P.H.M. Balm

Integrated physiological response of tilapia, Oreochromis mossambicus, to sublethal copper exposure

Abstract Juvenile and mature tilapia (Oreochromis mossambicus) were exposed to a range of sublethal copper (Cu) concentrations for 6 days to examine the mechanisms underlying the acclimation to the toxic effects of the metal. The study focuses on the gills, the primary target for waterborne pollutants. To obtain a comprehensive picture of the branchial acclimation processes operating, multiple biochemical and morphological parameters were studied. At all concentrations tested, Cu exposure resulted in the accumulation of the metal in mature fish. At 100 and 200 μg Cu l−1 only, chloride cell proliferation was observed, which was accompanied by an increase in average cell diameter in these groups. Whole body flux measurements in juvenile fish demonstrated a decrease in Na influx in fish exposed to 200 μg l−1 Cu, in the absence of an effect on Ca influx. Gill Na + K + - ATPase activity was also decreased in the crude branchial homogenates of the mature fish exposed to the highest Cu concentration only, but not in the purified branchial vesicle preparations of these fish, which may indicate reactivation of in vivo Cu-inhibited ATPase activity during the isolation process. Plasma pH, Na, Cl, K, glucose and ceruloplasmin concentrations were also affected in the 200 μg Cu l−1 group exclusively. In accordance with the gill accumulation data, plasma Cu levels were clearly elevated in all groups exposed to the metal. The results underscore the integrated response of the gills to Cu, which, however, does not come into play until challenged by relatively high ambient concentrations. These results indicate that, in comparison to the Cu-sensitive rainbow trout, tilapia is more Cu-tolerant. The most sensitive parameters affected by Cu are gill and plasma metal levels, followed by chloride cell number and diameter.

The ultrastructure of chloride cells in the gills of the teleost Oreochromis mossambicus during exposure to acidified water

SummaryBranchial chloride cells, which actively take up ions in the gills of freshwater fish, were studied in tilapia (Oreochromis mossambicus) exposed to sublethally acidified freshwater. Structural damage of cells, resulting in cell death by necrosis, only occurred transiently, when the reduction of water pH was acute rather than gradual. The most prominent effects of water acidification were the rapid increase in the number of chloride cells and the changes in frequency of the different stages of the chloride cell cycle. In the opercular inner epithelium, a twofold increase in cells occurred 48 h after gradual acidification. Cell density stabilized after 4 weeks at a level 5 times that of control fish. Four transitory stages were distinguished in the chloride cell cycle: accessory or replacement cells, immature, mature, and degenerating (apoptotic) cells. In control fish, mature chloride cells dominated (over 50%) with immature and apoptotic cells totalling about 40%. After 4 weeks in acid water, only 13% of the cells were mature. Immature and apoptotic cells dominated, each representing about 40% of the total number of chloride cells. Mature cells apparently age rapidly under these conditions. Thus, chloride cells turn over quickly in acid water, with a minor increase in ion transport capacity of the gills. This conclusion is supported by the observation that opercular and branchial Na+/K+ ATPase activities in treated fish are only 40%–50% higher than in controls.

Background colour influence on the stress response in cultured red porgy Pagrus pagrus

Red porgy Pagrus pagrus were placed and maintained in white, grey and black background fibreglass tanks for 2 weeks. Additionally, fish kept in white and black background tanks were then subjected to crowding stress. After 2, 9, 16 and 23 days, blood samples were taken and plasma cortisol, alpha melanocyte stimulating hormone (α-MSH) and glucose values were analysed and compared with values from uncrowded fish from white or black tanks. Measurements of plasma cortisol and α-MSH in unstressed red porgy from white, grey and black tanks revealed no significant differences among the three groups. However, the results show that background colour markedly affects the in vitro interrenal sensitivity to both α-MSH and ACTH, as interrenal cells from black adapted fish had become virtually unresponsive to both secretagogues. Crowded fish on a black background showed a prominent increase in plasma cortisol after 2 days, which was followed by a recovery. In fish crowded on a white background however, the increase of cortisol was lower but was maintained through the entire experiment. Plasma α-MSH levels increased at 23 days as a consequence of crowding; this increase was also dependent on the background, being more prominent in fish placed in white background tanks. Thus, in the red porgy, a white background appears to modify the stress response, particularly in the long term.

Substrate mobilization and hormonal changes in rainbow trout (Oncorhynchus mykiss, L) and common carp (Cyprinus carpio, L) during deep hypoxia and subsequent recovery

Common carp (at 20°C) and rainbow trout (at 15°C) were fitted with an indwelling cannula in the dorsal aorta. The fish were exposed to a controlled decline of waterpO2 followed by 90 min deep hypoxia at 0.3 kPa (carp) or 4.8 kPa (trout). Thereafter, normoxic recovery was monitored in both species for 48 h. At regular intervals blood samples were analysed for glucose, lactate, free fatty acids, adrenaline, noradrenaline and cortisol. The oxygen restriction was maximal in both species and resulted in a significant increase of plasma lactate levels. In carp, adrenaline, noradrenaline and cortisol levels increased to 2, 50, and 753 ng·ml-1 respectively during anoxia, whereas in trout these hormones increased to 12, 8 and 735 ng·ml-1 respectively during hypoxia. In hypoxic trout, the plasma levels of glucose (3 mol·l-1) were increased modestly whereas levels of free fatty acids (0.25 mmol·l-1) were decreased to 0.15 mmol·l-1. In carp, however, a marked and prolonged hyperglycaemia (from 5 to 10 mmol·l-1) and a significant continuous depression of plasma levels of free fatty acids (from 0.4 to 0.2 mmol·l-1) were observed indicating a difference in metabolic organization. It is suggested that hyperglycaemia is likely to be the result of hepatic glycogenolysis, stimulated by circulating catecholamines and a stimulation of gluconeogenesis by cortisol during recovery. The mechanism for the decline of plasma levels of free fatty acids is most probably a reduction of lipolytic activity, which appears to be an adaptation to hypoxia.

