Juan Miguel Mancera

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.

Rapid Activation of Gill Na + ,K + -ATPase in the Euryhaline Teleost Fundulus heteroclitus

The rapid activation of gill Na(+),K(+)-ATPase was analyzed in the mummichog (Fundulus heteroclitus) and Atlantic salmon (Salmo salar) transferred from low salinity (0.1 ppt) to high salinity (25-35 ppt). In parr and presmolt, Salmo salar gill Na(+),K(+)-ATPase activity started to increase 3 days after transfer. Exposure of Fundulus heteroclitus to 35 ppt seawater (SW) induced a rise in gill Na(+), K(+)-ATPase activity 3 hr after transfer. After 12 hr, the values dropped to initial levels but showed a second significant increase 3 days after transfer. The absence of detergent in the enzyme assay resulted in lower values of gill Na(+),K(+)-ATPase, and the rapid increase after transfer to SW was not observed. Na(+),K(+)-ATPase activity of gill filaments in vitro for 3 hr increased proportionally to the osmolality of the culture medium (600 mosm/kg > 500 mosm/kg > 300 mosm/kg). Osmolality of 800 mosm/kg resulted in lower gill Na(+),K(+)-ATPase activity relative to 600 mosm/kg. Increasing medium osmolality to 600 mosm/kg with mannitol also increased gill Na(+),K(+)-ATPase. Cycloheximide inhibited the increase in gill Na(+),K(+)-ATPase activity observed in hyperosmotic medium in a dose-dependent manner (10(-4) M > 10(-5) M > 10(-6) M). Actinomycin D or bumetanide in the culture (doses of 10(-4) M, 10(-5) M, and 10(-6) M) did not affect gill Na(+),K(+)-ATPase. Injection of fish with actinomycin D prior to gill organ culture, however, prevented the increase in gill Na(+),K(+)-ATPase activity in hyperosmotic media. The results show a very rapid and transitory increase in gill Na(+),K(+)-ATPase activity in the first hours after the transfer of Fundulus heteroclitus to SW that is dependent on translational and transcriptional processes. J. Exp. Zool. 287:263-274, 2000.

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.

Immunocytochemical study of the hypothalamic magnocellular neurosecretory nuclei of the snake Natrix maura and the turtle Mauremys caspica

SummaryAn immunocytochemical study of the magnocellular neurosecretory nuclei was performed in the snake Natrix maura and the turtle Mauremys caspica by use of antisera against: (1) a mixture of both bovine neurophysins, (2) bovine oxytocin-neurophysin, (3) arginine vasotocin, and (4) mesotocin. Arginine vasotocin- and mesotocin-immunoreactivities were localized in individual neurons of the supraoptic and paraventricular nuclei, with a distinct pattern of distribution in both species. The same cells appeared to be stained by the anti-oxytocin-neurophysin and anti-mesotocin sera. The supraoptic nucleus can be subdivided into rostral medial and caudal portions. In N. maura, but not in M. caspica, neurophysin-immunoreactive neurons were found in the retrochiasmatic nucleus. No immunoreactive elements were seen in the suprachiasmatic nucleus of both species after the use of any of the antisera. A dorsolateral aggregation of neurophysin-containing cells, localized over the lateral forebrain bundle, was present in both species. Magnocellular and parvocellular neurophysin-immunoreactive neurons were present in the paraventricular nucleus of both species. In the turtle, the paraventricular neurons were arranged into four distinct layers parallel to the ependyma; these neurons were bipolar with the major axis perpendicular to the ventricle, and many of them projected processes toward the cerebrospinal-fluid compartment. In N. maura a group of large neurons of the paraventricular nucleus was found in a very lateral position. The posterior lobe of the hypophysis and the external zone of the median eminence contained arginine vasotocin- and mesotocin-immunoreactive nerve fibers. The lamina terminalis of both species was supplied with a dense bundle of fibers containing immunoreactive neurophysin. Neurophysin-immunore-active fibers were also present in the septum, some telencephalic regions, including the cortex and the olfactory tubercule, in the paraventricular organ, and the periventricular and periaqueductal gray of the brainstem.

Chronic and acute stress responses in senegalese sole (solea senegalensis): The involvement of cortisol, crh and crh-bp

The hypothalamus-pituitary-interrenal (HPI) axis is pivotal in the endocrine stress response of fish. Hypothalamic corticotropin-releasing hormone (CRH) initiates the endocrine stress response and stimulates the release of adrenocorticotropic hormone (ACTH) from the pituitary pars distalis, which in turn activates cortisol production and release by the interrenal cells of the head kidney. CRH activity depends on the levels of a specific CRH binding protein (CRH-BP). We have characterized the cDNAs coding for CRH and CRH-BP in Senegalese sole (Solea senegalensis) and investigated their mRNA expression in juveniles that were submitted to a protocol that involved exposure to a chronic stressor (viz. increased cultivation densities) followed by an acute stressor (viz. transfer to increased ambient salinity). Juveniles were cultivated at three densities (1.9, 4.7 and 9.8 kg/m(2)) for 33 days, and then exposed to an osmotic challenge that involved transfer from seawater (39‰ salinity, SW) to hypersaline seawater (55‰, HSW). The highest density imposed stress as indicated by elevated cortisol levels and CRH mRNA expression compared to fish stocked at low density. Fish kept at high density differentially responded to a posterior transfer to HSW; no cortisol or CRH response was seen, but osmoregulatory and metabolic parameters were affected. No differences in CRH-BP mRNA expression levels were found at different stocking densities; transfer to HSW enhanced expression in both low and high density stocked animals, suggesting that CRH-BP acts as a modulator of the acute stress response, not so of the chronic stress response. We conclude that stocking of Senegalese sole at high density is a stressful condition that may compromise the capacity to cope with subsequent stressors.

