Thesis (Ph.D.)--Murdoch University, 2006. / Thesis submitted to the Division of Science and Engineering. Includes bibliographical references (leaves 149-162).

The aim of the current study was to determine the organic iron (Fe) requirements of gilthead sea bream (Sparus aurata). A total number of four experiments have been carried out each one for 12 weeks, in order to address and extend the knowledge on nutritional issues and challenges related with the culture of the gilthead sea bream in the Greek aquaculture industry and therefore enhance the fish health status under intensive culture conditions. These experiments aimed to determine the optimum level of organic Fe supplemented in commercial type diets of sea bream, the comparison between an organic Fe form and two inorganic Fe forms added in the diet of the fish, the effect of supplemented organic Fe on sea bream species exposed to oxygen deprivation stressors related to poor aquaculture husbandry practices and finally the interaction of organic Fe in the diet of sea bream with various levels of other trace minerals (Zn, Cu). The parameters evaluated were the growth performance of the fish, the Fe concentration in three selected tissues (the spleen, the liver and the muscle), the haematological status of the fish (the haematocrit, the red blood cell count, and the haemoglobin) and both the humoral and cellular immunology of the fish (the antibacterial activity of serum and the respiratory burst respectively). 150 mg/Kg of added organic Fe appears to be the recommended level as well as the minimum amount on fish exposed to overstocking conditions. The comparison between the two inorganic Fe forms (Ferrous Sulphate and Ferrous Carbonate) added in the diets show no significant effect on the fish. While, the fish fed the diets with 150 mg/Kg organic Fe and Cu levels lower that 5 mg/Kg had higher Hb values.

A series of five experiments were carried out to determine the effect of supplementary enzymes on growth performance and feed utilisation of juvenile gilthead sea bream, Sparus aurata, fed diets in which soybean meal (S8M) partially replaced fish meal (FM). In the first of these experiments the addition of cocktails containing 1 g/kg low pH active protease and 1 glkg a-galactosidase or 1 glkg high pH active protease and ] glkg a.-galactosidase to a 320 glkg SBM, 260 glkg FM pressed diet were both found to significantly (P<0.05) improve performance of fish fed these diets compared to fish fed the unsupplemented diet and a 320 glkg FM, 220 glkg SBM diet. This improvement in performance was not obtained when fish were fed 440 glkg SBM, 230 glkg FM diets with the same enzyme combinations. In some parameters performance of fish decreased as the SBM level in the diets was increased. The significant improvements observed in Experiment 1, with addition of enzyme cocktails to the 320 glkg S8M diet, were not repeated in any of the subsequent experiments. The second experiment was aborted due to ahnormal feeding hehaviour of the fish. In the third experiment, in which the enzymes employed in Experiment 1 were used individually at 1 glkg in 320 glkg SBM diets, no significant differences in specific growth rate (SGR), food conversion ratio (FeR) or protein efficiency ratio (PER) were noted in comparison to fish fed the unsupplemented diet. This was also the case with fish fed diets to which the two enzyme cocktails had been added at enzyme inclusion levels of 0.5 glkg each. Although no significant differences were found, feeding the diet with low pH protease alone appeared to increase performance compared to fish fed the unsupplemented diet, and the results of fish fed diets with high pH protease alone or with a.-galactosidase alone indicated that there was a decrease in performance compared to fish fed the unsupplemented diet. In Experiment 4 fish fed 320 g/kg SBM diets with 0.5, 1.0 and 1.5 g/kg low pH protease showed similar SGRs, FCRs and PERs which appeared to show an improved performance (although not significantly so) compared to fish fed diets with 1.0 glkg agalactosidase used together with either 0.5 or 1.0 glkg low pH protease. In the fmal experiment fish were fed 320 g/kg SBM extruded diets to which 0, 0.33, 0.66, 1.00 and 1.33 glkg of low pH protease had been added. Although no significant differences in SGR, FCR or PER were obtained, fish fed the diets containing 0.66 and 1.33 glkg protease appeared to improve performance