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.     

cDNA cloning and expression of a novel group IB phospholipase A2 in the intestine of the red sea bream,Pagrus (Chrysophrys) major

A cDNA encoding a novel phospholipase A₂ (PLA₂), which was named IN PLA₂, was cloned from the intestine of the red sea bream. The amino acid sequence of IN PLA₂ showed 49–75% homology with those of red sea bream group IB sPLA₂, hepatopancreas DE-1 and DE-2 PLA₂, and gill G-3 PLA₂. IN PLA₂ consists of a prepropeptide of 24 amino acid residues, followed by a mature protein. IN PLA₂ contains 14 cysteines, and includes Cys11, the calcium binding loop and the pancreatic loop that are commonly conserved in group IB sPLA₂ enzymes. In addition, IN PLA₂ is a cationic protein with a pI of 8.52. Therefore, IN PLA₂ was identified as a novel group IB sPLA₂ isoform in red sea bream. IN PLA₂ mRNA was found by northern blot analysis to be expressed mainly in the pyloric caeca and the intestine, and was detected in the goblet cells of the intestine by in situ hybridization. The expression level of IN PLA₂ mRNA was elevated by intravenous injection of lipopolysaccharide—the outer-membrane component of Gram-negative bacteria. These results suggest that IN PLA₂ is secreted from the goblet cells of the intestine in response to stimulus such as bacterial infection, and that it contributes to antimicrobial defense in addition to the digestion of dietary phospholipids in the gastrointestinal tract.     

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.     

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。     

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.     

Morphological aspects of feeding and improvement in feeding ability in the early larval stages of red sea bream Pagrus major

The present study examined the appearance, ossification and growth of the bones that form the oral cavity in early larval stages of laboratory reared red sea bream ( Pagrus major ) for 380 h after hatching. The fundamental elements of the oral cavity appeared 11 h after initial mouth opening (HAMO). Development in the red sea bream, based on the osteological development of the feeding apparatus, was divided into three phases following the first feeding (24 HAMO; mean total body length 3.3 mm). The first phase was the early sucking phase (24 to 80–100 HAMO; approximately 3.9 mm), during which the head and bones increased in size. The intensified sucking phase (to 200–220 HAMO; approximately 4.9 mm) was defined by the appearance of new structural elements and a continued enlargement of the head and bones. Finally, during the transition phase (beyond 300 HAMO; approximately 5.6 mm), larvae used grasping as well as sucking to feed, new elements appeared, ossification began, size increased and teeth were acquired. As the larvae advanced through these three phases, the ability to feed by sucking was enhanced by the appearance and growth of new bones. The developmental phases appear to be linked to the transition from endogenous to exogenous nutrition resources under laboratory rearing conditions and to diversification in the size and components of wild food organisms.     

Reproduction,growth and stress response in adult red sea bream,Pagrus major (Temminck & Schlegel) exposed to different photoperiods at spawning season

Adult red sea bream, Pagrus major (body weight, 1.0–2.0 kg) was exposed to three photoperiods [12 h light:12 h dark (12L:12D), 16 h light:8 h dark (16L:8D) and 24 h light:0 h dark (24L:0D)] from 2 months before spawning till the end of the spawning season to investigate growth, spawning and stress response. During the spawning season, tanks were checked every morning for spawned eggs. The growth performance in fish under 24L:0D was stimulated with significantly higher feed intake than those under other photoperiods (P<0.05). The number of eggs and gonadal histology confirmed that three and five females out of six in each of duplicate tanks of the 16L:8D treatment spawned. In contrast, only two out of six females in one tank of the 24L:0D treatment spawned, and no spawns were observed in the 12L:12D treatment. At the end of the spawning period, both 17β‐estradiol and testosterone levels were significantly higher in fish exposed to 16L:8D followed by 12L:12D and 24L:0D photoperiods (P<0.05). Photoperiod manipulation did not cause significant stress response in fish (P>0.05). The results suggest that stimulating the growth performance of red sea bream at reproductive stage with a 24L:0D photoperiod is possible if the fish are subjected to this photoperiod long before the onset of the spawning season.     

