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.     

Gonadal sex differentiation and sex control in red sea bream, Pagrus major

The gonadal sex differentiation in red sea bream, Pagrus major, which is one of the most important species for aquaculture in Japan, was revealed histologically. The suitable conditions for induction of all-male groups in the fish were investigated, and functional males were induced by the conditions of oral administration of 17α-methyltestosterone. The sex determination of this fish was also discussed.     

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.     

Acoustic pressure sensitivities and effects of particle motion in red sea bream Pagrus major

The auditory pressure thresholds of red sea bream were examined using cardiac response in the field by placing fish subjects far from the sound source to prevent particle motion. Pressure and particle motion thresholds were also obtained using the auditory brainstem response (ABR) technique. The thresholds at 100 and 200 Hz were significantly higher when measured using the cardiac response in the far field than those obtained in previously conducted experiments in experimental tub. However, thresholds obtained using ABR from 200 to 500 Hz were not remarkably lower, although significantly different (0.01 < P < 0.05), compared with those obtained using cardiac response in the far field. Furthermore, calculated particle velocity thresholds indicated that fish probably detected particle motion within the frequency range of 50–200 Hz, even in fish with a deactivated lateral line. Although the ABR method is widely applied in fish auditory study, hearing thresholds are apparently affected by particle motion.     

Vertebral deformities in cultured red sea bream, Pagrus major, Temminck and Schlegel

Vertebral deformities were investigated in cultured red sea bream, Pagrus major. In the field, deformities in seedlings were categorized and their incidence was calculated. In the laboratory, the symptoms of major vertebral deformities were examined morphologically using radiographs and by making transparent skeletal specimens. The internal structure of deformed vertebrae was examined histologically. The shortened body condition had the highest incidence (0.9–8.3%) of all deformities in the seedlings. In individuals with the shortened body condition, the ratio of trunk and caudal part length to body height was smaller. These fish had skeletal anomalies in the vertebrae, mainly centrum defects (64.3%) or undersized centrums (25.2%). The specimens with centrum defects had a characteristic anomaly in the vertebrae, with plural pairs of neural and haemal spines on a single centrum. This anomaly was frequently observed in the posterior abdominal vertebrae. The internal skeletal structure of such abnormal centrums was basically the same as that of normal centrums. In all the specimens with undersized centrums, both the centrum length and diameter were shorter than normal except for the first and second centrum, and urostyle.     

Optimum dietary taurine level in casein-based diet for juvenile red sea bream Pagrus major

ABSTRACT:   This study was conducted to investigate the effect of dietary taurine and cholyltaurine (C-tau) on growth and body composition of juvenile red sea bream Pagrus major . Semi-purified casein-based diets supplemented with 0 (control diet), 0.1, 0.3, 0.5 and 0.7% taurine and 0.5% C-tau were fed to red sea bream (average body weight 4.7 g) for 6 weeks at 20°C. The growth and feed efficiency were the lowest in fish fed the control diet. Taurine supplementation improved the growth and feed efficiency of fish dose-dependently, and the taurine requirement was estimated as 0.52% in terms of optimizing growth and 0.48% in terms of optimizing feed efficiency. Taurine content in the whole body and liver increased with the dietary taurine level. Supplemental C-tau at the 0.5% level had limited effects on the growth and no effect on body taurine, biliary bile salt and liver fat contents. From these results it can be inferred that the optimal dietary taurine requirement of juvenile red sea bream is 0.5% on a dry weight basis, and that the supplementation of taurine in the diet not only improves the growth but also increases hepatic lipid levels of red sea bream juveniles.     

Ontogeny of tolerance to and avoidance of ultraviolet radiation in red sea bream Pagrus major and black sea bream Acanthopagrus schlegeli

ABSTRACT:   Ontogenetic changes of tolerance to, and avoidance of, ultraviolet-B radiation (UV-B) were examined in red sea bream Pagrus major and black sea bream Acanthopagrus schlegeli . In the tolerance experiment, larvae and juveniles (age 13–46 days) were put in beakers, and were exposed to one of five different levels of UV-B radiation (1.8, 1.1, 0.2, 0.1, and 0 W/m²) for one hour. Their survival rates were calculated either 12 or 24 h later. In the avoidance experiment, fish (age 3–49 days) were put in a long experimental tank, half of which was covered with UV-blocking film and placed under two levels of UV-B radiation (1.1 and 0.2 W/m²), and their avoidance indices were calculated. Black sea bream had significantly better survival compared to red sea bream for most ages. Only black sea bream of ages 37 and 49 days showed significant avoidance of UV radiation under the higher level of UV-B, whereas both species did not show avoidance on any days at the lower level. The present results suggest that black sea bream are significantly better adapted to habitats with high UV-B radiation, than red sea bream, reflecting that back sea bream live in shallower waters through their early life stages.     

