Sjoerd E. Wendelaar Bonga

The interaction of temperature and salinity on growth and food conversion in juvenile turbot (Scophthalmus maximus)

Abstract The effects of temperature and salinity on growth and feed conversion of juvenile turbot (initial mean weight 14 g) were investigated by rearing fish at (mean±SD) 10±0.2°C, 14±0.2°C, 18±0.3°C and 22±0.2°C and 15±0.4‰, 25±0.4‰ and 33.5±0.1‰ for 3 months. Growth, food consumption, and food conversion efficiency were highest at 15‰, and lowest at 33.5‰. There was an interactive effect of temperature and salinity at the two highest temperatures (18°C and 22°C) but not at 10°C and 14°C. The optimal temperature for growth (Topt. G) varied with salinity: Topt. G at 33.5‰ was 19.6±0.3°C (±SEM), whereas the Topt. G at 15‰ was 22.9±1.0°C, and at 25‰ was 24.7±2.1°C. A similar trend was found for food conversion efficiency (FCE). The optimal temperatures for FCE were 17.4±0.5°C, 17.9±1.0°C and 19.0±0.9°C at 33.5‰, 25‰ and 15‰, respectively. Overall, we found the optimal temperature–salinity combination for growth to be 21.8±0.9°C and 18.5±0.8‰. The optimal temperature–salinity combination for food conversion efficiency was found to be 18.3±0.6°C and 19.0±1.0‰. It is concluded that growth and food conversion efficiency of juvenile turbot can be improved by rearing them at intermediate salinities in the upper temperature range.

The influence of temperature and ration on growth, feed conversion, body composition and nutrient retention of juvenile turbot (Scophthalmus maximus)

Abstract The influence of two water temperatures (16 and 22 °C) on growth, feed conversion, body composition and nutrient retention was investigated in juvenile turbot fed to satiation (0.9% and 1.1% bw day−1 at 16 and 22 °C, respectively) and at restricted rations of 65% and 35% of the satiation level at each temperature. Fish fed the same % rations at 16 and 22 °C did not differ in final mean weight or specific growth rate, which decreased at restricted rations. Feed restriction did not result in an increase in size heterogeneity over time at any temperature, as indicated by the stability of the coefficients of variation of weight (ΔCVw=1.00–1.13%). At both temperatures, the best feed conversion efficiency (FCE) was found at the 65% ration, and the FCE from fish fed the same rations was higher at 16 °C (1.30 g g−1) than at 22 °C (1.17 g g−1). A similar trend was found in energy and protein retention levels. At both temperatures, fish fed 35% rations had lower body lipid and higher ash and moisture content compared to fish fed to satiation, with the most pronounced effects on lipid (4.8% vs. 7.8% bw) and ash (4.1% vs. 3.6% bw) at 22 °C. Feeding ration proved to be the main differentiating factor in all growth, feed conversion and body composition parameters, whereas additional temperature and/or interaction effects were found in FCE, whole body protein, lipid, moisture and energy contents. Between fish fed 100% and 65% rations, only minor differences were found, but at 35% ration, the rearing temperature of 22 °C had a pronounced negative influence, and resulted in a reduction of available anabolic energy for growth and adaptive responses.

12 Calcium Transport Processes in Fishes

Publisher Summary This chapter discusses the calcium transport processes in fishes. Vertebrates are dependent on calcium for the formation of the skeleton and for many cellular functions. This requires the regulation, within narrow limits, of the calcium concentration, in particular ionic calcium, of the intracellular and intercellular fluids. Seawater fishes maintain a high, inside-positive, transepithelial potential across the branchial epithelium that is higher than the equilibrium potential for Ca 2+ , and therefore a passive influx of Ca 2+ from the seawater is also unlikely. For the transcellular uptake of Ca 2+ in the gills, the apical membrane of the ionocyte forms the primary barrier for Ca 2+ between the water and the fish. Taking into consideration the physiological conditions—millimolar concentrations of Ca 2+ outside and submicromolar concentrations in the cytosol—the importance of the apical membrane becomes fully apparent in keeping Ca 2+ out to maintain the physiological intracellular Ca 2+ concentration. A series of specific criteria have also been advanced to positively identify the calcium pump in the basolateral plasma membrane of the gills of fishes.

Toxic levels of dietary copper in Atlantic salmon (Salmo salar L.) parr

Abstract Atlantic salmon ( Salmo salar ) parr were exposed for 4 weeks to dietary Cu concentrations of 5 (control), 35 and 700 mg Cu kg −1 dry feed to establish maximum tolerable levels of dietary Cu. Parr exposed to 35 and 700 mg Cu kg −1 dry feed had significantly increased ( P P >0.05). There were no significant changes in gill Cu, plasma Cu or liver MT levels at any of the dietary Cu concentrations, indicating that Cu homeostasis was maintained. A significant increase in liver Cu (1.6-fold relative to controls), but no significant change in liver MT, were observed only at the highest copper concentration. Cu-exposed Atlantic salmon parr showed an onset of adaptive responses (increase of intestinal PCNA, apoptosis, and MT levels) at concentrations of 35 and 700 mg Cu kg −1 dry feed, indicating that stress responses are induced at these dietary Cu concentrations.

