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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparative assessment of purine nucleotides adenosine,guanosine and inosine monophosphates as functional supplements on growth and health performances of red sea bream,Pagrus major juvenile

Purine nucleotides regulate the cellular functions in the animal body. The current study evaluates the comparative efficacy of dietary purine nucleotides, that is, adenosine monophosphate (AMP), guanosine monophosphate (GMP) and inosine monophosphate (IMP) in red sea bream Pagrus major. Semi‐purified basal diet was formulated (Control, D1) containing 550 g/kg protein, supplemented with purine nucleotides AMP, GMP and IMP at their optimum supplementation level (2, 4 and 4 g/kg) to formulate the experimental diet groups D2, D3 and D4, respectively. Initial weight of 3.5 ± 0.01 g fish was randomly fed test diets in triplicate. After 56 days, % weight gain (p = .003), specific growth rate (p = .003) and apparent lipid digestibility (p = .04) were significantly higher in fish fed diet group D4 followed by D3 and D2. Supplemented groups showed significantly higher feed intake in comparison to control (p = .001). Supplemented groups showed significantly increased and decreased NBT (p = .003) and CAT (p = .003) activity, respectively. Fish fed IMP supplemented diets had significantly lower blood urea nitrogen (p = .04), glutamyl oxaloacetic transaminase (p = .04) and glutamic‐pyruvate transaminase (p = .001) followed by other supplemented groups. Supplemented diet groups showed enhanced stress resistance. Interestingly, D2 and D4 groups showed best oxidative stress status of fish. Therefore, these results indicated that among purine nucleotides, supplementation of IMP could be a more effective nucleotide as functional supplement in red sea bream diet.     

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.     

A natural infection by the red sea bream iridovirus‐type Megalocytivirus in the golden mandarin fish Siniperca scherzeri

An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)‐subgroup I. Additionally, this train clustered with the ehime‐1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV‐type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18 × 10² to 7.95 × 10⁶ copies/mg of tissue individually, suggesting that the infected fish were in the disease‐transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV‐type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.     

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.     

Studies on epizootic iridovirus infection among red sea bream,Pagrus major (Temminck & Schlegel), cultured in Taiwan

Since 1993, an epizootic viral disease has occurred in net-cage cultured red sea bream, Pagrus major (Temminck & Schlegel), in Peng-hu Island located on the south-western coast of Taiwan. The diseased fish exhibited abnormal swimming and were lethargic, but few visible external signs were observed. The cumulative mortality because of the disease sometimes reached 50-90% over 2 months. Histopathogical studies of the affected fish showed enlarged basophilic cells in the gill, kidney, heart, liver and spleen. These necrotic cells were Feulgen-positive and stained blue using Giemsa. Transmission electron microscopy revealed icosahedral virions in the cytoplasm of the necrotic cells. The viral particles consisted of a central nucleocapsid (75-80 nm) and envelope, and were 120-150 nm in diameter. These results suggest that the virus belongs to the Iridoviridae. Using polymerase chain reaction (PCR), approximately 570 bp fragments were produced from the viral DNA using as a template 1-F and 1-R primers derived from red seabream iridovirus (RSIV) from red sea bream in Japan. Similar results were also obtained using nested-PCR with different primer sets (1-F, 2-R and 2-F, 1-R). Although the size and some features of epizootics of this virus differed from RSIV in Japan, it shows close genetic affinities with the latter and it is suggested that RSIV has been introduced to Taiwan.     

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.