compared to fish fed any of the other diets or a diet containing 320 glkg FM and 220 glkg SBM. Fish fed the other 320 glkg SBM supplemented dietc; gave similar results. A histological study of the position of nuclei in hepatocytes and the presence of fat globules around hepatopancreatic tissue in liver samples taken from fish fed the various experimental diets failed to show any relationships with either SBM level or enzyme inclusion in the diet. A series of analyses on the distribution of activities of six enzymes in the digestive tract of sea bream indicated that relative activities differed from one enzyme to another and from one region to another. In an investigation into the variation of pH in various parts of the digestive tract after one or two feeds, it was observed that within the first 24 hours after feeding the pH in the stomach decreased to a minimum value of 2.5 and the pH in the rest of the intestine varied between 6.5 and 7.7. vi From a series of gastric evacuation trials which were performed, it was found that the time of day sea bream were fed a meal influenced the gastric evacuation rate, with fish fed in the afternoon taking longer to evacuate the meal than fish fed a similar meal in the morning. Doubling the size of a meal did not double the gastric evacuation time. Instead, the time to evacuate a given percentage of the larger meal only increased by 1.4 and 1.6 times in fish fed the pressed and extruded feeds respectively compared to fish fed the smaller meal. When the sea bream were fed multiple meals it was found that the evacuation rate of an earlier meal was increased by a subsequent meal. A series of trials investigating the distribution in consumption of a population of sea bream fed a single meal indicated that there was a wide variation in the amount of food consumed by each fish in the population and it was observed that even fish of the same size consumed very different quantities of food. Before any definite conclusions can be drawn regarding the use of the three enzymes tested in these experiments to improve growth and feed utilisation in FM-substituted diets, further investigations need to be carried out in an attempt to obtain more significant results. This thesis has shown that additional research into the mode of action of these enzymes is required as well as studies into how the digestive physiology of the sea bream may affect the use of these (and other) supplementary enzymes.

[EN] The gilthead sea bream (Sparus aurata) is the fish specie more produced in the Mediterranean countries, however, the real nutrient requirements have not been studied until recently. The aim of the present thesis was to develop a multi-factorial model that allow to estimate the nutritional requirements of the gilthead sea bream, considering several growth situations for the weight range between 20 and 450g, as well as to design diets with a optimum nutritive composition. With that purpose, 6 trials were made, with gilthead sea bream ranged from 23 to 433g, fish were fed with increasing feeding rates from starvation to apparent satiation, with the aim of define the response curves of the protein retention, energy recovery, and growth next to the increasing feeding intakes. With the results obtained, factorial models able to determine the growing requirements were developed. And with them diets optimizing the nutrient efficiency for several situations were defined. The actual commercial diets with a ratio of digestible protein/digestible energy between 20 and 25 g MJ-1 are underestimated and produce a fat accumulation in the gilthead sea bream. A ratio of DP/DE between 25-30 g MJ-1 seems more adequate to optimize efficiencies and for improve the feed conversion ratios. / [ES] La dorada (Sparus aurata) es la especie piscícola que más se produce en los países mediterráneos, sin embargo no ha sido hasta hace relativamente poco cuando se empezaron a estudiar sus necesidades nutritivas reales. El objetivo de la presente tesis fue desarrollar un modelo factorial que permita estimar las necesidades nutritivas en dorada considerando diversas situaciones de crecimiento para un rango de pesos de entre 20 y 450 gramos, así como diseñar piensos con una composición nutritiva óptima. Para ello se realizaron 6 experimentos, con doradas de entre 23 g y 433g, a las que se alimento con tasas de alimentación crecientes desde ayunas hasta saciedad aparente, con el fin de determinar las