Purification and characterization of phospholipase A2 isoforms from the hepatopancreas of red sea bream, Pagrus major1

Two phospholipase A₂ (PLA₂) isoforms, tentatively denoted as DE-1 and DE-2 PLA₂s, were purified from the hepatopancreas of red sea bream (Pagrus major) to near homogeneity by sequential column chromatography on S-Sepharose fast flow, DEAE-Sepharose fast flow and butyl-Cellulofine, and by ion-exchange, gel-filtration and reversed-phase HPLC. The purified DE-1 and DE-2 PLA₂s both showed a single band with the apparent molecular mass of approx. 13.5 kDa by SDS-polyacrylamide gel electrophoresis, and were found to be both related to group I PLA₂ based on the N-terminal amino acid sequences. DE-1 PLA₂ had a pH optimum in the alkaline region at around pH 10 and required approximately 10 mM of Ca²⁺ and 4-10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine and phosphatidylethanolamine as substrates. DE-2 PLA₂ also had a pH optimum in the alkaline region at around pH 8-9 and required >10 mM of Ca²⁺ and approximately 6 mM of sodium deoxycholate for its maximum activity with 2 mM of phosphatidylcholine as a substrate; its enzymatic activity towards phosphatidylethanolamine was greatly inhibited by the addition of sodium deoxycholate. The results demonstrate that red sea bream hepatopancreas contains two enzymatically distinct group I PLA₂ isoforms.     

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.     

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.     

Effect of in vitro exposure to Vibrio vulnificus on hydroelectrolytic transport and structural changes of sea bream (Sparus aurata L.) intestine

The everted gut sac technique has been used to investigate the effect of Vibrio vulnificus on water and electrolyte (Na⁺, K⁺, Cl⁻, HCO₃ ⁻) transport on the intestine of sea bream (Sparus aurata L.). Both the anterior and the posterior intestine were incubated in a medium containing 10⁸ V. vulnificus cells ml⁻¹ at 25°C for 2 h. The presence of V. vulnificus resulted in a significant reduction (P < 0.05) of water absorption in the anterior intestine, while sodium absorption in the anterior (P < 0.01) and posterior (P < 0.05) intestine was elevated. Chloride absorption was increased, but the changed was not significant, while potassium absorption decreased significantly (P < 0.05), but only in the posterior intestine. Incubation the sea bream intestine with V. vulnificus did not affect carbonate secretion in the anterior segment, whereas high secretion was stimulated in the posterior segment (P < 0.01). Histological evaluations demonstrated damage in the anterior intestine of sea bream that was characterized by the detachment of degenerative enterocytes, alterations in the microvilli, and the presence of a heterogenous cell population, indicating inflammation. Based on our results, we conclude that V. vulnificus caused cell damage to the intestine of sea bream and that the anterior intestine is more susceptible than the posterior part of the intestine. Several hypotheses are suggested to explain our observations, such as the presence of higher numbers of villosities in the anterior intestine than in the posterior one and/or the presence of endogenous bacteria in the posterior intestine which may have a protector role.     

Effects of Fish Size and Water Temperature on the Acute Toxicity of Copper for Japanese Flounder,Paralichthys olivaceus,and Red Sea Bream,Pagrus major

The acute toxicities of copper were examined for Japanese flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, in terms of fish size and water temperature. Artificial seawater of low pH of 5.4–6.7 was used as testing water to keep dissolved copper concentration at 0.04–41 mg Cu/L. Japanese flounder of 0.3–17 g and red sea bream of 0.5–13 g were exposed to different concentrations of copper for 96 h at 20 C under semistatic condition. Median‐lethal concentration for 96 h of Japanese flounder and red sea bream were 8.7–12.2 and 2.0–5.2 mg Cu/L, respectively. No significant relationships were observed between median‐lethal concentrations for 96 h and fish size for Japanese flounder, while the value decreased significantly with increasing fish size for red sea bream. Effect of water temperature on the acute toxicity was examined for Japanese flounder of 0.3 and 0.4 g at 10, 15, 20, and 25 C and red sea bream of 0.5 and 1.0 g at 12, 15, 20, and 25 C. Ninety‐six‐hour median‐lethal concentrations for Japanese flounder and red sea bream were 5.1–11.2 and 1.0–5.3 mg Cu/L, respectively. No significant relationships were observed between median‐lethal concentrations for 96 h and water temperature for both fish species.     

