María P. Martín del Río

Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus.

SUMMARY Changes in different osmoregulatory and metabolic parameters over time were assessed in gills, kidney, liver and brain of gilthead sea bream Sparus auratus transferred either from seawater (SW, 38 p.p.t.) to hypersaline water (HSW, 55 p.p.t.) or from SW to low salinity water (LSW, 6 p.p.t.) for 14 days. Changes displayed by osmoregulatory parameters revealed two stages during hyperosmotic and hypo-osmotic acclimation: (i) an adaptive period during the first days of acclimation (1–3 days), with important changes in these parameters, and (ii) a chronic regulatory period (after 3 days of transfer) where osmotic parameters reached homeostasis. From a metabolic point of view, two clear phases can also be distinguished during acclimation to hyperosmotic or hypo-osmotic conditions. The first one coincides with the adaptive period and is characterized by enhanced levels of plasma metabolites (glucose, lactate, triglycerides and protein), and use of these metabolites by different tissues in processes directly or indirectly involved in osmoregulatory work. The second stage coincides with the chronic regulatory period observed for the osmoregulatory parameters and is metabolically characterized in HSW-transferred fish by lower energy expenditure and a readjustment of metabolic parameters to levels returning to normality, indicative of reduced osmoregulatory work in this stage. In LSW-transferred fish, major changes in the second stage include: (i) decreased glycolytic potential, capacity for exporting glucose and potential for amino acid catabolism in liver; (ii) enhanced use of exogenous glucose through glycolysis, pentose phosphate and glycogenesis in gills; (iii) increased glycolytic potential in kidney; and (iv) increased glycogenolytic potential and capacity for use of exogenous glucose in brain.

Energy Metabolism in Fish Tissues Related to Osmoregulation and Cortisol Action

This is an overview of our recent studies of energy metabolism in fish brain and other organs regulated by exogenous (feeding, salinity) and endogenous (hormones) factors. To highlight our approach, we present latest results concerned osmoregulation in the gills of gilthead seabream, Sparus auratus. Our model, the seabream, is a euryhaline teleost capable of adaptation to extreme changes in environmental salinity. Treatment with cortisol allowed us to achieve circulating cortisol levels similar to those observed during osmotic adaptation and to assess how elevated hormonal levels affected simultaneously metabolic and osmoregulatory capacities of the gill tissue. Cortisol-implanted fish showed higher gill Na+,K+-ATPase activity than control fish but no changes were observed in plasma osmolality and ion levels. Plasma levels of glucose and lactate increased in cortisol-implanted fish while protein levels decreased. Cortisol treatment elicited metabolic changes in liver and brain reflecting an activation of the glycogenic and gluconeogenic potential in liver, and the glycogenic potential in brain, which are confirmatory of data obtained in previous experiments. In gills, we demonstrated that cortisol treatment elicited changes in their energy metabolism that can be summarized as a decreased capacity in the use of exogenous glucose (decreased HK activity), a decrease in the capacity of the pentose phosphate pathway (decreased G6PDH activity), and an increased glycolytic potential (increased PK activity). Observed metabolic changes in gills can be associated with those occurring in nature during osmotic adaptation in the same fish species.

Interactive effects of environmental salinity and temperature on metabolic responses of gilthead sea bream Sparus aurata.

The gilthead sea bream Sparus aurata is a euryhaline and euritherm species with the capacity of living under different environmental conditions of salinity and temperature. The influence of acclimation to different environmental salinities (5, 38 and 55 per thousand) and temperatures (12 degrees , 19 degrees and 26 degrees C) for seven weeks was analyzed in plasma and tissues (liver, gills and kidney) of gilthead sea bream assessing levels of metabolites and enzyme activities related to energy metabolism. Changes observed in specimens acclimated to different environmental salinities agree with previous results reported for this species. The temperature alone did also affect metabolic parameters in a way similar to that previously described. A significant interaction of salinity with temperature was found in most parameters assessed in tissues suggesting that the metabolic effects of salinity are different depending on the temperature of acclimation. The interactions were different among tissues and parameters displaying different patterns of changes. In general, the acclimation to extreme temperatures (especially low) alters the metabolic responses to different salinities thus suggesting that the energy demand of increased osmoregulatory work is not so important under temperature conditions different from those commonly found in nature and in those used in culture.

