Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.)

PKC activity was detected in spleen extracts from the turbot, Scophthalmus maximus, a teleost flatfish that is farmed commercially in several countries, in assays with the substrate EGF- R651–658 as phosphate acceptor. The activity was purified about 700-fold by a three-step chromatographic procedure (DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose). Maximal activity was obtained in the presence of the typical PKC cofactors Ca2+ (0.1 mM) PtdS (20 g ml–1) and either DAG (2 g ml–1) or PMA (2 g ml–1). Activity was dose-dependently inhibited by H7 and by the PKC-specific inhibitors PKC19–36 and N-myristoylated PKC19–31. The rate of phosphorylation was highest with the PKC-specific substrate MARCKS161–175. In immunoblotting, MC5 (a mouse monoclonal antibody raised against bovine PKC) recognized bands of 80 and 100 kDa. Immunoblotting with antibodies raised against mouse PKC isozymes (, , , , , , and ) indicated the presence of all these isozymes in turbot spleen.     

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.     

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.     

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.     

Influence of temperature on the hatching of eggs of Diplectanum aequans,a parasite of sea bass

This study deals with the influence of temperature on the hatching of eggs of Diplectanum aequans, a branchial ectoparasite of sea bass (Dicentrarchus labrax). At temperatures of 20 °C, 25 C and 30 °C hatching takes place within a few days of laying, whereas at 15 C and 10 C it occurs respectively between the 7th and the 12th day and between the 11th and the 19th day. No hatching has been observed at 5 C. Development is not completely inhibited at 5 C as the eggs can, at least partially, go on maturing and will subsequently hatch as soon as higher temperatures are encountered.     

Stimulation of ureogenesis in the perfused liver of an Indian air-breathing catfish, Clarias batrachus, infused with different concentrations of ammonium chloride

The pattern of changes of activity of the urea cycle enzymes and the rate of urea-N excretion were studied in the perfused liver of an Indian air-breathing ureogenic walking catfish, Clarias batrachus. The liver was perfused with different concentrations of NH₄Cl for a period of 60 min to determine the role of ammonia for stimulation of hepatic ureogenesis and the threshold level of ammonia loading needed to cause such stimulation. Both the urea-N excretion and the ammonia uptake by the perfused liver were found to be a saturable process. Ammonia accumulated significantly in the liver infused with 1.25 moles g liver ^–1 min^–1 of NH₄Cl, followed by a maximum accumulation of about 28.5 moles g wet wt^–1 with the infusion of 5.08 moles g liver^–1 min^–1. The Vmax of the urea-N excretion (0.47 mol g liver^–1 min^–1) was obtained with the addition of 5.08 moles g liver^–1 min^–1 of NH₄Cl. Both the tissue and the specific activity of the urea cycle enzymes, except ornithine transcarbamylase and arginase, were stimulated significantly with the infusion of either 1.25 or 5.08 moles g liver^–1 min^–1 of NH₄Cl. Maximum stimulation of tissue activity of carbamoyl phosphate synthetase (about 120%) was seen with the infusion of 5.08 mol g liver^–1 min^–1, and for argininosuccinate synthetase (about 135%), and argininosuccinate lyase (about 50%) with the infusion of 10.81 mol g liver^–1 min^–1 of NH₄Cl. Higher accumulation of ammonia of about 10–15 mol g wet wt^–1 from the physiological level in the perfused liver while infusing with NH₄Cl was suggested to be one of the major causes of stimulation of ureogenesis. The presence of such physiological adaptive strategy is probably necessary in this unique group of air-breathing walking catfish to survive under hyper-ammonia stress in their normal habitat or while living outside water or while burrowing inside mud.     

Oxygen consumption and responses to hypoxia of ammocoetes of the southern hemisphere lampreyGeotria australis

