Ionoregulatory and respiratory responses of brown trout,Salmo trutta,exposed to lethal and sublethal aluminium in acidic soft waters

Soft water acclimated (Ca²⁺ 0.02 mM; Na⁺ 0.03 mM; K⁺ 0.01 mM; pH 7.0), cannulated brown trout (Salmo trutta) were exposed to various pH and aluminium (Al) regimes (pH 7.0, pH 5.0, pH 5.0 plus Al: 50, 25, and 12.5 g l^–1) for up to 5 days in order to determine (i) the sublethal concentration of Al at pH 5.0 for this species (ii) their ionoregulatory and respiratory status. No mortality or physiological disturbances were evident at pH 7.0 or pH 5.0. All trout died within 48 h at pH 5.0 in the presence of Al at 50 g l^–1 and 67% died over the 5 day period at pH 5.0 in the presence of Al at 25 g l^–1. Fish at these lethal Al concentrations showed significant decreases in arterial blood oxygen content (C_aO₂) but no changes in plasma osmolarity or the concentrations of plasma Na⁺, K⁺ and Cl^–. Physiological disturbance was more marked at the 50 g l^–1 Al concentration. The surviving fish at 25 g l^–1 showed few signs of physiological recovery while continually exposed to this regime. No fish died during the exposure to water of pH 5.0 containing 12.5 g l^–1 Al, but physiological disturbance was still apparent. These sublethally-stressed trout showed a transient decline in the plasma concentrations of Na⁺ and Cl^–1. Although C_aO₂ decreased, recovery was evident. The data suggest that in the brown trout, environmental Al concentration is as important as pH and calcium concentration in determining the physiological status of the fish.     

Effects of sulfide on K+ flux pathways in red blood cells of crucian carp and rainbow trout

The effect of sulfide on K⁺ influx pathways was measured in red blood cells (RBCs) of sulfide-sensitive rainbow trout (Oncorhynchus mykiss) and sulfide-tolerant crucian carp (Carassius carassius). In trout RBCs, maximal inhibition of Na⁺, K⁺-ATPase was attained at 10 mol l^–1 sulfide and amounted to 32% without being influenced by pH between 6.7 and 8.3. Ouabain-resistant K⁺ influx in the absence and presence of sulfide was insignificant at pH values between 6.7 and 7.7. At higher pH values ouabain-resistant K⁺ influx increased, but was inhibited to about 15% by 30 mol l^–1 sulfide. In RBCs of crucian carp neither Na⁺, K⁺-ATPase nor ouabain-resistant K⁺ influx were affected by sulfide concentrations up to 850 mol l^–1. Differences in sulfide-sensitivity of K⁺ influx between both species can be based upon different properties of the membrane transporter themselves. The reduced Na⁺, K⁺-ATPase activity in trout RBCs may also result from a slightly reduced (by 9%) ATP level after sulfide exposure. In addition, intracellular sulfide concentrations were higher in trout RBCs as compared to crucian carp. In trout, intracellular sulfide concentrations reached extracellular levels within 5 min of incubation whereas sulfide concentrations in crucian carp RBCs remained about 2-fold lower than extracellular concentrations. Although the physiological basis of sulfide-insensitive K⁺ influx in crucian carp RBCs is currently unknown it may contribute to the extremely high sulfide-tolerance of this species.     

NAD+-linked isocitrate dehydrogenase in fish tissues

NAD⁺-linked isocitrate dehydrogenase was found in the brain, heart, gills, kidney, liver and muscle of trout, and in the liver and muscle of eel. A complex homogenization buffer containing 1 mM ADP, 5 mM MgSO₄, 5 mM citrate and 40% glycerol is required for retrieval of significant amounts of stable enzyme. The highest activities were found in brain of trout and the lowest in white muscle of trout and eel. The enzyme was partially purified from frozen trout heart to a final activity of 0.04 M/min/mg protein, and the kinetic properties of this partially purified enzyme were studied. The enzyme requires either Mn²⁺ or Mg²⁺ for activity, higher activities being observed with Mn²⁺. Saturation kinetics for DL-isocitrate were sigmoidal, apparent S_0·5=8.2±0.6 mM and n_H=1.8±0.2, in the absence of ADP, changing to hyperbolic, apparent S_0·5=1.4±0.3 mM and n_H=1.0, with 1 mM ADP added. Citrate and Ca²⁺ were found to activate the enzyme to a small extent. NADH strongly inhibited the enzyme, I₅₀=3.7±0.5 M. ATP was also found to be an inhibitor, I₅₀=7.2±1.4 mM. These properties are consistent with the role of the enzyme as a major control site of the tricarboxylic acid cycle.     