Ontogeny of adenohypophyseal cells in the pituitary of the American shad (Alosa sapidissima)

The distribution and ontogeny of adenohypophyseal cells have been studied in the pituitary gland of embryos, larvae, and juveniles of the clupeid American shad (Alosa sapidissima) using immunocytochemical techniques. In juvenile specimens, adenohypophysis was composed of rostral pars distalis (RPD), formed by cavities lined by prolactin (PRL), adrenocorticotropic hormone (ACTH), and gonadotropic hormone (GTH) cells; proximal pars distalis (PPD), containing growth hormone (GH), GTH, and putative thyroid stimulating hormone (TSH) cells; and pars intermedia (PI) with somatolactin (SL) and melanophore stimulating hormone (MSH) cells. At 3 days post-fertilization (3 days pre-hatching) the pituitary of embryos consisted of an oval mass of cells, close to the ventral margin of the diencephalon, divided in rostral and caudal regions. At this time PRL and ACTH cells appeared in the rostral region of the adenohypophysis, while SL cells were observed in the caudal region where MSH cells showed reactivity 1 day before hatching. At variance, GH cells showed a weak immunoreactivity in the rostral portion at hatching that increased 2 days latter. GTH cells also showed weak immunoreactivity in the rostral region of the adenohypophysis at hatching time. Two days later GTH cells were located in the rostral and central regions of the adenohypophysis. At hatching, the neurohypophysis was very small and no nerve processes were seen to penetrate the adenohypophysis tissue. After hatching, the pituitary gland elongated and in 7 days old larvae, the RPD showed a small lumen surrounded by a palisade of PRL, ACTH, and GHT cells; the PPD showed GH and GTH cells while the PI contained SL and MSH cells. The adenohypophysis and neural lobe increased in size with development and, in 42 days old larvae, they were similar to those of juvenile specimens.

Gene and protein expression for prolactin, growth hormone and somatolactin in Sparus aurata: Seasonal variations

The seasonal variation of PRL, GH and SL gene and protein expression has been analyzed in gilthead sea bream (Sparus aurata) pituitaries using Real-Time Q-PCR and Western Blots, respectively. Animals were cultured in earthen ponds under natural photoperiod, temperature and salinity conditions. Samples were taken during winter 2005 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Beta-actin, used as the housekeeping gene both for Q-RT-PCR and Western analysis, did not present significant differences among seasons. Higher expression was observed during spring and autumn for PRL, summer and winter for GH, and spring for SL. Expression of PRI, GH and SL, presented seasonal variation, suggesting that these hormones could play a role in the molecular signal transduction of environmental factors (especially of photoperiod and temperature) in eurythermal fish.

Pituitary gene and protein expression under experimental variation on salinity and temperature in gilthead sea bream Sparus aurata

Temperature and salinity are important factors that affect several physiological processes in aquatic organisms, which could be produced by variation of certain hormones. In this study, the expression of pituitary hormones involved in the acclimation to different temperatures and salinities was examined in Sparus aurata, a euryhaline and eurytherm species, by Q-Real Time RT-PCR and Western blot analyses for mRNA and protein expression, respectively. Three different experimental conditions were designed with specimens (10 per treatment) acclimated to: a) low salinity water; b) sea water; and c) high salinity water. Additionally, fish under different salinities were acclimated to three different temperatures: 12, 19 and 26 degrees C. Animals were maintained seven weeks before sampling pituitary glands. Our results provided enough evidence for a differential expression of PRL, GH and SL in the pituitary of gilthead sea bream, under different temperature and salinity regimes.