Assessment of the use of Oblada melanura (L. 1758) otolith fluctuating asymmetry as environmental disturbance indicator.

Human impact on the environment is of widespread concern. The majority of anthropogenic impacts are centred on coastal ecosystems, so surveying them is an important step in the protection of the marine environment. We have tested Oblada melanura (L. 1758) otoliths' fluctuating asymmetry as a bioindicator in a Mediterranean coastal zone. The French Riviera is characterised by a summer population increase leading in particular to more yachting, and seasonal climatic changes with reduced, more concentrated waterway flows and storm events causing soil erosion. The present three-year study compares nine sites, situated in three zones, and characterised by three types of chemical pollutant states (low; waterway mouth; recreational harbour). For O. melanura juveniles, we have not shown any significant difference in the otoliths' fluctuating symmetry between zones or types of sites. We hypothesize that high stress levels are needed to induce significant fluctuating asymmetry variation.

The stress axis, stanniocalcin, and ion balance in rainbow trout.

In teleosts, the stress hormone cortisol and the calcium regulatory hormone stanniocalcin (STC) are both involved in the regulation of ion balance. Under stressful conditions, ion balance is easily disturbed as stressors via the stress signals they evoke disturb easily and primarily gill function. The gills are key in fish gas exchange and ion regulation. The present work evaluates the effect of the pivotal stress signal cortisol, the eventual output of the stress axis on STC secretion in freshwater rainbow trout (Oncorhynchus mykiss). Plasma cortisol levels were manipulated by intraperitoneal injections of porcine ACTH(1-39) or dexamethasone (Dex), and plasma cortisol, STC and mineral status were assessed. A perifusion assay of trout Stannius corpuscles was validated and used to study the direct effects of stress-related signals on STC release. In perifusion, cortisol, adrenocorticotropic hormone (ACTH), and dexamethasone did not affect STC release. ACTH injections increase plasma cortisol (corresponding to an acute stress) and STC concentrations, but did not affect mineral status. Dexamethasone injections resulted either in a classical hypocortisolinemia or, unexpectedly, in hypercortisolinemia. However, independently of the resulting cortisol status Dex induced a chronic stress effect, as indicated by decreased plasma Na, Cl, and Ca levels, and increased plasma STC concentrations. The increased STC secretion cannot be explained by the classical elevation of plasma calcium concentration. Thus, plasma parameters other than calcium could be involved and we propose that STC secretion might be stimulated also by a decrease of NaCl concentrations, implying a broader function than the classical hypocalcemic action of STC.

Neurohypophysial hormone receptors and second messengers in trout hepatocytes.

Neurohypophysial hormone receptors and second messengers were studied in trout (Oncorhynchus mykiss) hepatocytes. Arginine vasotocin (AVT) and isotocin (IT) elicited a concentration-dependent inhibition of cAMP accumulation in the presence of 5x10(-8) M glucagon (maximal effect for 4.5x10(-7) M and 1.4x10(-7) M, half-maximal effect for 2.1x10(-8) M and 0.7x10(-8) M, AVT and IT respectively). The effect of glucagon was inhibited up to 90% by AVT and 80% by IT. While AVT inhibited (up to 50%) the basal cAMP production, IT had no such action. Specific V(1) or V(2) analogues (with reference to vasopressin in mammals) were used for pharmacological characterization of the type of neurohypophysial hormone receptor involved in this inhibition. The V(1) agonist [Phe(2), Orn(8)]-oxytocin inhibited the glucagon-stimulated cAMP production with a maximal effect for 6x10(-7) M and a half-maximal effect for 0.9x10(-8) M concentrations of the analogue. While the V(1) agonist reduced the glucagon-stimulated cAMP level by 70%, it showed only a tendency to reduce the basal level. The V(2) agonist [deamino(1), Val(4),d -Arg(8)]-vasopressin had no effect either on basal or on glucagon-stimulated cAMP production. The V(1) antagonist [d(CH(2))(5)(1), O-Me-Tyr(2), Arg(8)]-vasopressin totally reversed the 10(-8) M AVT-induced inhibition of 5x10(-8) M glucagon-stimulated cAMP production, whereas the V(2) antagonist [d(CH(2))(5)(1),d -Ile(2), Ile(4), Arg(8), Ala(9)]-vasopressin had no such effect. In this particular case, maximal and half-maximal effects of the V(1) antagonist were obtained for 2.3x10(-6) M and 1. 2x10(-6 )M respectively. Changes in intracellular calcium content were measured using the fluorescent probe FURA-2/AM. AVT and IT elicited a concentration-dependent increase in Ca(2+) accumulation. The comparison of the effect of 10(-8) M agonists versus AVT showed the following order of potency: AVT=IT>V(1) agonist>V(2) agonist. The V(1) antagonist reversed the AVT-induced Ca(2+) accumulation whereas the V(2) antagonist had no such effect. These results are taken as evidence for the presence in trout hepatocytes of neurohypophysial hormone receptors functionally close to the V(1a)-type linked to cAMP production and Ca(2+) mobilization.

