Effectiveness of a vaccine against red sea bream iridoviral disease in a field trial test.

Since 1990, red sea bream iridovirus (RSIV) has caused high mortalities in the summertime in cultured red sea bream Pagrus major in southwest Japan. To establish control measures for red sea bream iridoviral disease (RSIVD), the effectiveness of a formalin-killed viral vaccine was evaluated in a field trial. Two groups each consisting of 1000 juvenile red sea bream were either intraperitoneally inoculated with vaccine (vaccinated group) or were not vaccinated (non-vaccinated group). After vaccination, the fish were held for 1 wk, then transferred to a marine net pen and observed for 12 wk. The cumulative mortalities caused by RSIVD in the vaccinated group or control group were 19.2 and 68.5%, respectively. Additionally, the presence of virus antigen in the spleen was investigated and body weight was measured 6 and 12 wk post vaccination. In the vaccinated group, viral antigen was not detected. The increase in body weight of vaccinated fish was significantly (p < 0.05) greater than that of control fish. These results suggest that the vaccine against RSIVD was effective in 1 field trial.     

Red sea bream iridoviral disease

The first outbreak of red sea bream iridoviral disease caused by red sea bream iridovirus (RSIV) was recorded in cultured red sea bream Pagrus major in Shikoku Island, Japan in 1990. Since 1991, the disease has caused mass mortalities of cultured marine fishes not only red sea bream but also many other species. The affected fish were lethargic and exhibited severe anemia, petechiae of the gills, and enlargement of the spleen. The causative agent was a large, icosahedral, cytoplasmic DNA virus classified as a member of the family Iridoviridae and was designated as red sea bream iridovirus (RSIV). The genome of RSIV is liner dsDNA and considered to be circularly permitted and terminally redundant like other iridoviruses. The length of physical map of RSIV genome is 112,415bp. An indirect immunofluorescence test with a monoclonal antibody and PCR are commonly used for the rapid diagnosis of RSIV infected fish in the field. For the control of this disease, a formalin-killed vaccine against red sea bream iridoviral disease was developed and now commercially available.     

Effects of dietary protein and lipid levels on growth and feed utilization of wild‐caught striped sea bream,Lithognathus mormyrus

A feeding trial was carried out to determine the effects of dietary protein and lipid levels on the growth performance and feed utilization of wild‐caught striped sea bream (Lithognathus mormyrus). The experimental fish were collected from a local lagoon (Çardak Lagoon, Çanakkale, Turkey), transferred to the Marine Net Cage Unit and fed by hand to apparent satiation with a commercial sea bream feed (Biomar; 42% crude protein, 16% crude lipid). Approximately 4 weeks were needed to acclimate the fish to farming conditions. No pathological signs were observed and no fish losses occurred during the adaptation period. For the test trials four test diets with different levels of protein and lipid were formulated [low protein and low lipid (LP:LL), low protein and high lipid (LP:HL), high protein and low lipid (HP:LL), and high protein and high lipid (HP:HL)] and fed to L. mormyrus (mean weight 85.0 ± 4.6 g SEM) in the net cages (Ø 2 m, depth 2.5 m) for 60 days. During the experiment water temperature varied between 21.1 and 26.4°C; dissolved oxygen 8.4–9.6 mg L^?1; pH 7.2–8.6; and salinity 23.3–25.6‰. Growth performances of fish fed high protein diets were higher compared to fish fed low protein diets, irrespective of the dietary lipid level (P < 0.05). Feed conversion ratio (FCR) and protein efficiency ratio (PER) were not influenced by dietary protein or lipid levels (P > 0.05). Preliminary results indicate that striped sea bream can be easily adapted to farming conditions in net cages, and that a diet containing 50% crude protein and 15% crude lipid (HP:LL) levels with 23.0 g protein MJ^?1 gross energy of protein/energy ratio would be suitable for striped sea bream growth.     

