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In fish, vitellogenin is an important nutritional precursor protein produced solely in the liver and released into the blood where it binds calcium. In the gilthead sea bream (Sparus auratus) 17beta-Estradiol (E2) plays an important role in the synthesis of vitellogenin, but also the pituitary hormones prolactin (PRL) and growth hormone (GH) can stimulate vitellogenin induction in fish. Considering the emerging involvement of PTHrP in fish calcium metabolism and the importance of calcium regulation in reproduction, we investigated the possible role of PTHrP in vitellogenesis. E2-naïve and E2-primed sea bream hepatocytes were used in an in vitro primary hepatocyte culture and stimulated with a recombinant sea bream PTHrP (sbPTHrP) to establish the contribution of sbPTHrP alone or in combination with E2 to the regulation of hepatic vitellogenin synthesis. Hepatocytes stimulated solely with sbPTHrP were not affected in their vitellogenesis. However, in hepatocytes stimulated with E2 in combination with sbPTHrP a higher vitellogenin production was seen than with E2 alone. It is concluded that sbPTHrP has a potentiating effect on estradiol stimulation of vitellogenin production by sea bream hepatocytes. The sea bream provides a unique model where vitellogenesis regulation can be studied on E2-naïve liver cells, both in vivo and in vitro.

Parathyroid hormone-related protein (PTHrP) is a factor associated with normal development and physiology of the nervous, cardiovascular, immune, reproductive, and musculoskeletal systems in higher vertebrates. It also stimulates whole body calcium uptake in sea bream (Sparus auratus) larvae with an estimated 60% coming from intestinal uptake in seawater. The present study investigated the role of PTHrP in the intestinal calcium transport in the sea bream in vitro. Unidirectional mucosal-to-serosal and serosal-to-mucosal 45Ca fluxes were measured in vitro in duodenum, hindgut, and rectum mounted in Ussing chambers. In symmetric conditions with the same saline, bathing apical and basolateral sides of the preparation addition of piscine PTHrP 1-34 (6 nM) to the serosal surface resulted in an increase in mucosal to serosal calcium fluxes in duodenum and hindgut and a reduction in serosal to mucosal in the rectum, indicating that different mechanisms are responsive to PTHrP along the intestine. In control asymmetric conditions, with serosal normal and mucosal bathed with a saline similar in composition to the intestinal fluid, there was a net increase in calcium uptake in all regions. The addition of 6 nM PTHrP 1-34 increased net calcium uptake two- to threefold in all regions. The stimulatory effect of PTHrP on net intestinal calcium absorption is consistent with a hypercalcemic role for the hormone. The results support the view that PTHrP, alone or in conjunction with recently identified PTH-like peptides, counteracts in vivo the hypocalcemic effects of stanniocalcin.

Cortisol, and heat shock protein 70 (HSP70) are known to perform key roles as part of the fish stress response. In the present study, two in vitro systems were used to investigate a possible cortisol-HSP70-apoptosis regulatory relationship. Using a developed silver sea bream fibroblast cell line (SSF), cortisol was found to induce HSP70 synthesis with a concomitant protection against camptothecin induced apoptosis. The induction of HSP70 synthesis using azetidine was also found to protect SSF against apoptosis. A primary culture of silver sea bream macrophages (SSM) displayed reduced HSP70, underwent apoptosis and displayed reduced phagocytic activity upon exposure to cortisol. The effect of cortisol on HSP70 expression in both SSF and SSM were blocked by the glucocorticoid antagonist, RU486. Treatment of SSM with azetidine protected against apoptosis and also enhanced phagocytic activity. The data from this study demonstrates for the first time that cortisol can be either anti- apoptotic or pro-apoptotic in different fish cells and such actions can be mediated via HSP70 induction or suppression respectively.

The pineal gland via its secretory product, melatonin, influences the light-dark rhythm in most vertebrates including fish. Apart from the information concerning this circadian rhythm, the interrelation of the melatonin with other physiological processes has not been considered in fish. Thus, we evaluated the changes in the humoral innate immune system of seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) specimens exposed to a constant light-dark photoperiod (12 hr L:12 hr D). Serum was obtained from blood samples collected at 02:00, 08:00 hr (light-on), 14:00, 20:00 hr (light-off) and at 08:00 hr again. Among the humoral innate immune responses, complement, lysozyme and peroxidase activities were determined. Complement activity was higher during the day than during the night in both fish species. Seabream lysozyme activity reached its maximum at 20:00 and 02:00 hr but was hardly affected in sea bass. Finally, the peroxidase activity of seabream was significantly higher at 08:00 hr than during the rest of the cycle while, in sea bass, it showed little variation. The present results demonstrate that the humoral innate immune system has a circadian rhythm based on the light-dark cycle and that this cycle might be affected by the pineal gland.

The osmoregulatory and metabolic role of testosterone (T) in the euryhaline teleost Sparus auratus was examined. Fish were implanted with a slow-release coconut oil implant alone (control) or containing T (2 or 5microgg(-1) body weight) and sampled 1, 3, and 7 days after implantation. Gill Na(+),K(+)-ATPase activity increased in fish treated with the lower dose of T after 7 days of treatment. Kidney Na(+),K(+)-ATPase activity enhanced at first day post-implantation in the group treated with the higher dose of T but the values diminished by day 3. Plasma levels of metabolites (glucose, lactate, triglyceride, and protein) increased after T treatment. This higher availability of plasma metabolites was reflected in several metabolic changes within different tissues of T-treated fish such as (i) increased glycogen levels and capacity for gluconeogenesis, ketogenesis, glucose exporting, and amino acid catabolism in the liver, (ii) enhanced lipogenic capacity in the gills, (iii) increased glycogen levels and capacity for oxidizing amino acids in the kidney, and (iv) enhanced levels of glycogen, aceotacetate, glucose and triglycerides, and higher capacity of phosphorylating glucose in the brain. These results provide evidence regarding an osmoregulatory and metabolic role for T in S. auratus that could be related to changes in both processes during sexual maturation.

Juvenile gilthead sea bream were exposed to diluted seawater (2.5 per thousand salinity; DSW) for 3 h or, in a second experiment, acclimated to DSW and fed a control or calcium-deficient diet for 30 days. Branchial Ca2+ influx, drinking rate and plasma calcium levels were assessed. Sea bream plasma parathyroid hormone related protein (sPTHrP) was measured, and mRNAs of pthrp, its main receptor, pth1r, and the calcium-sensing receptor (casr) were quantified in osmoregulatory tissues and the pituitary gland. When calcium is limited in water or diet, sea bream maintain calcium balance; however, both plasma Ca2+ and plasma sPTHrP concentrations were lower when calcium was restricted in both water and diet. Positive correlations between plasma sPTHrP and plasma Ca2+ (R2 = 0.30, N = 39, P < 0.05), and plasma sPTHrP and body mass of the fish (R2 = 0.37, N = 148, P < 0.001) were found. Immunoreactive sPTHrP was demonstrated in pituitary gland pars intermedia cells that border the pars nervosa and co-localises with somatolactin. In the pituitary gland, pthrp, pth1r and casr mRNAs were downregulated after both short- and long-term exposure to DSW. A correlation between pituitary gland pthrp mRNA expression and plasma Ca2+ (R2 = 0.71, N = 7, P < 0.01) was observed. In gill tissue, pthrp and pth1r mRNAs were significantly upregulated after 30 days exposure to DSW, whereas no effect was found for casr mRNA expression. We conclude that in water of low salinity, declining pituitary gland pthrp mRNA expression accompanied by constant plasma sPTHrP levels points to a reduced sPTHrP turnover and that sPTHrP, through paracrine interaction, is involved in the regulation of branchial calcium handling, independently of endocrine pituitary gland sPTHrP.

