Dietary P regulates phosphate transporter expression,phosphatase activity,and effluent P partitioning in trout culture

Phosphate utilization by fish is an important issue because of its critical roles in fish growth and aquatic environmental pollution. High dietary phosphorus (P) levels typically decrease the efficiency of P utilization, thereby increasing the amount of P excreted as metabolic waste in effluents emanating from rainbow trout aquaculture. In mammals, vitamin D₃ is a known regulator of P utilization but in fish, its regulatory role is unclear. Moreover, the effects of dietary P and vitamin D₃ on expression of enzymatic and transport systems potentially involved in phosphate utilization are little known. We therefore monitored production of effluent P, levels of plasma vitamin D₃ metabolites, as well as expression of phosphatases and the sodium phosphate cotransporter (NaPi2) in trout fed semipu diets that varied in dietary P and vitamin D₃ levels. Mean soluble P concentrations varied markedly with dietary P but not with vitamin D₃, and constituted 40–70% of total effluent P production by trout. Particulate P concentrations accounted for 25–50% of effluent P production, but did not vary with dietary P or vitamin D₃. P in settleable wastes accounted for <10% of effluent P. The stronger effect of dietary P on effluent P levels is paralleled by its striking effects on phosphatases and NaPi2. The mRNA abundance of the intestinal and renal sodium phosphate transporters increased in fish fed low dietary P; vitamin D₃ had no effect. Low-P diets reduced plasma phosphate concentrations. Intracellular phytase activity increased but brushborder alkaline phosphatase activity decreased in the intestine, pyloric caeca, and gills of trout fed diets containing low dietary P. Vitamin D₃ had no effect on enzyme activities. Moreover, plasma concentrations of 25-hydroxyvitamin D₃ and of 1,25-dihydroxyvitamin D₃ were unaffected by dietary P and vitamin D₃ levels. The major regulator of P metabolism, and ultimately of levels of P in the effluent from trout culture, is dietary P.Communicated by: C.-H. Wang     

Accumulation and effects of aluminium in the minnow (Phoxinus phoxinus L.) at different pH levels

The accumulation and effects of 150 pg All^-1 on minnows ( Phoxinus phoxinus L.) were studied in soft water at pH 7, 6 and 5. The fish were kept in a flow-through water system for up to 48 days. Addition of aluminium to the water resulted in poor appetite, passivity and a tendency for the fish to gather as far as possible from the aluminium inlet. The mortality was high at pH 5 and also after addition of aluminium at pH 6. Severe lesions were noted in the gills and olfactory organs after exposure to aluminium. The concentration of aluminium in the gills, liver and kidney was increased at low pH. A large proportion of the aluminium was located on the surface of the gill epithelium. The recovery capacity was pronounced when the aluminium exposure was terminated and the pH was increased from 5 to 7. No further mortality occurred and gill and olfactory organ structure returned to near normality in about 36 days.     

Enzyme histochemistry of the sheep uterus during the oestrous cycle

Enzyme histochemical techniques were applied to frozen sheep uteri from different stages of the oestrous cycle. The localization and activities of succinate, lactate, glucose-6-phosphate, and isocitrate (NADP+) dehydrogenases and acid and alkaline phosphatases were studied in the luminal and glandular epithelia, caruncle and myometrium. Enzyme activity in the sections was scored on a scale of 0--5. In general the enzyme activity in the uterine caruncles and epithelia was higher than in the myometrium. The myometrium did not show any alkaline phosphatase activity and isocitrate dehydrogenase (NADP+) activity was negligible. The low activities of acid phosphatase and lactate dehydrogenase and the moderate levels of glucose-6-phosphate and succinate dehydrogenases in the myometrium were constant. The caruncular tissue showed high levels of phosphatases and glucose-6-phosphate dehydrogenase, moderate levels of lactate and succinate dehydrogenases, and low levels of isocitrate dehydrogenase (NADP+) throughout the oestrous cycle. Much lower phosphatase and isocitrate dehydrogenase (NADP+) levels were found in the epithelium of deep glands compared with superficial glands. The high activity of acid and alkaline phosphatases in the luminal epithelium and the superficial glands was constant from mid-cycle to ovulation, but a significant decrease was observed immediately after ovulation. The level of dehydrogenases in epithelia was generally high and did not change during the oestrous cycle.     