Mitochondrial NAD(P)-malic enzyme from herring skeletal muscle

Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD⁺ oxidoreductase (decarboxylating)] was purified from herring skeletal muscle to a specific activity of 8.2 mol/min/mg. The purification procedure involved chromatography on DEAE-cellulose, Red Agarose and a Sephacryl S-300 with a final recovery of 38% of enzyme activity. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of either NAD or NADP in the presence of Mn²⁺. Some kinetic characteristics of this enzyme were determined. The pH optimum of activity is 7.0. ATP was shown to be a competitive inhibitor with malate. The inhibition by ATP displayed hyperbolic competitive kinetics with a K_i (ATP) of 0.28 mM in the presence of NAD and 0.75 mM in the presence of NADP. Fumarate reversed ATP inhibition.In vivo, regulation of NAD(P)-dependent malic enzyme might respond to changing levels of mitochondrial ATP and fumarate with the enzyme undergoing kinetic activation by an increase in the concentration of mitochondrial fumarate which could reverse enzyme inhibition by ATP.     

Influence of exercise on the distribution of enzymes in trout white muscle and kinetic properties of AMP-deaminase from free and bound fractions

Effects of exercise on the distribution of phosphofructokinase (PFK), fructose-1,6-biphosphatase (FBPase), and AMP-deaminase between free and particulate-bound fractions was analyzed in white skeletal muscle of rainbow trout Oncorhynchus mykiss. With a widely used technique for the separation of free and bound enzyme fractions (homogenization in low ionic strength, high sucrose buffer), the data showed that the amount of bound PFK increased from 64 to 95% during burst swimming whereas other enzymes were unaffected. Since this data for AMP-deaminase contrasted with earlier reports, different methods of separating free and bound enzyme were evaluated. A clear effect of exercise on AMP-deaminase binding occurred when high ionic strength media (either KCl or KF) were used; in extraction media containing 150 mM KCl, the percent bound rose from 30% in controls to 97% after 1 min burst swimming. Exercise also produced stable changes to AMP-deaminase kinetic properties, including for the bound enzyme (compared with the free) a 2-fold higher K_m AMP, a 3-fold higher K_i for inorganic phosphate, and a 60% increase in K_a ADP after 1 min burst exercise. The data suggest that AMP-deaminase in working skeletal muscle is subject to combined controls by allosteric effectors, post-translational modification, and distribution between free and bound states.     

The effect of photoperiod, tank colour and light intensity on growth of larval haddock

In two separate experiments, haddock (Melanogrammus aeglefinus) larvae were raised under different photoperiods (24L : 0D or 15L : 9D), or different combinations of tank colour (black or white) and light intensity (1.1 mol s^–1 m^–2 or 18 mol s^–1 m^–2). Growth (0.8% day^–1 in standard length; 2.9% day^–1 in body area) and survival (2%) were not significantly different between photoperiod treatments after 35 days. Larval survival was greater in white versus black tanks after 41 days (2% versus l%, respectively). Growth of larvae was impaired in black tanks at low (1.1 mol s^–1 m^–2) light intensity (0.8% day^–1 in standard length and 2.2% day^–1 in body area versus 1.1% day 21 in standard length and 3.1% day^–1 in body area, for all other treatments). Transmission and reflection of light was low in black tanks at low incident light, and there was very little upwelling light. The resultant poor prey to background contrast probably resulted in larvae being unable to consume sufficient food to sustain a level of growth comparable to that in other treatments.     

