Characterization of a pancreatic DNase from pyloric caeca of atlantic cod (Gadus morhua L.)

An alkaline deoxyribonuclease (DNase) from cod pancreatic tissue has been characterized. The enzyme is a DNase I type endonuclease and hydrolyzes effectively both native and denatured DNA. Monomeric actin inhibits the enzyme reaction. The enzyme obeys Michaelis-Menten kinetics and the apparent K_m value for native linear duplex DNA is 33 µg/ml. The cod DNase opens supercoiled plasmid DNA, by introducing adjacent nicks in both strands, possibly separated by 5–10 nucleotides. DNA hydrolyzed by cod DNase functions as substrates both for DNA polymerase and ligase, and the nicks therefore contain 5-phosphoryl and 3-hydroxyl groups. Optimum concentrations of divalent cations are 5 mM Mg²⁺, 0.63 mM Mn²⁺ and 0.075 mM Ca²⁺. However, Ca²⁺ is apparently not essential for the enzymatic functions. The enzyme has a narrow temperature optimum at 42°C and is thermolabile above 50°C; however, Mn²⁺ shifts the optimum slightly to 45°C by causing increased temperature stability. The cod DNase reaction is inhibited by the DNA intercalating compounds actinomycin D and ethidium bromide. Histidine-modifying reagents such as tosyl phenylalanyl chloromethylketone and diethyl pyrocarbonate inhibit the enzyme activity, but the cod DNase is insensitive to disulfide-reducing agents.     

Purification and characterization of phospholipase A2 from the pyloric caeca of red sea bream, Pagrus major

A phospholipase A₂ was purified 55,000-fold in a yield of 10% from the lipid-free extract of powder of the pyloric caeca of red sea bream to near homogeneity by sequential column chromatography on S-sepharose fast flow, butyl-cellulofine, Asahipak ES-502C cation-exchange HPLC, TSK gel G3000SW gel-filtration HPLC, and Asahipak ODP-50 reversed-phase HPLC. The final preparation showed a single band with the apparent molecular mass of 14 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an estimated specific activity was 717 µmol min^-1 mg^-1 protein. The purified enzyme had a pH optimum in the range of pH 8.0–9.0 and required the presence of both 8 mM of Ca²⁺ and from 2 to 10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine as a substrate. The purified enzyme preferentially hydrolyzed the 2-acyl ester bonds of both phosphatidylglycerol and phosphatidylcholine in the presence of sodium deoxycholate, followed in order by phosphatidylethanolamine and phosphatidyl-serine. In contrast to porcine pancreatic PLA₂, pyloric caeca PLA₂ hydrolyzed mixed-micellar phosphatidylcholine substrate effectively, regardless of the kinds of bile salts used. These results indicate that Ca²⁺-dependent low molecular mass PLA₂, so called secretory PLA₂, occurs in the pyloric caeca of red sea beam.     

Glutathione and its Related Enzymes in the Nile Fish

Glutathione (GSH) and related enzymes, glutathione transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) form an important phase 2 biotransformation enzymes system. The objective of this study was to compare this enzymes system in three fish species from the river Nile, Oreochromis niloticus, Claris lazera and Cyprinus carpio in order to establish the main differences and to purify and characterize GST from the liver of O. niloticus.The level of GSH and the activity of GST, GPx and GR in the liver, kidney and gills of the three fish species were examined. A simple reproducible procedure for the purification of GST from the liver of O. niloticus to homogeneity, which includes chromatography on DEAE- cellulose followed by affinity chromatography on GSH-sepharose was established. The molecular mass was found to be 25,460 Da by SDS-PAGE. The Michaelis-Meneten constants (K_m) of the enzyme for GSH and CDNB were 0.35 mM and 0.42 mM, respectively. The affinity purified enzyme exhibited maximum pH at pH 8.0 and increasing pH above 8.0 did not affect the observed maximum. The purified enzyme acts readily on CDNB, less readily on some standard transferase substrates (1,2-dichloro-4-nitrobenzene and p-nitrophenethyl bromide) and not at all on others (bromosulphophthalein and p-nitrobenzyl chloride). Bromosulfophthalein, cibacron blue and hematin inhibited CDNB-conjugating activity of the purified enzyme with IC₅₀ 0.079, 3.98 and 0.126 μM, respectively.     