The gilthead sea bream (Sparus auratus) is an euryhaline fish where prolactin (PRL) and growth hormone (GH) play a role in the adaptation to different environmental salinities. To find out the role of these pituitary hormones in osmoregulation and energy metabolism, fish were implanted with slow release implants of ovine GH (oGH, 5 microg g(-1) body mass) or ovine prolactin (oPRL, 5 microg g(-1) body mass), and sampled 7 days after the start of the treatment. GH increased branchial Na(+),K(+)-ATPase activity and decreased sodium levels in line with its predicted hypoosmoregulatory action. GH had metabolic effects as indicated by lowered plasma protein and lactate levels, while glucose, triglycerides and plasma cortisol levels were not affected. Also, GH changed liver glucose and lipid metabolism, stimulated branchial and renal glucose metabolism and glycolytic activity, and enhanced glycogenolysis in brain. PRL induced hypernatremia. Furthermore, this hormone decreased liver lipid oxidation potential, and increased glucose availability in kidney and brain. Both hormones have opposite osmoregulatory effects and different metabolic effects. These metabolic changes may support a role for both hormones in the control of energy metabolism in fish that could be related to the metabolic changes occurring during osmotic acclimation.

Sharpsnout seabream ability for macronutrient self-selection was studied using gelatine capsules containing pure macronutrients. In particular, the existence of non-oropharyngeal chemosensory pathways involved on protein (P), fat (F) and carbohydrate (CH) selection, as well as sharpsnout seabream response to dietary protein dilution were investigated. In a sequence of experimental phases, sharpsnout seabream were fed a pelleted complete diet, an encapsulated complete diet or a combination of separately encapsulated pure macronutrients. In order to induce associative learning, capsules containing a given macronutrient were paired with a particular colour. The animals composed a diet containing 62.7% P, 21.3% CH and 16.0% F, in terms of macronutrient percentage intake, and this selection pattern was maintained throughout all experimental phases. In a second experiment, individually kept sharpsnout seabream were challenged with protein dilution. After protein capsules were diluted (from 91.9% P to 56.3% P) with cellulose, the animals increased their protein intake to compensate for dilution in such a way that their energy intake was not significantly modified (17.4 kJ/100 g BW vs. 17.6 kJ/100 g BW after dilution). These results show that sharpsnout seabream feeding on encapsulated diets are able to select and maintain a particular diet composition, as well as sustain their energy intake, without using the diet's oropharyngeal chemosensory properties. Moreover, they were also able to maintain their protein intake after dilution, which highlights the importance of this macronutrient in this omnivorous species.

In marine fish, meiosis resumption is associated with a remarkable hydration of the oocyte, which contributes to the survival and dispersal of eggs and early embryos in the ocean. The accumulation of ions and the increase in free amino acids generated from the cleavage of yolk proteins (YPs) provide the osmotic mechanism for water influx into the oocyte, in which is involved the recently identified, fish specific aquaporin-1o (AQP1o). However, the timing when these processes occur during oocyte maturation, and the regulatory pathways involved, remain unknown. Here, we show that gilthead sea bream AQP1o (SaAQP1o) is synthesized at early vitellogenesis and transported towards the oocyte cortex throughout oocyte growth. During oocyte maturation, shortly after germinal vesicle breakdown and before complete hydrolysis of YPs and maximum K(+) accumulation is reached, SaAQP1o is further translocated into the oocyte plasma membrane. Inhibitors of yolk proteolysis and SaAQP1o water permeability reduce sea bream oocyte hydration that normally accompanies meiotic maturation in vitro by 80% and 20%, respectively. Thus, yolk hydrolysis appears to play a major role to create the osmotic driving force, while SaAQP1o possibly facilitates water influx into the oocyte. These results provide further evidence for the role of AQP1o mediating water uptake into fish oocytes, and support a novel model of fish oocyte hydration, whereby the accumulation of osmotic effectors and AQP1o intracellular trafficking are two highly regulated mechanisms.

Sharpsnout seabream is a marine teleost of increasing interest for Mediterranean aquaculture, but there is still a lack of information regarding its circadian organization. In this study, we have investigated sharpsnout seabream locomotor activity, feeding and plasma melatonin daily rhythms under a 12:12-h LD cycle, as well as the persistence of locomotor activity circadian rhythmicity under constant light (LL) conditions. When submitted to an LD cycle, most sharpsnout seabream displayed a diurnal locomotor pattern, with an average 74% of activity recorded during daytime. However, along the experiment 40% of fish spontaneously changed their locomotor rhythm phasing and became nocturnal. Feeding behaviour, nevertheless, remained strictly diurnal in all cases, with 97% of food demands being made during the light period. Free-running locomotor rhythms were recorded in one third of the fish kept under LL. Daily plasma melatonin levels displayed a rhythmic profile, with low daytime values (111 pg/ml) and high nighttime concentrations (791 pg/ml). Taken together, these results evidence a high degree of plasticity for sharpsnout seabream activity patterns, as well as phasing independence of locomotor and feeding rhythms. Finally, the existence of a well-defined daily rhythm of plasma melatonin was found.

The influence of acclimation to different environmental salinities (low salinity water, LSW; seawater, SW; and hyper saline water, HSW) and feeding conditions (fed and food deprived) for 14 days was assessed on osmoregulation and energy metabolism of several tissues of gilthead sea bream Sparus auratus. Fish were randomly assigned to one of six treatments: fed fish in LSW, SW, and HSW, and food-deprived fish in LSW, SW, and HSW. After 14 days, plasma, liver, gills, kidney and brain were taken for the assessment of plasma osmolality, plasma cortisol, metabolites and the activity of several enzymes involved in energy metabolism. Food deprivation abolished or attenuated the increase in gill Na+,K+-ATPase activity observed in LSW- and HSW-acclimated fish, respectively. In addition, a linear relationship between renal Na+,K+-ATPase activity and environmental salinity was observed after food deprivation, but values decreased with respect to fed fish. Food-deprived fish acclimated to extreme salinities increased production of glucose through hepatic gluconeogenesis, and the glucose produced was apparently exported to other tissues and served to sustain plasma glucose levels. Salinity acclimation to extreme salinities enhanced activity of osmoregulatory organs, which is probably sustained by higher glucose use in fed fish but by increased use of other fuels, such as lactate and amino acids in food-deprived fish.