Impacts of short-term salinity and turbidity stress on the embryonic stage of red sea bream Pagrus major

Fisheries Science - Heavy rainfall can reduce salinity and increase turbidity in coastal waters, creating stressful conditions for the organisms found there, especially for the early stages of...     

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.     

Fish meal replacement by soy protein from soymilk in the diets of red sea bream (Pagrus major)

Six isoenergetic diets were formulated as follows: fish meal (FM) 700 g kg^–1 (control, C), FM 300 g kg^–1 + soy protein concentrate 300 g kg^–1 (SPC), FM 300 g kg^–1 + enzyme‐treated SPC 300 g kg^–1 (ESC), FM 170 g kg^–1 + soy protein isolate 300 g kg^–1 (SPI), FM 160 g kg^–1 + enzyme‐treated SPI 300 g kg^–1 (ESI) and FM 150 g kg^–1 + conglycinin 300 g kg^–1(CG). Forty fish (3.9 g) were randomly distributed into each of eighteen 300‐L tanks, fed twice daily until satiation for 8 weeks. The final body weight, specific growth rate and condition factor did not show significant differences among the fish fed with diets C, SPC, ESC and ESI (p > .05). The survival was significantly lower in fish fed with diets SPI and CG. Feed efficiency was significantly higher in fish fed with diets SPC and C than in fish fed with other diets (p < .05). There were no significant differences in nutrients retention efficiencies in fish fed with diets C, SPC, ESC and ESI. A significantly higher phosphorus retention efficiency in fish fed with soymilk protein diets resulted in lower phosphorus discharge to the environment (p < .05). These results suggest that the soymilk proteins can comfortably replace 570–770 g FM kg^–1 diet of red sea bream juvenile, which will ensure significant ecological benefits through reducing phosphorus load to the environment.     

Polymorphisms and haplotypes of the myostatin gene associated with growth in juvenile red sea bream Pagrus major

Polymorphisms of the myostatin gene (MSTN) have been studied in vertebrates including several aquaculture species, revealing their role in growth. We attempted to identify polymorphisms in MSTN associated with growth traits of juvenile farmed red sea bream Pagrus major, an important cultured fish species in Japan. Polymorphisms in the coding region of MSTN were screened, and six polymorphisms were found among three exons: a deletion in exon 1, a single nucleotide polymorphism (SNP) in exon 2 and four SNPs in exon 3. The deletion in exon 1 eliminated a codon (glutamine) from the region comprising the polyglutamine structure, but all SNPs were synonymous. We analysed the polymorphisms for association with growth phenotype in large and small P. major specimens obtained from commercial production at 50 days post hatching. Two SNPs located in exon 3 (c.+846T>C and c.+1140T>C) were significantly more frequent in the large group, while no SNPs were significantly associated with the small phenotype. Haplotypes were reconstructed using genotype information of the two phenotype groups, and nine haplotypes (Hap_1 to Hap_9) were successfully reconstructed. Hap_6 and Hap_7 were significantly more often observed in small and large groups respectively. Analysis of diplotypes (haplotype combinations) of individual specimens revealed 19 diplotypes. The Hap_7/Hap_7 diplotype was found in 46.7% of large phenotype specimens, significantly more frequently than in the small group (13.5%). These results will be useful for marker‐assisted selection of red sea bream to improve production with respect to growth.     

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.     

日本鹿儿岛湾真鲷的资源评估和增殖策略

通过世代分析对1990年至2002年鹿儿岛湾真鲷（Pagrus major）自然种群和放流种群的种群大小进行了评估,评估模型考虑了渔业资源管理和增殖放流措施。结果表明放流种群的规模有所减少,而自然种群的规模维持不变。真鲷自然种群的捕获量日趋减少,而放流种群的捕获量已超过50吨。研究结果表明增殖700000—800000尾真鲷种苗与其50吨的捕捞量相协调。在各种捕获量和放流量情况下,采用种群动力学模型预测了2017年真鲷的捕获量,其结果表明提高放流真鲷的数量能增加其捕获量。     

Hypoxic conditions induce centrum defects in red sea bream Pagrus major (Temminck and Schlegel)

