Sevenband Grouper Epinephelus Septemfasciatus Research Articles

An Oral Vaccination Method with the Aid of Capsaicin against Viral Nervous Necrosis (VNN)

To develop an oral vaccination method for viral nervous necrosis (VNN) caused by piscine nodavirus (betanodavirus), sevenband grouper Epinephelus septemfasciatus was immunized with inactivated redspotted grouper nervous necrosis virus (RGNNV) vaccine. ​Seven-day consecutive oral administration of the RGNNV vaccine via feed failed to induce protection in fish against the virus challenge at 21 days post-immunization. ​However, anal intubation of the vaccine conferred protective immunity on fish, although virus neutralizing antibodies could not be detected. Similar protective immunity was also confirmed by oral administration of vaccine via feed supplemented with capsaicin, suggesting that capsaicin modulated the intestinal mucosal barrier to induce immune response.

Genetic characterization of four hatchery populations of the seven-band grouper (Epinephelus septemfasciatus) using microsatellite markers

The seven-band grouper Epinephelus septemfasciatus is of considerable economic value and a candidate species for aquaculture in East Asia. Genetic characterization of hatchery populations is needed to maintain their genetic diversity and to develop more effective aquaculture practices. We analyzed the genetic structure of four cultured populations of E. septemfasciatus in Korea using 13 microsatellite markers. The number of alleles per locus ranged from 5 to 16, with an average of 9.6. The mean observed and expected heterozygosities were 0.699 and 0.679, respectively. Pairwise multilocus FST tests suggested the existence of two populations, although the isolation-by-distance model was rejected. An analysis of molecular variance (AMOVA) also revealed two genetic clusters. These results could reflect current aquacultural practices, with breeds and eggs exchanged between hatcheries, and/or genetic drift due to intensive breeding practices. Therefore, for the optimal management of resources, the genetic variation of hatchery stocks should be monitored and inbreeding controlled for the seven-band grouper stocks. This genetic information will be useful for the management of E. septemfasciatus fisheries and the aquaculture industry.

Cell Culture Medium Inhibits Antigen Binding Used in an ELISA for Detection of Antibodies against Nervous Necrosis Virus

Abstract We investigated the optimum dilution of nervous necrosis virus (NNV) for use as antigens to detect antibodies by an enzyme-linked immunosorbent assay (ELISA) in the Sevenband Grouper Epinephelus septemfasciatus. The ELISA values for a standardized suspension of antigens diluted with L-15 medium containing 1% fetal bovine serum decreased gradually with the dilution of the antigens, whereas those for the antigens diluted with distilled water (DW) initially increased with the dilution of the antigens, peaked at a 320-fold dilution, and then decreased thereafter. Additional studies revealed that binding of NNV antigens to ELISA wells was inhibited by fetal bovine serum and other substances in the L-15 medium. Sera obtained from Sevenband Grouper vaccinated with live NNV vaccine and survivors from natural NNV-infection were subjected to antibody detection by ELISA. All of the sera were positive by ELISA when the standardized suspension was diluted 320-fold, whereas sera from five out of the six survivors and two out of the six vaccinated fish were negative or weakly positive by ELISA using NNV antigens diluted 10-fold. We therefore concluded that cultured NNV solutions prepared in cell culture media may need to be diluted with distilled water for use in ELISA. Received January 28, 2014; accepted April 16, 2014.

Sperm cryopreservation of sex-reversed seven-band grouper, Epinephelus septemfasciatus