Effects of genetic strain and holding facility on the characteristics of alkaline phosphatase and brush border enzymes in silver perch (Bidyanus bidyanus)

The activity of the enzymes located in the digestive tract (pyloric caeca and intestine) in two strains and F₁ crosses of the Australian freshwater fish silver perch [Murray River (M) and Cataract Dam (C)] was evaluated. The effect of the fish holding system (cages or ponds) on the activity of alkaline phosphatase (AP) and the brush border enzymes: maltase, leucine amino peptidase (LAP) and γ‐glutamyl transferase (γ‐GT) was examined. The enzymatic activity was tested in three intestinal sections: the pyloric caeca, upper and lower intestine. All the tested enzyme activities were influenced by an interaction between the intestinal section and the strain or cross of silver perch. For fish maintained in ponds, there were two distinct enzymatic activity patterns, one for the pure strains (C × C and M × M) and one for the crosses (C × M and M × C). Significantly higher (P<0.05) enzymatic activities of the crosses compared with the pure strains were particularly noticeable in the pyloric caeca. In fish held in cages, there was a significantly higher (P<0.05) enzymatic activity in the pyloric caeca of the C × C strain. The total enzymatic activities in the fish reared in the ponds were significantly higher (P<0.05) than those in the fish held in cages. The results of the present investigation confirm that the different genetic background of the silver perch in the Murray River and in the Cataract Dam influences the activity of the brush border digestive enzymes. This activity also varies between maintenance systems.     

Do digestive enzymes set a physiological limit on growth rate and food conversion efficiency in the Atlantic cod (Gadus morhua)?

The objective of this study was to determine the potential sites of maximal growth limitation in Atlantic cod (Gadus morhua). Forty cod were reared in ten sea water tanks. Fish were randomly divided into 5 groups, a control group (injected with saline solution) and 4 experimental groups that received different levels of recombinant bovine somatotropin (rbST: 1.0, 2.0, 4.0 and 10.0 g g fish^–1 2 weeks^–1) by injection in the abdominal cavity. Fish were fed ad libitum 3 times a week during four weeks. We measured individual body mass, growth rate, food intake and food conversion efficiency. At the end of the experiment, we measured the activity of acid proteases in the stomach; trypsin and chymotrypsin in the pyloric caeca; alkaline phosphatase (ALP) and glutamyltransferase (GGT) in the intestine. No significant differences were observed in growth rate, food intake or food conversion efficiency among the five groups. Trypsin, chymotrypsin, ALP and GGT activities when expressed in U g fish^–1 were correlated with growth rate. These enzymes were also correlated with food ingestion except for trypsin when expressed in U mg protein^–1. Trypsin was the only enzyme that showed a significant correlation with food conversion efficiency. Our conclusion is that, at the level of digestion, trypsin is the only enzyme measured that could be suspected to potentially limit growth rate in cod.     

Digestive capacity and compensatory growth in Atlantic cod (Gadus morhua)

The objective of this study was to examine whether digestive capacity correlates with growth rate in Atlantic cod (Gadus morhua). Ninety fish (1035±240 g; 478±36 mm) were assigned to one of three treatments: deprived of food for 5 weeks and then re-fed for 24 days, deprived for 10 weeks and then re-fed for 24 days, or fed for 24 days without deprivation (controls). Three times a week during feeding, the fish were provided with meals of capelin (Mallotus villosus) in excess. Within each treatment, ten fish were sacrificed before and twenty after the feeding period. The relative masses of the pyloric caeca, intestine, and white muscle were determined. Cytochrome c oxidase (CCO), citrate synthase (CS), and nucleoside diphosphate kinase (NDPK) activities were assayed in the pyloric caeca, intestine, and muscle as was trypsin activity in the pyloric caeca. During the re-feeding period, fish that had been deprived of food for 10 weeks showed compensatory growth, growing 1.9 times faster than the controls (0.94±0.26 versus 0.50±0.31% body mass ⋅ day⁻¹). Fish that displayed compensatory growth had a lower relative white muscle mass than controls (66.5±4.1% vs. 69.6±7.4%) while the relative masses of the pyloric caeca (2.21±0.49% vs. 1.78±0.31%) and intestine (0.95±0.20% vs. 0.77±0.15%) were greater than the controls, suggesting that the sizes of these digestive tissues might correlate with compensatory growth capacity. Citrate synthase in the pyloric caeca was the only enzyme that showed higher activity during compensatory growth (8.57±0.94 U ⋅ g tissue⁻¹ compared to 7.13±1.03 U ⋅ g tissue⁻¹ in the control group). This suggests that aerobic catabolic capacity of pyloric caeca could be related to growth capacity during recovery, possibly via the energetic cost of digestive enzyme synthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Loma morhua (Microsporidia) infection on the cardiorespiratory performance of Atlantic cod Gadus morhua (L).