A V1-type receptor for mediating the neurohypophysial hormone-induced ACTH release in trout pituitary.

We analysed the effects of specific neurohypophysial analogues for pharmacological characterization of the type of vasotocin receptor involved in the control of the adrenocorticotrophin hormone (ACTH) release from the perifused pituitary in the rainbow trout, Oncorhynchus mykiss. Mammalian corticotrophin releasing factor (CRF) and teleostean neurohypophysial peptides (arginine vasotocin (AVT) and isotocin (IT)) stimulated ACTH release. Analysis of concentrations giving half-maximal effects (D50) showed that these peptides affected ACTH release in the following order of potency: CRF (8 x 10(-13) M) > AVT (2 x 10(-10) M) > IT (10(-7) M). Maximal responses (Dmax) were obtained for hormonal concentrations of 10(-10) M, 10(-8) M and 10(-6) M respectively. This suggests that AVT and IT have different roles in the control of ACTH release. The values obtained for AVT and IT were in agreement with the circulating levels we previously found for these peptides. Specific V1 or V2 agonists or antagonists (with reference to vasopressin in mammals) were used to define the specificity of the neurohypophysial peptide receptor involved in this stimulation. The V1 agonist, [Phe2, Orn8]-oxytocin, stimulated ACTH release while the V2 agonist, [deamino1, Val4, D-Arg8]-vasopressin, had no such effect. Maximal and half-maximal responses were obtained in the presence of the V1 agonist with 10(-7) M and 7 x 10(-9) M respectively, and were in the range of values obtained with natural peptides. The V1 antagonist, [d(CH2)5(1), O-Me-Tyr2, Arg8]-vasopressin, and the V2 antagonist, [d(CH2)5(1), D-Ile2, Ile4, Arg8, Ala9]-vasopressin, maximally reversed the 10(-9) M AVT-stimulated ACTH release by 60% and 25% respectively, for a 5 x 10(-10) M concentration of the analogues and a D50 approximately 2 x 10(-11) M. These results demonstrated the presence of only one V1-type receptor in fish pituitary, with some of the structural and functional peculiarities typically displayed by the mammalian V1a-type receptor, but distinct from it. In this sense, the fish pituitary vasotocin receptor may represent a novel type of neurohypophysial hormone receptor, more closely related to the mammalian V1b-type.

Enzyme linked immunosorbent assay for the neurohypophyseal hormones arginine vasotocin and isotocin.

A competitive enzyme linked immunosorbent assay (ELISA) using new polyclonal antibodies was developed for the first time for each of two neurohypophyseal hormones: arginine vasotocin (AVT, ubiquitous in vertebrates) and isotocin (IT, restricted to teleost fish). The antibodies obtained were highly specific, showed no cross-reaction between the two peptides and were able to suppress the activity of the peptides in in vitro bioassays. An original feature of the assays was the covalent binding of the peptidic antigen to Covalink microplates. Competition was thereafter made between this bound antigen and the antigen in samples, for a fixed amount of the relevant antibody. Revelation used peroxidase-labeled antibodies. These ELISAs were sensitive enough to detect 1 ng/ml and 0.1 ng/ml for AVT and IT respectively. Moreover, for the first time, the two hormones were measured separately, each in the presence of the other, and shown to exist as circulating hormones in fish.

ELISA measurements of vasotocin and isotocin in plasma and pituitary of the rainbow trout: effect of salinity.

The fish neurohypophysial hormones arginine vasotocin (AVT) and isotocin (IT) were measured for the first time by ELISAs (in comparison with other techniques) in plasma and hypophysis of rainbow trout adapted stepwise from freshwater (FW) to seawater (SW). AVT concentrations were higher than IT in plasma and, conversely, lower in hypophysis. No difference appeared between FW and SW conditions, but plasma hormone concentrations fell when FW fish were moved to 1/3 SW and increased progressively when fish were moved from 1/3 SW to SW. Peptide values obtained in 1/3 SW may correspond to the lowest osmoregulatory constraints occurring in an isosmotic medium in comparison to FW or full SW. The data suggest that storage and/or release of AVT and IT differ, but vary in a similar way with external salinity, and that these peptides should play a role in teleost fish osmoregulation.