Fisiología y bioquímica digestiva de la liseta (Chelon labrosus)

En los últimos años la acuicultura ha experimentado un progreso importante, aumentando el número de especies cultivadas y el volumen producido para hacer frente a la reducción de la captura de peces salvajes y poder satisfacer la demanda de de alimentos acuáticos. La mayoría de las especies cultivadas son carnívoras y, por este motivo, la alimentación sigue siendo el principal problema de la acuicultura al demandar una alta cantidad de harinas y/o aceites de pescado. Para paliar este problema, existe una tendencia a intentar producir especies herbívoras o capaces de alimentarse a partir de un alto contenido de proteína vegetal. En este sentido, la liseta (Chelon labrosus) es una especie que reúne los requisitos idóneos para una producción eficiente y una reducción de los costes alimenticios, siendo una especie omnívora, capaz de adaptarse a diferentes ambiente y está presente en las aguas someras de nuestras costas. El objetivo de esta Tesis Doctoral es incrementar el conocimiento sobre la fisiología y bioquímica digestiva de esta especie, con el fin de poder establecer unas pautas de alimentación específicas. Para ello se caracterizaron las principales enzimas digestivas presentes en la especie y se determinó su actividad en especímenes de tres tamaños diferentes, sugiriendo los resultados que las variaciones en las actividades de las distintas enzimas están ligadas a cambios en la composición de la dieta con el tamaño, pasando de tener un hábito alimenticio parcialmente carnívoro a una alimentación de tendencia herbívora. Este cambio posiblemente este asociado a cambios en la alimentación mostrados en su medio natural. Por otro lado, se determinó el efecto de diferentes salinidades ambientales en la expresión génica de estas enzimas digestivas y en la actividad bioquímica total de las mismas. Además, se evaluó, mediante un ensayo in vitro, el efecto de la salinidad sobre la hidrólisis proteica. A pesar de que la expresión y la actividad enzimática muestran cambios relacionados con las distintas salinidades, la liseta es capaz de vivir en un amplio rango de salinidad ambiental, pudiendo existir mecanismos compensatorios que la ayuden a adaptarse a estos cambios. Además, se ha encontrado una notable disparidad entre el transcriptoma y el proteoma de dichas enzimas digestivas, lo que sugiere que la expresión de estas se ve afectada por mecanismos post-transcripcionales. Los ensayos de digestibilidad in vitro indican que en entornos más salinos deben existir mecanismos de compensación que aseguren un proceso digestivo eficiente. Siguiendo esta línea de investigación, se analizó la influencia de diferentes salinidades ambientales sobre el metabolismo, órganos osmorreguladores y expresión de hormonas involucradas en el crecimiento. Los resultados confirman que la liseta es una especie fuertemente eurihalina, pero su aclimatación a salinidades extremas conlleva un gasto extra de energía y, probablemente, afecta negativamente al crecimiento. Por último, se evaluó el efecto de distintos hábitos alimenticios (ayuno y realimentación) sobre la actividad metabólica y composición de tejidos, el eje GH/IGF-I, y la actividad de las enzimas digestivas en ejemplares juveniles de liseta. Los datos obtenidos muestran que la liseta es capaz de ajustar fácilmente su metabolismo ante situaciones de ayuno y realimentación a corto plazo. Sin embargo, la medida de expresión de ARNm del eje GH/IGF-I, utilizada como indicador del estado nutricional, no muestra suficiente sensibilidad para ser aplicada en esta especie

Nuevos retos para la diversificación en acuicultura: la liseta (Chelon labrosus)

Chelon labrosus (Risso, 1827), commonly known as thicklip grey mullet, is a marine teleost fish from the Mugilidae family, very common in coastal and estuarine areas. It is characterized as an amphidromous species, migrating to brackish or lagoon waters to feedChelon labrosus (Risso, 1827), comúnmente conocida como lisa, liseta o corcón, es un teleós- teo marino de la Familia Mugilidae muy frecuente en zonas costeras y estuáricas. Se caracteriza por ser una especie anfídroma, migrando a aguas salobres o lagunares para alimentars

Acute stress response in gilthead sea bream (Sparus aurata L.) is time-of-day dependent: Physiological and oxidative stress indicators