The microsporidian Loma morhua infects Atlantic cod (Gadus morhua) in the wild and in culture and results in the formation of xenomas within the gill filaments, heart and spleen. Given the importance of the two former organs to metabolic capacity and thermal tolerance, the cardiorespiratory performance of cod with a naturally acquired infection of Loma was measured during an acute temperature increase (2 °C h^?1) from 10 °C to the fish's critical thermal maximum (CT_Max). In addition, oxygen consumption and swimming performance were measured during two successive critical swimming speed (U_crit) tests at 10 °C. While Loma infection had a negative impact on cod cardiac function at warm temperatures, and on metabolic capacity in both the CT_Max and U_crit tests (i.e. a reduction of 30–40%), it appears that the Atlantic cod can largely compensate for these Loma‐induced cardiorespiratory limitations. For example, (i) CT_Max (21.0 ± 0.3 °C) and U_crit (~1.75 BL s^?1) were very comparable to those reported in previous studies using uninfected fish from the same founder population; and (ii) our data suggest that tissue oxygen extraction, and potentially the capacity for anaerobic metabolism, is enhanced in fish infected with this microsporidian.     

Processed soybean meal as an alternative protein source for yellow perch (Perca flavescens) feed

The goal of this study was to compare conventional soybean meal (SBM) with modified SBM (MSBM) after chemical and enzyme pretreatment to potentially reduce the antinutritional factors (ANF), as a fishmeal (FM) replacer at 50% or 100% in the diets of yellow perch (Perca flavescens). Half of the SBM and MSBM diets contained an enzyme cocktail (of phytase and carbohydrate‐degrading enzyme), and the other half received no enzyme supplementations. Fingerlings (297; initial weight, 11.01 ± 0.19 g) were randomly distributed over nine treatments, in triplicate, and fed these isonitrogenous diets (crude protein 410 g/kg) that included replacing FM with SBM at 50% or 100% without or with enzyme supplementations (S50, S100, S50+E or S100+E, respectively), MSMB at 50% or 100% without or with enzyme supplementations (MS50, MS100, MS50+E or MS100+E, respectively) or a control FM‐based diet. After 10 weeks, the growth performance, feeding efficiencies, proximate composition, intestinal/pyloric caeca digestive enzymes and liver metabolic/antioxidant enzymes in P. flavescens were measured. The highest (p < 0.05) growth performance and nutrient utilization parameters (protein efficiency ratio and protein productive value, PPV) were observed for the MS_50+E group, which was not statistically different to parameters for the control and MS₅₀ groups, and significantly (p < 0.05) higher than all other groups. The lowest and highest growth performance and feed conversion ratios, respectively, were observed in the S100, S100+E and MS100 groups. The highest protease activity (in both intestine and pyloric caeca) was observed for the control group, but was significantly similar to MS₅₀, MS_50+E and S_50+E groups. The lowest value was observed for 100% replacement of FM protein by SBM and MSBM fed groups. However, inclusion of exogenous enzymes in feed showed positive effects in MS_50+E (compared to MS₅₀) for PPV, lipid productive value and amylase activity in the intestine. Activity of protein metabolism enzymes (i.e., alanine transaminase and aspartate aminotransferase) in the liver was the highest in the control group, which was similar (p < 0.05) to the MS_50+E and MS₅₀ groups. Antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) in the liver were the lowest in the control group, which was significantly similar (p < 0.05) to the group given the feed with 50% replacement of FM protein. Complete (100%) replacement of FM protein exhibited the highest antioxidant enzyme activity. Conclusively, performance of the MS₅₀ and MS_50+E groups was similar to the FM group and better than SBM group; therefore, MSBM with high protein and low ANFs has considerable potential as an alternative to FM in aquafeed.     