Toxicity of chlorothalonil to marine organisms

We evaluated the acute toxicity of the antifouling biocide 2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile (chlorothalonil, TPN) to an alga (Skeletonema costatum), crustacea (Tigriopus japonicus, kuruma prawn Marsupenaeus japonicus), and a teleost fish (red sea bream Pagrus major) commonly found in the coastal waters of Japan. We also performed acute toxicity and early-life-stage tests for TPN using mummichog Fundulus heteroclitus and used the results to predict the chronic toxicity of TPN to red sea bream. The acute toxicity values of TPN (based on measured concentrations) for S.?costatum, T.?japonicus, M.?japonicus, red sea bream, and mummichog were 0.95, 16, 290, 35, and 61???g/l, respectively. The early-life-stage test with mummichog embryos revealed that hatchability and growth were the most sensitive indicators, and the lowest- and no-observed-effect concentrations, based on the measured concentrations, were 32 and 11???g/l, respectively. We estimated the chronic toxicity value for red sea bream to be 11???g/l based on these toxicity values. Considering the current measured concentrations of TPN in coastal waters of Japan, its environmental impact would appear to be limited.     

Comparison of the swimming performance of farmed and wild gilthead sea bream, Sparus aurata

Farmed gilthead sea bream, Sparus aurata, frequently escape from the sea cages and interact with wild populations. The impact of these interactions on the wild populations will depend, in part, on differences in performance of the bream. This study compared the swimming performance of the wild and farmed fish in a current channel. The absolute critical swimming speed (U_crit) increased with increasing size while the relative U_crit decreased. Even at the same length there were noticeable performance differences between the individuals. The wild sea bream have significantly higher (P<0.05) absolute U_crit performance (0.86±0.01 m s⁻¹) than the farmed fish (0.79±0.01 m s⁻¹) and significantly higher (P<0.05) relative U_crit performance (4.52±0.05 BL s⁻¹) than the farmed fish (4.21±0.05 BL s⁻¹). The present study suggests that cultured sea bream may not have the ability to compete with wild sea bream in native seawaters.     

Effects of dietary mannan oligosaccharides in early weaning diets on growth,survival, fatty acid composition and gut morphology of gilthead sea bream (Sparus aurata,L.) larvae

Early weaning of marine fish larvae with dry diets delays gut maturation and reduces growth rates. In juvenile and adult forms of several marine fish species, inclusion of dietary mannan oligosaccharides (MOS) improves gut integrity and functionality, but the effects of MOS inclusion in gilthead sea bream (Sparus aurata, L.) larval diets have not been addressed yet. Thus, this study assesses the effects of dietary MOS inclusion on survival, growth performance, gut morphology, feed acceptance and quality of gilthead sea bream larvae. For that purpose, 16 days post‐hatched gilthead sea bream larvae were fed four graded levels of MOS (Biomos^®, Alltech, Nicholasville, KY, USA) in weaning diets as follows: 0 g kg^?1 MOS, 0.5 g kg^?1 MOS, 1.5 g kg^?1 MOS and 2 g kg^?1MOS. Dietary MOS did not affect feed acceptance in gilthead sea bream larvae (P > 0.05). MOS supplementation was correlated in a dose‐dependent way with higher larval survival (P = 0.026). After 15 days of feeding, dietary MOS increased whole larvae (P < 0.01) arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. Gilthead sea bream larvae fed 2 g kg^?1 MOS presented higher gut occupation with goblet cells after feeding compared with larvae fed the other dietary treatments. Overall, the results suggest that inclusion of MOS in early weaning diets for gilthead sea bream improves essential fatty acid utilization and may promote growth and final survival.     

