The role of dietary lipids and essential fatty acids in the processes of skeletogenesis and pigmentation in Senegalese sole (Solea senegalensis) larvae = Efecto de los lípidos y ácidos grasos esenciales de la dieta sobre el desarrollo esquelético y la pigmentación en larvas del lenguado senegalés (Solea senegalensis)

Skeletal deformities and pigmentary disorders, occurring from the early larval stages, affect the external appearance of fish, downgrade the market value of the final product and reduce the intensive production of flatfish juveniles. Among other factors, lipid nutrition is known to be involved in skeletogenesis and pigmentation, and unbalanced levels of lipids or essential fatty acids (EFA) or their inappropriate form of supply in the diet during larval development may lead to skeletal deformities and pigmentary disorders. This thesis aimed to study the role of dietary lipids and EFA in the incidence of skeletal and pigmentary disorders in Senegalese sole (Solea senegalensis), a flatfish of high market value produced in the Iberian Peninsula. In a first part, larvae were fed different commercial enriching products for Artemia metanauplii, presenting distinct FA profiles. Absolute levels of DHA, EPA and ARA of 9.5, 3.1 and 0.7% TFA, respectively, and n−3/n−6 PUFA, DHA/EPA, ARA/ DHA and OA/PUFA ratios of 5.2, 3.0, 0.1, 0.5, respectively, were recommended as more suitable for Senegalese sole growth performance, larval development and ossification, although the incidence of skeletal deformities was not significantly affected by the dietary FA composition. A variation, even slight, of these proportions might have modified the metabolism of lipids and disrupted the profile of lipid accumulation in the target tissues, leading to severe intestinal and hepatic steatosis and reduced larval growth and development. In a second part, larvae were fed increasing amounts of dietary peroxidized lipids (34.5 to 78.8 nmol MDA g-1 w.w.). Although no physiological alterations were detected on their FA profile, oxidative stress status, survival, growth performance, metamorphosis and ossification, fish seemed to activate antioxidant defense mechanisms in response to the dietary oxidative stress, through the consumption of the dietary vitamin E and the activation of antioxidant enzymes. Feeding fish with highly oxidized diets caused an increase of fat accumulation in the enterocytes and a reduction in bone mineralization. Vitamin E was suggested to interact with ossifying cartilage. In a third part, the effects of dietary arachidonic acid (ARA) on the incidence of skeletal malformations and pigmentary disorders were evaluated on Senegalese sole larvae. The amount of 4.5% TFA of ARA fed to larvae during the Artemia feeding period was optimal for proper growth and ossification, whereas 1 and 7.5% TFA of ARA led to imbalanced ARA/EPA and n-6/n-3 ratios that was suspected to disrupt the relative proportions of prostaglandins PGE2 and PGE3, and significantly delayed somatic growth and skeletal ossification. None of the dietary ARA contents affected the incidence of skeletal deformities in vertebral and caudal regions, but feeding larvae with high levels of dietary ARA (10.2% TFA in rotifer and 7.1% in Artemia metanauplii) during the pre-, pro- and/or post-metamorphosis induced an impaired eye migration in larvae, altered their head shape and cranial bone remodeling and induced malpigmentations. The ontogeny of the pigmentation of the ocular side skin at both morphological and molecular levels was studied in Senegalese sole larvae and the appearance of the ARA-induced pseudo-albino phenotype was characterized. A high “sensitivity window” to ARA-induced malpigmentation was identified during pre- and pro-metamorphosis stages, resulting in pseudo-albino specimens characterized by the absence of differentiation of post-metamorphic populations of chromatophores and the inhibition of the melanogenesis, reflected by the disrupted expression of genes involved in these processes (asip, pax3, cKit, mitf, tyr, trp1 and slc24a5). Supplying high ARA amounts to larvae altered their dietary ARA/EPA ratio and relative concentrations of PGE2 and PGE3, causing pigmentary disorders. This work provides helpful knowledge to understand how dietary lipids and EFA affect skeletogenesis and pigmentation processes, to improve actual Senegalese sole larval rearing and juveniles’ quality.

Las malformaciones esqueléticas y problemas pigmentarios son dos de los problemas más importantes que afectan actualmente a la producción masiva de juveniles de peces planos. Estas anomalías, que ocurren desde las etapas iniciales del desarrollo larvario, afectan su apariencia externa, disminuyen su precio final en el mercado y representan un importante cuello de botella para la acuicultura. Entre los factores implicados en la aparición de los citados desórdenes, la nutrición lipídica es uno de los parámetros más importantes afectando a la esqueletogénesis y a la pigmentación. Un desequilibrio en los niveles de lípidos y/o ácidos grasos esenciales (AGE) puede conducir a un desarrollo anormal del esquelético y despigmentación. El objetivo de la presente tesis doctoral investigó el efecto de los lípidos y AGE (particularmente el ácido araquidónico, ARA) administrados en la dieta, sobre la esqueletogénesis y pigmentación en larvas del lenguado senegalés (Solea senegalensis), especie de pez plano producido en la península Ibérica, con alto precio en el mercado. El efecto de diferentes productos comerciales con distintos perfiles de ácidos grasos (AG) usados comúnmente para enriquecer Artemia, fue evaluado sobre el crecimiento, la maduración del sistema digestivo, la deposición de lípidos en diversos tejidos objetivos, la osificación y la incidencia de deformaciones esqueléticas en larvas de lenguado senegalés, trabajos que fueron completados con el estudio de la regulación molecular de dichos procesos durante el proceso de metamorfosis. Los efectos de los lípidos oxidados en la dieta también fueron evaluados sobre el crecimiento larvario, la actividad de enzimas antioxidantes, la deposición intestinal de lípidos, la osificación y la incidencia de deformidades esqueléticas en el lenguado senegalés. Finalmente, los efectos del ARA de la dieta sobre el crecimiento, la osificación, la incidencia de malformaciones esqueléticas y los desórdenes pigmentarios fueron evaluados y relacionados con las proporciones de prostaglandinas PGE2 y PGE3. La ontogenia de la pigmentación de la piel del lado ocular fue estudiada a nivel morfológico y molecular con el fin de detectar posibles alteraciones de un perfil normal de desarrollo de la pigmentación, mientras que la aparición del fenotipo pseudo-albino inducido nutricionalmente por un exceso de ARA fue también caracterizada.