In the last decades, marine reserves have dramatically increased in number worldwide. Here I examined the potential of no-take marine reserves to reestablish lost predatory interactions and, in turn, cause community-wide changes in Mediterranean rocky reefs. Protected locations supported higher density and size of the most effective fish preying on sea urchins (the sea breams Diplodus sargus and D. vulgaris) than unprotected locations. Density of sea urchins (Paracentrotus lividus and Arbacia lixula) was lower at protected than at unprotected locations. Size structure of P. lividus was bimodal (a symptom of predation on medium-sized urchins) only at the protected locations. Coralline barrens were less extended at protected than at unprotected locations, whereas turf-forming and erect-branched algae showed an opposite pattern. Erect-unbranched and erect-calcified algae and conspicuous zoobenthic organisms did not show any pattern related to protection. Tethering experiments showed that predation impact on urchins was (1) higher at protected than at unprotected locations, (2) higher on P. lividus than on A. lixula, and (3) higher on medium-sized (2-3.5 cm test diameter) than large-sized (>3.5 cm) urchins. Sea urchins preyed on by fish in natural conditions were smaller at unprotected than at protected locations. The analysis of sea urchin remains found in Diplodus fish stomachs revealed that medium-sized P. lividus were the most frequently preyed upon urchins and that size range of consumed sea urchins expanded with increasing size of Diplodus fish. These results suggest that (1) depletion and size reduction of predatory fish caused by fishing alter patterns of predation on sea urchins, and that (2) fishing bans (e.g., within no-take marine reserves) may reestablish lost interactions among strongly interactive species in temperate rocky reefs with potential community-wide effects.

To elucidate the involvement of aromatase in sex reversal, the gilthead seabream ovarian P450 aromatase (cyp19a1a) cDNA and its 5'-flanking region were isolated and characterized. Northern blot analysis revealed that only one cyp19a1a transcript (2.0 kb) is expressed in the ovary. Four cAMP-responsive elements were identified at the 5'-flanking region of seabream cyp19a1a indicating a high potential to respond to gonadotropin signaling. Studying the seasonal profile, two expression peaks of cyp19a1a transcripts in the ovarian tissues were found in July (about 15000 copies/ng total RNA) for ambisexual fish and in December (about 12000 copies/ng total RNA) for spawning females. Starting from September, transcript levels of cyp19a1a in the ovarian portions of the male-developing gonads gradually decreased. Furthermore, the ovarian portions of the female gonads expressed cyp19a1a at a significantly higher level than the ovarian portions of the male gonads after November. Taken together with levels of plasma estradiol in reversing females being significantly higher than those in developing males, the above results reinforce the importance of cyp19a1a in sex reversal. In vitro exposure of ovarian fragments to gonadotropins (hCG) at 1, 10, and 100 IU/ml significantly (P < 0.05) upregulated cyp19a1a expression. Additionally, expression of cyp19a1a displayed a stronger and significant correlation with the transcript expression of ovarian Lh receptor rather than Fsh receptor during the ambisexual stage. Our results indicate that the differential expression of cyp19a1a gene is associated with sex reversal and that gonadotropin signals (particularly Lh) may serve as major players in regulating the expression of cyp19a1a during the process of sex reversal.

The isopod parasite Ceratothoa oestroides becomes hard to eradicate in intensive fish rearing systems and in addition to causing weight loss in adult fish it causes mortalities in larval and juveniles stages of sea bass and sea bream. Faced with the fast emergence of this infection in Adriatic finfish facilities, an effective compound had to be tested for bath treatment. Ivermectin was chosen for this purpose on the basis of extensive practice in northern European countries which used ivermectin administered in food with good results in treatment of Lepeophtheirus sp. and Caligus sp. infections. For determination of toxicity of ivermectin, a short-term toxicity bioassay on 40-day-old sea bream, Sparus aurata, L., was conducted, following FAO and UNEP procedures for methodology in aquatic toxicity trials. Median survival time, slope functions, and 95% confidence limits for ivermectin showed moderate toxicity in sea bream, with first 100% mortalities during 96h at the concentration of 1.8 mg/L.

Whole animal studies have indicated that Ca(2+) uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using amino-terminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of (125)I-(1-35(tyr)) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (K (D)=2.59 nM; B (max)=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1-34)PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The amino-terminal peptides (2-34)PTHrP, (3-34)PTHrP and (7-34)PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1-34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca(2+) uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.

The present study was undertaken to evaluate whether the age of brood stock females of Sparus aurata affects the mobilization of lipids from muscle and liver towards the gonads to aid in oocyte development. Taking into account the role some hormones play in lipid mobilization the production of 17beta-estradiol (E2) and testosterone (T) was also measured throughout the spawning season. Four groups of fish were established consisting of 3-, 4-, 5- and 6-year-old females (1.3, 1.5, 2.3 and 2.8 kg average weight, respectively), maintained in separate tanks together with a number of two-year-old males. The results for all four groups showed no variations in fish total length between the beginning and end of the experimental period. However, losses were recorded both of body weight and condition factor. In general terms, there were no significant differences in the plasma levels of E2, T and the gonadosomatic (GSI) and hepatosomatic (HSI) index among the four groups throughout the spawning season. In all groups, the plasma levels of E2, T, GSI and HSI were at their lowest at the end of spawning. Between December (pre-spawning) and March (mid-spawning), all groups suffered depletion of the lipid content of liver and muscle, while gonad lipid content remained constant. The mobilization of lipids from liver and muscle to contribute to the upkeep of the gonadal lipid involved the mobilization of all the fatty acid groups, saturates, monoenes, n-6, n-3, and n-3 HUFA. A good correlation existed between the percentage of the various fatty acid groups transferred from muscle and the age of the reproductive females. However, the greater lipid mobilization from muscle matching the greater age of the reproductive females did not result in greater lipid gonadal reserves or greater body size, suggesting that reproduction on the part of older females requires greater effort. Despite this, the results as a whole indicated that lipogenic capacity, considered as the mobilization of lipids from muscle and liver towards the gonad for the development of oocytes, is unaffected by age in reproductive 3- to 6-year-old gilthead seabream females.

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.

This morphodynamic study provides an insight on how the architecture of the heart ventricle of the gilthead seabream (Sparus auratus) is designed to accomplish the functional performance typical of an active teleost species. Using an in vitro working heart preparation, mechanical performance was analyzed under loading (i.e., preload and afterload) challenges. The hearts were very sensitive to filling pressure increases. Maximum cardiac output (CO: 55.66+/-4.54 ml/min/kg body weight; mean+/-SEM) and maximum stroke volume (VS: 0.42+/-0.027 ml/kg body weight; mean+/-SEM) were obtained at an input pressure of 1 kPa. When exposed to output pressure (OP) changes, the hearts maintained constant CO and SV up to about 4 kPa; further increases of afterload significantly compromised mechanical performance. Surprisingly, this "athletic" pumping performance was achieved by an entirely trabeculated pyramidal ventricle. The ventricular architecture was characterized by a system of small luminae and trabecular sheets radiating outward from the central lumen. The most peripheral part of the ventricular chamber contained single trabeculae and the corresponding lacunary spaces. The ventricular cavity was bounded by an outer myocardial monolayer "shell" to which the peripheral trabeculae were attached. Myofibril organization differed in the trabeculae and in the outer monolayer. The structural features challenge common beliefs regarding the typical "athletic" teleost heart design., (Copyright (c) 2005 Wiley-Liss, Inc.)