Microsatellite multiplex panels for population genetic analysis of red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

In this study, multiplex PCR panels were developed to amplify 20 microsatellite markers for red sea bream, Pagrus major, with the aim of reducing labor and material costs associated with genetic analysis of this species. The usefulness of these panels was validated in 200 fish collected at five sampling locations. The occurrence of null alleles was suggested at five markers, which were dropped from further analysis. The remaining 15 microsatellite loci showed high levels of heterozygosity (H _E range 0.34–0.97, H _O range 0.32–1) and a wide range in the number of alleles per locus (A; range 5–46). Increasing the number of microsatellite loci from three to ten and 15 improved the performance of the panels for population differentiation (F _ST) and estimation of Nei’s (D _S) genetic distance. The results of this study confirm the usefulness of genotyping a large number of microsatellite DNA markers to expand our knowledge of red sea bream population genetics.     

Cloning and expression analysis of innate immune genes from red sea bream to assess different susceptibility to megalocytivirus infection

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL‐8 and COX‐2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL‐1β, IL‐8 and COX‐2 and the antiviral factor like Mx genes performed with in vivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream and rock bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream were markedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)‐induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation‐related genes. The in vitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species.     

Pattern of feed intake in four species of fish under commercial farming conditions: implications for feeding management

Meal duration and feed ingestion rate were measured in sea cage-reared Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, yellowtail Seriola quinqueradiata and red sea bream Pagrus major fed dry extruded feed in discrete meals. At the population level, satiation times in yellowtail, salmon and trout were typically about 15–25 min, but time to satiation was longer (60–90 min) in red sea bream. In all species, feed ingestion rate declined progressively during the course of the meal as the fish became satiated. Initial feed ingestion rates in salmon were ≈ 0.3–0.5 kg feed tonne fish^–1 min^–1 and in trout 0.5–0.9 kg feed tonne fish^–1 min^–1, although the capacity to deliver feed may have restricted ingestion. Water temperature had little effect on ingestion rates, possibly because the number of meals per day (1–3) was varied with water temperature, and this may have standardized hunger level at the start of meals. Yellowtail ingested feed at ≈ 3.5 kg feed tonne fish^–1 min^–1 at water temperatures of 18 °C and 28 °C, whereas red sea bream ingested feed at initial rates of 0.6 and 1.4 kg feed tonne fish^–1 min^–1at 26.5 °C and 18 °C respectively. The findings are discussed in relation to feeding strategies to minimize interfish competition for feed and to improve the ability of fish farmers to detect the point at which fish are satiated.     

Analyzing the economic performance of the red sea bream Pagrus major offshore aquaculture production system in Korea

This study aimed to analyze the economic effect of the red sea bream Pagrus major offshore aquaculture production system in Korea. Based on the data collected, revenue and expenses throughout the culture period were calculated, and the net present value and internal rate of return of the ten-year cash flow and cash outflow were estimated in order to determine the economic validity of red sea bream offshore aquaculture production. Results showed that the red sea bream offshore aquaculture production system had high profitability based on the current production and market conditions. This is because of its relatively high survival rate, relatively low feed conversion ratio, and good market price level. However, sensitivity analyses of the main variables indicated that the profitability of red sea bream offshore aquaculture is highly vulnerable to production conditions and market condition changes.     

Establishment and characterization of a fibroblast cell line derived from the dorsal fin of red sea bream,Pagrus major (Temminck & Schlegel)

The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF‐2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 °C in Leibovitz L‐15 medium with 10% foetal bovine serum. Propagation of RSBF‐2 cells was serum dependent and exhibited low plating efficiency (<1.7%). Aside from long‐term cryopreservation, the cells could also be kept at 4 °C for 72 days. The distribution of the chromosome number was 38–98 with a mode of 48. The RSBF‐2 cell line was susceptible to red sea bream iridovirus but only produced a few rounded and refractory cells. Virus‐inoculated RSBF‐2 cells were then subcultured to generate a persistently infected cell line. RSBF‐2 was also very sensitive to the extracellular products of Photobacterium damselae ssp. piscicida and produced significant fluorescent signals after transfection with pEGFP‐C3. Analysis of mitochondrial cytochrome b gene sequences revealed 99% identity between the cell line and Pagrus major.     