Dehydration is more effective for the control of embryonic development and larval hatching of Diplectanum aequans (Monogenea, Diplectanidae) than formalin and trichlorphon

Embryonic development and larval hatching of the monogenean Diplectanum aequans, gill parasite of sea bass Dicentrarchus labrax, was studied in relation to different prophylactic treatments. Groups of eggs of D. aequans were submitted to different in vitro treatments: formalin (300 and 100 L L^–1 per 1 hour), Neguvon^® (trichlorphon 0.2 mg L^–1 per 48 hours) and dehydration for 4 and 8 hours. Percentages of hatched larvae, aborted larvae and undeveloped embryos were estimated in comparison with the control group. Results showed that 300 L L^–1 formalin and dehydration treatments were able to reduce larval hatching significantly, while Neguvon^® and 100 L L^–1 formalin treatments had no effect.     

Characterisation of head kidney and liver microsomal 17-hydroxyprogesterone 21-hydroxylase activity in rainbow trout (Oncorhynchus mykiss) and the lack of regulatory effects of ACTH

The formation of 11-deoxycortisol from 17-hydroxyprogesterone was studied in rainbow trout (Oncorhynchus mykiss) head kidney and liver microsomes. The K _m for head kidney microsomal 17-hydroxyprogesterone 21-hydroxylase measured in pooled microsomes from juvenile rainbow trout was 3.75 M, and the V _max was 41.4 pmol/(mg protein × min). The K _m for liver microsomal 17-hydroxyprogesterone 21-hydroxylase was 31.0 M, and the V _max was 51.0 pmol/(mg protein × min). The intrinsic clearance values suggest that liver, in addition to head kidney, could significantly contribute to the formation of 11-deoxycortisol in rainbow trout. Progesterone inhibited both the head kidney and the liver microsomal 17-hydroxyprogesterone 21-hydroxylase activity competitively. The K _i for progesterone in head kidney was 1.8 M and in liver 144 M. The different K _m and K _i values for microsomal 21-hydroxylation suggest that 21-hydroxylation of 17-hydroxyprogesterone in head kidney and liver is catalysed by different enzyme systems. Treatment of rainbow trout by adrenocorticotrophic hormone (ACTH) for 1.5 or 3 h (single injections) or three weeks (injections every other day) did not affect head kidney or liver17-hydroxyprogesterone 21-hydroxylase activity. ACTH may thus not regulate this enzyme activity. The increase in plasma cortisol levels 1.5 and 3 h after ACTH injection but not following repeated injections suggests that the hormone regulates the initial secretion of cortisol, but does not have a long-term regulatory effect upon cortisol production in rainbow trout.     

Effects of purified microcystin-LR and cell extracts of Microcystis strains PCC 7813 and CYA 43 on cardiac function in brown trout (Salmo trutta) alevins

Sub-lethal cardiac responses of brown trout alevins (Salmo trutta L.) were determined in response to aqueous extracts of the cyanobacterium Microcystis strains PCC 7813 (microcystins detectable by HPLC) and CYA 43 (no microcystins detectable by HPLC) and to the purified cyanobacterial hepatotoxin, microcystin-LR (MC-LR) at concentrations of 5, 50 and 500 g microcystin-LR equivalents l^–1. Responses were determined using a flow chamber and video camera attached to a low power microscope. Heart rate in brown trout alevins was acutely sensitive to cyanobacterial extracts and significant increases were observed within 15–60 sec of exposure to aqueous extracts, although no change was observed on exposure to purified MC-LR. Stroke volume increased in all treatments at 50 and 500 g MC-LR equivalents l^–1, which may, at least in part, have been due to vasodilation of the yolk-sac blood vessels. Cardiac output increased significantly at all three concentrations of cyanobacterial cell extracts but not at the lowest concentration of MC-LR, although the rate increased at levels at/or above 50 g l^–1. Increased heart rate, stroke volume and cardiac output occurred at environmentally relevant microcystin concentrations of Microcystis PCC 7813 and CYA 43 aqueous extracts.     