Comparison of UV-B tolerance between wild and hatchery-reared juveniles of red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegeli)

The amount of ultraviolet (UV)-B radiation reaching the sea surface has increased due to ozone depletion. Several laboratory studies have highlighted the negative impacts of UV radiation on fish using hatchery-reared specimens. However, potential differences in UV tolerance between wild and hatchery-reared fish have been given little consideration. Wild and reared juveniles of red sea bream and black sea bream were exposed to one of four different UV-B radiation levels (1.8; 1.1; 0.4; 0?W/m²) for 4?h. Survival rate was measured every 2?h for a period of 24?h (red sea bream) or 48?h (black sea bream) following exposure. Wild and reared juvenile red sea bream were characterized by similar survival rate, with survival declining to almost 0?% 24?h after exposure at the 1.1 and 1.8?W/m² levels. In black sea bream, wild individuals showed significantly higher survival than reared fish in levels 1.1 and 1.8?W/m². Melanophore density was also measured since melanin absorbs UV radiation. Wild black sea bream showed higher melanophore density compared to reared individuals, while no such difference was observed in red sea bream. We conclude that wild black sea bream juveniles acquire higher UV tolerance partly by increasing melanophore density through exposure to UV radiation. Our results indicate that the predicted impacts of UV radiation on fish populations solely based on experimentation with hatchery-reared specimens may be overestimated for some species.     

Morphometric comparison and differentiation between artificially-released and wild red sea bream (Pagrus major)

The objective of this study is to develop a method of differentiation of hatchery-reared and artificially-released red sea bream (Pagrus major) from wild fish, based upon their morphometric differences. Morphometric measurements were done on fork length and 14 other characters. Among these charac)ters, significant differences between hatchery-reared and wild red sea bream were observed in body height, height at eye, eye diameter and upper jaw length. Discriminant functions were effective in differentiating artificial fish from wild fish.     

Phenotypic Diversity of Infectious Red Sea Bream Iridovirus Isolates from Cultured Fish in Japan

Megalocytivirus is causing economically serious mass mortality by infecting fish in and around the Pacific region of Asia. The recent emergence of many new iridoviruses has drawn attention to the marked taxonomic variation within this virus family. Most studies of these viruses have not included extensive study of these emergent species. We explored the emergence of red sea bream iridovirus (RSIV) on a fish farm in Japan, and we specifically endeavored to quantify genetic and phenotypic differences between RSIV isolates using in vitro and in vivo methods. The three isolates had identical major capsid protein sequences, and they were closely related to Korean RSIV isolates. In vitro studies revealed that the isolates differed in replication rate, which was determined by real-time quantitative PCR of viral genomes in infected cells and cell culture supernatant, and in cell viability, estimated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay for infected cells. In vivo studies showed that the isolates exhibit different virulence characteristics: infected red sea bream showed either acute death or subacute death according to infection with different isolates. Significant differences were seen in the antigenicity of isolates by a formalin-inactivated vaccine test. These results revealed that variant characteristics exist in the same phylogenetic location in emergent iridoviruses. We suggest that this strain variation would expand the host range in iridoviral epidemics.Iridoviruses are designated as icosahedral cytoplasmic DNA viruses; this group of viruses has different hosts, including fish, amphibians, and insects, causing economic and environmental problems. The family Iridoviridae includes five genera: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus, and Megalocytivirus (45). Piscine iridoviruses belong to the genera Ranavirus, Lymphocystivirus, and Megalocytivirus. In recent years, the genotypical variation between newly found iridovirus strains included in the genus Ranavirus has been studied in this viral family (5). However, the properties of and variation between iridovirus species have not been well characterized except for a few iridoviruses isolated from amphibians (11, 46), fish (8, 18, 19), and insects (23, 42, 44). Most studies attempting to differentiate variants have relied on genotypic rather than phenotypic properties.Members of the Megalocytivirus genus produce characteristic basophilic inclusion bodies in the enlarged cells of host fish organs, which have been collected from mass mortalities occurring in wild and cultured fish species (14, 37). Red sea bream (Pagrus major) aquaculture has suffered great losses from the prevalence of red sea bream iridovirus (RSIV) infection in Japan. RSIV has been assigned to the Megalocytivirus genus. The virus was first isolated from cultured red sea bream in western Japan in 1990 (22).Many other piscine iridoviruses have been reported in Asian countries (7, 24, 25) from more than 100 different species (41), including freshwater and marine fish (19). RSIV is closely related genetically to viruses isolated from ornamental fish in Southeast Asia, based on nucleotide sequence studies (21, 39). A formalin-inactivated RSIV vaccine has been used in juvenile marine fish against this disease (31, 32).The incidence of iridovirus infection has been increasing among cultured fish in Japan (29, 30). In addition, genetic and phenotypic iridovirus variants suggest the presence of diverse variants in this virus group (16). In the present study, we examined the detailed properties of three RSIV isolates from two fish species collected from fish farms in western Japan. Using in vitro and in vivo processes, we focused close examination specifically on quantitative genetic and phenotypic differences between the three isolates. The aim of this study was to increase understanding of the epidemiology of RSIV.     