Measurement of the viability of arbuscular-mycorrhizal fungi using three different stains; relation to growth and metabolic activities of soybean plants

Histochemical staining of alkaline phosphatase (ALP) and succinate dehydrogenase (SDH) activities in four arbuscular mycorrhizal fungi (Glomus intraradices, G. fasciculatum, G. monosporum and G. mosseae) and their relation to growth and metabolic activities of soybean plants were investigated in a greenhouse experiment. In general, mycorrhizal inoculation significantly increased the growth responses, phosphorus and nitrogen contents, acid and alkaline phosphatases as well as total soluble protein of soybean compared to non-mycorrhizal plants. Stimulation was related to the viability of each mycorrhizal fungus. The localization of succinate dehydrogenase (as a vital stain of metabolically active fungus) and alkaline phosphatase activity (as a potential marker of efficiency of the symbiosis) in the arbuscular mycorrhizal fungi were variable. The activity appeared in young arbuscles and intercellular hyphae, whereas the collapsed arbuscules were inactive. The histochemical staining results demonstrated that the activity of alkaline phosphatase fungi was lower than succinate dehydrogenase. The use of nitroblue tetrazolium chloride as a vital stain for SDH activity showed that all mycorrhizal infection revealed by trypan blue staining was not physiologically active. Thus, the possible utilization of these enzymes to assess the activity of mycorrhizal fungi and its relation with effectively for plant growth and mineral contents is discussed.     

Isoenzymes of carbonic anhydrase of an euryhaline fish. Variations related to the osmoregulation]

A chemical study of carbonic anhydrase (EC 4.2.1.1) from the red blood cells and the gills of an euryhaline fish (Anguilla anguilla) is presented. Animals adapted to fresh water were compared to those adapted to sea water. The physiochemical constants of the various molecular formsisolated by chromatography and isoelectric focusing were determined; isoelectric pH, molecular weight, and the Km and V/E of the enzyme dehydration activity were compared. In both red cells and gills of fish adapted in either media various forms were isolated, characterized by different enzymatic kinetics (high- and low- activity forms) but having the same molecular weight (27 250). Some isoenzymes isolated from the gills differed significantly from those isolated from the red cells. Adaptation to fresh water or sea water is accompanied by modifications in the distribution of the isoenzymes in both red cells and gills: adaptation to sea water is characterized by a shift of the molecular forms towards an isoelectric pH higher than pH equals 6. The role of these enzymes is discussed under both a physiological and biochemical point of view in relation to the electrolyte exchange across fish gill. The origin of the different molecular forms of the carbonic anhydrase is discussed in relation to the prevailing theories on this subject.     

Application of different test systems and biochemical indicators for environmental monitoring of the Troitsa Bay,Sea of Japan

The DNase express test system and the SF-bioassay (sea urchin sperm fertilization bioassay) were used to assess the quality of sea and bottom sediment water sampled at 8 stations of the Troitsa Bay (Possyet Bay, Sea of Japan). The specific activities of 8 enzymes (alkaline and acid DNases, RNases, phosphatases and phosphodiesterases) were determined for the liver and gill tissues of the mussel Crenomytilus grayanus sampled at the same stations. The activities of all enzymes were higher in the liver than in the gills, and the activity of acid nucleases in both tissues was significantly higher than that of alkaline nucleases. The specific activities of some nucleases in the mussel tissues correlated to the level of seawater pollution. The activities of acid RNases and phosphodiesterases decreased in both tissues upon an increase in water pollution, up to 1.5–3 times in the gills, while the activities of alkaline and acid phosphatases and phosphodiesterases increased 1.5–4 times. Thus, the specific activities of acid RNases, phosphatases and phosphodiesterases from mussel liver can be a useful indicator of marine pollution. This approach can be used for biotesting of ocean water and for assessment of the biological condition of invertebrates adapting to various ecological and anthropogenic effects.     