Presence and ontogeny of intestinal and pancreatic phospholipase A2-like proteins in the Red Sea bream,Pagrus major. An immunocytochemical study

We have studied the location and the ontogeny of the digestive enzyme, phospholipase A₂ (PLA₂) immunohistochemically in the adult and larvae/juvenile of the red sea breamPagrus major by using an antiserum against theNaja naja venom PLA₂. The antiserum reacts with at least one enzyme among the PLA₂s purified from the fish hepatopancreas or intestine. Although the reactivities were comparatively low, it labelled zymogen granules of the pancreatic acinar cells and secretory materials of certain epithelial cells in the depths of epithelial crypts in the pyloric caeca of the adult. The immunoreactivities of PLA₂s were investigated in the viscera of larvae and juveniles of the 0 to 85^th day after hatch. In the larvae of the 13^th day, accumulation of PLA₂-positive zymogen granules in the pancreatic acinar cells were first recognized by the immunostaining. The intensity of the labelling subsequently became stronger and dramatically increased between the 20^th and 30^th day. This increase appeared to be one of the physiological changes associated with the transition to a new benthic life as juveniles. Lack of PLA₂ in the pancreas before the 13^th day may suggest the possibility that larvae utilized exogenous PLA₂, inherent in their prey, to digest the phospholipids. On the other hand, no reactivity was found in the intestine until the 85^th day.     

Trypsin from the viscera of Bogue (<Emphasis Type="Italic">Boops boops</Emphasis>): isolation and characterisation

Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS–PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-l-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants K _m and k _cat on BAPNA were 0.13 mM and 1.56 s⁻¹, respectively, while the catalytic efficiency k _cat /K _m was 12 s⁻¹ mM⁻¹. Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.     

Gluconeogenesis in trout (Oncorhynchus mykiss) white muscle: purification and characterization of fructose-1,6-bisphosphatase activity in vitro

Studies of the enzyme fructose-1,6-bisphosphatase (FBPase) of rainbow trout (Oncorhynchus mykiss) have been undertaken in order to illuminate aspects of skeletal muscle gluconeogenesis in these animals. Maximal activities in crude homogenates of several organs suggest that the liver possesses the greatest FBPase activity on a unit g^–1 tissue basis but that the white muscle, owing to its bulk, contributes substantially to whole body FBPase activity. Studies of fructose-6-phosphate-1-kinase (PFK) and FBPase in crude homogenates of several organs suggests an important role for intracellular pH in regulating the relative carbon flux through the FBPase/PFK locus in vivo. Furthermore, a three-step purification scheme is described for trout white muscle FBPase by which a stable and homogeneous (by SDS PAGE) enzyme preparation (isoelectric point = 7.2; molecular weight = 37.6 kd) was obtained. Kinetic studies of the purified enzyme were undertaken at 20°C under conditions reflective of "rest" and "exercise/recovery" intramuscular pH in vivo. Affinity for substrate (F-1,6-P₂) was increased (Km = 6.88 versus 2.44 mol 1-^–1 as was enzyme activity when pH was lowered from 7.0 to 6.5. Various inhibitor metabolites are identified including F-2,6-P₂ (mixed-type inhibitor, K_i = 0.201 mol 1^–1, pH 7.0) and AMP (non-competitive inhibitor, K_i = 0.438 mol 1^–1, pH 7.0). Inhibition by F-2,6-P₂ was strongly alleviated by a reduction in pH from 7.0 to 6.5 (I₅₀ increased from 0.14 to 0.32 mol 1^–1). AMP on the other hand was a more potent inhibitor at pH 6.5 but this inhibition was totally reversed under conditions of citrate, NH₄ ⁺ and AMP typical of muscle during recovery from exercise in vivo. In purified white muscle enzyme preparations, FBPase demonstrated maximal activity at pH 6.5 whereas the optimal pH of PFK was 7.0 or greater. Indeed, it appears from these in vitro data that regulation by metabolite levels as well as pH are required for net FBPase flux in vivo. It is concluded, therefore that trout white muscle FBPase demonstrates the potential to play an important enzymatic role in the control of intramuscular gluconeogenesis in these animals. The results are discussed in relation to present knowledge regarding the metabolic responses of trout white muscle to, and its subsequent recovery from, exhaustive exercise.     