A previous study elucidated that an extreme hypoxia during somitogenesis induced the most frequent skeletal malformation centrum defects in red sea bream (RSB), Pagrus major. In this study, details of the hypoxic conditions to induce them in RSB, dissolved oxygen (DO) concentration and exposure time to hypoxia, were investigated. Fertilized eggs were exposed to seawater of six DO concentrations (0%, 10%, 25%, 50%, 75% and 100% of saturation) for seven different periods (5, 10, 30, 60, 120, 240 and 360 min) during somitogenesis. Somitic disturbances in newly hatched larvae were induced by exposure to 0% and 10% DO concentration for 10 and 120 min and longer respectively. Rearing eggs exposed to hypoxic condition of 10% DO for 240 min for 40 days post‐hatch showed that the location and the frequency of somitic disturbances in larvae and centrum defects in juveniles were significantly correlated (P<0.01). Dissolved oxygen concentration of the interstitial water in the egg high density layer formed at the water surface in a stationary state abruptly decreased to 3.7% within 7 min. Centrum defect induction by exposure of eggs to extreme low DO concentrations for a short period, which is the probable situation in the practical juvenile production, suggests that careful maintenance of DO concentration is important in the incubating water of fertilized eggs during egg sorting and transportation, where eggs are made into a pile and undergo hypoxia, for the prevention of centrum defects.     

The microhabitat and size of gammarid species selectively predated by young red sea bream Pagrus major

SUMMARY: Mechanisms of prey selection by young red sea bream Pagrus major were examined in Shijiki Bay by comparing the species composition of gammaridean amphipods in the diet with that in the environment. Although gammarids were the major source of food for young red sea bream, their species composition in fish stomachs changed with the growth of fish from June to October and always differed from that in the environment. The patterns of selection of gammarids were related most closely to the microhabitat of each species before mid-July. The order of selectivity of species in terms of the microhabitat was as follows: epifaunal species > shallow-burrowing species > infaunal tube-dwelling species > deep-burrowing species. After mid-July, however, selection was determined by the body size of gammarid species. Byblis japonicus that were large enough as a prey species were mainly consumed and other smaller species were not selectively eaten. Thus, the microhabitat and the body size of gammarids play dominant roles in prey selection by young red sea bream.     

Genetic diversity of red sea bream Pagrus major in western Japan in relation to stock enhancement

SUMMARY: Stock enhancement is used in Japan as a tool to help the replenishment of wild populations of red sea bream Pagrus major . In this study, we analyzed the genetic diversity and composition of wild red sea bream at seven locations around Shikoku Island, South-west Japan, using three microsatellite loci. This analysis was done to test the hypothesis that: (i) red sea bream comprises a single Mendelian population along Japan; and (ii) stock enhancement programs around Shikoku Island are causing genetic differentiation among wild stocks. The results indicated that some locations from the Shikoku area were not significantly different from the rest of Japan, supporting the hypothesis of a single Mendelian population. Significant departures from Hardy–Weinberg equilibrium and significant pairwise F_ST among locations indicated genetic instability within this region. We suggest that the stock enhancement programs made in the region are the possible cause of this genetic instability. A management scheme for the hatcheries involved in the stock enhancement of red sea bream is presented.     

Effect of dietary taurine enhancement on growth and development in red sea bream Pagrus major larvae

This study investigated the effect of feedings taurine‐enriched rotifers on the growth and development of larval red sea bream (RSB). Rotifers incubated in taurine‐enriched water at a taurine concentration of 800 mg L^?1 (T‐800) and 0 mg L^?1 (T‐0) were fed to larvae from 3 to 20 days after hatching (DAH). Notochord length, body weight and specific growth rate of T‐800 group were significantly greater than those of T‐0 at 14, 17, 9–11 and 18–20 DAH. Taurine content of larvae in the T‐800 group increased rapidly from 11 DAH and thereafter remained significantly higher than T‐0. Flexion larvae firstly appeared in both groups at 8 DAH, however, at 20 DAH post‐flexion larvae were significantly more abundant in T‐800 than T‐0. While nucleic acid and protein contents (μg mg^?1 wet fish) showed remarkable changes, ontogenetic growth in RSB larvae stage was observed to switch from hyperplastic growth to hypertrophic growth with the start of the flexion stage. Although a similar change in nucleic acid contents was observed between the two groups, the protein content (μg fish^?1) and protein/DNA ratio of T‐800 remained higher than that of T‐0 during the hypertrophic growth period. These results suggest that dietary taurine accelerates the growth and development in RSB larvae especially during hypertrophic growth (flexion stage) after the early hyperplastic growth.