Coordinated regulation of chromatophore differentiation and melanogenesis during the ontogeny of skin pigmentation of Solea senegalensis (Kaup, 1858)

Abnormal pigmentation of Senegalese sole has been described as one problem facing the full exploitation of its commercial production. To improve our understanding of flatfish pigmentation of this commercially important species we have evaluated eleven genes related to two different processes of pigmentation: melanophore differentiation, and melanin production. The temporal distribution of gene expression peaks corresponds well with changes in pigmentation patterns and the intensity of skin melanization. Several gene ratios were also examined to put in perspective possible genetic markers for the different stages of normal pigmentation development. Further, the phenotypic changes that occur during morphogenesis correspond well with the main transitions in gene expression that occur. Given the dramatic phenotypic alterations which flatfish undergo, including the asymmetric coloration that occurs between the ocular and the blind side, and the synchrony of the two processes of morphogenesis and pigmentation ontogenesis, these species constitute an interesting model for the study of pigmentation. In this study we present a first approximation towards explaining the genetic mechanisms for regulating pigmentation ontogeny in Senegalese sole, Solea senegalensis.Ministry of Science and Innovation (MICIIN) of the Spanish Government [AGL2008-03897-C04-01/ACU]; European Community [FP7/2007-2013-222719-LIFECYCLE]; Spanish Governmentinfo:eu-repo/semantics/publishedVersio

Characterization of the different behaviours exhibited by juvenile flathead grey mullet (Mugil cephalus Linnaeus, 1758) under rearing conditions

Aim of study: To describe the common behaviour of flathead grey mullet (Mugil cephalus) under rearing conditions. Area of study: Tepic, Mexico. Material and methods: Behaviours exhibited by mullets were videorecorded with submersible cameras installed inside of three tanks. A total of 690 min per day (07:30 - 18:30 h) were recorded per tank during a week. Afterwards, the different behaviours exhibited by juvenile M. cephalus were described, identified and characterized in an ethogram and grouped into two categories: a) locomotion, including three different observed behaviours (resting, swimming and fast swimming) and b) feeding, including three behaviours (surface feeding, bottom feeding and rubbing). Each of the behavioural variables were quantified. Main results: M. cephalus is a species with a constant locomotion associated to feeding, since fish showed continuous movement during most of day light period. On the contrary, fish exhibited reduced movement during dark periods. Mullets were observed to be a non-aggressive fish species under conditions of the present study, since the absence of dominance and aggression towards conspecifics was observed, which suggested a high predisposition for adaptation to captivity. Finally, behavioural frequencies of grey mullet juveniles were similar among the three tanks for most of the behavioural variables analysed (p>0.05) except for the variable bottom feeding (p=0.02). Research highlights: Results from this study could be of interest for the aquaculture industry to optimize rearing techniques and welfare for the production of grey mullet.info:eu-repo/semantics/publishedVersio

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)

[spa]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.[eng]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

Morphological and Molecular Characterization of Dietary-Induced Pseudo-Albinism during Post-Embryonic Development of Solea senegalensis (Kaup, 1858)

16 páginas, 5 tablas, 8 figurasThe appearance of the pseudo-albino phenotype was investigated in developing Senegalese sole (Solea senegalensis, Kaup 1858) larvae at morphological and molecular levels. In order to induce the development of pseudo-albinos, Senegalese sole larvae were fed Artemia enriched with high levels of arachidonic acid (ARA). The development of their skin pigmentation was compared to that of a control group fed Artemia enriched with a reference commercial product. The relative amount of skin melanophores, xanthophores and iridophores revealed that larval pigmentation developed similarly in both groups. However, results from different relative proportions, allocation patterns, shapes and sizes of skin chromatophores revealed changes in the pigmentation pattern between ARA and control groups from 33 days post hatching onwards. The new populations of chromatophores that should appear at post-metamorphosis were not formed in the ARA group. Further, spatial patterns of distribution between the already present larval xanthophores and melanophores were suggestive of short-range interaction that seemed to be implicated in the degradation of these chromatophores, leading to the appearance of the pseudo-albino phenotype. The expression profile of several key pigmentation-related genes revealed that melanophore development was promoted in pseudo-albinos without a sufficient degree of terminal differentiation, thus preventing melanogenesis. Present results suggest the potential roles of asip1 and slc24a5 genes on the down-regulation of trp1 expression, leading to defects in melanin production. Moreover, gene expression data supports the involvement of pax3, mitf and asip1 genes in the developmental disruption of the new post-metamorphic populations of melanophores, xanthophores and iridophores.This work was funded by the Ministry of Science and Innovation (MICIIN) of the Spanish Government (project AGL2008-03897-C04-01/ACU). MD was supported by a Juan de la Cierva post-doctoral contract (MICIIN, Spain)Peer reviewe