Since fish show daily rhythms in most physiological functions, it should not be surprising that stressors may have different effects depending on the timing of exposure. In this study, we investigated the influence of time of day on the stress responses, at both physiological and cellular levels, in gilthead sea bream (Sparus aurataL.) submitted to air exposure for 30 s and then returned to their tank. One hour after air exposure, blood, hypothalamus and liver samples were taken. Six fish per experimental group (control and stressed) were sampled every 4 h during a 24-h cycle. Fish were fed in the middle of the light cycle (ML) and locomotor activity rhythms were recorded using infrared photocells to determine their daily activity pattern of behaviour, which showed a peak around feeding time in all fish. In the control group, cortisol levels did not show daily rhythmicity, whereas in the stressed fish, a daily rhythm of plasma cortisol was observed, being the average values higher than in the control group, with increased differences during the dark phase. Blood glucose showed daily rhythmicity in the control group but not in the stressed one which also showed higher values at all sampling points. In the hypothalamus of control fish, a daily rhythm ofcorticotropin-releasing hormone(crh) gene expression was observed, with the acrophase at the beginning of the light phase. However, in the stressed fish, this rhythm was abolished. The expression ofcrh-binding protein(crhbp) showed a peak at the end of the dark phase in the control group, whereas in the stressed sea bream, this peak was found at ML. Regarding hepatic gene expression of oxidative stress biomarkers: (i)cytochrome c oxidase 4showed daily rhythmicity in both control and stressed fish, with the acrophases located around ML, (ii)peroxiredoxin(prdx) 3 and5(prdx5) only presented daily rhythmicity of expression in the stressed fish, with the acrophase located at the beginning of the light cycle and (iii)uncoupling protein 1showed significant differences between sampling points only in the control group, with significantly higher expression at the beginning of the dark phase. Taken together, these results indicate that stress response in gilthead sea bream is time-dependent as cortisol level rose higher at night, and that different rhythmic mechanisms interplay in the control of neuroendocrine and cellular stress responses

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Presentació

Expresión y actividad de enzimas digestivas en juveniles de liseta (Chelon labrosus) sometidos a diferentes salinidades ambientales

Trabajo presentado en el XIV Congreso Nacional de Acuicultura, celebrado en Gijón del 23 al 25 de septiembre de 2013.The aim of this study was to evaluate the influence of environmental salinity on the mRNA expression and protein activity of the main digestive enzymes of thick-lipped grey mullet (Chelon labrosus). Specimens of thick-lipped grey mullet were acclimated to different environmental salinities (0, 12, 40 and 55 ppt) during 21 days. Samples of complete digestive system were obtained for mRNA expression analysis and for determining enzymatic activities. Results showed a clear effect of environmental salinity on digestive activity as well as on gene expression of these enzymes. Low salinities had a positive influence on digestive acid proteases, which showed a higher activity and expression in 12 ppt-acclimated specimens salinity, whereas higher mRNA expression levels of alkaline proteases were found in fish maintained at 40 ppt.Este trabajo ha sido financiado por el proyecto AGL2010-14876 del MICINN otorgado a JMM. JAM-S está financiado por una beca predoctoral FPU (Referencia AP2008-01194) del Ministerio de Educación (España).Peer reviewe

Pepsin activity and pepsinogen mRNA expression during protein digestion in gilthead seabream juveniles

Trabajo presentado en el XV International Symposium on Fish Nutrition and Feeding, celebrado en Molde (Noruega) del 4 al 7 de junio de 2012.Pepsin is activated from its precursor, the pepsinogen, at low gastric pH environment. In fish with stomach, low pH values are eventually attained during the postprandial period. Previous results indicated that meal timing and frequency affected the daily acidification rhythm. These gastric pH variations may affect the potential digestive capacity in relation to the pepsin activity. In this study we have examined the daily pepsin activity pattern in gilthead seabream Sparus aurata juveniles (5-7 g wet weight) maintained under 12L/12D photoperiod and three different feeding regimes: A) one daily meal at 9:00, B) two meals at 9:00 and 17:00, respectively, and C) continuous feeding during the light period, from 9:00 to 21:00. In all cases the same amount of food was supplied. Specifically we have determined the pepsin activity at the actual working pH and compared with the gene expression of mRNA codifying pepsinogen in samples taken periodically during a 24 h cycle.This work was financed by projects Aquagenomics CDS2007-00002 and AGL2007-64450-C02-01/02 (MICINN, Spain, and FEDER/ERDF).Peer Reviewe