Anionic Trypsin from the Pyloric Ceca of Pacific Saury (Cololabis saira): Purification and Biochemical Characteristics

Anionic trypsin from Pacific saury (Cololabis saira) pyloric ceca was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. It was purified to 53.7-fold with a yield of 6.1%. The apparent molecular weight of the enzyme was about 24 kDa, as determined by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On native-PAGE, trypsin showed a single band. The purified anionic trypsin displayed optimal activity at pH 8.5 and 55°C. The enzyme was stable at neutral and alkaline pH and in the temperature range of 20–50°C. The stability was affected by the calcium ion. The activity of purified anionic trypsin was completely inhibited by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and partially inhibited by ethylenediaminetetraacetic acid (EDTA). NaCl (0–30%) decreased the activity in a concentration-dependent manner. The kinetic trypsin constants K_m and K_cat were 0.19 mM and 210 s^?1, respectively, while the catalytic efficiency (K_cat/K_m) was 1105.26 s^?1 mM^?1. The N-terminal amino acid sequences of anionic trypsin, IVGGYECQAH, were found and were homologous to those of trypsin from other fish species.     

Growth and nutrient utilization of Murray cod Maccullochella peelii peelii (Mitchell) fingerlings fed diets with varying levels of soybean meal and blood meal

Abstract The Australian native freshwater fish Murray cod, Maccullochella peelii pellii (Mitchell), currently supports a fledgling inland aquaculture industry, which is thought to have considerable growth potential. The aim of this study was to evaluate the suitability of two alternate protein sources [blood meal (BM) and defatted soybean meal (SBM)] as substitutes for fish meal at various levels of inclusion in diets for juvenile Murray cod. The growth performance of juvenile Murray cod in response to nine isonitrogenous and isocalorific diets (50% protein, 14% lipid, 20.2 kJ g^?1) consisting of a control diet in which protein was supplied from fish meal, and test diets in which the fish meal protein was substituted at levels of 8%, 16%, 24%, and 32% with BM or SBM was evaluated from a 70‐day growth experiment. The per cent apparent dry matter (% ADC_dm) and percentage protein digestibility (% ADC_p) of the test diets were also determined using Cr₂O₃ as a marker. Survival in all the SBM dietary treatments was high but that of fish on the BM dietary treatments was significantly (P < 0.05) lower than in all the other dietary treatments. Specific growth rate (% day^?1) of Murray cod fed SBM incorporated diets ranged from 1.63 ±  0.06 to 1.78 ±  0.10 and even at the highest level tested (32% of the dietary protein from SBM) was not significantly different (P > 0.05) from the fish fed the control diet (1.65 ±  0.09). Feed conversion ratios of the SBM dietary treatments ranged from 1.36 ±  0.08 to 1.45 ±  0.07. The protein efficiency ratios and protein conversion efficiencies of Murray cod in the soybean meal treatments were also good and for a majority of the SBM diets were better than those for the control diet. Per cent ADC_dm and ADC_p of the SBM diets tested ranged from 70.6 ±  1.46 to 72.3 ±  1.81% and 88.6 ±  0.57 to 90.3 ±  0.17%, respectively, and was not significantly different (P > 0.05) from the control diet (% ADC_dm 74.3 ±  1.63; % ADC_p 91.3 ±  0.55). The reasons for significantly poor survival and growth of Murray cod reared on BM incorporated diets, and relatively poor digestibility of these diets are discussed. The study shows that for Murray cod diets in which fish meal protein is substituted up to 32% performance or carcass composition is not compromised.     

Mitochondrial NAD(P)-dependent malic enzyme from herring testicular tissue: Purification,kinetic behaviour and regulatory properties

Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD⁺ oxidoreductase (decarboxylating)] was purified from herring testicular tissue to a specific activity of 26.4 μmol NADH/min/mg protein. Herring testicular tissue is one of the most abundant sources of this enzyme. The purification procedure involved differential centrifugation of mitochondria and then chromatography on DEAE-Sephacel, Red Agarose and Sephacryl S-300. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of Mn²⁺ and either NAD or NADP. Under V_max conditions the ratios for the rate of NAD/NADP reduction was 1.8. A study of the reductive carboxulation reaction indicated that this enzyme reaction is reversible; at pH 7.0 the reverse reaction exhibited 22% of the activity of forward reaction. Some kinetic characteristics of the enzyme were determined. ATP was found to be a competitive inhibitor with respect to malate. Fumarate reversed ATP inhibition. Regulation of NAD(P)-dependent malic enzyme from herring testicular tissue mitochondria could respond to changing levels of mitochondrial ATP and fumarate in vivo.     