Characterization of a new hemolytic protein toxin from the sea anemone Anthopleura asiatica

A 20-kDa protein toxin (bandaporin) was isolated from the sea anemone Anthopleura asiatica (Actiniidae) using the precipitation of ammonium sulfate saturation, and ion-exchange and gel-permeation chromatographies. Bandaporin showed potent hemolytic activity (EC₅₀ value: 8.8 ng/ml to 0.8% suspension of sheep red blood cells) and lethal toxicity to crayfish (LD₁₀₀ value: 0.58 mg/kg). The hemolytic activity of bandaporin was inhibited specifically by sphingomyelin. Eight other tested membrane lipids, including cholesterol, and gangliosides GM1 and GM2, did not inhibit bandaporin hemolytic activity. Antimicrobial and phospholipase A₂ (PLA₂) activities were not detected in bandaporin. The complementary DNA (cDNA) encoding bandaporin was sequenced and the deduced amino acid sequence of bandaporin revealed that this actinoporin had not been previously characterized. Actinoporins are cytolytic peptide toxins that are widely distributed in Actiniidae and Stichodactylidae sea anemones. This study was the first characterization of an actinoporin from a sea anemone of the genus Anthopleura.     

Effect of Soluble CO2 Stabilization on the Quality of Fillets from Farmed Gilthead Sea Bream (Sparus aurata) and European Sea Bass (Dicentrarchus labrax)

ABSTRACT

Soluble gas stabilization (SGS) is a relatively recent methodology of active packaging that has been proposed to extend the shelf life of packaged fish. The aim of the present work was to study the effect of SGS applied at different extents (2 bars: 30 and 60 min) on the shelf life of sea bream and sea bass fillets packed in air during chilled storage. Quality changes were evaluated by sensory assessment, microbiological analysis (TVC), TBARs, pH value, TVB-N, and TMA-N. No significant extension of sensory shelf life was visible as a function of SGS treatment. The treatment of fillets in CO₂ at 2 bars during 60 min had a positive effect on the microbiological shelf life of both species. On the other hand, TMA-N and TVB-N showed similar changes during storage period and were not affected by the CO₂ treatment. Sea bream treated with SGS always presented higher TBARs than control samples. Nevertheless, oxidation of sea bream and in particular, of sea bass fillets, did not appear to be a significant problem during chilled storage.     

Spawning season,spawning grounds,and egg production of red sea bream in Hiuchi-nada,Seto Inland Sea

The spawning season and grounds of red sea bream in Hiuchi-nada, the central part of the Seto Inland Sea, were described using a new method based on monoclonal antibodies for identifying Pagrus major eggs, and the daily egg production (standardized by the incubation time and survival rate) was estimated. At the peak of spawning (May), the ranges of sea temperature, salinity, and chlorophyll a where red sea bream eggs occurred were 14.8–17.4°C, 32.0–33.0, 0.5–4.4 μg/L, respectively. The main spawning grounds of the red sea bream were confirmed as being the areas near the Geiyo Islands, Misaki Peninsula, Saijyo, Niihama. The spatial spread of red sea bream eggs increased with the egg developmental stage. The ranges of daily egg production in 2005 and 2006 were 0.3–19.3 and 0.2–6.7 × 10⁹ eggs/day, respectively. In Hiuchi-nada, aquaculture farms are located close to the spawning grounds, and the potential spawning population from the aquaculture farms equaled or exceeded that of the estimated spawning population obtained by the egg production method. Red sea bream eggs in Hiuchi-nada might be produced by both wild and aquaculture-based spawning populations.     