curvas de respuesta, retención de proteína y retención de energía, y de crecimiento ante dosis crecientes de alimento. Con los resultados obtenidos, se pudieron desarrollar modelos factoriales capaces de determinar las necesidades de crecimiento y con ellas definir los piensos que optimizan la eficiencia de los nutrientes ante diversas situaciones. Los piensos actuales tienen una proporción de proteína digestible /energía digestible (PD/ED) subestimada, de entre 20 y 25 g MJ-1, lo que produce un engrasamiento paulatino de la dorada. Un ratio de PD/ED de entre 25-30 g MJ-1, parece más adecuado para optimizar las eficiencias y mejorar los índices de conversión alimentarios. / [CAT] L'orada és l'espècie d'aqu¿icultura més produïda en el països de la mediterrània. No obstant, no fou fins fa ben boc quan es van començar a estudiar les seues necessitats nutritives reals. L'objectiu d'aquesta tesi ha estat desenvolupar un model factorial que permeta estimar les necessitats nutritives de l'orada, considerant diferents situacions de creixement per a un interval de pesos entre els 20 i els 450 grams, a més de dissenyar pinsos amb una composició nutritiva òptima. Per tal d'aconseguir aquests objectius, es realitzaren 6 experiments, amb orades d'entre 23 i 433 grams, les quals foren alimentades amb taxes d'alimentació creixents, des del dejuni fins la sacietat aparent, amb la finalitat de determinar les corbes de resposta, de retenció de proteïna i d'energia i de creixements amb dosis creixents d'aliment. Amb els resultats obtinguts, es pogueren desenvolupar models factorials capaços de determinar les necessitats de creixement, amb les quals es puguen dissenyar els pinsos que optimitzen l'eficiència dels nutrients davant de diverses situacions. Els pinsos actuals tenen una proporció de proteïna digestible / energia digestible (PD/ED) subestimada, d'entre 20 i 25 g MJ-1, produint un engreixament gradual de l'orada. Una ràtio PD/ED d'entre 25 i 30 g MJ-1 sembla més adequada per optimitzar l'eficiència i millorar els índexs de conversió alimentària. / Jauralde García, I. (2016). Necesidades de mantenimiento y crecimiento de la Dorada (Sparus aurata L.) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62406 / TESIS

De nombreuses espèces de poissons marins côtiers à forte valeur économique ont un cycle de vie complexe impliquant des migrations saisonnières entre l'environnement marin et les habitats côtiers. Une meilleure compréhension de leur cycle de vie, en particulier l'identification de leurs nourriceries clés, est vitale pour la gestion durable des stocks de poissons exploités. Cette étude évalue la fonction de nourricerie des lagunes côtières du Golfe du Lion (Méditerranée NO) pour la daurade royale, Sparus aurata, une espèce d'intérêt commercial, dont les juvéniles colonisent ces habitats chaque année pendant leur premier été de vie. La qualité en tant que nourriceries de quatre lagunes aux conditions environnementales contrastées a été estimée par des mesures de la condition (indices de Fulton et TAG:ST) et du taux de croissance (par otolithométrie) des juvéniles. Les résultats montrent une condition et une croissance des juvéniles plus importantes dans les deux lagunes les plus dessalées et peu profondes (Bages-Sigean et Mauguio) que dans les deux lagunes profondes aux salinités proches de celle de la mer (Salses-Leucate et Thau). Les différences spatiales, stables dans le temps, de signatures élémentaires des otolithes ont permis de valider ces dernières comme tag naturel pour discriminer les habitats des juvéniles de daurade royale. Ainsi, l'identification a posteriori des nourriceries colonisées au stade juvénile chez des adultes pêchés le long de la côte du Golfe du Lion a pu être réalisée. Les estimations de la contribution relative des habitats des juvéniles au stock d'adultes (selon la contribution absolue ou la contribution par unité de surface) révèlent l'importance globale des lagunes pour le maintien des populations. Les lagunes les plus dessalées ont été identifiées comme les meilleures nourriceries de la zone d'étude. Le rôle important de la lagune de Thau pour le maintien des stocks de poissons a également été mis en évidence. Les résultats dans leur ensemble montrent une certaine convergence entre les estimations d'exports potentiel et réel des habitats des juvéniles. Les conclusions de cette étude sont primordiales pour la gestion et la protection des habitats clés