Viscera of Labeo rohita: A Potential Source of Trypsin for Industrial Application

ABSTRACT

The serine protease trypsin was isolated and purified from the digestive system of carp Labeo rohita rohu by ammonium sulphate precipitation, ion exchange, and affinity chromatography. The purified enzyme showed high activity between pH 7.0 and 9.0. The activity was maximum at 40°C. Incubation of the purified enzyme with CaCl₂ (2 mM) stabilized the enzyme activity for 8 h. The enzyme showed stability at 30 and 40°C for 1 h, but above 40°C, enzyme activity was reduced. The kinetic constants were recorded as K_m (0.104 mM), k_cat (44.25 s^?1), and catalytic efficiency (427.54 s^?1 mM^?1). Monovalent, bivalent, and trivalent ions (Li⁺, K⁺, Hg²⁺, Al³⁺, Mg^2+, Cd^2+, Co^2+, Zn²⁺, and Al³⁺) influenced the enzyme activity. Phenylmethylsulfonylflouride, soybean trypsin inhibitor, and N-α-p-tosyl-_L-lysine chloromethyl ketone completely inhibited the enzyme activity, while ethylenediaminetetraacetate caused partial inhibition. Molecular mass of the purified enzyme was 22.46 kDa. The pH and temperature stability of enzyme may be useful for its industrial applications.     

Yersiniosis in Atlantic cod,Gadus morhua (L.), characterization of the infective strain and host reactions

A disease outbreak in farmed Atlantic cod caused by Yersinia ruckeri is reported. Mortality started following vaccination of cod reared in two tanks (A and B). The accumulated mortality reached 1.9% in A and 4.8% in B in the following 30 days when treatment with oxytetracycline was applied. Biochemical and molecular analysis of Y. ruckeri isolates from the cod and other fish species from fresh and marine waters in Iceland revealed a high salinity‐tolerant subgroup of Y. ruckeri serotype O1. Infected fish showed clinical signs comparable with those of Y. ruckeri ‐infected salmonids, with the exception of granuloma formations in infected cod tissues, which is a known response of cod to bacterial infections. Immunohistological examination showed Y. ruckeri antigens in the core of granulomas and the involvement of immune parameters that indicates a strong association between complement and lysozyme killing of bacteria. Experimental infection of cod with a cod isolate induced disease, and the calculated LD₅₀ was 1.7 × 10⁴ CFU per fish. The results suggest that yersiniosis can be spread between populations of freshwater and marine fish. Treatment of infected cod with antibiotic did not eliminate the infection, which can be explained by the immune response of cod producing prolonged granulomatous infection.     

Does the aerobic capacity of fish muscle change with growth rates?

To ascertain whether growth rate modifies the oxidative capacity of fish white muscle, we examined the effects of individual growth rate on the activities of four mitochondrial enzymes in white muscle of the fast growing Atlantic cod,Gadus morhua. Growth rates were individually monitored in cod held at three acclimation temperatures during experiments repeated in four seasons. The size dependence of citrate synthase (CS), cytochrome C oxidase (CCO) and β-hydroxyacyl CoA dehydrogenase (HOAD) activities was established using wild cod ranging from 115 to 17,350 g. Given their negative allometry, CS and CCO activities in the experimental cod were corrected to those expected for a 1.2 kg animal. HOAD activities did not change with size. The specific activities of CCO and CS were positively correlated with growth rate. However, for both enzymes, season explained more of the variability than growth rate or temperature. Season was the only factor to significantly affect the activity of HOAD, while temperature and season interacted to determine glutamate dehydrogenase activity. CS activity was positively correlated with the initial condition of the cod, which differed among the seasons. The other enzymes did not show this relationship. The independent changes of these enzymes suggest that mitochondria undergo qualitative modifications with changes in growth rate, season and size. Although growth rate and the activities of CCO and CS are positively correlated, the activity of the mitochondrial enzymes is more affected by size, physical condition and season.     