Swimming performance,whole body ions,and gill Al accumulation during acclimation to sublethal aluminium in juvenile rainbow trout (Oncorhynchus mykiss)

Juvenile rainbow trout (2–5 g) were chronically exposed (for 22 days) to acidified softwater (Ca²⁺ = 25 Eq/l, pH 5.2) in the presence or absence sublethal Al (30 g/l). Al-exposed fish (5.2/Al group) suffered 20% whole body Na⁺ and Cl^– losses and a 30% reduction in the maximum sustainable swimming speed (U_crit) over the initial 7 days. These disturbances were approximately 2 fold greater than those observed in the fish exposed to low pH alone (5.2/0 group). However, whole body ion levels were completely restored in the 5.2/Al fish by day 22, whereas they merely stabilized at a new reduced level in the 5.2/0 group. Increased resistance to acutely lethal Al (200 g/l at pH 5.2) was observed from day 17 onwards in the 5.2/Al fish. Despite this acclimation and recovery of whole body ions, U_crit remained significantly lower than in the 5.2/0 group throughout. Growth on a restricted diet of 1% body wt. /day was normal in the 5.2/0 group compared with controls maintained in pH 6.5 softwater, whereas 5.2/Al fish suffered a 50% reduction in growth rate on the same diet. The 5.2/Al fish accumulated large amounts of Al on the gills, reaching an initial peak after 4 days, followed by a decline at 7 days, and a secondary rise thereafter. Therefore acclimation and recovery of whole body ionic status was not associated with a reduction in the gill Al burden. Some of the metabolic costs of acclimation to Al, namely a continued impairment of swimming speed and growth, are discussed in light of the physiological and structural changes reported to occur at the gills.     

Effects of sublethal, acidic aluminum exposure on blood ions and metabolites, cardiac output, heart rate, and stroke volume of rainbow trout, Oncorhynchus mykiss

Doppler flow probes were fitted around the ventral aorta of rainbow trout, which were exposed to combinations of pH and aluminum (pH 5.1–6.2, Al 0–80 g l^–1) for 60 h. Fish accumulated Al at the gill and exhibited decreased blood Na⁺, Cl^–, and Ca²⁺ concentrations, and increased K⁺, hematocrit, hemoglobin, glucose, and lactate, indicating increasing ionoregulatory disturbance with increasing Al concentration. Fish exposed to ambient water (6.2) or low-pH (5.3) water without Al exhibited slight reductions in heart rates, as well as increased stroke volume, resulting in little variation in cardiac output. In the presence of Al (20 to 40 g l^–1) at low pH (5.1–5.3), fish increased their heart rate slightly and generally maintained their stroke volume, resulting in increased cardiac output in the first two days of exposure. At the highest Al concentration (80 g l^–1, pH 5.1), tachycardia was observed, concomitant with a decrease in stroke volume. The ionoregulatory imbalance and resulting increased blood viscosity explain these increases in heart rate rather than stroke volume in fish exposed to high concentrations of Al.     

Creatine turnover in the starry flounder,Platichthys stellatus

Starry flounder (Platichthys stellatus) were cannulated and a bolus of 9 Ci¹⁴C-creatine in saline was injected into the caudal vein. The fish were sacrificed at intervals ranging from 1h to 36d after label injection. Creatine pool size (PCr+Cr) and creatinine (Crn) content in blood, muscle, gills and liver were analyzed and specific activities (SA) determined.Mean concentrations of PCr+Cr/Crn in PCA-extracts of muscle, gills, liver and blood of experimental fish (at rest) were 38.1/2.40, 4.1/0.25, 5.6/0.45 and 0.3/n.d. mol.g^–1 respectively.Within 10 min, plasma SA had decreased by approximately 90%. In white muscle, the rate of¹⁴C–Cr appearance as well as label disappearance was slow compared to gills and liver. In fish examined 36d postinjection, mean SA in muscle had decreased to 23% of maximum SA which occurred 24h after injection.¹⁴C–Cr was incorporated into the liver tissue at a very high rate, SA being two orders of magnitude higher in liver than in white muscle. Over the first 6d, retention of label was observed in liver; after 36d only 3% of the original label was detected.Creatine pool size (PCr+Cr) in white muscle decreased with food deprivation. In flounder sacrificed after 36d, PCr+Cr was only 52% that of fed control fish, suggesting that creatine or precursors for its biosynthesis are supplied with the diet.     