It has been shown previously that professional phagocytic granulocytes are present in the testis of the gilthead seabream, a seasonal breeding teleost that offers an excellent model for studying the testicular regression process that occurs in seasonal testicular involution and sex change. It is unexpected that testicular granulocytes produce interleukin-1beta, a regulator for spermatogonia proliferation in mammals, but are not involved in the elimination of degenerative germ cells. Here, we show that phagocytosis and reactive oxygen intermediate (ROI) production were suppressed dramatically in testicular phagocytic granulocytes, compared with their level of activity in the head-kidney, the main hematopoietic organ in fish. Furthermore, testicular-conditioned media modulated migration, phagocytosis, and ROI production of head-kidney phagocytic granulocytes, and the addition of testicular cells impaired their ROI production capacity. Until now, monocytes/macrophages were believed to be the only innate immune cells able to develop into functional subsets, whereas neutrophils only infiltrate the tissues upon infection or inflammation. Our findings demonstrate, however, that fish professional phagocytic granulocytes also display functional adaptation to different microenvironments and strongly suggest a role for these cells in the reorganization of the testis during post-spawning.

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.

Cryopreservation produces several types of damage in spermatozoa, leading to fertility impairment. The reduction arises both from a lower viability post-thaw and from sublethal dysfunctions in some of the surviving cells. In the present study, we have analysed the effect of cryopreservation in 5 ml macrotubes on the quality of post-thawed gilthead sea bream sperm. Several standard sperm quality parameters were determined: pH and osmolarity of seminal plasma, sperm concentration, and motility. An exhaustive determination of sperm quality before and after cryopreservation was investigated. Several parameters related with spermatozoal status were determined: ATP content, plasma membrane integrity and functionality, mitochondrial functionality, and sperm fertility. Our results demonstrated that gilthead sea bream spermatozoa suffer several types of damage after freezing/thawing. The percentage of viable cells slightly decreased after cryopreservation, however plasma membrane was affected by cryopreservation, since cells could not resist the hyperosmotic shock. Mitochondrial status was affected by cryopreservation since there was a decrease in the parameters of sperm motility, ATP content (3.17 nmol ATP/10(5) spermatozoa to 1.7 nmol ATP/10(5) spermatozoa in 1:20 frozen samples) and an increase of the percentage of cells with mitochondrial depolarized membranes (11% for fresh and 27% for 1:20 frozen samples). Fertility rate was similar either using fresh or frozen/thawed sperm (77 and 75% hatched larvae, respectively).

The genes encoding heat shock cognate 70 (hsc70) and inducible heat shock protein 70 (hsp70) were cloned and characterized from silver sea bream liver. Upon acute heat shock (+7 degrees C), the transcript abundance of hsc70 was increased 1.7-fold whereas the transcript abundance of hsp70 increased 6.7-fold. The chronic acclimation of sea bream to cold temperature (12 degrees C) resulted in a downregulation of hsc70 and an upregulation of hsp70 in comparison to levels in sea bream kept at a warmer temperature (25 degrees C). The expression of heat shock transcription factor I was also increased during cold temperature acclimation. Increased amounts of hepatic insulin-like growth factor 1 transcript, serum thyroxine (T4), and triiodothyronine (T3) were also found during cold temperature acclimation whereas serum cortisol remained unchanged. The results from this study demonstrate how temperature acclimation, in fish, can affect the regulation of the hsp70 multigene family and hormonal factors that are associated with anabolism.

Numerous studies have described the presence of an intragonadal IGF system involved in regulation of gametogenesis in teleost fish. In the present study, the in vivo effects of estradiol-17beta (E2) and growth hormone (GH) exposure on IGF-I, IGF-II, IGF1R, and IGFBP2 gene expression in sea bream ovary were monitored by RT-PCR during prereproductive and reproductive periods. The evidence demonstrates that both hormones investigated here affect the ovarian IGF system, showing that it is not only under GH control, but also can be regulated by sexual hormones; this hormonal modulation is related to reproductive phase.

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the testicular regression process that occurs in both seasonal testicular involution and sex change. Insofar as fish reproduction is concerned, little is known about cell renewal and elimination during the reproductive cycle of seasonal breeding teleosts with asynchronous spermatogenesis. We have previously described how acidophilic granulocytes infiltrate the testis during postspawning where, surprisingly, they produce interleukin-1beta, a known growth factor for mammalian spermatogonia, rather than being directly involved in the elimination of degenerative germ cells. In this study, we are able to discriminate between spermatogonia stem cells and primary spermatogonia according to their nuclear and cytoplasmic diameters and location in the germinal epithelium, finding that these two cell types, together with Sertoli cells, proliferate throughout the reproductive cycle with a rate that depends on the reproductive stage. Thus, during spermatogenesis the spermatogonia stem cells, the Sertoli cells, and the developing germ cells (primary spermatogonia, A and B spermatogonia, and spermatocytes) in the germinal compartment, and cells with fibroblast-shaped nuclei in the interstitial tissue proliferate. However, during spawning, the testis shows few proliferating cells. During postspawning, the resumption of proliferation, the occurrence of apoptotic spermatogonia, and the phagocytosis of nonshed spermatozoa by Sertoli cells lead to a reorganization of both the germinal compartment and the interstitial tissue. Finally, the proliferation of spermatogonia increases during resting when, unexpectedly, both oogonia and oocytes also proliferate. This proliferative pattern was correlated with the gonadosomatic index, testicular morphology, and testicular and gonad areas, suggesting that complex mechanisms operate in the regulation of gonocyte proliferation in hermaphrodite fish.

The positive buoyancy of marine fish eggs in sea water, allowed by hydration of the oocyte, is critical for their survival and dispersion in the ocean. We isolated an aquaporin, SaAQP1o, that belongs to a unique subfamily of aquaporin-1-like channels specifically evolved in teleosts and mainly expressed in the ovary. We further show that hormone-induced fish oocyte hydration is a highly controlled process based on the interplay between protein hydrolysis and the translocation of SaAQP1o to the plasma membrane, indicating a specialized physiological role for this aquaporin.

Immature Sparus aurata L. (gilthead seabream) were exposed to 17beta-estradiol (E(2)) 4000 ng/l and to the same E(2) concentration mixed with 50,000 ng/l 4-nonylphenol (E(2) + NP) during 4, 8, 12 and 16 h. E(2) availability and E(2) plasma level variations were assessed. Liver biotransformation capacity was measured as ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST) activities. Plasma cortisol, lactate and glucose were also determined. Genotoxicity was assessed through erythrocytic nuclear anomalies (ENA) frequency. Liver EROD activity significantly decreased during the whole experiment for both treatments, with the exception of 16 h exposure to E(2). Liver GST activity was significantly increased after 8 and 12 h of exposure either to E(2) or E(2) + NP. An endocrine disruption expressed as plasma cortisol decrease was observed after 16 h exposure under both tested conditions, concomitantly with a plasma lactate increase. No genotoxic responses, measured as ENA frequency, were detected. Analyzing the E(2) water concentration in aquaria without fish it was demonstrated an intense and fast E(2) loss, considerably reducing its availability to fish. In the presence of fish, E(2) water levels were drastically reduced after 4 h exposure, being this reduction more pronounced in E(2) aquarium when compared to E(2) + NP aquarium. In addition, it was demonstrated a rapid E(2) uptake from the water since the highest E(2) plasma concentrations were observed after 4 h exposure, followed by a continuous decrease, which became more pronounced between 8 and 12 h of exposure. Furthermore, during the first 8 h exposure to E(2) and E(2) + NP, seabream plasma E(2) concentrations were higher than the initial water exposure concentration. Comparing the E(2) plasma levels in both seabream-exposed groups, it was clear that its concentration is always higher in E(2) + NP-treated fish. Despite the previous results, no significant differences were found in the measured responses between E(2) and E(2) + NP.

