Effect of cortisol on some osmoregulatory parameters of the teleost, Oreochromis niloticus L., after transference from freshwater to seawater

Efeito do cortisol sobre parâmetros de osmorregulação do teleósteo, Oreochromis niloticus L., após a transferência de água doce para água salgada

VETERINARY MEDICINE

Effect of cortisol on some osmoregulatory parameters of the teleost, Oreochromis niloticus L., after transference from freshwater to seawater

Efeito do cortisol sobre parâmetros de osmorregulação do teleósteo, Oreochromis niloticus L., após a transferência de água doce para água salgada

A. Fontaínhas-Fernandes^I; E.F. Gomes^II; Mª.A. Reis-Henriques^II; J. Coimbra^II

^IUniversidade de Trás-os-Montes e Alto Douro-ICETA Apt. 1013, 5000-911 Vila Real, Portugal

^IIICBAS e CIIMAR. Lg Prof. Abel Salazar nº 2, 4050 Porto, Portugal

ABSTRACT

This trial was conducted in order to determine the effects of cortisol on salt water acclimation of tilapia Oreochromis niloticus (L.). Tilapia (n=42) were injected intraperitoneally with cortisol and then were directly transferred from freshwater (FW) to 15‰ salt water (SW). Changes in plasma osmolality, chloride ion concentration (Cl-), plasma level of cortisol and gill Na⁺, K⁺-ATPase activity were measured at 6, 12, 24, 48, 72 and 168 hours after transference to 15‰ SW. Plasma osmolality and Cl- increased immediately after transference until 12-24 h. The fish injected with cortisol (F) showed higher plasma levels of cortisol than those from control group (C) that maintained the initial levels during the experiment. Gill Na⁺, K⁺-ATPase activity of C fish began to increase at first hours after transference and peak at 48h. The differences between gill Na⁺, K⁺-ATPase activity of F and C groups were significant (P<0.05) in FW, which confirm the effect of exogenous cortisol.

Keywords: tilapia Oreochromis niloticus, freshwater, salt water, cortisol

RESUMO

Este estudo foi realizado com o objectivo de testar os efeitos do cortisol na aclimatação da tilápia Oreochromis niloticus (L.) à água salgada. As tilápias (n=42) foram injectadas intraperitonealmente com cortisol e directamente transferidas de água doce para água salobra (15‰). As alterações da osmolaridade, concentração em cloretos (Cl-), os níveis plasmáticos de cortisol e a actividade branquial da Na⁺, K⁺-ATPase foram medidas (6, 12, 24, 48, 72 e 168 horas) após a transferência para água salobra. A osmolaridade e a concentração em Cl- aumentou imediatamente após a transferência até às 12-24h. O grupo injectado com cortisol (F) mostrou níveis plasmáticos de cortisol mais elevados do que o grupo controlo (C) que manteve os níveis iniciais durante a experiência. A actividade branquial da Na⁺, K⁺-ATPase dos peixes do grupo C começou às primeiras horas após a transferência e teve um pico às 48h. As diferenças entre a actividade enzimática da Na⁺, K⁺-ATPase dos grupos F e C foram significativas (P<0,05) em água doce, o que confirma o efeito exógeno do cortisol.

Palavras-chave: tilápia Oreochromis niloticus, água doce, água salgada, cortisol, osmorregulação

INTRODUCTION

Tilapia species are a good biological model for studying the mechanism of osmoregulation in teleost fish because they can survive to direct transference from freshwater (FW) to salt water (SW). In tilapia, the transition from FW to SW is associated with a temporary elevation in plasma osmolality and chloride (Cl-)ion concentration (Assem & Hanke 1979; Hwang et al., 1989; Fontainhas-Fernandes et al., 2001), followed by a transient increase in plasma cortisol and growth hormone (GH) levels (Assem & Hanke 1979; Yada et al., 1994), and an increase in gill Na⁺, K⁺-ATPase activity (Morgan et al., 1997). However, plasma prolactin levels decrease (tPRL₁₇₇ and tPRL₁₈₈) (Morgan et al., 1997). Several studies have examined the effects of temperature, metabolic state (Jürss et al., 1984; Johnsson & Clark 1988; Vijayan et al., 1996), size, sexual maturation, (McCormick & Naaiman 1984a,b), and body weight (Iwata et al., 1982) on SW acclimation. Some authors have studied the effect of sexual hormones on the osmoregulatory capacity in salmonids and have shown that estradiol decrease the gill Na⁺, K⁺-ATPase activity and induces a lower number of MR cells (Madsen & Korsgaard 1989; Coimbra et al., 1993).