The effect of seawater flow and temperature on metamorphosis and postlarval development in great scallop

Variations in growth and survival of hatchery-reared post-metamorphicjuveniles of great scallop Pecten maximus prompted anexamination of settlement and postlarval development. The effects ofseawater flow and temperature on great scallop metamorphosis andpostlarvae were studied over a 4–5 week period. In allexperiments, and regardless of environmental conditions, great scallopmetamorphosed after a 2–3 week period with values of 35 to70%. Subsequently, spat numbers increased slightly. Spatmortality generally occurred from the third week onward and reachedlevels as high as 30% by the fifth week under standardconditions. At 20 °C, however, 60% mortality levels wererecorded. Differences in spat growth rate, ranging from 37 to 45 mday^–1, were noticed at different seawater flow ratesbut no clear tendency could be discerned. Temperature affected spatgrowth with an increase in size from 24 m day^–1 at15 °C to 35 m day^–1 at 18 °C. Conversely,growth was suppressed at 20 °C (14 m day^–1).For optimal metamorphosis and postlarval development in great scallop, aseawater flow of 4.3 L h^–1 per sieve and a temperatureof 15 °C are recommended.     

Quantification of presumptive Na+/H+ antiporters of the erythrocytes of trout and eel

The presumptive Na⁺/H⁺ exchange sites of trout and eel erythrocytes were quantified using amiloride-displaceable 5-(N-methyl-N-[³H]isobutyl)-amiloride (³H-MIA) equilibrium binding to further evaluate the mechanisms of i) hypoxia-mediated modifications in the trout erythrocyte -adrenergic signal transduction system and ii) the marked differences in the catecholamine responsiveness of this system between the trout and eel. MIA was a more potent inhibitor of both trout apparent erythrocyte proton extrusion (IC₅₀ = 20.1 ± 1.1 mol l^–1, N = 6) activity (as evaluated by measuring plasma pH changes after addition of catecholamine in vitro) and specific ³H-MIA binding (IC₅₀ = 257 ± 8.2 nmol l^–1, N = 3) than amiloride, which possessed a proton extrusion IC₅₀ of 26.1 ± 1.6 mol l^–1 (N = 6) and a binding IC₅₀ of 891 ± 113 nmol l^–1 (N = 3). The specific Na⁺ channel blocker phenamil was without effect on adrenergic proton extrusion activity or specific ³H-MIA binding. Trout erythrocytes suspended in Na⁺-free saline and maintained under normoxic conditions possessed 37,675 ± 6,678 (N = 6) amiloride-displaceable ³H-MIA binding sites per cell (B_max, presumptive Na⁺/H⁺ antiporters) with an apparent dissociation constant (K_D) of 244 ± 29 nmol l^–1 (N = 6). Acute hypoxia (PO₂ = 1.2 kPa; 30 min) did not affect the K_D, yet resulted in a 65% increase in the number of presumptive Na⁺/H⁺ antiporters. Normoxic eel erythrocytes, similarly suspended in Na⁺-free saline, possessed only 17,133 ± 3,716 presumptive Na⁺/H⁺ antiporters (N = 6), 45% of that of trout erythrocytes, with a similar K_D (246 ± 41 nmol l^–1, N = 6). These findings suggest that inter- and intra-specific differences in the responsiveness of the teleost erythrocyte -adrenergic signal transduction system can be explained, in part, by differences in the numbers of Na⁺/H⁺ exchange sites.     

Quantitation of inert feed ingestion in larval silver sea bream (Sparus sarba) using auto-fluorescence of alginate-based microparticulate diets

The ingestion of an inert feed as a sole food source was investigated in larval silver sea bream (Sparus sarba) fed an alginate-based microparticulate diet. Using the auto-fluorescent properties of pigments associated with the alginate base, ingestion and gut content were investigated over a 6 h experimental period in fed and unfed larvae. By extracting and measuring chlorophyll a (Chl a) and phaeopigment content of feeding larval fish and relating this to standardized Chl a and phaeopigment content of the diet, relative to diet weight, it was determined that individual fed 7-day old larvae had a maximum gut content of 1.05±0.09 g diet while 14-day old fed fish had a maximum gut content of 3.17±0.90 g diet. On average, the gut content of 14-day old fish was 2.89 times greater than the gut content of 7-day old fish. The dry weight of larval sea bream increased from 43±4.2 g at day 7 to 134.3±20.4 g at day 14 indicating that growth of fish fed this inert feed was substantial. Gut pigment dynamics suggested that Chl a was degraded to phaeopigments by 7-day but not 14-day old larvae and the individual gut dietary content varied considerably in 14-day old fish. The maximum Chl a and phaeopigment content in larval sea bream was 0.4 ng ind^–1 and 0.55 ng ind^–1 for 7-day old fish and 1.54 ng ind^–1 and 2.81 ng ind^–1 for 14-day old fish respectively. The present method may potentially allow simple and direct assessment of larval fish feed ingestion in both an experimental and commercial setting.     