Activity of some enzymes during growth and sporulation of Clostridium botulinum type E

The activities of alkaline and acid phosphatases, glucose dehydrogenase and NADH oxidase were assayed in cell-free extracts of sporogenic and asporogenic mutants of Clostridium botulinum. During growth of both mutants, the activities of alkaline and acid phosphatases were relatively constant, but during sporulation of the sporogenic mutant, the alkaline phosphatase activity rose to a maximum of 70 mol/min·mg protein whereas the acid phosphatase decreased rapidly before it increased, indicating a possible role in sporogenesis. Glucose dehydrogenase activity was detected only in cell-free extracts of the sporogenic mutant and reached a maximum of 7 mol/min·mg protein during the endospore maturation stage. The NADH oxidase activity was detected in both mutants. The NADH oxidase seems to stimulate glucose oxidation in both mutants during growth and the dehydrogenation processes of the butyric type of fermentation during spore formation in the sporogenic mutant. The findings suggest that increased glucose dehydrogenase activity in C. botulinum, as in Bacillus species, may serve as a spore event marker and that alkaline and acid phosphatases may play a regulatory role in anaerobic sporulation metablolism.This work was supported by the Aquatic Biology Research Unit of the University of Manitoba from a Federal Fisheries Research Grant.     

Regulation of succinate oxidation by NAD+ in mitochondria purified from potato tubers

NAD⁺ supplied to purified Solanum tuberosum mitochondria caused progressive inhibition of succinate oxidation in State 3. This inhibition was especially pronounced at alkaline pH and at low succinate concentrations. Glutamate counteracted the inhibition. NAD⁺ promoted oxaloacetate accumulation in State 3; supplied oxaloacetate inhibited O₂ uptake in the presence of succinate much more severely in State 3 than in State 4. NAD reduction linked to succinate oxidation by ATP-dependent reverse electron transport was likewise inhibited by oxaloacetate. We conclude that NAD⁺-induced inhibition of succinate oxidation is due to an inhibition of succinate dehydrogenase resulting from increased accumulation of oxaloacetate generated from malate oxidation via malate dehydrogenase. The results are discussed in the context of the known regulatory characteristics of plant succinate dehydrogenase.     

Isoenzymes de l'anhydrase carbonique d'un poisson euryhalin: Variations en relation avec l'osmoregulation

A chemical study of carbonic anhydrase (EC 4.2.1.1) from the red blood cells and the gills of an euryhaline fish (Anguilla anguilla) is presented. Animals adapted to fresh water were compared to those adapted to sea water.The physicochemical constants of the various molecular forms isolated by chromatograhy and isoelectric focusing were determined; isoeleletric pH, molecular weight, and the K_m and V/E of the enzyme dehydration activity were compared.In both red cells and gills of fish adapted in either media various forms were isolated, characterized by different enzymatic kinetics (high- and low-activity forms) but having the same molecular weight (27 250). Some isoenzymes isolated from the gills differed significantly from those isolated from the red cells.Adaptation to fresh water or sea water is accompanied by modifications in the distribution of yhe isoenzymes in both red cells and gills: adaptation to sea water is characterized by a shift of the molecular forms towards an isoelectric pH higher than pH = 6.The role of these enzymes is discussed under both a physiological and biochemical point of view in relation to the electrolyte exchange across fish gill. The origin of the different molecular forms of the carbonic anhydrase is discussed in relation to the prevailing theories on this subject.     

Enzymatic responses of transplanted tumour cells towards estrogen, progesterone and testosterone.

The influence of estrogen, progesterone and testosterone on the activities of alkaline and acid phosphatases, adenosine triphosphatase and succinate dehydrogenase were determined by cytochemical methods in sarcoma 180 and Ehrlich's carcinoma cells transplanted in male and female Swiss mice. The results revealed differential effects of the sex hormones on different enzymes which seemed to depend on the type of tumour cell studied and the sex of the host mice.     