Previous studies have shown that tilapia cannot tolerate an abrupt transference from FW to 32‰ SW, and need a gradual acclimation period, that has a physiological effect on some euryhaline teleosts (Stickney, 1986). Tilapia O. mossambicus transferred to SW with pre-acclimation to lower salinity (20‰) for 24h, exhibited a more rapid increase in gill Na⁺, K⁺-ATPase activity with less dehydration than those that were transferred directly to 30‰ SW (Hwang et al., 1989).

It has been shown that cortisol increases hypoosmoregulatory capacity in fish (Mommsen et al., 1999). One of the mechanisms by which cortisol adapts fish to SW is by cellular differentiation of the MR cells and by stimulating the gill Na⁺, K⁺-ATPase activity (Madsen, 1990a; McCormick, 1995). Madsen et al. (1995) have demonstrated that this cortisol-induced increase in gill Na⁺, K⁺-ATPase activity is partially due to the expression of the Na⁺, K⁺-ATPase a -subunit mRNA. Studies in salmonids report that gill Na⁺, K⁺-ATPase activity responds positively to injections of cortisol in Atlantic salmon, Salmo salar (Bisbal & Specker 1991), rainbow trout, Oncorhynchus mykiss (Madsen, 1990a), and sea trout Salmo trutta (Madsen, 1990b). However, in coho salmon, Oncorhynchus kisutch and in Atlantic salmon, exogenous cortisol has no effect on gill Na⁺, K⁺-ATPase activity in vivo (Langdon et al., 1984; Redding et al., 1984). Given the differences in experimental approaches, it is not surprising that inconsistencies exist in the literature (Mommsen et al., 1999).

The main objective of the present study was to characterize the effects of cortisol on the physiological and biochemical mechanisms involved in SW acclimation in the tilapia Oreochromis niloticus. Plasma cortisol levels, gill Na⁺, K⁺-ATPase activity, and plasma osmolality and Cl- concentration were the main investigated parameters.

MATERIALS AND METHODS

Adult male tilapia Oreochromis niloticus were obtained from a laboratory stock at the University of Trás-os-Montes and Alto Douro, originated from a population that was introduced from the Fish Laboratory of INRA (Rennes, France). The fish were checked to ensure that no brooding females were used in the experiment.

Fish were sorted out into two groups: saline (C) and cortisol-injected (F). Fish were injected with 0.2 µg cortisol g^-1 body weight administered intraperitoneally on days 1, 3 and 5, before transition from FW to 15‰ SW. Cortisol was administered as hydrocortisone hemisuccinate (Sigma, H-4881. Sigma Chemical Co - USA), dissolved and diluted in saline (NaCl 0,15M) immediately before its use. The injection volume was 100µl. The last injection was administered 24h before the transition from FW to SW. In order to minimize handling stress, the fish were briefly anaesthetized in ethylene glycol monophenylether (0.4ml l^-1) prior to each injection. Fish were fasted during the experiment.

After three injections (FW 5 day), subgroups of 42 tilapia (30-40 g body weight) were directly transferred to 15‰ SW in similar tanks with a water flow rate of 0.5 l min^-1. Water temperature was kept similar in each treatment tank (25±1°C) and supplemental aeration was provided to maintain dissolved oxygen near saturation. Other water quality parameters such as ammonia, nitrite, nitrate and suspended solids were maintained at acceptable levels by mechanical and biological filtration. A diurnal light:dark cycle of 12L:12D was provided by fluorescent lighting controlled by timer.

Prior to transfering to 15‰ SW six fish were sampled. Sampling of six fish of each group was performed in 15‰ SW at 6, 12, 24, 48, 72 and 168h after transference. The fish were anesthetized by immersion in an ethylene glycol monophenylether bath, stunned by a blow to the head, and the blood was drawn from the caudal vessels into a heparinized syringe and centrifuged at 3000 ´ g for 10 min. Plasma was immediately stored at -20°C for later analysis. After blood collection, fish were decapitated and gill filaments were dissected free from the second gill arch on the left side of the fish, washed in ice-cold sucrose buffer (0.25 M, pH 7.4) to remove blood, quickly frozen in liquid nitrogen and then, stored at -70°C until the measurement of Na⁺, K⁺-ATPase.