Effects of growth hormone on plasma ionic regulation,respiration and extracellular acid-base status in trout (Oncorhynchus mykiss) transferred to seawater

The effects of trout recombinant growth hormone (rtGH) treatment (0.25 g g^–1 by intraperitoneal implant) on plasma ionic regulation, extracellular acid-base status and respiration were investigated in freshwater rainbow trout and during a 4-day period after direct transfer into seawater (35 g 1^–1).In freshwater, rtGH treatment resulted in a significant increase in gill (Na⁺, K⁺) ATPase activity and in standard metabolism (MO₂). The latter would mainly result from a higher rate of protein synthesis. Direct transfer from freshwater to seawater induced a decrease in arterial blood pH, far more pronounced in controls than in treated fish. This effect could be regarded in both groups mainly as a metabolic acidosis resulting from extracellular ion composition changes (i.e., an increase higher in chloride than in sodium, more marked in controls than in treated fish). As the rise in PaCO₂, in spite of an increase in ventilatory activity, is more significant in controls than in treated fish, it can be assumed that rtGH treatment lightened the decrease in the gas diffusing capacity of gills induced by transfer to seawater. The initial increase in MO₂ in both controls and treated fish could be the consequence of an increase in energetic cost of ventilation and osmoregulation. Then, in treated fish, the persistent high level of M may indicate a stimulation of intermediary metabolism by rtGH. In addition, the absence in treated fish of an increase in plasma lactate concentration, as observed in controls, would indicate that rtGH attenuated the decrease in O₂ affinity of haemoglobin foreseeable from the metabolic acidosis.This article is dedicated to Professor Claude Peyraud, whose recent death has deeply saddened us. We respectfully pay a tribute to his memory.     

Basal H2-receptor stimulated and pH-dependent acid secretion from an isolated stomach mucosa preparation of the cod,Gadus morhua,studied using a modified pH-static titration method

Gastric acid secretion from isolated cod stomach mucosa was measured using a pH-static titration method. A basal acid secretion rate (BASR) of 6.0±0.6 nEqH⁺min^–1cm^–1 was measured when using 0.9% NaCl as luminal solution. There was a dose-dependent increase in response to histamine between 0.12 and 0.20 M (EC₅₀=0.15 M), above which gastric acid secretion plateaued at 13.5±1.8 nEqH⁺min^–1cm^–1. Ranitidine, a H₂-receptor antagonist, completely blocked the stimulatory effect of histamine and reduced the BASR. The H₁-receptor antagonist, clemastine, did not inhibit the response to histamine. Acid secretion rates decreased significantly when the pH of the luminal side of the mucosa was lowered from pH 5.75 to pH 4.50, indicating that a negative feedback mechanism was operating. Histological staining showed that oxynticopeptic cells were uniformly distributed throughout the cardiac stomach.It is concluded that the acid secretion in the isolated stomach mucosa of cod can be measuredin vitro with a pH-static titration method. The method was used to demonstrate that the BASR is downregulated by a decrease in pH. Furthermore, we conclude that the histamine receptor in the cod stomach mucosa resembles the mammalian H₂-receptor and that histamine is secreted under basal conditions.     