Biomarker responses in Solea senegalensis exposed to sodium hypochlorite used as antifouling

The time-course stress responses (0, 1, 2, and 7 d) was assessed in plasmatic, branchial and renal parameters of juveniles Solea senegalensis exposed to different concentrations of the antifouling sodium hypochlorite (0.1, 0.2, and 0.5mgL(-1)). These stress responses were only assessed for the total length of exposure (7d) at the lowest NaClO concentration due to the high toxicity of this chemical. In addition, the xenobiotic metabolism responses were evaluated by means of enzymatic activities of ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPX), catalase (CAT), and carboxylesterase (CbE) in liver; as well as GST, GPX, CAT and acetylcholinesterase (AChE) in gill. Oxidative stress damage due to sodium hypochlorite exposure was measured by lipid peroxidation levels in liver and gill. Concentrations of 0.2 and 0.5mgL(-1) produced lethal effects after 1d and 2h of exposure, respectively. After 1d of exposure to sublethal concentration of sodium hypochlorite (0.1mgL(-1)) osmoregulatory (osmolality and chloride) and stress (cortisol, glucose and lactate) plasmatic parameters were enhanced to respect at control fish. However after 3 or 7d these parameters returned to control values. No effects were observed on plasma protein and triglyceride levels or on gill and kidney Na(+)/K(+)-ATPase activities. Diverse gill pathologies such as hypertrophy, lamellar fusion and an increase in goblet cell number and size were observed after 7d of exposure. Most biochemical parameters related to xenobiotic metabolism and oxidative stress were also significantly affected which suggests that seawater affected by sodium hypochlorite discharges from power plants, is able to alter the fish xenobiotic metabolism and generate oxidative stress.

A stereological study of copper toxicity in gills of Oreochromis niloticus

Stereological methods were used to estimate the volumetric density (V(V)) of the filamentar epithelium (FE, 39%), lamellae (L, 28%), central venous sinus (CVS, 14%), central axis (16%), mucous cells (MC, 2%) and chloride cells (CC, 1%) in the gill filament of control Nile Tilapia. The relative volumes of FE and L, and the relative volumes of CVS and central axis, varied inversely under exposure to copper, with high copper toxic levels declanching a chronic defence mechanism that was, nevertheless, overcome, and low copper toxic levels causing adaptation within a moderate acute phase type of response. Copper also induced a decrease of the V(V) (MC, gill filament) due to reduction of surface MC, despite the marked increase of stem MC at chronic exposure to high copper toxic levels. Diminution of the numerical density of filamentar CC was responsible for the decreased V(V) (CC, gill filament), although lamellar CC significantly increased at chronic exposure to low copper toxic levels. The present results demonstrate that cell relative volumes, mean volumes and numerical densities are dependent on the variations of the FE and L, which without a quantitative approach may be misinterpreted, thus stressing the importance of using stereological tools for analyzing histopathological patterns.

Daily Rhythms in the Hypothalamus-Pituitary-Interrenal Axis and Acute Stress Responses in a Teleost Flatfish, Solea senegalensis

The endocrine axis controlling the stress response displays daily rhythms in many factors such as adrenal sensitivity and cortisol secretion. These rhythms have mostly been described in mammals, whereas they are poorly understood in teleost fish, so that their impact on fish welfare in aquaculture remains unexplored. In the present research, the authors investigated the daily rhythms in the hypothalamus-pituitary-interrenal (HPI) axis in the flatfish Solea senegalensis, which has both scientific and commercial interest. In a first experiment, hypothalamic expression of corticotropin-releasing hormone (crh) and its binding protein (crhbp), both pituitary proopiomelanocortin A and B (pomca and pomcb) expression, as well as plasma cortisol, glucose, and lactate levels were analyzed throughout a 24-h cycle. All variables displayed daily rhythms (cosinor, p < .05), with acrophases varying depending on the factor analyzed: crh and cortisol peaked at the beginning of the dark phase (zeitgeber time [ZT] = 14.5 and 14.4 h, respectively), pomca and pomcb as well as glucose at the beginning of the light phase (ZT = 1.2, 2.4, and 3.4 h, respectively), and crhbp and lactate at the end of the dark phase (ZT = 22.3 and 23.0 h, respectively). In a second experiment, the influence of an acute stressor (30 s of air exposure), applied at two different time points (ZT 1 and ZT 13), was tested. The stress response differed depending on the time of day, showing higher cortisol values (96.2 ± 10.7 ng/mL) when the stressor was applied at ZT 1 than at ZT 13 (52.6 ± 11.1 ng/mL). This research describes for the first time the daily rhythms in endocrine factors of the HPI axis of the flatfish S. senegalensis, and the influence of daytime on the stress responses. A better knowledge of the chronobiology of fish provides a helpful tool for understanding the circadian physiology of the stress response, and for designing timely sound protocols to improve fish welfare in aquaculture. (Author correspondence: jflopez@um.es)