Regulation of branchial Na + /K + -ATPase in common carp Cyprinus carpio L. acclimated to different temperatures

SUMMARY Isogenic carp Cyprinus carpio L. were acclimated to water temperatures of 15, 22 and 29°C for at least 8 weeks. The acclimations consistently resulted in slightly, but significantly, different plasma osmolality, sodium, potassium and chloride concentrations between the groups studied. Plasma total and ionic calcium levels were unaffected, indicating successful adaptation. The apparent changes in set point for plasma ion levels are explained by altered sodium pump activity and hormonal control of branchial permeability to water and ions. It appears that in 15°C-acclimated fish, a lower apparent Na+/K+-ATPase activity is compensated by strongly enhanced Na+/K+-ATPase expression (determined biochemically and immunohistochemically). In 29°C-acclimated fish, the higher ambient temperature activates the enzyme. Arrhenius plots for branchial Na+/K+-ATPase preparations of the three groups of fish suggest the occurrence of different enzyme isoforms or protein (in)stability as explanations for differences in apparent enzyme activities, rather than temperature-dependent changes in membrane fluidity. As for hormonal control over permeability, prolactin mRNA expression (and anticipated production and release) is lower in fish kept at 29°C, suggesting that control over branchial permeability to water and ions needs to be downregulated at higher temperatures. In so doing, enhanced sodium pump activity is balanced by a controlled passive ion loss to fine-tune plasma sodium levels. Basal plasma cortisol levels did not correlate positively with Na+/K+-ATPase expression, but doubling plasma cortisol levels in control fish by administering exogenous cortisol (for 7 days, using implanted minipumps and thus stress-free) enhanced Na+/K+-ATPase expression. This effect must be the result of a glucocorticoid action of the steroid: in fish, mineralocorticoid receptors have higher affinity for cortisol than glucocorticoid receptors. At a lower ambient temperature, branchial Na+/K+-ATPase expression is upregulated to counteract the temperature-inhibited activity of the sodium pump, perhaps via a mineralocorticoid receptor.

Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

SummaryThe inhibition of Ca2−-ATPase, (Na++K+)-ATPase and Na+/Ca2+ exchange by Cd2+ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven 45Ca2+ uptake into inside-out membrane vesicles displayed a Km for Ca2+ of 88±17 nm, and was extremely sensitive to Cd2+ with an IC50 of 8.2±3.0 pM Cd2+, indicating an inhibition via the Ca2+ site. (Na++K+)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd2+, displaying an IC50 of 2.6±0.6 μm Cd2+. Cd2+ ions apparently compete for the Mg2+ site of the (Na− +K+)-ATPase. The Na+/Ca2+ exchanger was inhibited by Cd2+ with an IC50 of 73±11 nm. Cd2+ is a competitive inhibitor of the exchanger via an interaction with the Ca2+ site (Ki = 11 nm). Bepridil, a Na+ site specific inhibitor of Na+/Ca2+ exchange, induced an additional inhibition, but did not change the Ki of Cd2+. Also, Cd2+ is exchanged against Ca2+, albeit to a lesser extent than Ca2+. The exchanger is only partly blocked by the binding of Cd2+. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na+/Ca2+ exchanger. We conclude that intracellular Cd2+ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.

Cortisol protects against copper induced necrosis and promotes apoptosis in fish gill chloride cells in vitro

Abstract In order to distinguish between toxic actions of copper (Cu) and the indirect actions of the metal mediated via the stress hormone cortisol, a 24 h in vitro gill filament culture was used to investigate the effects of this heavy metal and hormone, singly and in combination, on apoptosis and necrosis of chloride cells in the cichlid fish, tilapia (Oreochromis mossambicus). Cell death was identified after fluorescent double-labelling using a confocal laser scanning microscope. Incubation of filaments with 50 μM and 100 μM CuSO1 caused an approximate 5- and 16-fold increase, respectively, in chloride cell necrosis when compared to control, but had no significant effect on apoptosis. A 12 h incubation with 0.28 μM cortisol prior to exposure to 100 μM CuSO1 reduced necrosis by about 75%. The apparent protection provided by cortisol against copper toxicity could be blocked by the glucocorticoid receptor blocker RU 486. Incubation with 0.83 μM cortisol induced apoptosis to the same extent as that of camptothecin, a topoisomerase I inhibitor. We conclude that Cu directly causes necrosis of chloride cells, whilst cortisol protects against copper toxicity at lower concentrations, and induces apoptosis at higher concentrations, typical for severely stressed fish.

Tissue metallothionein, apoptosis and cell proliferation responses in Atlantic salmon (Salmo salar L.) parr fed elevated dietary cadmium.