Stimulatory and inhibitory effects of 5-HT1A receptors on adrenocorticotropic hormone and cortisol secretion in a teleost fish, the Arctic charr (Salvelinus alpinus)

Abstract The serotonin1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) was administrated to juvenile Arctic charr either by intraperitoneal (i.p.) injections or through a permanent i.p. implanted catheter. 8-OH-DPAT induced an elevation of plasma cortisol in non-stressed fish (receiving the drug through a permanent i.p. catheter), whereas 8-OH-DPAT had the opposite effect when administrated through a standard i.p. injection, dampening the rise in cortisol induced by handling and injection, an effect which was most pronounced at low doses of 8-OH-DPAT. In both cases, plasma levels of adrenocorticotropic hormone followed the same general pattern as plasma cortisol. The results suggests that 5-HT1A receptors are involved in the regulation of hypothalamic–pituitary–interrenal axis activity in Arctic charr, but the effect of 5-HT1A stimulation seems to vary depending on the state of the fish.

Interactions between copper and cadmium modify metal organ distribution in mature tilapia, Oreochromis mossambicus.

Sexually mature female tilapia were exposed to sublethal concentrations of waterborne Cu and/or Cd over 6 days, and subsequent body concentrations of these metals were determined in several organs. The results show that the distribution of Cu and Cd was metal and organ specific. This is demonstrated, for example, by the observation that in tilapia, Cu exposure did not result in Cu accumulation in the liver, whereas in the intestinal wall, notably high concentrations of Cu and Cd were measured in metal exposed fish. In addition to single metal exposed fish, we also determined Cu and Cd body distribution in Cu?Cd co-exposed fish. The observed interactions in metal accumulation were most pronounced in the organs of fish exposed to low, environmentally realistic, metal concentrations.

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.

Morphological and metabolic changes in common carp, Cyprinus carpio, during short‐term copper exposure: Interactions between Cu2+ and plasma cortisol elevation

The effects of increased endogenous cortisol levels were compared with those of sublethal copper exposure in the freshwater common carp, Cyprinus carpio. Fish were exposed to either increased levels of endogenous cortisol (200 ng/ml) or sublethal copper (1.9 microM) alone or were pretreated by elevating plasma cortisol levels prior to copper exposure to assess whether interactions between both treatments occurred. Effects induced by increased cortisol levels included increased Na+/K(+)-adenosine triphosphate (ATPase) activity and increased plasma Na+ and plasma osmolarity, while copper exposure induced anaerobic metabolism, gill damage, decreasing Na+/K(+)-ATPase activity, decreasing plasma ion levels, and blood thickening. Pretreatment of copper-exposed fish with cortisol partially protected these fish by reducing the copper-induced decrease in Na+/K(+)-ATPase activity. Overall, the results obtained in this study argue against a major role for cortisol as an intermediate for the toxic effects of copper.

Endorphin and MSH in concert form the corticotropic principle released by tilapia (Oreochromis mossambicus; Teleostei) melanotropes

HPLC characterization of tilapia pituitary endorphins using an antibody specific for N-terminally acetylated endorphins yielded three major peaks in the neurointermediate lobe, but none in the pars distalis. The melanotropes secreted two of the immunoreactive products in vitro, one of which coeluted with Xenopus laevis N-ac-beta-END(1-8). This immunoreactive fraction also coeluted with diacetyl-alpha-MSH. Evidence is presented that the noteworthy corticotropic potency of this HPLC fraction, previously attributed to diacetyl-alpha-MSH, results from END and MSH acting in a coordinated fashion. Confinement stress had no effect on plasma N-ac-beta-END immunoreactivity, but led to a decrease in plasma alpha-MSH levels. Therefore, it seems unlikely that the corticotropic action of the peptides regulates the elevation of cortisol production that takes place during confinement, but it may play a role during other forms of stress that are known to activate the melanotropes.

Bacterial lipopolysaccharide (LPS) and interleukin 1 (IL-1) exert multiple physiological effects in the tilapia Oreochromis mossambicus (Teleostei)

To gain insight in immuno-endocrine communication in teleosts the physiological effects of interleukin 1 and bacterial lipopolysaccharide in teleosts were investigated. Tilapia (Oreochromis mossambicus) were treated with murine interleukin 1 and E. coli lipopolysaccharide in vivo, and lipopolysaccharide was administered to pituitary lobes and head kidneys in vitro. The integument of the fish appeared to be a sensitive target for the preparations tested, since proliferation of chloride cells and of epidermal mucous cells was observed as well as an increase in epidermal thickness. These effects may relate to an acute phase-like reaction caused by the treatments. Lipopolysaccharide administration furthermore resulted in an increase in plasma free fatty acids levels. Lipopolysaccharide, but not interleukin 1, stimulated the interrenal axis of the fish, as judged by the increase in cortisol production measured in superfusion of head kidneys. In addition to these in vivo effects, lipopolysaccharide also displayed several effects in vitro. Pituitary adrenocorticotropic hormone, as well as α-melanocyte stimulating hormone, release was inhibited, and the head kidney responsiveness to adrenocorticotropic hormone was inhibited after pretreatment of the tissue with the E. coli product. This latter effect coincided with the release of an unidentified α-melanocyte stimulating hormone immunoreactive fraction by the head kidneys which could be stimulated by lipopolysaccharide. The data strongly support the notion that the immune system is involved in adaptive regulations in teleosts, and that immuno-endocrine interactions are phylogenetically old mechanisms.