Mortality, changes in blood osmolality, and pO2 in the goldlined seabream (Rhabdosargus sarba) on exposure to a subbloom concentration (2,000 cells/ml) of a toxic red tide alga, Chattonella marina, were investigated and related to quantitative ultrastructural alterations of the gill. The median lethal time (LT50) was 6 h. Significant induction of filamental chloride cells (CCs) (increases in CC density, apical opening area, fractional area, volume densities of CCs, and mitochondria within CCs), concomitant with a significant reduction in blood osmolality, was found within 3 h of exposure to C. marina. Further reduction in blood osmolality (67%) and a drastic decline of pO2 (70%) were detected in moribund fish after 6 h. Fish were also subjected to severe salinity stress (abrupt transfer to 0 and 60% salinities), and the same parameters were measured. Our quantitative ultrastructural and physiological data suggest that fish exposed for 6 h to C. marina (2,000 cells/ml) suffered similar but more severe osmotic distress as compared to that induced by abrupt transfer to 60% hypersaline water. Results of the salinity stress experiment also showed that suffocation was not a secondary response induced by osmotic impairment in the moribund fish. Osmoregulatory failure in conjunction with suffocation may be the cause of death following exposure to C. marina. The findings of this study provide evidence that C. marina, even in concentrations below visible blooms, can pose a significant threat to marine fish.

It is widely believed that FSH and LH, which are known to play key roles in controlling the production of functional oocytes in vertebrates, are synthesized and secreted exclusively by the anterior pituitary. Here we present evidence for the novel expression of FSHbeta, LHbeta, and the common glycoprotein-alpha (Cgalpha) in the gilthead seabream ovary. Using in situ hybridization and immunocytochemistry, FSHbeta was detected in primary-growth and secondary-growth-I oocytes, LHbeta was found in secondary-growth oocytes, and Cgalpha was observed in both primary and secondary-growth oocytes. Northern blot analyses demonstrated that Fshbeta transcript is 0.6 kb in both pituitary and ovary, whereas the ovarian Lhbeta transcript (1.1 kb), unexpectedly, is longer than the known pituitary Lhbeta transcript (0.6 kb). Sequence analyses revealed that ovarian Lhbeta is driven by a different promoter than pituitary Lhbeta, which generates an additional 459 bases at the distal portion of the 5'-untranslated region of the ovarian Lhbeta. Furthermore, using in vitro ovarian fragment incubation, we demonstrated that mammalian GnRH analog agonist enhanced the expression of ovarian Fshbeta (up to 2.7-fold), Lhbeta (up to 1.4-fold), Cgalpha (up to 1.8-fold), and the secretion of ovarian LH (up to 2.2-fold). In contrast, GnRH antagonist, analog E, suppressed the secretion of ovarian LH. Our findings suggest that a GnRH-gonadotropin axis is present in the gilthead seabream ovary and that FSH and LH, the well-characterized pituitary hormones, may have prominent novel roles in teleost intraovarian communication between oocytes and ovarian follicle cells.

The action of a number of growth hormone secretagogues (GHS) on growth hormone (GH) secretion and gene expression was studied in a primary culture of pituitary cells isolated from the black seabream Acanthopagrus schlegeli. The peptide GHS employed included growth hormone-releasing peptide (GHRP)-2, ipamorelin, and human ghrelin. The nonpeptide GHS employed included the benzolactam GHS L692,585 and the spiropiperidine GHS L163,540. Secreted GH was measured in the culture medium by an enzyme-linked immunosorbent assay (ELISA) method using a specific antibody against seabream GH. The GH mRNA content in the incubated cells was assessed by reverse transcription polymerase chain reaction (RT-PCR) using a pair of gene-specific primers designed from the cloned black seabream GH cDNA sequence. A dose-dependent stimulation of GH release was demonstrated by all the GHS tested, except human ghrelin, with EC(50) values in the nanomolar range. Simultaneous measurement of GH mRNA levels in the incubated seabream pituitary cells indicated that the GHS-stimulated increase in GH secretion was not paralleled by corresponding changes in GH gene expression. In contrast to the situation previously reported in the rat, no change in GH gene expression was noticed in the seabream pituitary cells even though the time of stimulation by GHS was increased up to 48 h, confirming that the GHS-stimulated GH secretion in seabream is independent of GH gene transcription.

The osmoregulatory action of 17beta-estradiol (E2) was examined in the euryhaline teleost Sparus auratas. In a first set of experiments, fish were injected once with vegetable oil containing E2 (1, 2 and 5 microg/g body weight), transferred 12h after injection from sea water (SW, 38 ppt salinity) to hypersaline water (HSW, 55 ppt) or to brackish water (BW, 5 ppt salinity) and sampled 12h later (i.e. 24 h post-injection). In a second experiment, fish were injected intraperitoneally with coconut oil alone or containing E2 (10 microg/g body weight) and sampled after 5 days. In the same experiment, after 5 days of treatment, fish of each group were transferred to HSW, BW and SW and sampled 4 days later (9 days post-implant). Gill Na+,K+ -ATPase activity, plasma E2 levels, plasma osmolality, and plasma levels of ions (sodium and calcium), glucose, lactate, protein, triglyceride, and hepatosomatic index were examined. Transfer from SW to HSW produced no significant effects on any parameters assessed. E2 treatment did not affect any parameter. Transfer from SW to BW resulted in a significant decrease in plasma osmolality and plasma sodium but did not affect gill Na+,K+ -ATPase activity. A single dose of E2 attenuated the decrease in these parameters after transfer from SW to BW, but was without effect on gill Na+,K+ -ATPase activity. An implant of E2 (10 microg/g body weight) for 5 days significantly increased plasma calcium, hepatosomatic index, plasma metabolic parameters, and gill Na+,K+ -ATPase activity. In coconut oil-implanted (sham) fish, transfer from SW to HSW or BW during 4 days significantly elevated gill Na+,K+ -ATPase. Gill Na+,K+ -ATPase activity remained unaltered after transfer of E2-treated fish to HSW or BW. However, in E2-treated fish transferred from SW to SW (9 days in SW after E2-implant), gill Na+,K+ -ATPase activity decreased with respect to HSW- or BW-transferred fish. Shams transferred to HSW showed increased levels of lactate, protein, and trygliceride in plasma, while those transferred to BW only displayed increased trygliceride levels. E2-treated fish transferred to HSW showed higher protein levels without any change in other plasmatic parameters, while those transferred to BW displayed elevated plasma glucose levels but decreased osmolality and protein levels. These results substantiate a chronic stimulatory action of E2 on gill Na+,K+ -ATPase activity in the euryhaline teleost Sparus auratas., (Copyright 2004 Wiley-Liss, Inc.)