Juvenile bisexuality in the red sea bream,Pagrus major

The histology of the gonad of the red sea bream,Pagrus major, was examined in order to study the early gonadal development, sexual maturation and sex ratio in a natural population. A total of 1,117 fish between the ages of 4 months and 8 years were examined. Gonads of 4-month-old fish were either sexually undifferentiated with a central cavity, or ovarian in form. Gonads of 12- and 18-month-old fish were ovaries or bisexual gonads, while those of 2-year-old fish were ovaries, bisexual gonads or testes. Fish aged between 3 and 8 years had ovaries or testes, except for a few bisexual gonads found in 3- and 4-year-old fish. The chronological appearance of females, hermaphrodites and males in that order, and histological evidence, suggested that the testis originates from the ovary via a bisexual gonad in the juvenile stage. The sex ratio of females to males at the age of 2 years and over was about 1:1, suggesting that hermaphroditic red sea bream appear in about 50% of the juvenile population. The sexual pattern in this species, therefore, is concluded to be gonochorism with a bisexual juvenile stage.     

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer‐assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time‐consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm² vs 4.50 ± 0.04 μm² and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm² vs 3.18 ± 0.06 μm² and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.     

Gilthead sea bream liver proteome altered at low temperatures by oxidative stress

Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold‐stress response was reproduced in gilthead sea bream acclimated to 20°C (Warm group) when the water temperature was lowered to 8°C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2‐DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine‐homocysteine‐methyl transferase, glutathione‐S‐transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin‐methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen‐like protein indicated an increase in proteolysis. Increases in elongation factor‐1α, the GAPDH oxidative form, tubulin and Raf‐kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.     

Application of the arming system for the expression of the 380R antigen from red sea bream iridovirus (RSIV) on the surface of yeast cells: a first step for the development of an oral vaccine

The cell surface is a functional interface between the inside and the outside of the cell. Moreover, cells have systems for anchoring surface specific proteins and for confining surface proteins to particular domains on the cell surface. For use in bioindustrial processes applied to oral vaccination, we consider that cell-surface display systems must be useful and that the yeast Saccharomyces cerevisiae, the most suitable microorganism for practical purposes, is available as a host for genetic engineering because it can be subjected to many genetic manipulations. In particular, the rigid structure of the cell makes the yeast suitable for several of the applications. In this study, we describe the expression of one of the target antigens, 380R, from the red sea bream iridovirus (RSIV), which is one of the most common viral diseases in the cultured marine fish Pagrus major in Japan, using the arming yeast system and aiming at its application for oral vaccination. We first performed the molecular cloning and expression of the 380R antigen from RSIV in Escherichia coli. The nucleotide sequence of the 380R antigen was composed of an open reading frame (ORF) of 1360 bp encoding a protein of 453 residues. To prepare a specific antibody against the 380R antigen, the recombinant protein was overexpressed and purified in E. coli. As a result of indirect immunofluorescence with the specific antibody, we could observe the expression of the 380R antigen on the surface of the yeast cells. Thus, we have successfully prepared the source of an oral vaccine using cell-surface display technology in yeast.     

Organization of the lipoprotein lipase gene of red sea bream Pagrus major

Lipoprotein lipase (LPL) is a key enzyme of lipid deposition and metabolism. To investigate the mechanism of lipid deposition in fish, as a first step, we have characterized the LPL gene of a marine teleost red sea bream Pagrus major by cDNA and genomic structure analysis. The red sea bream LPL gene encodes 511 amino acids and spans approximately 6.3 kb of the genome. The coding region is organized into ten exons and nine introns. In comparison with the LPL of other animals, the deduced amino acid sequence shows a high degree of similarity with a conservation of functional domains, e.g. catalytic triad, N-glycosylation sites, lipid and heparin binding regions. The 1.1 kb of 5′ flanking region contains two CCAAT, sequences homologous to Oct-I site and response elements for hormones including glucocorticoid, insulin and thyroid hormone. The results of the present study will facilitate further study of the function and regulation of the LPL in non-mammalian vertebrates.     