The standard rate of oxygen consumption of ammocoetes (larvae) ofGeotria australis with a mean weight of c. 0.5 g was 9.6, 31.4 and 59.4l g^–1 h^–1 at 4.5, 15.5 and 25.0°C respectively, which gives an overall Q₁₀ of 2.4. The regression coefficient for the logarithmic relationship between oxygen consumption and body weight at 15.5°C was 0.704. The ammocoetes ofG. australis have a much lower rate of oxygen consumption at 15.5 and 25.0°C than those of holarctic lampreys. This presumably reflects the lower oxygen delivery pressure to their tissues and helps account for their slow growth rate. At 15.5°C, ammocoetes ofG. australis emerged from the substrate at 21–25 mm Hg and, unlike those of the Northern HemisphereIchthyomyzon greeleyi, died at 14–17 mm Hg. Thus, despite having a thinner water/blood barrier in the gills and blood with a higher oxygen affinity and capacity than holarctic ammocoetes, the larvae ofG. australis cannot survive very low dissolved oxygen tensions. This is apparently related to an inability of larvalG. australis to meet the high oxygen requirements of the respiratory pump at these oxygen tensions. During metamorphosis, oxygen consumption at 15.5°C rose from approximately 27l g^–1 h^–1 at the beginning of transformation to 33.2l g^–1 h^–1 by Stage 3 and then rapidly to 66l g^–1 h^–1 at Stage 6. It remained near this level in Stage 7 and the downstream migrant.     

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.     

Influence of environmental temperature and water oxygen concentration on gas diffusion distance in sea bass (Dicentrarchus labrax, L.)

Gas diffusion distance (GDD) of sea bass was measured in fish bred under farm conditions, at different dissolved oxygen concentrations (DO): normoxia condition (80–100% of the saturation value) and `mild' hyperoxia condition (120–130% of the saturation value). Measures were carried out two times in a year (beginning of summer and autumn) in order to evaluate the effect of water temperature on GDD at the two different dissolved oxygen concentrations. There was a significant influence of both dissolved oxygen concentration (p 0.001) and environmental temperature (p 0.001) on GDD. In summertime it was 1.75 m and 2.31 m for fish reared under normoxia and hyperoxia, respectively, and in autumn 2.51 m and 2.96 m for fish reared under normoxia and hyperoxia, respectively. When DO was reduced at the higher temperatures, GDD decreased as well. Results lead to the conclusion that GDD increased with the increasing of DO, both due to reduced water temperature and to the mild oxygen hypersaturation following application of pure oxygen. The advantage for fish may be found in the compromise between maximising O<sub>2</sub> diffusion at the gills and ions/water intake/loss, known as `osmoregulatory compromise'.     

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.     

Angiotensins stimulate in vitro ovulation and contraction of brook trout (Salvelinus fontinalis) follicles

The effects of salmon angiotensin I (sAI) and human angiotensin II (hAII) on in vitro ovulation of preovulatory (preOV) brook trout (Salvelinus fontinalis) follicles were investigated. Both angiotensins increased levels of ovulation above that in controls after 12 hours of incubation. The increase was statistically significant in incubates with greater than 1 M hAII. The effects of the angiotensins on follicle contraction were also studied indirectly by measuring the decrease in weight of punctured follicles taken prior to germinal vesicle breakdown. At 1 M, both angiotensins significantly decreased the weight of punctured follicles after 16 hours of incubation. The angiotensin-stimulated decrease in weight was not blocked by indomethacin (10 g/ml), indicating that follicle contraction was not prostaglandin-dependent. The data indicate that angiotensins might be directly involved in brook trout ovulation and the stimulatory effects of angiotensins on ovulation may be attributed to their effects on follicle contraction.This work was supported by NIH grant #HD25924-02.     

Influence of Algal Exudates and Bacteria on Growth and Survival of White Shrimp Larvae Fed Entirely on Microencapsulated Diets

Intact microencapsulated protozoeal diets (MED), added to 5 m filtered hatchery water at the recommended concentration of 4 mg L^-1 gave no significant difference in bacterial numbers after 6 h, over a 48 h period compared to control groups in autoclaved hatchery water. Disruption of the MED resulted in a significant increase in bacterial numbers up to 12 h compared to controls with intact microcapsules. Protein leach rates were similar in sterile water and 5 m filtered water, indicating that leaching was independent of microbial activity. MED leached significantly less dietary solubles compared to a microparticulate diet (MPD). There was no evidence to suggest significant attachment of bacteria to diet particles for at least 48 h.In low-quality water after algal blooms, survival of white shrimp (Penaeus indicus) larvae was poor when reared in both sterile and 5 m filtered water. The addition of bacteria filtered from xenic algal cultures together with the algal exudates to such water gave significant improvements in survival to larvae fed MED in 5 m filtered water but not in autoclaved culture water. Live algal diets promote high larval survival and growth irrespective of the inclusion of bacteria.It is suggested that algal exudates act as selective bacteriocides and total replacement of algae with MED requires a balanced bacterial community which can be attained at times from natural sea water, or when the natural bacterial community balance is disturbed, corrected by the use of algal exudates.     