Brush border membrane vesicles (BBMV) enriched in sucrase, maltase and alkaline phosphatase, and impoverished in Na(+)-K(+)-ATPase, were isolated from proximal and distal intestine of the gilthead sea bream (Sparus aurata) by a MgCl(2) precipitation method. Vesicles were suitable for the study of the characteristics of D-glucose apical transport. Only one D-glucose carrier was found in vesicles from each intestinal segment. In both cases, the D-glucose transport system was sodium-dependent, phlorizin-sensitive, significantly inhibited by D-glucose, D-galactose, alpha-methyl-D-glucose, 3-O-methyl-D-glucose and 2-deoxy-D-glucose, and showed stereospecificity. Apparent affinity constants of D-glucose transport (K(t)) were 0.24 +/- 0.03 mM in proximal and 0.18 +/- 0.03 mM in distal intestine. Maximal rate of influx (Jmax) was 47.3 +/- 2.2 pmols. mg(-1) protein for proximal and 27.3 +/- 3.6 pmols. mg(-1) protein for distal intestine. Specific phlorizin binding and relative abundance of an anti-SGLT1 reactive protein were significantly higher in proximal than in distal BBMV. These results suggest the presence of the same D-glucose transporter along the intestine, with a higher density in the proximal portion. This transporter is compatible with the sodium-dependent D-glucose carrier described for other fish and with the SGLT1 of higher vertebrates.

In this study the influence of the dietary level of the fatty acid arachidonic acid (ArA, 20:4n-6) was determined on the acute stress response and osmoregulation of adult gilthead seabream Sparus aurata L. Seabream were fed a diet containing either 0.9% or 2.4% of total fatty acids as ArA for 18 days before being subjected to a 5 min period of net confinement. Prior to this stressor, a subgroup of fish from both dietary treatment groups was treated with acetylsalicylic acid (ASA), an irreversible blocker of cyclooxygenase (COX). This would indicate whether any effects were caused by an enhanced synthesis of prostaglandins derived from ArA. The highest ArA levels were found in the kidneys, and these were further enhanced by dietary ArA-supplementation. In gill tissues, there were significant changes in all selected fatty acid classes 24 h after confinement, except for the docosahexaenoic acid (DHA, 22:6n-3): eicosapentaenoic acid (EPA, 20:5n-3) ratio. ArA feeding strongly reduced the cortisol response to confinement, which was partially counteracted by ASA treatment. ArA also attenuated the stress-associated increase in plasma osmolality and, in combination with ASA, enhanced the osmolality and plasma chloride levels, but reduced plasma sodium levels after confinement. Furthermore, ArA enhanced the branchial Na(+), K(+)-ATPase activity both before and after confinement, whereas feeding ASA diminished this effect. It appeared that the effects of ArA-supplementation could not always be ascribed to an increase in prostaglandin synthesis. It is advisable to determine the long-term effects of replacing fish oils in commercial diets with vegetable oils that contain no long-chain fatty acids, particularly in carnivorous/marine species with low fatty acid elongation and desaturation activities. The effects of a low dietary intake of ArA (and other polyunsaturated fatty acids) should be studied over a longer term, taking into account any consequences for the health of the fish.

To address the complexity of the origin of the GnRH system in perciforms, we investigated the ontogenic expression of three GnRHs in gilthead seabream. Using in situ hybridization, chicken (c) GnRH-II mRNA-expressing cells were detected in the hindbrain at 1.5 days postfertilization (DPF) and in the midbrain at 2 DPF and thereafter; the hindbrain signals became undetectable after 10 DPF. Salmon (s) GnRH mRNA-expressing cells were first seen in the olfactory placode at 3 DPF, started caudal migration at 14 DPF, and reached the preoptic areas at 59 DPF. Seabream (sb) GnRH mRNA-expressing cells were first detected in the terminal nerve ganglion cells (TNgc), ventral part of the ventral telencephalon, nucleus preopticus parvocellularis, and thalamus at 39 DPF, and extended to the nucleus preopticus magnocellularis at 43 DPF, ventrolateral hypothalamus at 51 DPF, and nucleus lateralis tuberis and posterior tuberculum at 59 DPF. Coexpression of sbGnRH and sGnRH transcripts was found in the TNgc. Using real-time fluorescence-based quantitative polymerase chain reaction, transcript levels of cGnRH-II and sGnRH were first detected at 1 and 1.5 DPF, respectively, and increased and remained high thereafter. Transcript levels of sbGnRH remained low after first detection at 1 DPF. Furthermore, these GnRH expression profiles were correlated with the expression profiles of reproduction-related genes in which at least four concomitant increases of GnRH, GnRH receptor, gonadotropin, gonadotropin receptor, and Vasa transcripts were found at 5, 8, 14, and 28 DPF. Our data provide an expanded view of the ontogeny of the GnRH system and reproductive axis in perciforms.

The potential for input of two common antibacterial agents in Mediterranean fish farms, oxytetracycline (OTC) and oxolinic acid (OA), was estimated from measurements of these drugs in the faecal excretions of two important farmed sparids, gilthead sea bream, Sparus aurata and sharpsnout sea bream Diplodus puntazzo. Oxolinic acid was found to be well absorbed by gilthead sea bream (92%) and sharpsnout sea bream (88%) while the absorption of OTC was found to be considerably lower in both species (27 and 40%, respectively). These data were integrated with production records for sparids, drug dosage regimes and treatment frequency information to calculate potential annual drug release to the aquatic environment from Greek fish farms. These calculations suggest potentially significant quantities of unmetabolised OTC can be passed unabsorbed through the body of treated sparids and excreted via the faeces into the local marine environment. The situation with OA was much less pronounced. It was estimated that potentially more than 1900 kg of OTC and more than 50 kg of OA may be released via faecal excretion into the environment by sparid farms per year. Further drug may also be released via uneaten medicated feed, leached drugs and other routes of fish elimination (renal excretion, branchial secretions). Drug pollution of the marine environment in the vicinity of fish farms can have adverse ecological effects, including development of resistant bacterial populations and exposure with potential drug accumulation in aquatic fauna and flora.

To examine the relationship between structure and function of glucose-6-phosphatase (G6Pase) in fish, we undertook molecular cloning and modulation of G6Pase expression by starvation and refeeding on diets with different nutrient composition in the liver of the carnivorous fish, Sparus aurata. A cDNA encoding the full-length G6Pase catalytic subunit from the liver of S. aurata was isolated. This cDNA encodes a 350-amino acid protein, with low homology to the mammalian G6Pase, although it contains most of the key residues required for catalysis. Based on hydrophobicity and membrane structure prediction, we propose a model containing nine-transmembrane regions for S. aurata G6Pase. Northern blots showed that refeeding after a prolonged starvation rapidly reverses the glucose/glucose-6-phosphate substrate cycle flux in the fish liver through decreased G6Pase expression and strong glucokinase (GK) induction. The effect of refeeding different diets on G6Pase and GK expression, indicated that hepatic intermediary metabolism of fish fed diets with low protein/high carbohydrate diets is impelled towards utilization of dietary carbohydrates, by means of modulation of GK mRNA levels rather than G6Pase expression. These findings challenge the role attributed to dysregulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolise glucose.

