Steffen S. Madsen

The role of cortisol and growth hormone in seawater adaptation and development of hypoosmoregulatory mechanisms in sea trout parr (Salmo trutta trutta).

The role of growth hormone (GH) and cortisol in the development of hypoosmoregulatory mechanisms in sea trout parr, Salmo trutta trutta, was investigated by injecting freshwater (FW) yearlings every second day with saline, ovine growth hormone (oGH, 2.0 micrograms/g), cortisol (hydrocortisone hemisuccinate, 8.0 micrograms/g), or oGH + cortisol for a maximum of 14 days. Subgroups of the treated fish were transferred to three-fourths seawater (SW) after 7 or 15 days of treatment and the effects on plasma Na+, Cl-, muscle water content, gill Na+/K(+)-ATPase activity, and gill interlamellar chloride cell density were examined. In FW, gill Na+/K(+)-ATPase chloride cell density, and chloride cell apical to basal length increased by all hormone treatments, most significant by oGH + cortisol treatment. Plasma ions and muscle water content were unaffected in FW. Both SW transfers resulted in considerable mortality (50%) in control fish, whereas few cortisol-treated and no GH-treated or GH + cortisol-treated fish died. Plasma Na+ and Cl- levels increased dramatically (greater than 50%) in control fish and muscle water content decreased (8%) on Day 2 after both transfers. All hormone-treated groups regulated plasma ions and muscle water significantly better than controls in SW, indicating the physiological significance of the treatment. Notably, the oGH + cortisol-treated fish showed only insignificant changes in ion-osmotic homeostasis after SW transfer, suggesting a synergistic effect of the two hormones. It is concluded that treatment with the two hormones increases the salinity tolerance of sea trout parr at a developmental stage where FW life is obligatory.

Osmoregulation and Salinity Effects on the Expression and Activity of Na + ,K + -ATPase in the Gills of European Sea Bass, Dicentrarchus labrax (L.)

The European sea bass, Dicentrarchus labrax, tolerates salinities ranging from freshwater (FW) to hypersaline conditions. In two experiments, we analysed changes in plasma ions, muscle water content (MWC), gill Na+,K(+)-ATPase activity, and alpha-subunit mRNA expression during the course of acclimation from 15 ppt salt water to FW or high salinity seawater (HSSW). In Experiment 1, fish (6.2 +/- 1.1 g) were acclimated from 15 ppt to either FW, 5, 15, 25, 50, or 60 ppt SW and sampled after 10 days. Gill Na+,K(+)-ATPase activity was stimulated in FW- and in 50 and 60 ppt SW-groups relative to the 15 ppt control group. In Experiment 2, subgroups of fish (89 +/- 7 g) were transferred from 15 ppt SW to FW or 50 ppt SW, and sampled 1, 2, 4, and 10 days later. Plasma osmolality, [Na+] and [Cl-] decreased in the FW-group and increased in the HSSW-group one day after transfer and lasting until day 10. This was accompanied by a pronounced increase in MWC in the FW-group and an insignificant decrease in the HSSW-group. The plasma [Na+]:[Cl-]-ratio increased markedly in the FW-group and decreased slightly in the HSSW-group, suggesting acid-base balance disturbances after transfer. Gill Na+,K(+)-ATPase activity was unchanged in 15 ppt SW but doubled in FW- and HSSW-groups after transfer. In both groups, this was preceded by a 2- to 5-fold elevation of the gill alpha-subunit Na+,K(+)-ATPase mRNA level. Thus increased expression of alpha-subunit mRNA is part of the molecular mechanism of both FW and SW acclimation in sea bass. Gill Na+,K(+)-ATPase Na(+)-, K(+)-, and ouabain-affinity were similar in fish acclimated to FW, 15 ppt, and HSSW, suggesting that identical isoforms of the catalytic subunit of the enzyme are expressed irrespective of salinity.

Effects of 17β-estradiol and 4-nonylphenol on smoltification and vitellogenesis in Atlantic salmon (Salmo salar)