Phosphatases; origin,characteristics and function in lakes

Phosphatases catalyze the liberation of orthophosphate from organic phosphorus compounds. The total phosphatase activity in lake water results from a mixture of phosphatases localized on the cell surfaces of algae and bacteria and from dissolved enzymes supplied by autolysis or excretion from algae, bacteria and zooplankton. External lake water phosphatases usually have pH optima in the alkaline region. Acid phosphatases generally seem to be active in the internal cell metabolism. The synthesis of external alkaline phosphatases is often repressed at high phosphate concentrations and derepressed at low phosphate concentrations. Phosphatase activity has therefore been used as a phosphorus deficiency indicator in algae and in natural plankton populations. The possibilities for this interpretation of phosphatase activity in lake water are limited, however, and this is discussed. The in situ hydrolysis capacity, i.e. the rate by which orthophosphate is released from natural substrates, is unknown. However, we advocate that this process is important and that the rate of substrate supply, rather than phosphatase activity, limits the enzymatic phosphate regeneration.     

Active ammonia excretion in the giant mudskipper, Periophthalmodon schlosseri (Pallas), during emersion

The main objective of this study was to determine whether active NH(4) (+) excretion occurred in the giant mudskipper, Periophthalmodon schlosseri, during emersion. Our results demonstrated that continual ammonia excretion in P. schlosseri during 24 hr of emersion resulted in high concentrations ( approximately 30 mmol l(-1)) of ammonia in fluid collected from the branchial surface. For fish injected intraperitoneally with 8 mumol g(-1) ammonium acetate (CH3COONH4) followed by 24 hr of emersion, the cumulative ammonia excreted was significantly greater than that of the control injected with sodium acetate. More importantly, the ammonia excretion rate at hour 2 in fish injected with CH3COONH4 followed by emersion was greater than that in fish immersed in water as reported elsewhere, with the greatest change in the ammonia excretion rate occurring at hour 2. Assuming that the rate of endogenous ammonia production remained unchanged, 33% of the exogenous ammonia was excreted through the head region, presumably through the gills, during the first 6 hr of emersion. Indeed, at hour 6, the ammonia concentration in the branchial fluid increased to an extraordinarily high concentration of >90 mmol l(-1). Therefore, our results confirm for the first time that P. schlosseri can effectively excrete a high load of ammonia on land, and corroborate the proposition that active NH(4) (+) excretion through its gills contributes in part to its high tolerance of aerial exposure. Only 4.6% of the exogenous ammonia was detoxified to urea. The glutamate contents in the muscle and liver also increased significantly, but the glutamine contents remained unchanged.     

Water pH and metabolic parameters in silver catfish (Rhamdia quelen)

This study evaluated the effects of an acute change in water pH (from pH 7.5 to 4.0, 5.0, 6.0, 7.5, 8.0 or 9.0) on several biochemical parameters in juveniles of the silver catfish, Rhamdia quelen. Ammonia levels decreased in the liver and increased in the muscle with increasing water pH. In the kidney, lower ammonia levels were observed at neutral pH. An increase in water pH decreased the glucose, glycogen and lactate levels in the liver and kidney (except for glycogen levels in the kidney and lactate levels in the liver, which presented lower levels at neutral pH). In muscle, the glucose and glycogen levels decreased with increasing water pH, whereas lactate levels tended to be lower at neutral pH. Gill and kidney Na⁺/K⁺-ATPase activities tended to increase in alkaline water, and the highest value was observed in fish exposed to pH 9.0. The optimal levels of the analyzed biochemical parameters occurred at neutral pH. In conclusion, exposure to acidic and alkaline pH changes the metabolic parameters of silver catfish as well as gill Na⁺/K⁺-ATPase activity.     

The effects of Heterosigma akashiwo on juvenile Oncorhynchus tshawytscha and its implications for fish culture

The effects of natural blooms of Heterosigma akashiwo on freshwater-and saltwater-acclimated juvenile chinook salmon were assessed. Rates of fish mortality in the blooms were independent of acclimation of fish to seawater and the ambient oxygen levels, but were dependent on concentration of algae and ambient water temperatures. No pathological abnormality to gills or other internal organs in the fish were evident. Aeration or oxygenation of fish cages did not enhance or inhibit fish survival in a H. akashiwo bloom. Cause of death was considered to be due to a labile ichthyotoxic agent.     