Atlantic salmon parr were reared for 4 months on experimental diets supplemented with 0 (control), 0.5, 5, 25, 125, or 250 mg Cd x kg(-1) feed to establish a threshold concentration for dietary cadmium exposure by assessing early adaptive cellular responses. At the end of the experiment, the lowest dietary Cd concentration that caused significant accumulation in the gut, kidney and muscle was 5 mg Cd x kg(-1) compared to the control group. Over time, dietary Cd accumulated first in the gut (after 1 month), followed by the kidney (2 months), and later by muscle (4 months). Highest Cd accumulation (100-fold) was found in the gut. A significant increase in regulated cell death and proliferation in salmon fed 125 mg Cd x kg(-1) compared to control fish appeared efficient in preventing gross histopathological damage in the intestine. The highest increase in metallothionein levels was found in the kidney, and metallothionein (MT) levels increased disproportionally to Cd accumulation at increased exposure concentrations. It was concluded that MT was not directly associated with long-term Cd accumulation. Atlantic salmon showed increased metallothionein levels in the kidney at a median effective concentration (concentration of dietary Cd giving 50% of the maximum increase in metallothionein, EC50) of 7 mg Cd x kg(-1), indicating toxic exposure at this concentration.

Metallothionein response in gills of Oreochromis mossambicus exposed to copper in fresh water.

Freshwater Oreochromis mossambicus (tilapia) were exposed to 3.2 micromol/l Cu(NO(3))(2) in the water for up to 80 days, and copper (Cu) and immunoreactive metallothionein (irMT) were localized in the branchial epithelium. Cu was demonstrated in mucous cells (MC), chloride cells (CC), pavement cells (PC), respiratory cells (RC), and basal layer cells (BLC) via autometallography combined with alcian blue staining for MC and Na(+)-K(+)-ATPase immunostaining for CC and, on the basis of their location in the epithelium of PC, RC, and BLC. In control fish (water with Cu concentration </=90 nmol/l) incidentally irMT was observed in the area where progenitor cells of the branchial epithelia reside, as demonstrated by proliferating cell nuclear antigen staining. This was also the area where the first increase irMT expression of the Cu exposure was observed. After 2 days of exposure to Cu, irMT was found in CC and PC. From 5 days on, a pronounced irMT staining was observed in BLC of branchial epithelium, which then appeared to migrate and differentiate into mature CC, PC, and RC. We conclude that MT expression in mature CC, PC, and RC requires exposure to Cu in a earlier stage of development of these cells. Once expression is initiated in undifferentiated cells, MT remains expressed throughout the life cycle of the cell.

Distribution and quantification of corticotropin-releasing hormone (CRH) in the brain of the teleost fish Oreochromis mossambicus (tilapia)

The recent characterization of the corticotropin‐releasing hormone (CRH) prehormone of the fish tilapia (Oreochromis mossambicus) showed that more variation exists between vertebrate CRH amino acid sequences than recognized before. The present study investigates whether the deviating composition of tilapia CRH coincides with an atypical distribution of CRH in the brain. For this purpose we applied immunohistochemistry, as well as radioimmunoassay (RIA) quantification in brain slices. The results are plotted in a new atlas and reconstruction of the tilapia brain. The largest population of CRH‐immunoreactive (ir) neurons is present in the lateral part of the ventral telencephalon (Vl). Approximately tenfold less CRH‐ir neurons are observed in the preoptic and tuberal region. The CRH‐ir neurons observed in the preoptic region are parvocellular and do not, or hardly, display arginine‐vasotocin (AVT) immunoreactivity. CRH‐ir neurons are also present in the glomerular layer of the olfactory bulb, in the periventricular layer of the optic tectum, and caudal to the glomerular nucleus. A very dense plexus of CRH‐ir terminals is located in the most rostral part of the dorsal telencephalon. This region has not been described in other teleosts and is in the present study subdivided into the anterior part of the dorsal telencephalon (Da) and the anterior part of the laterodorsal telencephalon (Dla). High densities of CRH‐ir terminals were observed in and around Vl, in the tuberal region, around the rostral part of the lateral recess, and in the caudal part of the vagal lobe. In the pituitary, CRH‐ir terminals are concentrated in the neuro‐intermediate lobe. Overall, the immunohistochemical and quantitative data correlated well, as the RIA CRH profile in serial 160‐μm slices revealed four peaks, which corresponded with major ir‐cell groups and terminal fields. Our results strongly suggest that the CRH‐ir cells of Vl project to the rostro‐dorsal telencephalon. Consequently, they may not be primarily involved in regulation of pituitary cell types but may subserve other functions. The presence of a CRH‐containing Vl‐Da/Dla projection seems to be restricted to the most modern group of teleosts, i.e., the Acanthopterygians. Further anatomic indications for non‐pituitary‐related functions of CRH are found in the vagal lobe and the optic tectum of tilapia. Although the low CRH content of the preoptic region reported here for tilapia may be typical for unstressed fish, the fact remains that remarkably few CRH‐ir neurons are involved in regulating the pituitary. Overall, the CRH distribution in the brain of tilapia is more widespread than previously reported for other teleosts. J. Comp. Neurol. 453:247–268, 2002.