represent resistance to chemical stressors. European flounder (Platichthys flesus) from some waterways which have a long history of severe sedimentary contamination do not show elevated levels of CYP1A. The aim of the current study was to investigate whether any heritable differences were apparent between offspring from parents inhabiting long-term polluted and pristine areas. Flounder were obtained from a highly polluted estuary in the UK and crossed with fish from a relatively pristine environment. Offspring were raised in communal tanks in order to standardise environmental conditions, and allow investigations into the genetic variation of CYP1A. To allow identification of offspring to parental fish, polymorphic microsatellite loci were isolated and characterised for the flounder. Novel cDNA probes to transcription factors in the detoxification pathway (AhR2 and ARNT2) were cloned for flounder, and RT-PCR / Southern blot methods were developed for quantitation of gene transcript levels. A novel method of CYP1A quantification using real-time PCR was developed. PAH and PCB exposure trials were carried out on mixed batch offspring, and CYP1A gene transcript levels assessed using Northern blot and real-time PCR techniques. Offspring were genotyped to their parents using the microsatellites obtained, and CYP1A transcript levels were correlated with clean and polluted areas. CYP1A was further correlated to transcription factor expression, and data are presented. Following exposure to the commercial PCB mixture, Aroclor 1254, CYP1A transcript levels were found to be significantly lower in families whose parents originated from a polluted area. This observation indicates that there is a possible genetic component to variation in CYP1A levels, and that these fish may have acquired a heritable tolerance to polluted areas. The lack of induction, or correlation with CYP1A levels, of AhR2 and ARNT2 expression indicates a possible AhR independent pathway for the metabolism of PCBs in the flounder. © Tom Dixon 2003 http://www.tomdixon.org