Stimulation of growth performance without causing stress response in young red sea bream, Pagrus major (Temminck & Schlegel), by photoperiod manipulation

Three photoperiods (12L:12D, 16L:8D and 24L:0D) were used to investigate the growth performance and stress response in red sea bream, Pagrus major (body weight 200–400 g). Fish were fed a commercial diet to apparent satiation, two times a day for 8 weeks. Fish exposed to a 24L:0D photoperiod showed a significantly higher weight gain (%) than those exposed to other photoperiods (P<0.05). The best specific growth rate and feed conversion efficiency were achieved at 24L:0D and 16L:8D, without significant differences among them. Although fish exposed to 16L:8D showed a significantly higher plasma level of cortisol than those exposed to other photoperiods in the third week, there was no major variation in the cortisol level among the treatments either at the sixth week or at the end of this study. There were no significant differences either in the haematocrit level or the plasma levels of glucose, total cholesterol and total protein among the treatments during this study. The results revealed that the growth performance of red sea bream reared from 200 to 400 g can be stimulated significantly using a continuous (24L:0D) photoperiod without any measurable significant stress response in fish.     

Ultrasonic immunization of sea bream, Pagrus major (Temminck & Schlegel), with a mixed vaccine against Vibrio alginolyticus and V. anguillarum

In order to clarify the effectiveness of ultrasonication on vaccine delivery, juvenile sea bream, Pagrus major , were treated with eight different ultrasonic methods. A mixed vaccine against Vibrio alginolyticus and V. anguillarum was used to immunize the fish . The intensity and frequency of the ultrasound were 280 mW cm^–2 and 35 kHz, respectively. The ultrasonic methods included continuous or pulsed ultrasound for 3 min, and continuous or pulsed ultrasound for 3 min before and/or after immersion for 3 min. Of all the eight ultrasonic methods tested, `pulsed ultrasound followed by immersion' and `immersion, pulsed ultrasound, and followed by immersion again' provided the best protection, which were comparable with protection of fish immunized by intraperitoneal injection. Moreover, the convenience of applying these two ultrasonic methods for immunization was comparable with the immersion method and was much better than intraperitoneal injection. If 2 × 10⁸ CFU mL^–1 of this mixed vaccine was used for vaccination repeatedly five times by ultrasonic methods, it could still produce good protection for the immunized sea bream. Therefore, the ultrasonic method is an effective and practical approach for fish vaccination on a large scale.     

Effects of dietary taurine levels on epidermal thickness and scale loss in red sea bream,Pagrus major

Taurine is often added to artificial fish diets to compensate for a reduction in fish meal (FM). However, the taurine content of FM‐based diets is typically lower than in diets consisting of raw fish, even in diets where FM is the only protein source. We evaluated the effects of dietary taurine in FM‐based diets on epidermal thickness and scale detachability in red sea bream Pagrus major. We compared the effect of diets containing 0% (control), 0.3% (Tau‐0.3%), 0.6% (Tau‐0.6%) and 1.0% (Tau‐1.0%) taurine. Red sea bream (average body weight, 39 g) were fed these diets for 7 weeks. Taurine supplementation had no effect on growth, feed intake, feeding efficiency, or survival. However, the epidermal thickness was higher in fish in the Tau‐0.6% and Tau‐1.0% groups than in the control and Tau‐0.3% groups. Similarly, scale loss was significantly higher in the control group than in the Tau‐0.6% and Tau‐1.0% groups. Our results suggest that supplementation with >0.6% taurine (1.0% in diet) improves skin condition.     