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.     

Dopamine inhibits gonadotropin secretion in the Chinese loach (Paramisgurnus dabryanus)

The effects of dopamine on gonadotropin (GtH) secretion in sexually mature Chinese loach were investigated. Spontaneous secretion of GtH was inhibited within 1 h following an intramuscular injection of dopamine (100 g/g body wt). Similarly, dopamine (50 and 100 g/g body wt) caused a significant reduction in serum GtH in fish with elevated GtH levels as a result of pretreatment with gonadotropin-releasing hormone (GnRH) analogs either alone or in combination with the dopamine receptor antagonist domperidone. In summary, the present study provides direct evidence that dopamine functions as a gonadotropin-release inhibitory factor in the Chinese loach by blocking spontaneous and GnRH-stimulated GtH release.     

The effects of elevated winter temperature and sub-lethal pollutants (low pH, elevated ammonia) on protein turnover in the gill and liver of rainbow trout (Oncorhynchus mykiss)

Appetite, growth, and protein turnover (synthesis, growth and degradation) of liver and gills were measured in juvenile rainbow trout (Oncorhynchus mykiss) fed to satiation, and exposed for 90 days to elevated winter temperatures (+2 °C above ambient) and either low pH (5.2) in softwater or 70 M total ammonia (TAmm) in hardwater. All fish increased in weight during the experiments, but those exposed to +2°C grew significantly more than those at ambient temperature due to a stimulation of appetite. During the relatively constant temperature of the first 75 days, +2 °C caused a significant increase in the rates of protein synthesis and degradation in the liver of hardwater-acclimated fish, as a result of an increase in RNA translational efficiency (KRNA). The elevated temperature also induced an increase in gill protein synthesis in softwater-acclimated fish but in this case the underlying mechanism was an increase in Cs, the capacity for protein synthesis (RNA:protein) rather than in KRNA. The addition of 70 M TAmm had no effect on protein turnover in either liver or gills of hardwater-acclimated fish. Low pH inhibited protein growth in the liver of softwater-acclimated fish at day 90 under both temperature regimes. This inhibition was effected via a decrease in protein synthesis at control temperature but via an increase in protein degradation when the fish were exposed to both low pH and +2 °C. From these results we conclude that a simulated global warming scenario has potentially beneficial rather than detrimental effects on protein turnover and growth of freshwater fish during winter.     

Catecholestrogens inhibit dopamine methylation in the gonadotrops of the African catfish,Clarias gariepinus

Isolated gonadotrops of the African catfish,Clarias gariepinus, were incubated with dopamine (DA) and/or catecholestrone and the activity of the enzyme catechol-O-methyltransferase (COMT) was determined by measuring the methylated products. From the apparent Km values for DA and catecholestrone of 0.4–1.3 M and 17.9–25.2 M respectively, it was concluded that catecholestrone is a better substrate for the enzyme COMT, compared to DA. Moreover, the methylation of DA is inhibited by comparatively low concentrations of catecholestrone.     

The efficacy of a single intraperitoneal injection of either flumequine or oxytetracycline hydrochloride in prevention of outbreaks of atypical Aeromonas salmonicida infection in goldsinny wrasse, Ctenolabrus rupestris L., following stress.

In this investigation, the efficacy of a single intraperitonealinjection of either flumequine or oxytetracycline hydrochloride to preventoutbreak of atypical Aeromonas salmonicida infection ingoldsinny wrasse, Ctenolabrus rupestris, following stresswas studied. Six groups of goldsinny wrasse, each of 50 individuals, weretreated with an intraperitoneal injection of either propylene glycol : saline(50 : 50), 200 mg kg^–1 of oxytetracycline or 50mg kg^–1 of flumequine dissolved in propyleneglycol : saline (50 : 50). Three days following medication the fish in allgroups were stressed by an intraperitoneal injection of prednisolone acetate(0.05 ml) and a rise in seawater temperature from 9 to 11°C. Mortality was observed daily for 21 days. Flumequine wasthe more effective with a mean cumulative mortality of 5% compared tooxytetracycline with 54%. The mean cumulative mortality in the unmedicatedcontrol groups was 84%. Bacterial examination of kidneys from dead fishconfirmed the presence of atypical A. salmonicida as theprobable cause of death. Minimum inhibitory concentration (MIC) values forflumequine and oxytetracycline against the isolated A.salmonicida were determined to 0.13 gml^–1, and 2.0 g ml^–1,respectively.