Sorting grid trials to improve size selectivity of red mullet (Mullus barbatus) and annular sea bream (Diplodus annularis) in Turkish bottom trawl fishery

Sorting grids with two different bar spacings (12 mm and 14 mm) were tested to improve size selectivity of the commercially important fish species, red mullet (Mullus barbatus) and annular sea bream (Diplodus annularis), in Turkish bottom trawl fishery. Fishing trials were carried out with R/V ‘Egesüf’ between April and May 2003 in Izmir Bay, Aegean Sea, using a traditional bottom trawl. Selectivity data were collected by the top cover method and analyzed by means of a logistic equation with the maximum likelihood method. Selectivity parameters for individual hauls were obtained with the software program cc 2000 . Mean selectivity was also estimated and compared using the EC Model software that takes between‐haul variations into account. The codend catch size as an additional explanatory variable was used in the comparison. The Kolmogorov–Smirnov (K–S) test was also applied to detect differences between length‐frequency distributions in the upper and lower bags of the 12 and 14 mm bar spacings for red mullet and annular sea bream. The mean L₅₀ values of red mullet were 8.7 and 10.0 cm with the 12 and 14 mm grids, respectively. The L₅₀ value of 14 mm was comparable with the L₅₀ value of the codend mesh size; however, the 12 mm value was rather low. The mean L₅₀ values of 12 and 14 mm bar spacings were 8.8 and 10.4 cm for annular sea bream, respectively; the value of 14 mm bar spacing was very close to 50% size at sexual maturity of the species (10.5 cm). The K–S test indicated length distributions of red mullet and annular sea bream in the 12 and 14 mm upper and lower bags as significantly different (P < 0.05). These results show that improving the size selectivity in a multi‐species fishery using a single selective device is rather difficult. However, higher size selectivity can be obtained when considering the minimum landing size or the 50% sexual maturity size for a given species.     

Mineral composition in fillets of sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata): a comparison of cultured and wild fish

The objective of this study was to determine some of the trace mineral elements in four commercial feeds commonly available in Turkey for marine culture and in fillets of cultured and wild sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). The feeds and cultured fish were from four different fish farms operating in the same region but with four slightly different feeds. Concentrations of Iron (Fe), Zinc (Zn), Manganese (Mn), Copper (Cu), Lead (Pb), Cobalt (Co), Nickel (Ni), Chromium (Cr), and Cadmium (Cd) were analyzed in the feeds and fillets of cultured as well as wild fish. Significant differences in the mineral concentrations existed within feed groups, cultured fish groups as well as between cultured and wild fish. Fe, Zn, and Mn in the feeds, Fe, Co, and Zn in sea bass and Fe, Zn and Co in sea bream were predominant among the nine analyzed minerals. Fe, Zn, Mn, Cr, and Ni contents in the fillets of wild sea bass were significantly, (P < 0.05) lower than those of the cultured sea bass groups. Co, Cr, Pb, and Ni in the fillets of wild sea bream were also significantly (P < 0.05) lower than those of cultured sea bream groups. These differences in trace elements in the cultured and wild fish were probably related to differences in their dietary mineral concentrations.     

Genetic integrity of black sea bream (Acanthopagrus schlegeli) sperm following cryopreservation

Although semen cryopreservation has been applied successfully in many fish species, extensive variation in post‐thaw semen quality exists between species and individuals. AFLP (amplified restriction fragment length polymorphism) is a powerful method for detecting DNA polymorphisms at the individual, population, and species levels. The method has been successfully applied to boars (Sus domestica, Suidae, Artiodactyla, Mammalia) to detect and evaluate differences in DNA sequences that correspond with semen integretiy after employing various freezing techniques. Freezing and thawing of semen has also an effect of selecting for freezing‐resistant (or intact) and eliminating non‐viable or defective sperm. Only the fully intact and functional sperm, despite potential compromise by adverse freezing and osmotic stresses, retain fertility after thawing. The objective of this study was to use AFLP to assess any genetic changes associated with the effect of employed cryo‐methodology on the genetic integrity of sperm of the black sea bream (Acanthopagrus schlegeli) under different cryopreservation treatments. The cryopreservation protocols had no significant effect on sperm motility or survival rate of fertilized ova regardless of using fresh (% motile sperm 89.6 ± 3.0; % embryonic survival rate 54.4 ± 2.9) and frozen‐thawed semen (% motile sperm 80.2 ± 2.0; % embryonic survival rate 51.8 ± 2.0). The post‐thaw sperm motility and survival rates were not significantly different among the sperm samples of the five black sea bream males examined. In the present study, the remaining black sea bream sperm that survive the cryopreservation limit the power of AFLP to trace the genetic markers which correlate with the differences in the sensitivity of sperm to cryo‐injury. It is also possible that point mutations outside the AFLP priming sites may not have been detected. More thorough investigations are needed to determine whether such DNA fingerprints would be found in fish species.     