Commercial products for Artemia nauplii enrichment are not equivalent for lipid accumulation, growth and development in Senegalese sole (Solea senegalensis) larvae

Trabajo presentado en el V Workshop The cultivation of the Soles, celebrado en Faro (Portugal) del 5 al 7 de abril de 2011.First feeding nutrition in marine fish larvae is one of the most important factors influencing the success of larval rearing. Dietary lipids are particularly important for normal growth and larval development, since they represent the main energy source in developing fish larvae and a source of highly unsaturated fatty acids (HUFAs) indispensable for building new cellular structures and developing organs during morphogenesis. Marine fish larvae are dependent on dietary lipid intakes to get essential HUFAs, such as eicosapentaenoic acid (20:5n-3, EPA), docosahexaenoic acid (22:6n-3, DHA) and arachidonic acid (20:4n-6, ARA), since they are unable to synthesize these FA from their precursors 18:3n-3 and 18:2n-6. The important morphological and physiological changes occurring during the larval development noticeably affect dietary lipid utilization by larvae and make them very susceptible to dietary lipid form and composition. The quality of the diet ingested by larvae in terms of lipid content and composition is reflected by the structural organization of target tissues/ organs; the intestine (digestion and absorption), the vascular system (transport in the form of VLDL or chylomicrons) and liver and muscular tissues (metabolism and storage). When dietary lipids and HUFAs, particularly essential fatty acids (EFAs), are provided to the fish larvae in insufficient/excessive amounts or inadequate form, they can become harmful inducing steatosis, delayed growth, increased mortality, delayed maturation of the digestive tract and bone ossification, apparition of skeletal deformities or others anatomical alterations and pigmentary disorders. Nowadays, many enriching products used to improve live prey nutritional value are commercially available, but they present considerable differences in their physical appearances (emulsions, pastes, spray-dried powders), ingredients (fish oils, vegetal oils, single-cell algal and fungal heterotrophic or phototrophic organisms) and lipid compositions (lipid classes, absolute and relative amounts of saturated fatty acids, MUFAs, HUFAs (n-3 and n-6), EFAs (DHA, EPA, and ARA)). Up to now, no study has been conducted to compare the effect of these different enriching products on lipid absorption, transport, utilization and storage in fish larvae. Thus, the objective of this work was to determine whether any of these products was more suitable for Senegalese sole larval development and to test their influence on lipid accumulation in intestine, liver and trunk muscle by analyzing histological sections. A structural alteration of the histomorphological organization of these organs can provide useful information about the quality of the diet, the metabolism, and the nutritional condition of the larvae. Senegalese sole larvae were fed from 8 to 38 days post hatching (dph) with Artemia nauplii previously enriched with the following six commercial enrichments: Easy Selco© (INVE), Easy Selco© (INVE) half diluted with olive oil, Multigain© ( BioMar ), Red Pepper© ( Bernaqua ), Aquagrow Gold© ( ABN ) and Aquagrow DHA© (ABN). At the end of the experiment, larvae fed Artemia enriched with Aquagrow Gold presented significant higher growth and more advanced intestinal maturation and ossification than larvae fed Artemia enriched with Easy Selco. Lipid accumulation in the different studied tissues evolved differentially among dietary treatments and throughout larval development, revealing that larvae modified lipid utilization in function of the diet they ingested. Larvae from the Easy Selco group showed signs of steatosis in liver and muscle. The significance of these results is discussed in terms of HUFAs and EFAs profiles.Peer Reviewe