des juvéniles ainsi que les ressources exploitées qui en dépendent. / Many coastal marine species of major socio-economic importance exhibited complex life histories include seasonal migrations between offshore marine environments and inshore coastal habitats. An adequate scientific understanding of their life cycle and, in particular the identification of the most significant juvenile nursery grounds are urgently needed for the sustainable management of fish stocks. This present study investigated the nursery function of coastal lagoons in the Gulf of Lions (NW Mediterranean) for a valuable fish species, the gilthead sea bream Sparus aurata, whose juveniles colonize these habitats each year for feeding and growing over their critical first summer of life. Habitat quality of four contrasted lagoons was measured with body condition (Fulton and muscle TAG/ST indices) and otolith growth rate of juveniles. Results showed better condition and higher growth rates in juveniles inhabiting the two shallow and brackish lagoons (Bages-Sigean, Mauguio) compared to the two deeper and more saline lagoons (Salses-Leucate and Thau). Otolith elemental signatures were temporally validated as natural tags to discriminate among juvenile habitats, allowing the retrospective identification of juvenile origin of S. aurata adults captured along the Gulf of Lions coastline. The estimations of relative contributions of juvenile habitats to adult stocks revealed the overall significance of lagoons for the maintenance of population. While, brackish lagoons were identified as the major contributors and best nursery habitats in the area, the importance of Thau lagoon to sustain fish stocks was also highlighted. Combined results of this research revealed some agreement between potential and effective export estimates from juvenile habitats. Information derived from this study is highly valuable for the sustainable management and protection of juvenile habitats, and the fishery resources that depend on them.

Η παρούσα μελέτη αναφέρεται στη βιολογία και την ηθολογία του είδους Sparus aurata στη διάρκεια της διαβίωσής του στο σύμπλεγμα των λιμνοθαλασσών Μεσολογγίου Αιτωλικού. περιλαμβάνει τέσσερις ενότητες 1. βιοτικοί και αβιοτικοί παράγοντες του συμπλέγματος, και οι χωρικές και χρονικές διαφοροποιήσεις του 2.στοιχεία της βιολογίας και της ηθολογίας του είδους στις διαφορετικές λιμνοθάλασσες του συμπλέγματος 3.πρότυπα εκμετάλευσης Λ/Θ και η επίδρασή τους στα αποτελέσματα της αλιείας 4.μια πρώτη αξιολόγηση της επίδρασης που έχει η ανάπτυξη της εντατικής καλλιέργειας στην ευρύτερη περιοχή πάνω στους φυσικούς πληθυσμούς του είδους στο λιμνοθαλάσσιο σύμπλεγμα. / The study concerns the biology and behaviour of Sparus aurata during its presence in the lagoons of the Messolonghi-Etoloko (Western Greece). The following four components are included: 1. spatial variability and temporal fluctuations of the abiotic parameters in the area of the lagoons 2.elements of the biology and behaviour of the species in the different lagoons of the area 3.exploitation pattern of the biology of the lagoons and their contribution to the recorded landings 4.a first approach to estimate the influence of the intensive fish farming in the area on the wild populations of the species in the lagoons

La daurade royale (Sparus aurata, L.) est une espèce migratrice, connue pour coloniser les lagunes côtières, fortement anthropisées, du printemps à l'automne. Identifier les lagunes clés pour l'accomplissement du cycle de vie doit donc permettre de mieux gérer et protéger les populations de daurades du Golfe du Lion. Dans ce cadre, la microchimie de l'otolithe est un outil précieux. Au cours de sa croissance, cette concrétion de l'oreille interne incorpore des éléments chimiques prélevés dans le milieu. Piégés dans le réseau cristallin, ils sont une image des conditions environnementales rencontrées par le poisson à une date donnée ce qui rend possible, en théorie, de reconstituer ses migrations. Néanmoins, pour cela, il faut (i) connaître précisément la croissance du poisson et de ses otolithes, (ii) mettre en évidence des différences chimiques entre les zones clés pour le cycle de vie en identifiant les outils statistiques appropriés pour la discrimination des habitats. Ainsi, (i) la comparaison objective de modèles de croissance, associée à l'analyse d'un spécimen exceptionnellement âgé a permis de remettre en cause les données classiquement