Cloning and extression of growth hormone and insulin-like growth factor in response to different environmental salinities in thick-lipped grey mullet (Chelon labrosus)

Trabajo presentado en el 8º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Madrid del 5 al 7 de septiembre de 2011.Thick-lipped grey mullet, Chelon labrosus (Risso, 1827), is a marine teleost from the family Mugilidae, very common in the estuaries and natural earthen ponds of the Bay of Cádiz, and of great importance for extensive aquaculture in this area. This euryhaline species is characterized by amphidromic life cycles, migrating to estuarine areas for feeding and other physiological processes. Growth hormone (GH) and insulin-like growth factor I (IGF-I) present a role in fish osmoregulation processes, being important pieces for the adaptation of specimens to changes in environmental salinity. We cloned pituitary GH and hepatic IGF-I in C. labrosus by cDNA library screening, using a species specific probe developed by PCR with degenerated primers, designed from the conserved regions of other teleost sequences from GenBank. In addition, changes in expression were measured in specimens maintained under different environmental salinities (0, 12, 40 and 55 ppt) during 21 days using real time RT-PCR. Moreover, cortisol levels were evaluated in plasma samples in order to assess the osmotic stress of specimens. Fish acclimated to lower salinities enhanced plasma cortisol levels, while specimens maintained at the highest salinity decreased this value compared to control. Previous studies have shown the existence of a positive interaction between GH and cortisol for improving osmoregulatory capacity in teleosts, but this relation is species-dependent. Pituitary GH and hepatic IGF-I expression will be presented and discussed under this hypothesis.Peer Reviewe

Acute stress response in gilthead sea bream (Sparus aurata L.) is time-of-day dependent: Physiological and oxidative stress indicators

Since fish show daily rhythms in most physiological functions, it should not be surprising that stressors may have different effects depending on the timing of exposure. In this study, we investigated the influence of time of day on the stress responses, at both physiological and cellular levels, in gilthead sea bream (Sparus aurata L.) submitted to air exposure for 30s and then returned to their tank. One hour after air exposure, blood, hypothalamus and liver samples were taken. Six fish per experimental group (control and stressed) were sampled every 4h during a 24-h cycle. Fish were fed in the middle of the light cycle (ML) and locomotor activity rhythms were recorded using infrared photocells to determine their daily activity pattern of behaviour, which showed a peak around feeding time in all fish. In the control group, cortisol levels did not show daily rhythmicity, whereas in the stressed fish, a daily rhythm of plasma cortisol was observed, being the average values higher than in the control group, with increased differences during the dark phase. Blood glucose showed daily rhythmicity in the control group but not in the stressed one which also showed higher values at all sampling points. In the hypothalamus of control fish, a daily rhythm of corticotropin-releasing hormone (crh) gene expression was observed, with the acrophase at the beginning of the light phase. However, in the stressed fish, this rhythm was abolished. The expression of crh-binding protein (crhbp) showed a peak at the end of the dark phase in the control group, whereas in the stressed sea bream, this peak was found at ML. Regarding hepatic gene expression of oxidative stress biomarkers: (i) cytochrome c oxidase 4 showed daily rhythmicity in both control and stressed fish, with the acrophases located around ML, (ii) peroxiredoxin (prdx) 3 and 5 (prdx5) only presented daily rhythmicity of expression in the stressed fish, with the acrophase located at the beginning of the light cycle and (iii) uncoupling protein 1 showed significant differences between sampling points only in the control group, with significantly higher expression at the beginning of the dark phase. Taken together, these results indicate that stress response in gilthead sea bream is time-dependent as cortisol level rose higher at night, and that different rhythmic mechanisms interplay in the control of neuroendocrine and cellular stress responses. © 2014 Informa Healthcare USA, Inc. All rights reserved.This research was supported by the project AQUAGENOMICS (Consolider-Ingenio 2010 Programme) and CRONOSOLEA from the Spanish Ministry of Economy and Competitiveness (MINECO).Peer Reviewe

Effect of different salinities on gene expression and activity of digestive enzymes in the thick-lipped grey mullet (Chelon labrosus)

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquacultureThis work was funded by project AGL2010-14876 from MICINN to JMM.Peer reviewe