Molecular regulation of both dietary vitamin A and fatty acid absorption and metabolism associated with larval morphogenesis of Senegalese sole (Solea senegalensis)

The present study aimed to deepen the understanding of molecular mechanisms governing the absorption and metabolism of some nutrients, growth and development in larvae of Senegalese sole (Solea senegalensis) fed with Artemia enriched with Easy Selco© (ES, INVE) or Aquagrow Gold© (AGG, ABN), which mainly differed in their vitamin A (VA) content and fatty acid composition. The expression profile of genes involved in VA metabolism (crbp2, rbp, crabp1), lipid transport (i-fabp, l-fabp), nuclear receptors for VA and fatty acids (rarα1, rxrα, pparβ), growth (igf1, igf2 and their receptor igf1r) and development (bgp) was analyzed at 22, 30 and 38 days post hatching. The main results suggested that the amount of VA absorbed by larvae is controlled at the intestinal level by crbp2 in both groups, preventing excessive accumulation of this vitamin in the target tissues. The stable expression of i-fabp in the ES group with age could cause an excessive fat accumulation in the intestine inducing, in turn, the steatosis found in the liver and vascular system of these specimens. In liver, the regulation of rbp and fabp expression reflected the status of the physiological functions demanding VA and lipids. The findings revealed that dietary composition induced different strategies for VA and lipid absorption and metabolism affecting, in turn, larval development, growth and health.This work was funded by the Ministry of Science and Innovation (MICIIN) of the Spanish Government (project AGL2008-03897-C04-01/ACU). M.J. Darias was supported by a Juan de la Cierva post-doctoral contract.Peer reviewe

Isolipidic diets differing in their essential fatty acid profiles affect the deposition of unsaturated neutral lipids in the intestine, liver and vascular system of Senegalese sole larvae and early juveniles

How lipid content and composition in the diet is utilized by the various organs and tissues of fish is reflected in their structure, such as the intestine through which dietary lipids are digested and absorbed, the vascular system which is involved in their transport, and the liver where lipids are stored and metabolized. However, no study has been conducted to compare the effect of different diets containing different levels of highly unsaturated fatty acids (HUFA) and essential fatty acids (EFA) on lipid deposition in fish larvae and early juveniles. Thus, we evaluated the effects of six isolipidic diets (enriched Artemia salina), differing in their fatty acid profile, on the lipid accumulation patterns in selected target tissues (intestine, liver and vascular system) in Senegalese sole (Solea senegalensis) larvae and early juveniles. Results showed that the profile of fat accumulation in these three tissues was significantly affected by the dietary treatments, the developmental stage of the fish (premetamorphosis, metamorphosis or postmetamorphosis), as well as by the interaction between these two factors that were responsible for changes in the histological organization of the tissues. Histological results revealed that a slight variation in the EFA levels (e.g. EPA, DHA or ARA) or in their ratios (EPA/DHA, ARA/EPA, ARA/DHA, (n-3)/(n-6) HUFA, OA/PUFA) modified the metabolism of lipids and disrupted the pattern of lipid accumulation in the target tissues, leading to intestinal and hepatic steatosis.This work was funded by the Ministry of Science and Innovation (MICIIN) and the Spanish National Research Council (CSIC) of the Spanish Government (projects AGL2008-03897-C04-01/ACU, AGL2008-03897-C04-04/ACU and PIF-200930I128). M.J. Darias was supported by a Juan de la Cierva post-doctoral contract.Peer reviewe

Selecting Suitable Behavioural Tests to Identify Proactive and Reactive Stress Coping Styles in Flathead Grey Mullet (Mugil cephalus) Juveniles