acceptées sur la croissance de la daurade et le schéma de croissance de ses otolithes a été établi ; (ii) la comparaison de 4 méthodes statistiques de discrimination a permis de sélectionner la méthode du random forest pour différencier les signatures chimiques des otolithes de S.aurata de différentes lagunes. Ces avancées ont permis de confirmer le schéma migratoire précédemment décrit, tout en révélant une variabilité des comportements entre les individus et au cours de la vie du poisson. Les juvéniles semblent préférer les lagunes dessalées, suggérant un rôle de nurserie pour ces habitats, alors que les adultes préfèrent des lagunes profondes comme l'étang de Thau. Le maintien de la diversité des caractéristiques des différentes lagunes est donc nécessaire, afin d'assurer la pérennité de la population de S.aurata dans le Golfe du Lion. / The Gilthead seabream (Sparus aurata L.) is a migratory species, known to colonize coastal lagoons with high anthropogenic load, from spring to autumn. It is necessary to identify key lagoons for the seabream life cycle to allow better management and protection of gilthead seabream populations in the Gulf of Lions. For this, otolith microchemistry is a powerful tool. As it grows, this inner ear concretion absorbs chemical elements taken in its surrounding. Traped in the crystal net, these elements are an image of the environmental conditions met by the fish individual. It is then possible to rebuild fish past migrations. For this it is necessary to (i) precisely know the fish and otoliths growth calendars and (ii) identify chemical differences between the key habitats for the life cycle by choosing statistical methods for habitat discrimination.(i) The objective comparison of many growth models, combined to the analysis of an exceptionally old specimen allowed to propose a new scheme for the gilthead seabream growth and its otolith growth pattern was established ; (ii) the comparison of 4 statistical discrimination methods allowed to select the random forest method to differentiate the otolith chemical signatures of gilthead seabreams from various lagoons. These insights confirmed the already described migration pattern and emphasized a behavioural variability between individuals and between periods of life. The juveniles seem to prefer brackish lagoons, suggesting a nursery rôle for these habitats, whereas the adults prefer deeper lagoons as the Thau lagoon. The maintenance of the lagoons diversity is thus necessary for the preservation of the Gilthead seabream population in the Gulf of Lions.

La daurade royale (Sparus aurata) est une espèce de sparidé dont l’aire de répartition s’étend des côtes du Sénégal à l’Irlande, et est commune en mer Méditerranée. Elle est considérée comme rare en limite nord de répartition (Manche, mer d’Irlande et mer du Nord). Depuis une dizaine d’années, l’abondance d’individus pêchés en limite nord ne cesse d’augmenter. Ce phénomène peut être expliqué par l’évolution septentrionale de son aire de répartition, en lien avec le réchauffement climatique. Aucune donnée écologique sur les populations de cette espèce en limite nord de répartition, devenues une ressource de pêche en Manche, n’est actuellement publiée dans la littérature. Les traits de vie de S. aurata ont été étudiés face à la modification spatio-temporelle de son aire de répartition, via l’analyse du régime alimentaire, de la croissance et des déplacements côtiers. D’autre part, l’étude a conduit à l’analyse de la structure de ces populations septentrionales par une approche multi-marqueur couplant la génétique et la microchimie des otolithes.L’analyse des contenus stomacaux des populations de S. aurata en limite nord de répartition confirme un régime opportuniste, avec une forte proportion d’espèces-proies du genre Mytilus. Malgré un régime alimentaire similaire, la croissance des individus est, quant à elle, plus faible que celle observée sur des individus de mer Méditerranée, ce phénomène étant conditionné par les paramètres du milieu (température, salinité). Les variations élémentaires des otolithes ont permis de caractériser les migrations côtières lors des premières années de vie des poissons avec un passage en mer en hiver et la fréquentation de zones côtières. Cela suggère la présence de zones de nourricerie le long du pourtour atlantique et de la Manche. Une variabilité inter-individuelle a été mise en évidence suggérant une plasticité comportementale