Interrelationships between gill chloride cell morphology and calcium uptake in freshwater teleosts

The involvement of the freshwater fish gill chloride cells (CCs) in trans-branchial calcium uptake (JinCa²⁺) was investigated. This was accomplished by assessing the interspecific relationships between the apical surface area of CCs exposed to the external environment and JinCa²⁺. Three species of freshwater teleosts, the rainbow trout (Oncorhynchus mykiss), the American eel (Anguilla rostrata) and the brown bullhead catfish (Ictalurus nebulosus), were used. Chronic (ten-day) treatment with cortisol in each species was used as a tool to evoke variations in both JinCa²⁺ and gill CC morphology in order to assess intraspecific relationships between CC surface area and JinCa²⁺. The results of quantitative morphometry, based on analysis of scanning electron micrographs, demonstrated that catfish possessed the lowest fractional area of exposed CC (CCFA) on the gill filament epithelium (12,744 ± 2248 m²/mm²) and was followed, in increasing order, by American eel (21,355 ± 981 m²/mm²) and rainbow trout (149,928 ± 26,545 m²/mm²). With the exception of catfish, chronic treatment with cortisol caused significant increases in CCFA owing to proliferation of CCs and/or enlargement of individual CCs (eel only). The rates of JinCa²⁺ closely reflected the CC fractional area in each species. The results of correlation analysis revealed significant correlations between CC fractional area and JinCa²⁺ in trout and eel. Owing to the absence of an effect of cortisol treatment, there was no significant correlation in catfish because of insufficient variation in CC fractional area in this species. CC fractional area was significantly correlated with JinCa²⁺ among the three species examined. These results suggest that CC is involved in calcium uptake in freshwater teleosts and that both intra- and interspecific differences in the rates of calcium uptake can be accounted for by variability in the surface area of exposed CCs on the gill epithelia.     

The biliary accumulation of corticosteroids in rainbow trout,Oncorhynchus mykiss,during acute and chronic stress

The accumulation of immunoreactive corticosteroids in the bile of rainbow trout during stress was monitored by radioimmunoassay and GUMS. Although plasma cortisol levels were elevated by confinement for 1 hour, biliary levels of free and conjugated steroids in the bile were unaffected. However, after 24 hours confinement, in addition to elevated plasma cortisol levels, free and conjugated steroids in the bile were also significantly higher than in control, unstressed fish. The time-course of change in plasma and biliary corticosteroid levels was determined in rainbow trout subject to 96 hours confinement stress. Free steroid levels in the bile of stressed fish were elevated within 2 hours of the onset of stress, while levels of conjugated steroids were significantly elevated within 4 hours of the onset of confinement. Analysis of bile from stressed fish, by GC/MS, established the major conjugated steroids present to be tetrahydrocortisone (230 g ml^–1 bile), tetrahydrocortisol (75 g ml^–1), cortisone (33.5 g ml^–1), cortisol (25 g ml^–1) and -cortolone (5 g ml^–1). The data are discussed with reference to the role of cortisone and conjugating enzymes in the clearance of cortisol, and further data are presented to suggest that the analysis of biliary steroid content may provide a suitable means of identifying stressed fish under conditions in which an additional sampling stress is unavoidable.     

Relationship between metabolic rate in vitro and body mass in a marine teleost,porgy Pagrus major

The rate of oxygen consumption of minced whole body was determined volumetrically, as an indication of metabolic rate in vitro (M _{in vitro} ), at 20°C in porgy Pagrus major ranging from 0.0002 g (just after hatch) to 2.9 g (67 days old) in body mass. A triphasic relationship was found between M _{in vitro} of individual fish (l.min^–1) and wet body mass W (g). During the prolarval stage accompanied with the transitional period to the postlarval stage (0.00020–0.00023 g, 0–6 days old), the mass-specific metabolic rate in vitro (M _{in vitro} /W in l.g^–1.min^–1) increased with age (D in days) as expressed by an equation M _{in vitro} /W = 3.88 + 0.74/D. During the postlarval stage (0.00031–0.003 g, 8–22 days old), M _{in vitro} /W remained almost constant, independent of body mass following an equation M _{in vitro} /W = 5.24 W^–0.085. During the juvenile and adolescent stages (0.0047–2.9 g, 30–67 days old), M _{in vitro} /W decreased with increasing body mass following an equation M _{in vitro} /W = 1.66 W^–0.235. These results correspond with the triphasic relationship between metabolism in vivo and body mass observed in intact porgy of 0.0002–270 g (Oikawa et al. 1991). It is concluded, therefore, that the dependence of mass-specific metabolic rate on body size exists in vitro as well as in vivo, during the early stages in the porgy. Based on these results, factors controlling the metabolism-size relationship are discussed.