The impact of 17β-estradiol (E2) and the putative estrogenic compound, 4-nonylphenol (4-NP), on smoltification and vitellogenesis in Atlantic salmon (Salmo salar) was investigated during a 30 day period starting late April. Three groups of fresh water (FW) fish (1 year old, mixed sexes, average weight 23 g) were injected once a week with 50 µg (0.18 µmol) 17β-estradiol, 3 mg (13.6 µmol) 4-nonylphenol dissolved in peanut oil, or peanut oil alone as control. Every ten days, subgroups were challenged with 28 ppt seawater (SW) for 24h, and sampled together with subgroups of FW fish. Treatment effects were examined on vitellogenic and osmoregulatory parameters. E2 and 4-NP treatment increased the total calcium and protein level in plasma and the hepatosomatic index of FW fish, both indicating an activated vitellogenesis in the liver. The presence of vitellogenin in the plasma of 4-NP- and E2-treated groups was further indicated by the appearance of a high molecular weight vitellogenin band (550 kDa) in electropherograms produced by native gel electrophoresis. This band appeared in exactly the same position in both the E2- and the 4-NP-treated groups but could not be detected in controls. During the 30 day treatment period, control fish approached the peak of smoltification, as indicated by a distinct silvery appearance, decreasing condition factor, increasing levels of gill Na+,K+-ATPase and improved hypoosmoregulatory performance in the SW-challenge test. Both E2 and 4-NP treatments significantly inhibited the progress of smoltification, as judged by a significant reduction of gill Na+,K+-ATPase activity, relative α-subunit Na+,K+-ATPase mRNA expression, gill chloride cell density and a poorer hypoosmoregulatory performance of treated fish. The impaired SW-tolerance of E2- and 4-NP-treated fish was strongly correlated with a decreased gill Na+,K+-ATPase activity. Despite a difference in relative potency, the present study shows that 4-nonylphenol and 17β-estradiol may have qualitatively similar inhibitory effects on smoltification and hypoosmoregulatory physiology of Atlantic salmon. Both 4-NP and E2 activated the vitellogenic system, and the study supports the hypothesis that sexual maturation and smoltification are antagonistic, developmental phenomena in salmon. It is suggested that the presence of estrogenic compounds in the environment may negatively influence smoltification and migration in wild stocks of salmon.

Effect of repetitive cortisol and thyroxine injections on chloride cell number and Na+/K+-ATPase activity in gills of freshwater acclimated rainbow trout, Salmo gairdneri

Abstract 1. 1. Freshwater nonanadromous rainbow trout, Salmo gairdneri, were injected three times a week with either saline, 10μg cortisol/g, 1.0μg thyroxine/g or 10μg cortisol/g + 1.0μg thyroxine/g during a period of 28 days (12 injections). A separate group was derived as a subgroup from the thyroxine group on day 14 and received Cortisol + thyroxine from day 14 until day 28 (six injections). 2. 2. Gill chloride cell number and Na+/K+-ATPase activity increased by cortisol treatment, the changes being significant on days 7 and 14, respectively. 3. 3. Thyroxine treatment did not affect gill Na+/K+-ATPase activity or chloride cell number directly. Neither did it modify the stimulatory effect of cortisol on these parameters. 4. 4. Muscle water decreased in cortisol-treated fish and increased in thyroxine-treated fish, while no changes were observed in the combined hormone groups. 5. 5. No changes were observed in plasma chloride in any group during the experiment. 6. 6. The results demonstrate a putative role of cortisol in stimulating hypo-osmoregulatory mechanisms and suggest that thyroxine is without a direct or a supportive effect for cortisol action.

Cortisol treatment improves the development of hypoosmoregulatory mechanisms in the euryhaline rainbow trout, Salmo gairdneri.

The effect of cortisol on osmoregulatory parameters was studied in rainbow trout, (Salmo gairdneri), kept in freshwater (FW) and/or transferred to seawater (SW). Repeated injections of 20 µg cortisol/g fish stimulated gill and gut Na+/K+-ATPase activity and reduced plasma Na+ and Cl− levels after 2 weeks of treatment in FW-adapted fish. Cortisol doses of 0.05 and 1.0 µg/g were without effect. Repeated injections of 10 µg cortisol/g stimulated gill Na+/K+-ATPase activity and reduced plasma Na+ and Cl− levels in fish in FW, and significantly improved ion regulation after their transfer to 28SW. Higher doses of cortisol (10 and 20 µg/g) induced hyperglycemia, whereas low doses (0.05 and 1.0 µg/g were without effect or induced hypoglycemia. Plasma glucose levels decreased in cortisol-treated fish transferred to SW, whereas transient hyperglycemia was seen in the control fish.

Enhanced hypoosmoregulatory response to growth hormone after cortisol treatment in immature rainbow trout, Salmo gairdneri

The growth-independent effect of ovine growth hormone (oGH) and oGH + cortisol treatment on seawater (SW) adaptation in immature rainbow trout, Salmo gairdneri was investigated. Fish were injected every second day with saline, 2.0 μg oGH/g or 2.0 μg oGH + 8.0 μg cortisol/g for a maximum of 8 injections in freshwater (FW). Subgroups were transferred to 28‰ SW after 4 or 8 injections, and changes in plasma Na+ and Cl−, muscle water content and gill Na+/K+-ATPase activity were measured. In both of the hormone-treated groups retained in FW, gill Na+/K+-ATPase activity and interlamellar chloride cell density increased. The effects were most pronounced in the oGH + cortisol group after 2 weeks of treatment. After transfer to SW most of the control fish died due to the osmotic stress, whereas in the hormone-treated groups, mortality was low and there was a positive correlation between pretransfer gill Na+/K+-ATPase and the ability to maintain ionic-osmotic homeostasis after SW transfer. After two weeks of oGH + cortisol treatment, gill Na+/K+-ATPase activity was maximal. In contrast, after SW transfer, Na+/K+-ATPase activity increased further in the oGH-treated group. This group regulated ionic-osmotic parameters less effectively than the oGH + cortisol-treated group. The data indicate that GH and cortisol are important hormones in the regulation of hypoosmoregulatory mechanisms in S. gairdneri.