des individus. Les approches de génétique, combinant l’emploi de marqueurs mitochondriaux et des microsatellites, et de microchimie des otolithes ont mis en évidence l’absence de structure au sein des populations en limite nord de répartition. Ce phénomène concorde avec la colonisation récente des populations. Cependant, une différenciation entre les individus échantillonnés le plus au sud et ceux en limite nord d’échantillonnage a été mise en évidence, suggérant peu de mélange génétique. Les allèles communs entre les individus échantillonnés évoquent une colonisation des individus de proche en proche depuis la mer Méditerranée. Des différences génétiques et de traits de vie ont été observées entre les individus échantillonnés en Manche, suggérant l’existence d’une barrière biogéographique au sein de cet environnement. La présence de conditions environnementales favorables constitue un atout majeur pour cette espèce prédatrice qui semble présenter une capacité d’acclimatation importante. L’ensemble des approches abordées dans le cadre de ces travaux ont permis d’apporter les premières données sur les traits de vie et la structure des populations d’une espèce à fort intérêt commercial dans une zone d’expansion récente en lien avec des modifications globales des conditions environnementales. / The gilthead seabream (Sparus aurata) is a sparidae species whose natural distribution stretches from Senegal to Ireland coasts, with its common habitat in the Mediterranean Sea. This species is still considered as rare in its northern limit of distribution. For a decade, the abundance of individuals caught in the northern limit (English Channel, Irish Sea and North Sea), has increased. This species is now a fishing resources in the English Channel. This phenomenon, in link with global warming, is explained by the northern expansion of its distribution range. No ecological data about this species in northern distribution range is currently published. Life history traits of S. aurata in relation to the spatio-temporal modification of its distribution range has been studied, through diet, growth and sea/coastal movement analysis. On the other hand, the population structure analysis of these northern populations has been done with a multi-marker approach coupling the genetics and otolith microchemistry.Analysis of the stomach contents of S. aurata at the northern range confirms an opportunistic diet with a high proportion of prey species from the genus Mytilus. The growth of individuals is lower than that observed on individuals in the Mediterranean Sea. This phenomenon is conditioned by environmental parameters (temperature, salinity) at the distribution range limit. Elemental composition of the otoliths allowed us to characterize the sea/coastal migrations during the first year of life, with a sea transition in winter and the occupation of coastal zones. A variability between individuals has been observed as a behavioral plasticity of individuals. This suggests the presence of nursery areas along the Atlantic and Channel Sea coasts. Otolith microchemistry and genetic approaches, combining the use of mitochondrial and microsatellites markers, have demonstrated the lack of structure within populations at the northern distribution range. This phenomenon matches with the recent population colonization. However, a differentiation was identified between the most southern individuals sampled and those at the northern sampling boundary, suggesting little genetic mixing. Common alleles between individuals suggest a “step by step” colonization of individuals from the Mediterranean Sea. Genetic and life history traits differences were observed between individuals sampled in the Channel Sea, suggesting a biogeographic barrier within this environment.Favorable environmental conditions are a major asset for this predatory species, which appears to have a huge acclimatization ability. All the various approaches discussed in this work have then made it possible to provide the first data on the life characteristics and the population structure of a species with a high commercial interest on the northern range of its distribution.