Effects of Dietary Vitamin C on Blood Chemistry and Nonspecific Immune Response of Juvenile Red Sea Bream,Pagrus major

A trial was conducted to determine the effect of ascorbyl‐2‐monophosphate Na/Ca (AMP‐Na/Ca) on blood chemistry and nonspecific immune response of red sea bream juveniles. Test diets with three levels of AsA (free, 107, and 325 mg/kg diet) were fed to juvenile red sea bream (36.0 ± 1.3 g) two times a day for 3 wk. There were no significant differences in hematocrit, glucose, and blood urea nitrogen. Total cholesterol and triglyceride in plasma of fish fed AsA‐free diet was significantly (P < 0.05) higher than that of fish fed two other diets. There were no significant differences in serum albumin, total bilirubin, and total serum protein. Glutamyl oxaloacetic transaminase in serum of fish fed diets containing 107 and 325 mg of AsA were significantly (P < 0.05) lower than that of fish fed AsA‐free diet. Serum lysozyme activity (LA) of fish fed diets containing 107 and 325 mg of AsA were significantly (P < 0.05) higher than that of fish fed AsA‐free diet. There was no significant difference in mucus LA. The results mentioned above demonstrated that AMP‐Na/Ca is a bioavailable AsA source for red sea bream juveniles. Supplement of more than 107 mg AsA/kg in diets improved blood chemistry and nonspecific immune function of red sea bream juveniles.     

Dietary inorganic phosphorus or microbial phytase supplementation improves growth,nutrient utilization and phosphorus mineralization of juvenile red sea bream,Pagrus major,fed soybean‐based diets

A 2 × 3 factorial design with triplicates examined the interaction between dietary inorganic phosphorus (IP) and phytase on growth, mineral utilization and phosphorus (P) mineralization in juvenile red sea bream. The treatments were three levels of dietary IP supplementation at 0, 2.5 and 5 g kg^?1, either without or with phytase supplementation [2000 FTU kg^?1; phytase unit is defined as the amount of enzyme activity which liberates 1 micromol of inorganic phosphorus per minute at pH 5.5 and 37 °C at a substrate concentration (sodium phytate) of 5.1 mmol L^?1]. Juvenile red sea bream (IBW = 1.3 g ± 0.1) were stocked twelve fish per tank and fed for 50 days. Growth and feed efficiency were significantly (P < 0.05) enhanced by both dietary P and phytase supplementation. Feed intake and survival rate were not significantly affected by the dietary treatments. Both dietary IP and phytase supplementation significantly increased plasma IP and Mg levels. Concentration of vertebral mineral and scale P was significantly increased by both dietary treatments. A skeletal malformation syndrome of scoliosis occurred in fish fed both non‐IP and non‐phytase supplemented diet. Interaction between main dietary effects was detected for vertebral Zn, scale P and whole‐body ash and Mg content. With regard to growth and other examined productivity traits, phosphorus requirement of juvenile red sea bream can be met if supplemented with 2000 FTU phytase kg^?1 or in the absence of phytase, by dietary inclusion of 2.5–5 g kg^?1 of IP.     

Assessment of reference dietary amino acid pattern for juvenile red sea bream, <Emphasis Type="Italic">Pagrus major</Emphasis>

Four semi-purified diets, containing crystalline amino acids (CAAs), were fed to juvenile red sea bream, Pagrus major in order to ascertain the ideal dietary amino acid pattern for this species. A control diet containing 50% casein–gelatin as protein sources, but no CAAs were fed to the fish. The other diets contained 30% casein–gelatin and 20% CAAs. CAAs were added to diets to simulate with amino acid pattern of the red sea bream eggs protein (REP), red sea bream larvae whole body protein (RLP), red sea bream juvenile whole body protein (RJP), and brown fishmeal protein (BFP). The juveniles (average initial body weight, 1.58 ± 0.01 g) were maintained in triplicate tanks and fed twice daily for 30 days. The highest weight gain was observed in juveniles fed the RJP diet. No significant difference was observed in juveniles fed the RLP and BFP diet. Feed efficiency ratio, protein efficiency ratio and amino acid retention in the whole body were significantly (p < 0.05) affected by the simulated dietary amino acid patterns. The essential amino acid profile and A/E ratios of the whole body after the growth trial showed little difference among the dietary treatments. The results suggest that red sea bream juveniles are able to utilize high amounts of CAA in coated form. The amino acid pattern of RJP could be used as an appropriate of reference dietary amino acid for this species.