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.     

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.     

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.     

Oxygen deficiency during somitogenesis causes centrum defects in red sea bream, Pagrus major (Temminck et Schlegel)

Vertebral deformities in red sea bream, Pagrus major, remain serious obstacles to the improvement of seedling quality for its aquaculture. However, the causalities of the deformities remain unclear and prevention methods have not yet been established. In this paper, oxygen deficiency during somitogenesis was demonstrated to cause centrum defects (formerly called fused vertebrae in many cases), which are the major vertebral deformity in cultured red sea bream. An induction experiment of centrum defects was conducted by placing fertilized red sea bream eggs under low dissolved oxygen conditions (10.3–16.6%). The low oxygen treatment was carried out for five different developmental stages of embryo: two‐cell stage to blastula stage; gastrula stage; three to 10 somites stage; 11–17 somites stage and 18–24 somites stage. Oxygen deficiency during somitogenesis induced a high incidence of centrum defects. In contrast, it hardly induced centrum defects during the other stages. The dissolved oxygen concentration in the rearing water should be carefully regulated for fertilized eggs, especially during somitogenesis to reduce the incidence of vertebral deformities in the red sea bream.     

Effects of protein and lipid sources on the growth and survival of red sea bream Pagrus major and Japanese flounder Paralichthys olivaceus receiving micro-bound diets during larval and early juvenile stage

The effects of dietary protein and lipid sources on growth and survival of the red sea bream Pagrus major (30‐days old) and Japanese flounder Paralichthys olivaceus (15‐days old) were examined using four zein‐microbound diets (MBD) containing the following nitrogen and lipid sources: MBD‐1, SBP‐1000 (soybean peptides with molecular weights of about 1000) + fish oil calcium soap (FOCS); MBD‐2, SBP‐3000 (molecular weights of about 3000) + FOCS; MBD‐3, soybean protein isolate (SPI) + FOCS; MBD‐4, SBP‐1000 + stearoyl calcium lactate (SCL). Six groups of the test fish were maintained on the following diets for 30 days; group 1, live food; group 2, 1/2 live food + 1/2 MBD‐1; group 3, MBD‐1; group 4, MBD‐2; group 5, MBD‐3; group 6, MBD‐4. In the red sea bream, high performance in terms of total length (TL) and body weight gain (BWG) was obtained in groups 1, 2, 3 and 4. However, the fish receiving MBD‐3 (group 5) and MBD‐4 (group 6) had shorter TL and lower BWG than those receiving the other diets. Regarding the Japanese flounder, high performance in terms of TL, BWG, and survival rate (SR) was obtained in groups 1 and 2. Group 3 receiving MBD‐1 gave the BWG and SR close to those of group 2 but lower than group 1. In contrast, groups 5 and 6 receiving MBD‐3 or MBD‐4 alone had significantly (P < 0.05) shorter TL and lower BWG than groups 1 and 2. The present study thus indicated that soybean peptides with molecular weights of 1000–3000 and FOCS are superior to SPI and SCL as nitrogen and lipid sources of MBD for the red sea bream and Japanese flounder at larval and early juvenile stage.     

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.     

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.     

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.     

Daily change of genetic variability in hatchery offspring of red sea bream during spawning season

ABSTRACT:   In order to assess a daily change of genetic variability during spawning season, hatched larvae of red sea bream sampled on different dates were assayed by polymorphic markers such as microsatellite DNA (msDNA) and mitochondria DNA (mtDNA) control region. Based on the microsatellite loci, the average number of alleles per locus ranged between 13.7 and 18.3. The expected heterozygosities ranged between 0.843 and 0.919. A total of 23 mtDNA haplotypes were detected via digestion of mtDNA D-loop sequences with five endonucleases: Taq  I, Alu  I, Mbo  I, Rsa  I and Hinf  I. Significant fluctuation of genetic variability during spawning season was detected by both types of DNA markers. It was suggested that the genetic variability was maintained by pooling the seed fish collected on different spawning dates in a hatchery.     

Interactive effects of dietary vitamin C and phospholipid in micro‐bound diet for growth,survival, and stress resistance of larval red sea bream,Pagrus major

This study was conducted to examine the effects of dietary ascorbic acid (AsA) and phospholipid (PL) and their interaction on growth, survival, and stress resistance in red sea bream larvae. Twenty‐six days old red sea bream were fed nine micro‐bound diets supplemented three levels of AsA (0, 800 and 1600 mg kg^?1 diet) and PL (0, 20 and 40 g kg^?1 diet) for 15 days. Dietary AsA and PL were both significant factors on survival rates. There was also an interaction between dietary AsA and PL on survival rate (P < 0.05). The larvae fed 800 or 1600 mg kg^?1 AsA with 40 g kg^?1 PL diets showed the highest survival rate, with values similar to those of the live‐food supplemented group. Stress resistance against low salinity exposure significantly increased with increased dietary level of AsA and PL. However, significant interaction of AsA and PL was not detected. The larvae fed 1600 mg kg^?1 AsA with 40 g kg^?1 PL diet showed the highest stress resistance among all diets, but it was not significantly different than that of larvae fed 800 mg kg^?1 AsA with 40 g kg^?1 PL diet. This study clearly demonstrated that combined use of AsA and PL can improve survival of 26–40 days posthatching red sea bream larvae. Moreover, the present study suggested that 800 mg kg^?1 AsA with 40 g kg^?1 PL in diet was needed for producing high quality seedling under the stressful conditions.     

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 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.     

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.     

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.     

Polymorphisms of the growth hormone gene in domesticated red sea bream populations (Pagrus major) based on minisatellite genotypes and nucleotide sequences

The genetic diversity of the growth hormone gene in domesticated red sea bream (pmaGH) was evaluated using a minisatellite DNA marker located in intron 3 (pmaGH22) and nucleotide sequences. The number of alleles of pmaGH22 was largely decreased in domesticated strains of red sea bream, and the possibility of selection pressures was also detected based on the analysis of the Hardy–Weinberg equilibrium in some strains. However, each strain inherited a small number of alleles of pmaGH22, and the entire domesticated population (combining all strains) showed a large number of alleles (n = 17), similar to the allelic richness of the wild population (n = 18.5). Based on nucleotide sequencing analysis, three synonym mutations were found in the coding regions, and also several SNPs and indels were found in the noncoding regions. In addition, four genealogies of growth hormone haplotypes were identified based on principal coordinate analysis, and these genealogies of pmaGH partly reflected allele size ranges of pmaGH22. Several haplotypes shared alleles of pmaGH22, and also fragment size homoplasy in pmaGH22 was suspected. These alleles of pmaGH22 and the haplotypes will be a useful indicator for divergence of pmaGH and for broodstock individual selection with minimum inbreeding effect.