Identification of the causative gene of a transparent phenotype of juvenile red sea bream Pagrus major

Deformities in cultured fish species may be genetic, and identifying causative genes is essential to expand production and maintain farmed animal welfare. We previously reported a genetic deformity in juvenile red sea bream, designated a transparent phenotype. To identify its causative gene, we conducted genome-wide linkage analysis and identified two single nucleotide polymorphisms (SNP) located on LG23 directly linked to the transparent phenotype. The scaffold on which the two SNPs were located contained two candidate genes, duox and duoxa, which are related to thyroid hormone synthesis. Four missense mutations were found in duox and one in duoxa, with that in duoxa showing perfect association with the transparent phenotype. The mutation of duoxa was suggested to affect the transmembrane structure and thyroid-related traits, including an enlarged thyroid gland and immature erythrocytes, and lower thyroxine (T₄) concentrations were observed in the transparent phenotype. The transparent phenotype was rescued by T₄ immersion. Loss-of-function of duoxa by CRISPR–Cas9 induced the transparent phenotype in zebrafish. Evidence suggests that the transparent phenotype of juvenile red sea bream is caused by the missense mutation of duoxa and that this mutation disrupts thyroid hormone synthesis. The newly identified missense mutation will contribute to effective selective breeding of red sea bream to purge the causative gene of the undesirable phenotype and improve seed production of red sea bream as well as provide basic information of the mechanisms of thyroid hormones and its related diseases in fish and humans.Subject terms: Agricultural genetics, Animal breeding     

Isolation and characterization of polymorphic microsatellite loci from an EST‐library of red sea bream (Chrysophrys major) and cross‐species amplification

Microsatellite markers have been developed from a complementary DNA (cDNA) library of red sea bream, Chrysophrys major. Twenty‐eight microsatellites were selected for designing microsatellite primers, of which 11 gave working primer pairs. Observed and expected heterozygosities varied from 0.33 to 1.00 and from 0.38 to 0.83, respectively. Five additional fish species assessed for cross‐species amplification revealed between one and six positive amplifications and between 0 and 6 polymorphic loci per species.     

Exposure of eggs to solar UV‐B leads to reduced hatching rates in two sparid fishes,red sea bream Pagrus major and black sea bream Acanthopagrus schlegeli

Hatching success was examined under exposure to solar ultraviolet radiation (UVR) using filters to give three different light conditions [C1: UV‐B, UV‐A and photosynthetically active radiation (PAR), C2: UV‐A and PAR, C3: PAR] in red Pagrus major and black Acanthopagrus schlegeli sea bream. Hatching rate of both species was reduced by an exposure over a 2 day period to UVR and was not significantly different between two species under the three light conditions.     

A comparison of fatty acid,cholesterol and vitamin composition in sea bass [Dicentrarchus labrax (Linnaeus, 1758)] and sea bream [Sparus aurata (Linnaeus, 1758)] from three cage farm areas: Antalya and Muğla (Turkey) and İskele (Northern Cyprus)

In the present study the fatty acids, cholesterol and vitamin composition in farmed sea bass (8 fish per species per farm; weight range: 389.6–395.8 g, total length range: 297–316 mm) and sea bream (8 fish per species per farm; weight range: 386.8–391.7 g, total length range: 263–268 mm) from three cage farms (?skele in northern Cyprus, Antalya and Mu?la in Turkey) were compared during the harvesting period in June–July 2011. The results showed that the muscles of D. labrax and S. aurata farmed fish were rich in n‐3 fatty acids, but with important differences. For example, the muscles of sea bass farmed in ?skele were rich in docosahexaenoic acid (DHA) and n‐3 polyunsaturated fatty acids (PUFA). Palmitic acid (C16:0) was the primary saturated fatty acid, and oleic acid (C18:1 n‐9) the primary monounsaturated fatty acid in the muscle and liver samples of the cage‐farmed sea bass and sea bream. There were no significant differences in the cholesterol content in the muscles of sea bream farmed in ?skele, Antalya or Mu?la. In conclusion, the n‐3/n‐6 ratio in the muscle of farmed S. aurata and D. labrax is within the recommended limits for a healthy human diet, being very suitable for human nutrition.