Prolactin Antagonizes the Seawater-Adaptive Effect of Cortisol and Growth Hormone in Anadromous Brown Trout (Salmo trutta)

Abstract Two experiments are described in which the interaction of prolactin, cortisol and growth hormone on hypoosmoregulation in the anadromous brown trout was studied. In experiment 1, fish at the postsmolt stage in freshwater (FW) were given four injections on alternate days of 5 μg cortisol (F)/g in combination with 0, 0.2, 1 or 2 μg ovine prolactin (oPRL)/g. Additional groups received 2 μg oPRL/g or saline as control. In experiment 2, FW parr were given four injections on alternate days with 5 μg F/g and 2 μg ovine growth hormone (oGH)/g in combination with 0, 0.2, 1 or 2 μg oPRL/g. Additional groups received 2 μg oPRL/g, 2 μg oGH/g, 5 μg F/g or saline as control. In both experiments, the fish were subjected to a 48 hr seawater (SW) challenge test 24 hr after the last injection. Muscle water content, plasma osmolality and ion levels, kidney and gill Na+, K+-ATPase activity were measured. In experiment 1, F-treated fish had better hypoosmoregulatory capacity than control fish as judged by a higher level of muscle water content and lower plasma osmolality after 48 hr in SW. All three doses of oPRL completely abolished this action of F. Gill Na+,K+-ATPase activity was stimulated by F and unaffected by oPRL at any dose. In experiment 2, oPRL impaired, whereas F and oGH (injected individually or together) improved performance in the 48 hr SW test relative to control fish, judged by plasma osmolality and muscle water content. Ovine PRL inhibited the combined action of F and oGH in a dose-related manner but could not completely counteract the combined effect of these hormones. F and oGH had additive stimulatory effects on gill Na+,K+-ATPase activity. This activation was counteracted by coinjection of oPRL in a dose-related manner but not below the level found in the F-treated fish. We conclude that exogenous oPRL inhibits the hypoosmoregulatory action of F. However, oPRL does not antagonize the F-dependent stimulation of gill Na+,K+-ATPase activity. The observed antagonism between PRL and F in SW-acclimation may therefore occur on a target different from gill Na+,K+-ATPase.

Opposite effects of 17β-estradiol and combined growth hormone-cortisol treatment on hypo-osmoregulatory performance in Sea Trout presmolts, Salmo trutta

In March, sexually immature sea trout presmolts (Salmo trutta trutta) were injected every second day with saline, 2 micrograms 17 beta-estradiol (E2)/g, 2 micrograms ovine growth hormone (GH) + 6 micrograms cortisol (F)/g, or all three hormones (E2-GH-F) simultaneously. A SW-challenge test was performed after six injections. At the time of SW-transfer, high total plasma calcium levels in E2- and E2-GH-F-treated fish indicated activated vitellogenesis in these groups. All control, GH-F, and E2-GH-F-treated fish survived SW-transfer, whereas 43% of the E2-treated fish died after transfer. On Day 2 after transfer, there were marked differences among groups in their osmoregulatory response. Changes in ion-osmotic parameters (plasma Na+, Cl-, Mg2+, and total calcium and muscle water) indicated the following degree of osmotic stress: E2 greater than control greater than E2-GH-F greater than GH-F, which was inversely correlated with pretransfer gill Na+/K(+)-ATPase activity: GH-F greater than E2-GH-F greater than control greater than E2. On Day 7 after transfer there were no major differences among the groups with regard to plasma ions and muscle water content. The detrimental influence of elevated plasma E2 levels on hypo-osmo-regulatory physiology may indicate an important role of E2 during development.

Extrathyroidal effects of thiourea treatment in rainbow trout (Salmo gairdneri) rapidly transferred from fresh water to dilute sea-water

Abstract 1. 1. Exposure of yearling rainbow trout, Salmo gairdneri , to either 0.3 or 0.5‰ thiourea (TU) in fresh water (FW) or subsequently in dilute sea-water (SW); impaired osmoregulatory performance in both media. 2. 2. TU treatment resulted in depression of gill Na + /K + -ATPase activity in SW (in 0.5‰ TU) or delayed adaptive increase in ATPase activity (in 0.3‰ TU) following SW entry as compared to control fish. 3. 3. Plasma Na + and Cl − levels decreased in FW fish and increased in SW-acclimated fish exposed to TU. 4. 4. T 4 replacement did not abolish these effects. 5. 5. Neither did TU treatment depress circulating levels of total T 4 , and the observed effects were probably associated with TU toxicity in extrathyroidal sites. 6. 6. The effectiveness of TU to block thyroid function in fish is discussed.