Fate of plasma glucose in tissues of brown trout in vivo: effects of fasting and glucose loading

We report the fate of glucose, both as a source of energy and as a temporary store, in the tissues of brown trout (Salmo trutta) in control, fasted and glucose-loaded fish. Tissue glucose utilization (³H-2-deoxyglucose phosphorylation) and storage (conversion of ¹⁴C-glucose into glycogen, protein, and lipid) were measured in immature brown trout, and the oxidation rate was calculated as glucose utilization minus storage and ¹⁴C-ionic metabolites remaining in the tissue. The glucose utilization rate is tissue-specific, the highest values being found in spleen, kidney, hindgut, brain, and gill. All these tissues also showed a highly active glycolytic pathway. The lowest utilization indices were observed in white and red muscles, skin, stomach and caeca, which also presented the largest proportion of glucose converted into stores (mainly protein and glycogen). Fasting reduced the glucose disappearance rate by 24%, although there were no significant variations in glucose utilization indices or distribution profile. After a glucose load, plasma glucose and insulin levels rose and the rates of glucose utilization, storage, and oxidation also increased in all tissues (from 1.5- to 4-fold). The relative importance of each tissue in glucose disposal was similar to that in normoglycaemia. In liver, only glucose storage was measured reliably; the conversion of glucose to glycogen was higher than in other tissues, and rose markedly (35-fold) in glucose-loaded fish. In most tissues glucose flux into lipids, glycogen and protein increased. The distribution of glucose may not be a merely substrate-mediated process because fasting in glucose-loaded fish caused lower tissue glucose utilization, particularly in gut, red muscle and gills. Conversion of glucose to tissue stores was reduced, lipids being the most affected.     

Trypsin Enzyme from Viscera of Common Kilka (Clupeonella cultriventris caspia): Purification,Characterization, and Its Compatibility with Oxidants and Surfactants

The purification of trypsin from the common kilka (Clupeonella cultriventris caspia) viscera (pyloric caeca) resulted in a 28.3-fold increase and 12% yield by ammonium sulfate precipitation (30–60%), Sephadex G-75, and DEAE-cellulose chromatography. Trypsin showed a molecular weight of 23.2 kDa and appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, and zymography. The trypsin had optimal activity at pH 8.0 and 60°C for the hydrolysis of α-N-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) substrate. Trypsin was stable up to 50°C and at pH range of 7.0–10.0. Activity was significantly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) inhibitors (p < 0.05). The enzyme was relatively stable toward oxidizing agents, retaining 59.7 and 98.0% of its initial activity after 1 h incubation in the presence of 15% H₂O₂ and 1% sodium perborate, respectively. Trypsin was significantly activated by surfactants and Ca²⁺, Mg²⁺, and Mn²⁺ and inactivated by Fe²⁺, Zn²⁺, Cu²⁺, Al³⁺, Ba²⁺, and Co²⁺ (p < 0.05). Nevertheless, Na⁺ and K⁺ had no significant effect on trypsin activity (p > 0.05). The purified trypsin showed significantly higher catalytic efficiency (k_cat/K_m) than porcine pancreatic trypsin against BAPNA and N-α-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) substrates (p < 0.05).     

Growth and lipid composition of Atlantic cod (Gadus morhua) larvae in response to differently enriched Artemia franciscana

Considerable progress has been achieved in the intensive culture of Atlantic cod (Gadus morhua). However, there is little information concerning optimum live-feed enrichments for cod larvae, since many of the techniques used during the larviculture have been borrowed from other fish species and adapted for the production of Atlantic cod. The present study compared four different protocols for the enrichment of Artemia to be used as live feed for cod larvae. The protocols tested were: (1) AlgaMac 2000, (2) AquaGrow Advantage, (3) Pavlova sp. + AlgaMac 2000, and (4) DC DHA Selco + AlgaMac 2000. Larvae were fed differently enriched Artemia between 37 and 59 days post hatch. At the end of the experiment, larvae from treatment 1 [specific growth rate (SGR) = 10.4 ± 0.4% day⁻¹] grew faster than larvae from treatments 3 (SGR = 6.9 ± 0.2% day⁻¹) and 4 (SGR = 4.9 ± 0.4% day⁻¹, P < 0.0001). However, treatments 3 and 4 resulted in better larval survival at the end of the experimental period, estimated to be 3 on a scale from 1 to 5, whereas the survival estimates for the two other groups were 2. The treatments affected the fatty-acid composition of Artemia and of cod larvae. Larvae from treatment 1 had a higher percentage of AA (20:4ω6, P < 0.0001) and ω6DPA (22:5ω6, P < 0.0001) than the other larvae. Levels of DHA (22:6ω3) were similar in larvae from treatments 1 and 4, and higher than in the other larvae (P < 0.0001). Our results suggest that Artemia containing a DHA/EPA/AA ratio of 7/2/1 result in good larval performance. Joseph A. Brown—Deceased September 2005.