La fraîcheur est un paramètre clé de la qualité du poisson. Les méthodes actuelles appliquées en routine pour déterminer la fraîcheur du poisson ne sont pas applicables à toutes les espèces et reflètent davantage un début d'altération du produit. Ainsi, la recherche d'indicateurs précoces de fraîcheur du poisson représente encore un défi majeur et d'actualité dans l'industrie de la pêche. Le but de ces travaux de thèse était de démontrer que les fonctions et l'intégrité mitochondriales étaient susceptibles de constituer des indicateurs précoces de la fraîcheur de filets de poisson. En effet, la mitochondrie est la "centrale" énergétique de la cellule eucaryote et joue un rôle clef dans les mécanismes de mort cellulaire tels que l'apoptose et la nécrose. Les fonctions et l'intégrité mitochondriales de cellules musculaires de filets de poisson ont été étudiées à différents temps de conservation post mortem à 4°C. Le modèle d'étude était la daurade royale (Sparus aurata) (lignée cellulaire de fibroblastes (SAF-1) et muscles de poisson). Dans un premier temps, la structure des mitochondries de poisson a été étudiée par microscopie électronique à transmission. De nombreuses dégradations de la structure des mitochondries ont été observées dans les filets à partir de 72 heures (J3) de conservation à 4°C. Ces altérations se sont accentuées à J4 et J6. La fonctionnalité des mitochondries a ensuite été évaluée selon deux approches : la respiration mitochondriale (oxygraphie) et le potentiel membranaire mitochondrial (ΔΨₘ) estimé avec la sonde fluorescente Rhodamine 123. A partir de 96 heures de conservation à 4°C (J4), ces deux paramètres ont été significativement impactés témoignant d'une altération des fonctions et de l'intégrité mitochondriales.Ces résultats sont ainsi en corrélation avec l'altération structurale observée par microscopie. En parallèle, une méthode d'évaluation du potentiel membranaire a été développée avec un fluorimètre à microvolume à partir d'un modèle bactérien puis de mitochondries isolées. Ces travaux de thèse ont démontré que l'étude des fonctionnalités mitochondriales constitue un marqueur fiable et précoce de la fraîcheur des filets de poisson. Des connaissances supplémentaires sur les mécanismes cellulaires post mortem ont également été apportées. Ces résultats constituent ainsi le point de départ pour le développement d'un kit d'évaluation de la fraîcheur et ouvrent la voie pour la recherche de marqueurs de fraîcheur et de congélation/décongélation basés sur les fonctionnalités et intégrité mitochondriales. / Freshness is a key parameter of fish quality. Current routine techniques to determine fish freshness are not applicable to all species and reflect a late stage of alteration. Thus, research on early indicators of fish freshness still represents a major and topical challenge in fishing industry. This PhD research project aimed to demonstrate that mitochondrial functions and integrity constitute early indicators of fish fillet freshness. Mitochondria are the powerhouse of the cell and play a central role in cell death mecanisms such as apoptosis and necrosis. Mitochondrial function and integrity in fish filet muscle cells were studied at different times of storage post mortem at 4°C. The species studied as a model was the gilthead seabream (Sparus aurata) (gilthead seabream fibroblast cell line (SAF-1) and fish fillets). Firstly, the structure of fish mitochondria was studied by transmission electron microscopy. Numerous mitochondrial structural alterations have been observed in fish fillet from 72 hours (D3) of storage at 4°C. These alterations were more pronounced at D4 and D6. Then, mitochondrial functionality was assessed with two approaches: mitochondrial respiration (oxygraphy) and mitochondrial membrane potential (ΔΨₘ) estimated with the fluorescent probe Rh123. From 96 hours of storage at 4°C (D4), these two parameters were significantly disrupted demonstrating the alteration of mitochondrial function and integrity. The results are in correlation with the mitochondrial structural alterations described by microscopy. In parallel, a method of mitochondrial membrane potential evaluation has been developed with a micro-volume fluorimeter, first using bacteria and then isolated mitochondria. This work demonstrated that the mitochondrial functionality study constitutes a reliable and early fish filet freshness indicator. Additional knowledge on cell mechanisms in post mortem condition has been brought. These results constitute the starting point for the development of a fish freshness assay kit and pave the way to research on others freshness and freeze-thawing indicators based on mitochondrial integrity and functionality.