Plasma chloride concentration was measured by electrometric titration using a chloride meter (Jenway,model PCLM3 - USA). Plasma osmolality was measured with a micro-osmometer(model 3MO plus. Advanced Instruments, Lda, USA) and expressed in mOsm kg^-1. Each sample was examined at least three times. Plasma cortisol levels were determined using a commercial radioimmunoassay kit cortisol (¹²⁵ I) (Coat-a-Count, Diagnostic Products Corporation, USA), according to Iwama et al. (1989). The intra-assay coefficient was 5.8% and inter-assay variation was avoided by measuring all samples in the same assay. Cross-reactivity with cortisone, the other major corticosteroid in teleostean fishes, was 7.0%. Gill Na⁺, K⁺-ATPase activity expressed as µmoles phosphate (Pi) mg prot.^-1.h^-1 was determined according to the method of Epstein et al. (1967), modified by Lasserre et al. (1978). The amount of inorganic phosphate (Pi), liberated from ATP, was determined according to the method of Fiske & Subbarow (1925) and the concentration of the total protein was determined by the method of Lowry et al. (1951), using crystalline bovine albumin as standard.

Data are presented as means±standard error (SE). Two-way analysis of variance (ANOVA) was used to test the treatment and time effects. Significant differences between the treatment means were identified using Student-Newman-Keuls multiple comparison test (P<0.05).

RESULTS AND DISCUSSION

Following direct transference of O. niloticus from FW to 15‰ SW, plasma osmolality and Cl- concentration increased (Fig. 1). At 12 h after transition to 15‰ SW the plasma osmolality and Cl- levels decreased in both groups, and reached the basal level (FW) at 168 h. At all sampling periods, the differences of plasma osmolality and Cl- values between F and C groups were not statistically significant (P>0.05). Morgan et al. (1997) have shown that following transference of O. mossambicus from FW to SW, plasma Na⁺ and Cl- peaked after the first day and declined to FW levels on day 4. Assem & Hanke (1981) and Hwang et al. (1989) observed similar patterns for these ions after transference of O. mossambicus from FW to 27 and 20‰ SW, respectively.

At all sampling periods, the fish injected with cortisol showed significantly higher (P<0.05) plasma levels of cortisol after the transference to SW when compared to the C (Fig. 2), particularly during the first 24h, probably because they were in stress. However, the cortisol values of the control group did not increase when transferred to SW, although the gill Na⁺, K⁺-ATPase activity have raised. F group showed significantly higher (P<0.05) levels of gill Na⁺, K⁺-ATPase activity than the control in FW and at 6h after transference to SW (Fig. 2).

It is evident from these results that there is an effect of exogenous cortisol, because gill Na⁺, K⁺-ATPase activity of the F group increased 3.2 fold compared to the control in FW (0 h). These results are in accordance with other studies that showed that cortisol promotes an increase of gill Na⁺, K⁺-ATPase activity in vivo (Dangé 1986; Olsen et al., 1993), and in vitro (McCormick & Bern 1989; McCormick et al., 1991). Gill Na⁺, K⁺-ATPase activity of the control group (C) increased after transference to 15‰ SW. The time of the decrease in plasma Cl- and osmolality and the increase in Na⁺, K⁺-ATPase observed in this study are consistent with the known role of this enzyme in salt secretion to maintain an ionic balance in SW fish (Zadunaisky, 1984). Avella et al. (1993) also analyzed this parameter to test the SW tolerance of O. niloticus and O. aureus. They observed that transfer to 20‰ SW significantly enhanced the gill Na⁺, K⁺-ATPase activity 1.6 times in O. niloticus and 3 times in O. aureus. The present results are in accordance with those obtained by Luke et al. (1994) in the European eel (Anguilla anguilla). These authors reported that changes in the activity gill Na⁺, K⁺-ATPase increased immediately after transference, by a factor of three, within 6h, to activities found in FW groups. The initial peak of Na⁺, K⁺-ATPase was followed, some three days after transference, by a gradual and more prolonged increase in Na⁺, K⁺-ATPase activity, leading to levels at 21 days which matched the 6h peak.

In conclusion, the present investigation confirms the putative role for cortisol as a hypoosmoregulatory hormone. It is suggested that cortisol treatment prepares FW adapted tilapia O. niloticus to acclimate to a hyperosmotic environment.

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Publication Dates

• Publication in this collection
  13 Jan 2004
• Date of issue
  Oct 2003