Mammalian interleukin-1beta (IL-1beta) is a secretory cytokine lacking a signal peptide, which does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1beta-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. Little information is available on the production and release of fish IL-1beta, but the IL-1beta gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor/bacterial DNA (VaDNA)-primed immune cells of the marine fish gilthead seabream (Sparus aurata) accumulate intracellular IL-1beta as a approximately 30 kDa polypeptide (proIL-1beta). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor. More importantly, addition of extracellular ATP does not promote IL-1beta secretion by immune cells and fails to induce phosphatidylserine flip. In contrast, gilthead seabream SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1beta form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1beta by SAF-1 cells is abrogated by a specific ICE inhibitor.

Whole-body calcium uptake was studied in gilthead sea bream larvae (9-83 mg) in response to changing environmental salinity and [Ca2+]. Calcium uptake increased with increased fish size and salinity. Fish exposed to calcium-enriched, diluted seawater showed increased calcium uptake compared with fish in diluted seawater alone. Calcium uptake was unchanged in Na(+)-enriched, diluted seawater. Overall, [Ca2+], and not salinity/osmolarity per se, appears to be the main factor contributing to calcium uptake. By contrast, drinking was reduced by a decrease in salinity/osmolarity but was little affected by external [Ca2+]. Calculations of the maximum contribution from drinking-associated calcium uptake showed that it became almost insignificant (less than 10%) through a strong decrease in drinking rate at low salinities (0-8 per thousand ). Diluted seawater enriched in calcium to the concentration present in full-strength seawater (i.e. constant calcium, decreasing salinity) restored intestinal calcium uptake to normal. Extra-intestinal calcium uptake also benefited from calcium addition but to a lesser extent.

In the present study, the sea-bream Sparus aurata, a pelagic egg spawner, was used as experimental model, in order to establish the occurrence of apoptosis in vertebrates with external reproduction. The same female ovulates floating and nonfloating eggs, but only the former, after fertilization, proceed to embryo development. The eggs were divided into floating and nonfloating and both were analyzed for the presence of several apoptosis markers. The results here reported provide evidence that the nonfloating cells present severe shrinkage and highly express both FAS receptor and FAS ligand on their surface. Furthermore, DNA fragmentation and mitochondria swelling were found, suggesting that the nonfloating eggs were cells programmed to die., (Copyright 2003 Wiley-Liss, Inc.)

The potential for intraspecific chemical communication in the gilthead seabream (a marine perciform) was investigated by assessing the olfactory sensitivity to conspecific body-fluids (water occupied by conspecifics, intestinal fluid, urine, semen, egg fluid) by multiunit electrophysiological recording from the olfactory nerve. The olfactory system was responsive to water previously occupied by conspecifics, and the active compound(s) could be extracted by solid-phase extraction. The olfactory system was extremely sensitive to body fluids of sexually mature conspecifics: thresholds of detection were 1:10(7.4) (intestinal fluid), 1:10(6.1) (gametes), and 1:10(4.2) (urine). The olfactory system was also sensitive to amino acids with thresholds of detection from 10(-8.1) M (L-leucine) to 10(-6.1) M (L-phenylalanine). However, a range of other known fish odorants (steroids, bile acids, and prostaglandins) failed to evoke significant responses. Given the high olfactory sensitivity to intestinal fluid and the low urine release rates of marine compared with freshwater fish, we suggest that chemical communication is likely to be mediated via compounds present in the intestinal fluid rather than urine. Furthermore, the types of chemicals involved are likely to be different from those of freshwater fish. Their exact chemical identity and biological roles remain to be established.

Heavy metal pollution in rivers and its impact on aquatic ecosystems is a dynamic process. Fish are ideal indicators of heavy metal contamination in aquatic systems because they occupy different trophic levels and are of different sizes and ages. In particular, copper is an essential trace metal for living organisms and it is present in all natural waters and sediments. In this paper, we report data on the effect of copper on DNA erythrocytes from the teleost gilthead sea bream Sparus aurata and the bivalve mollusk Scapharca inaequivalvis. In particular, the effect of 0.1 ppm of Cu2+ on the nucleated erythrocytes was analyzed using the "comet assay." This test is a promising tool for estimation of DNA damage at the single cell level. The data obtained show that the in vivo treatment with 0.1 ppm of copper increased the susceptibility of DNA to be damaged. Exposure to Cu2+ produces a more evident effect on Sparus aurata, as all three comet parameters significantly increased (tail length, tail intensity, and tail moment). The higher comet parameters measured in Scapharca inaequivalvis compared to Sparus aurata could be due to the difference in stability of the respective hemoglobins. The comet assay could represent a useful test to evaluate the biological consequences of environmental contamination by metals on marine organisms.

Pahutoxin (PHN, choline chloride ester of 3-acetoxypalmitic acid) is a natural fish-killing (ichthyotoxic) agent derived from the defensive secretions of trunkfish. In spite of its obvious structural resemblance to synthetic cationic long-chain quaternary ammonium detergents, we show that PHN's action does not rely on its surfactant properties and is in fact, receptor-mediated. The above conclusion is supported by the following data: 1. Ichthyotoxicity is not related to its detergency or surfactivity, as indicated by the fact that the lethal concentration is about 1.5 orders of magnitude below its critical micelle concentration value (69 microM) and its liposomal/seawater partition coefficient is low (62-85); 2. The trunkfish is tolerant to its own pahutoxin; 3. Ichthyotoxicity occurs only upon application to the surrounding water, suggesting the existence of externally located receptors; 4. The receptor hypothesis was supported by the aid of equilibrium saturation binding assays revealing the presence of specific binding sites to PHN on the fish gill membranes; 5. The PHN tolerant trunkfish was shown to be devoid of PHN-binding sites. Some chemo-ecological, and environmental implications are discussed.

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as V(max) and K(m) determination were carried out in tissue extracts of Cu-exposed or control animals. V(max) and K(m) were also determined with or without addition of Cu(2+) in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu(2+) addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis.

Oxidative stress in fish (Sparus aurata) as a consequence of food restriction and fasting, has been studied. Four groups of fish were maintained for 46 days under different conditions of food supplementation: a control group with no food restriction (ratio of food/fish of 2% w/w), two groups of animals with restricted food supplement (1 and 0.5%) and a fasting group (no meal addition). Finally, all the fish were provided with food at the same ratio as the control group for the last 7 days. Sampling and weighing of fish were carried out every week and their livers were used for the analysis of known biomarkers of oxidative stress. Malondialdehyde and oxidized glutathione levels increased at the third week in fish with partial or total food deprivation, but these levels returned to normal values when the fish readapted to the control conditions. Antioxidant enzymes were also analyzed and significant increases in superoxide dismutase (SOD), glutathione reductase and glutathione peroxidase activities were found in parallel with food restriction; however catalase activity decreased in fasting fish. New SOD isoforms were detected by isoelectrofocusing in fish under food restriction at the second week, which disappeared when starved fish returned to the control conditions. These new SOD isoforms were detected before the appearance of other usual oxidative stress biomarkers.