The redox properties of Copper (Cu) make it both an ideal cofactor for many enzymes, and, in its free form, a highly toxic molecule capable of stimulating production of reactive oxygen species or binding to protein thiol groups. Therefore, living organisms have evolved homeostatic systems to “handle” Cu avoiding dangerous and wasteful aspecific interactions. These systems comprise uptake, carrier, storage and excretion proteins. The importance of Cu-homeostatic systems was initially discovered in humans where alterations of Cu-excretory proteins were shown to be responsible for two lethal genetic disorders; the Wilson and Menkes diseases. The levels of bioavailable Cu in the aquatic environment is important because concentrations in oceanic waters tend to be minute, whilst in some fresh and coastal waters, particularly around areas of mineral extraction, viniculture and farming operations, concentrations can be excessive. In contrast to terrestrial vertebrates, fish are not only exposed to dietary sources of copper but are also exposed to dissolved ionic copper that may enter via the skin and gills. Indeed, the latter route is important in fish and it has been demonstrated in physiological studies that under conditions of dietary deficiency, fish can satisfy their own body requirements by uptake from water. Therefore, fish must have systems relating to both gill and gut to enable maintenance of body homeostasis of this essential, yet toxic, metal. In an attempt to understand the mechanisms of Cu homeostasis in fish, whether under conditions of deficiency, adequacy or excess, it is essential to consider the expression of known Cu-homeostasis proteins. Thus, cDNAs for sea bream (Sparus aurata) homologues of copper transporter 1 (Ctr1), antioxidant protein 1 (Atox1), Menkes protein (ATP7A), Wilson protein (ATP7B), and metallothionein (MT), which are responsible for the uptake, delivery to the secretory pathway and scavenging of intracellular Cu, were cloned and their mRNA tissue expression levels measured. To investigate the molecular basis of the different homeostatic and toxic responses to waterborne or dietary Cu, sea bream were exposed to sub-toxic levels of Cu in the diet (130 mg/Kg of dry diet) or water (0.3 mg/L) and tissue mRNA and Cu levels were measured. Moreover, to discriminate between the effect of different metals on the transcriptional regulation of Cu homeostasis genes in fish, Sparus aurata fibroblast (SAF1) cells were exposed to sub-toxic levels of Cu (25 μM), Zn (100 μM) and Cd (10 μM). In addition, a microarray was used to gain a broader overview of the transcriptional response of SAF1 cells to Cu (25 μM). Waterborne or dietary Cu resulted in distinct expression profiles of Cu-homeostasis genes and markers of oxidative stress. After dietary exposure, Cu increased in intestine and liver, whilst after waterborne exposure Cu increased in gill and liver. Exposure to dietary Cu resulted in decreases in Ctr1 and ATP7A mRNA in both liver and intestine. Renal Ctr1 levels remained unchanged, whilst ATP7A mRNA decreased. In contrast, waterborne Cu exposure increased intestinal Ctr1 and ATP7A mRNA, and increased renal Ctr1 and decreased renal ATP7A mRNA. Both dietary and waterborne Cu increased ATP7B mRNA in liver. Metallothionein (MT) mRNA increased in liver and gill after waterborne Cu. Glutathione reductase (GR), a marker of oxidative stress, increased expression in liver and gill after waterborne Cu exposure, but decreased in intestine. Thus, exposure to Cu via water or diet has different, often opposite effects on Cu-homeostasis genes. The decrease in expression of both Cu-transport genes in intestine after dietary exposure may indicate a defensive mechanism to limit uptake of Cu. The opposite effects in intestine after waterborne exposure are more difficult to explain, but again may reflect a defence mechanism against excess bloodborne Cu coming from the gill. Since both dietary and waterborne Cu increased Cu levels in liver and increased hepatic ATP7B it is likely that well-characterised mammalian route of Cu excretion to bile is active in sea bream. However, only hepatic Cu derived from gill increased the expression of the stress markers MT and GR. This suggests that Cu is delivered to liver in a different form from gill as that from intestine, the intestinally derived pool being less toxic. Thus the increase in copper transport gene expression in intestine after gill exposure might be a mechanism to enable incorporation of excess bloodborne Cu into the intestinal pathway of Cu delivery to liver, thus minimizing toxicity. The in vitro exposure of SAF1 cells to Cu showed a similar response to liver of fish exposed to waterborne Cu indicating similar Cu availability and complexation. ATP7A mRNA levels were induced by Cu but not by Zn or Cd suggesting Cu-specific regulation. Conversely, MT and GR were induced by all metals tested. The transcriptomic analysis highlighted that the biological processes most significantly affected by Cu were secretion, protein trafficking and stress. Overall, these results show that in fish copper has distinct effects on tissue Cu transporter genes and oxidative stress depending on whether it is taken up via the gill or gut and that intestinal absorption may be required for normal uptake and metabolism of Cu, regardless of the route of uptake. Moreover, changes in mRNA levels indicate that Cu homeostasis genes, at least in fish, may be regulated at the transcriptional level. Although more work needs to be done to identify genes that are robust predictors of Cu toxicity, the microarray results presented here show a clear transcriptional fingerprint which may characterize Cu toxicity in fish.