Identifying Stress Coping Styles (SCS) in new species of interest for aquaculture has important implications for its future domestication and adaptation to captivity. Individual variability allows to select the potential positive characteristics for fish production. The main aim of this study was to identify phenotypic individual differences and characterize proactive and reactive SCS in flathead grey mullet (Mugil cephalus) juveniles by exposing fish to different stress situations and evaluating their individual and group responses to level behavioural and physiology. Juveniles were subjected to one group test (risk-taking) and five individual tests (predator, first feeding after stress, restraining, new environment and confinement). All assays were repeated twice, with a one-month interval between tests. Blood samples were taken from each individual (before and after stress) to quantify cortisol and glucose plasma concentrations. Flathead grey mullet juveniles exhibited a high inter-individual variability with two extremes of behaviours: proactive and reactive profiles that were characterized by opposed behavioural (activity time and escape attempts) and physiological (levels of cortisol and glucose) responses to stress and were consistent over time and across contexts. The flathead grey mullet juveniles showed differences in their predisposition for risk taking. Likewise, the Principal Component Analysis showed that three individual stress tests (predator, restraining and confinement tests) were reliable to characterize SCS in this fish species. This work reported for the first time the existence of stress coping styles in M. cephalus juveniles and the selection of a set of reliable behavioural tests to identify phenotypic profiles in flathead grey mullet. These results might be of interest for the aquaculture industry to improve fish welfare and health and to adjust management protocols for rearing this fish species in captivity.info:eu-repo/semantics/acceptedVersio

The effect of dietary oxidized lipid levels on growth performance, antioxidant enzyme activities, intestinal lipid deposition and skeletogenesis in Senegalese sole (Solea senegalensis) larvae

20 pages, 3 figures, 7 tablesFish tissues, particularly rich in n-3 PUFA, are prone to lipid peroxidation that can damage cellular membranes, cause severe lesions and subsequently incidences of disease and mortality. However, fish possess antioxidant defences, such as vitamin E (VE) and antioxidant enzymes, to protect them against oxidative damage. This study investigated the effects of an increasing gradient of oxidized dietary lipid on the survival, growth performance, skeletogenesis and antioxidant defensive processes occurring in Senegalese sole (Solea senegalensis) larvae. Four groups of fish were fed live prey enriched with experimental emulsions containing an increasing gradient of oxidized oil: non-oxidized, NO+VE, 34.5 nmol MDA g-1 w.w.; mildly oxidized, MO+VE, 43.1 nmol MDA g-1 w.w.; highly oxidized, HO+VE, 63.3 nmol MDA g-1 w.w. and highly oxidized without VE, HO-VE, 78.8 nmol MDA g-1 w.w. The oxidation levels increased in enriched rotifers following the oxidation gradient of the emulsions, but were not affected in enriched Artemia metanauplii. The oxidation status of Senegalese sole larvae increased during development, but this was not related to the dietary treatments. The increasing dietary oxidation levels did not affect the fatty acid profile, survival, growth performance and metamorphosis processes of sole larvae. Senegalese sole seem to activate antioxidant defence mechanisms in response to the increasing amounts of dietary peroxidized lipids, in a manner efficiently enough to prevent detection of any alterations of these physiological processes. Antioxidant systems and detoxification mechanisms appeared to occur through the consumption of dietary α-tocopherol, the activation of the antioxidant enzymes (catalase, superoxide dismutase, glutathione S-transferase, glutathione reductase) and the retention of oxidized fat in the intestinal enterocytes for detoxification prior to their utilization. However, fish fed the highest oxidized diet presented a reduction in bone mineralization, but lower incidence of deformities in the vertebral and caudal regions than fish fed the other diets. This study exemplifies the importance of rearing Senegalese sole larvae on non-oxidized diets during the early larval development to avoid detrimental consequences in older fish, most notably in the process of skeletogenesis. © 2014 John Wiley & Sons Ltd.This work was funded by the Ministry of Science and Innovation (MICIIN) and the Spanish National Research Council (CSIC) of the Spanish Government (project AGL2008-03897-C04-01/ACU, PIF-200930I128 and CTM 2010-1611)Peer Reviewe

Number of xanthophores (X) associated to one melanophore (M) during the post-metamorphosis period.

<p>Values are expressed in means ± SD. Superscript letters denote significant differences in the number of X related to one M between developmental ages (One-way ANOVA, <i>P</i><0.05).</p