Permethrin- and malathion-induced macromolecular abnormalities in adult Tribolium castaneum (herbst)

In Tribolium castaneum adults, sublethal doses of 1 and 2 ppm permethrin and 300 ppm malathion led to significant changes in amylase, trehalase, acid phosphatase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and isocitrate dehydrogenase activities. Malathion at 150 ppm did not affect phosphatases and lactate dehydrogenase. Both malathion and permethrin significantly increased cholinesterase activity. Mixing of the two insecticides resulted in antagonistic action with reference to various enzymatic activities. Glucose and glycogen contents were at first mobilized for energy supply under insecticidal stress conditions followed by lipid and cholesterol. Soluble protein, total protein, free amino acids, and urea contents remained unaltered under all experimental conditions.     

Quantitative succinate-dehydrogenase histochemistry. I. A Methodological study on mammalian and fish muscle.

In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are chosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.     

Acid and alkaline phosphatase activities in the clam Scrobicularia plana: kinetic characteristics and effects of heavy metals

The acid and alkaline phosphatase activities of the clam Scrobicularia plana have been partially characterised in different organs and tissues (digestive gland, gills, foot, siphon and mantle) and the 'in vitro' effect of heavy metals on both types of enzymatic activity have been analysed. The optimal pH ranged between 4.0 and 5.5 for acid phosphatase activity and 8.5 and 9.5 for alkaline phosphatase activity. The apparent optimum temperature was in the 30-60 degrees range for acid phosphatase activity and in the 30-40 degrees C range for alkaline phosphatase activity. The effect of substrate concentration on enzymatic activities in the tissues showed a good fit to the Michaelis-Menten model. For both types of enzymatic activity, the highest values were found in the digestive gland. The effect of heavy metals was dependent on the tissue analysed. Mercury showed the highest inhibition in the organs/tissues and the parameters Km and Vmax were modified when the inhibitor concentration increased, thus indicating a mixed type of inhibition.     

Physiological response of the fish, Cyprinus carpio, to formalin exposure--I. Effects of formalin on urine flow, heart rate, respiration.

1. Carp were exposed to 280 ppm formalin. Haematocrit and plasma glucose and lactic acid increased. In moribund fish, blood pH was remarkably lower and plasma protein increased. 2. When exposed to formalin, heart rate (HR) and respiration increased briefly, and then decreased. 3. Some fish increased urine flow (UF) immediately and maintained higher values for a while, followed by gradual decrease, and others decreased UF consistently. UF stopped at 30 min or longer prior to fish death. Urine osmotic pressure was higher at the 1st to 2nd hour and immediately before UF stopped. 4. The relationship between UF, HR and respiration was also discussed.     

Effect of battery-manufacturing effluent on endogenous antioxidant in freshwater fish

Abstract

This study monitors and assesses the effect of battery-manufacturing effluent containing metals Pb, Zn and Cd on endogenous antioxidants. Malonaldehyde (MDA), reduced glutathione sulfyhydryl (GSH) and catalase (CAT) which are known biomarkers of effluent were exposed to 0%, 25%, 50%, 75% and 90% amendments for 74h on the gills, liver and kidneys of C. punctata. There was more metal Zn accumulation in the gills and GSH contents increased significantly in the gills (P<0.01), liver accumulation of Pb was found to be more (P<0.05), whereas lowest accumulation of Pb was found in kidneys and the highest accumulation of Cd (P<0.05). Over all amendments of the effluents, MDA contents were increased in the gills, liver and kidneys (P<0.01). GSH levels were decreased among the liver and kidneys compared to the gills (P<0.01) at 90% amendment. Effluent exposure caused a significant decrease in the activities of CAT in the gills, liver and kidneys (P<0.01, 0.05 and 0.05) of fish. Increased MDA activity was indicative of the formation of free radicals in the fish with exposure to amendments of battery manufacturing effluent, while increased levels of GSH pointed to the occurrence of a scavenging mechanism of free radicals.