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.

The effects of GnRH agonist (GnRHa) on the hypothalamus-pituitary-gonadal axis were studied in female pre-pubertal red seabream. Sexually immature 16-month-old fish were implanted intramuscularly with cholesterol pellets containing GnRHa or GnRHa in combination with domperidone, putative dopamine antagonist, and reared for 10-20 days. In both GnRHa and GnRHa+domperidone implanted groups, vitellogenesis was observed on Day 10 and ovulation was observed on Day 20, while ovarian development was not observed in the control fish throughout the experimental period. The levels of GnRH receptor mRNA were significantly higher in both GnRHa implanted groups than in the control. The expressions of all three gonadotropin subunit genes were up-regulated and serum luteinizing hormone levels were increased by the GnRHa implantation. Serum testosterone and estradiol-17beta levels were also increased on Day 10 and maintained high levels on Day 20. On the other hand, seabream (sb) GnRH mRNA levels in the brain were relatively low and unchanged in all experiment groups. The present study first shows that GnRH alone can induce precocious puberty in red seabream. These results indicate that the system of pituitary-gonadal axis has already been developed in 16-month-old fish and the commencement of sbGnRH secretion may be an important physiological event for the onset of puberty in the red seabream.

The temperature dependence for Na(+)-K(+)-ATPase has been examined in the proximal-distal axis of the intestine of gilthead seabream (Sparus aurata), i.e. pyloric caeca (PC), anterior intestine (AI) and posterior intestine (PI). Data derived from the Arrhenius plots showed differences in terms of temperature discontinuity points ( Td) (13.29 degrees C, 16.39 degrees C and 17.48 degrees C for PC, AI and PI, respectively) and activation energy ratios (Ea(2)/Ea(1)) obtained at both sides of Td (2.38, 1.98 and 1.78, for PC, AI and PI, respectively). The analyses of polar lipids showed differences in the levels of certain fatty acids among intestinal regions. The content of each fatty acid and different fatty acid ratios were correlated with the corresponding Td and Ea(2)/Ea(1) values. Regression analyses revealed the existence of strong negative correlations between docosahexaenoic acid (22:6n-3, DHA) or the DHA/monoenes ratio and Td. No obvious relationships were observed for other polyunsaturated fatty acids (PUFA) nor saturated fatty acids. The results obtained in the present study indicate that the heterogeneous values of Td displayed by the Na(+)-K(+)-ATPase along the intestinal tract could be related to a modulatory role of certain fatty acid within the lipid microenvironment of the enzyme.

The effect of the concentration of tributyltin (TBT) in feed on the dietary bioaccumulation of tributyltin chloride (TBTCl) was studied in an 8-week uptake experiment and a 4-week elimination experiment using red sea bream (Pagrus major). The biomagnification factor (BMF) and the assimilation efficiency (AE) decreased from 0.30 to 0.15 and from 13% to 5.9%, respectively, as the TBT concentration in feed increased from 1.3 to 20 microg/g. The elimination rate constant (k(2)) was independent of the TBT concentration in the fish. Laboratory measurements of the BMF and AE of TBTCl underestimate actual field values if highly contaminated feed is used. Judging from the BMF and AE, the risk of the bioaccumulation of TBTCl through the food chain might be smaller than that of polychlorinated biphenyls., (Copyright 2002 Elsevier Science Ltd.)

The osmoregulatory actions of ovine prolactin (oPRL), ovine growth hormone (oGH), and cortisol were tested in the euryhaline gilthead seabream Sparus aurata. Acclimated to sea water (SW, 40 ppt salinity, 1000 mOsm/kg H(2)O) or brackish water (BW, 5 ppt, salinity, 130 mOsm/kg H(2)O), injected every other day for one week (number of injections, 4) with saline (0.9% NaCl), oPRL (4 microg/g body weight), oGH (4 microg/g body weight) or cortisol (5 microg/g body weight), and transferred from SW to BW or from BW to SW 24h after the last injection. Fish were sampled before and 24h after transfer. Gill Na(+), K(+)-ATPase activity, plasma osmolality, plasma ions (sodium and chloride), plasma glucose, and muscle water moisture were examined. SW-adapted fish showed higher gill Na(+), K(+)-ATPase activity, plasma osmolality, and plasma ions levels than BW-adapted fish. Transfer from SW to BW decreased plasma osmolality and ions levels after 24h, while transfer from BW to SW increased these parameters, whereas gill Na(+),K(+)-ATPase activity was unaffected. oPRL treatment significantly decreased gill Na(+),K(+)-ATPase activity and increased plasma osmolality and ions in SW- and BW-adapted fish. This treatment minimizes loss of osmolality and ions in plasma after transfer to BW and increased these values after transfer to SW. No significant changes were observed in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions in oGH-treated group with respect to saline group before or after transfer from SW to BW or from BW to SW. Treatment with cortisol induced, in SW-adapted fish, a significant increase of gill Na(+),K(+)-ATPase activity and decrease of plasma osmolality and plasma ions. In BW-adapted fish this treatment induced a significant increases in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions. After transfer to SW cortisol-treated fish had higher plasma osmolality than the saline group. Our results support the osmoregulatory role of PRL in the adaptation to hypoosmotic environment in the gilthead seabream S. aurata. Further studies will be necessary to elucidate the osmoregulatory role of GH in this species. Cortisol results suggest a "dual osmoregulatory role" of this hormone in S. aurata.

The steroid synthesis pathway in the ovarian follicles of the red seabream during final oocyte maturation (FOM) was investigated by incubating intact follicles with different radioactively labeled steroid precursors. During FOM, the steroidogenic shift from estradiol-17beta to 20 beta-hydroxylated progestin production occurred mainly due to a combination of inactivation of C 1720-lyase and activation of 20 beta-hydroxysteroid dehydrogenase. Of the steroids produced, 1720 beta-dihydroxy-4-pregnen-3-one (1720 beta-P) and 1720 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) exhibited the greatest effect on germinal vesicle breakdown (GVBD) in vitro. 1720 beta-P was further converted to its 5 beta-reduced form, 1720 beta-dihydroxy-5 beta-pregnan-3-one (1720 beta-P-5 beta), which had lower GVBD activity, suggesting that 5 beta-reduction plays a role in the inactivation of the maturation-inducing ability of 1720 beta-P. In contrast, no 5 beta-reduced metabolite of 20 beta-S was found. Serum levels of 1720 beta-P and 20 beta-S, measured by ELISA, showed that circulating levels of both progestins increased during FOM, and 20 beta-S levels were approximately twice as high as 1720 beta-P levels. This study clarified the complete steroidogenesis pathway during FOM in red seabream ovarian follicles and showed that two 20 beta-hydroxylated progestins, 1720 beta-P and 20 beta-S, act as maturation-inducing hormones in this species. The catabolites of these two progestins and their physiological roles in reproduction are also discussed.