Purification of gonadotropins (PmGTH I and II) from red seabream (Pagrus major) and development of a homologous radioimmunoassay for PmGTH II

Two gonadotropic glycoproteins (PmGTH I and II) were purified by ion-exchange chromatography, gel filtration and preparative SDS-PAGE, from pituitaries of red seabream, a marine teleost which has an asynchronous-type ovary and spawns almost daily during the spawning season. The glycoproteins were composed of distinct subunits and the molecular weights were estimated to be 32 and 38 kDa for PmGTH I and PmGTH II, respectively. Both PmGTH I and II were active in two homologous bioassays: in vitro oocyte maturation and/or in vitro estradiol-17 production assays. These two GTHs were distinct in electrostatic properties, molecular weight, stability and yields from pituitaries during the spawning season. These properties suggest that PmGTH I and II correspond to salmon GTH I and II, respectively.A homologous radioimmunoassay with which to measure PmGTH II was developed using a rabbit antiserum against the subunit of PmGTH II and intact PmGTH II as standards and radioactive competitors. Competition curves for red seabream plasma and pituitary extract were parallel to the standard curve, while PmGTH I had low cross-reactivity (3.1 %) with the antibody. This specific RIA system showed an in vivo LHRHa induced GTH surge in the plasma of female red seabream.     

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.     

Effect of rotifer enrichment with herbal extracts on growth and resistance of red sea bream,Pagrus major (Temminck & Schlegel) larvae against Vibrio anguillarum

The effects of the dietary medicinal herbs Massa medicata, Crataegi fructus (Cf), Artemisia capillaries, Cnidium officinale and their mixture (HM), on growth and resistance against Vibrio anguillarum were examined using larval red sea bream, Pagrus major. The methanol extracts of Cf and HM inhibited the proliferation of seven infectious bacterial strains including the genera Aeromonas, Edwasiella, Photobavterium, Pseudomonas and Vibrio. Rotifers enriched with emulsified bonito oil containing extracts of Cf and HM lowered the bacterial counts in their body on TCBS medium for the genus Vibrio in comparison with rotifers enriched by emulsified bonito oil alone. Although statistically not significant, larvae fed rotifer fortified with Cf from 3 to 20 days after hatching showed the best growth and final body weight followed by those given rotifer with HM and control enrichment. However, final body length of larvae fed rotifer with Cf was significantly larger than larvae fed on rotifers enriched with other herb extracts. After a challenge test with V. anguillarum, survival of larvae fed rotifer with Cf and HM was significantly higher than for the control treatment. These results revealed that Cf and HM in rotifer is useful to promote growth and resistance against V. anguillarum in red sea bream larvae, providing a new technology for mass production of disease‐resistant fry and fingerlings.     

Inosine supplementation effectively provokes the growth,immune response,oxidative stress resistance and intestinal morphology of juvenile red sea bream,Pagrus major

A feeding trial was conducted to determine effects of dietary inosine on growth, immune responses, oxidative stress resistance and intestinal morphology of juvenile red sea bream. A semi‐purified basal diet supplemented with 0 (D1, control), 2 g kg^?1 (D2), 4 g kg^?1 (D3), 6 g kg^?1 (D4) and 8 g kg^?1 (D5) dietary inosine, respectively, to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (initial weight: 8 g). After 50 days of feeding trial, fish fed diets with 4 g kg^?1 inosine had the highest (p < .05) final weight, weight gain and specific growth rate. Similarly, improved feed intake, feed conversion ratio and protein efficiency ratio were also found at 4 g kg^?1 inosine supplemented group. Some non‐specific immune parameters such as total serum protein, lysozyme activity and bactericidal activity tended to be higher for fish fed diets supplemented with inosine. Peroxidase and catalase activity also influenced (p < .05) by dietary inosine supplementation. Fish fed 2 g kg^?1 and 4 g kg^?1 inosine supplemented diets showed the least oxidative stress condition. Inosine supplementation significantly increased (p < .05) anterior enterocyte height and posterior fold height, enterocyte height and microvillus height compared with control. We concluded that 4 g kg^?1 dietary inosine supplementation effectively provokes growth and health performance of red sea bream by increasing growth, immune response, oxidative stress resistance and intestinal health condition.     

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.