Seven-band grouper Epinephelus septemfasciatus is a protogynous hermaphrodite. The male individuals’ number is more less than the female one. Thus, it is necessary for artificial reproduction and crossbreeding to research the sperm cryopreservation of sex-reversed seven-band grouper. In present study, the spermatozoa of sex-reversed immature fish were frozen using the different cryopreserving solutions for cryopreservation. The several factors that may affect the freezing survival rate of seven-band grouper spermatozoa such as the spermatozoa diluent, the concentration and composition of cryoprotective agent have been studied. The results showed that ES1-3 (60g/L glucose+10g/L NaCl+0.5g/L NaHCO3) was significantly better as a diluent compared with MPRS, TS-2 and other series of diluent ES1. The further experiment revealed that the optimal cryoprotectants were 10% dimethyl sulfoxide (DMSO) or 10% 1,2-propylene glycol (PG) with the post-thaw sperm motility was 76.67±0.00% and 75.00±5.00%, respectively. In addition, salinity of seawater is an important motility stimulator because that the highest motility of 96.00±1.73% was obtained at salinity 30‰. In crossbreeding test with fresh unfertilized eggs of kelp grouper Epinephelus moara, the ratio of fertilization and hatchability had no significant differences between the cryopreserved sperm and fresh sperm of seven-band grouper. It is suggested that the frozen sperm of seven-band grouper could be applied in artificial reproduction and crossbreeding.

Development of the musculature and muscular abnormalities in larval seven-band grouper Epinephelus septemfasciatus

There are a number of reports documenting skeletal development in groupers, but surprisingly little is known about muscle development. We have documented muscle development in the seven-band grouper Epinephelus septemfasciatus (Thunberg), focusing primarily on the cranial muscles from 0 days post-hatching (dph) to 28 dph, using a modified whole mount immunohistochemical staining method. The eye muscles developed first followed by the cheek muscles, which completed development prior to first feeding. The muscles associated with the opercle and the levator internus 1 developed gradually and were not linked to first feeding. Later, between 12 and 16 dph, the muscle composition in the dorsal branchial arches changed to the adult form. We observed a number of cultured larvae that exhibited abnormal musculature in the body and jaw. Our results provide the first documentation of muscle development in larval seven-band grouper. This information can be used to detect abnormalities during development and provide a marker to assess the effect of changes in larval rearing techniques.

Trash fish can be a source of betanodaviruses for cultured marine fish

A total of 360 samples of trash fish/mollusk used for feeding cultured marine fish were collected from 4 stations of National Research Institute of Aquaculture, Fisheries Research Agency, Japan. The brain or eyes of the samples were examined by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR assays to detect the coat protein gene of betanodavirus. Two species of trash fish/mollusk samples: Japanese jack mackerel Trachrus japonicus and Japanese common squid Todarodes pacificus were positive for betanodavirus and the sequences of the nested PCR products (177 nucleotides) of these two species were closely related (98%) to the redspotted grouper nervous necrosis virus (RGNNV) genotype. Sevenband grouper Epinephelus septemfasciatus were intramuscularly injected (10 6 TCID 50/fish) with betanodavirus isolates from trash fish/mollusk samples. The fish displayed abnormalities typical of viral nervous necrosis (VNN) infection, and mortalities ranged from 90 to 100%. These results suggest that trash fish/mollusk can be a source of betanodaviruses for cultured fish and that they pose a serious risk for outbreaks of VNN in susceptible cultured fish.

Fish immunization using a synthetic double-stranded RNA Poly(I:C), an interferon inducer, offers protection against RGNNV, a fish nodavirus

Viral nervous necrosis (VNN), caused by a fish nodavirus, is one of the most serious fish diseases worldwide. Here we report a unique vaccination method in sevenband grouper Epinephelus septemfasciatus using a synthetic double-stranded RNA polyinosinic polycytidylic acid (Poly(I:C)), an interferon inducer, followed by challenge with a live fish nodavirus. Fish injected with Poly(I:C) at 200 microg fish(-1) were highly protected from artificial challenge with red-spotted grouper nervous necrosis virus (RGNNV) (relative percentage survival, RPS: 100%), and specific antibodies against RGNNV were detected in sera from survivors. Moreover, the surviving fish were protected from rechallenge with RGNNV (relative percent survival RPS: 100%). Thus, it was confirmed that specific immunity against RGNNV was established in sevenband grouper by injection with live RGNNV following Poly(I:C) administration. Antiviral state was induced in fish by injection with Poly(I:C) at > or = 50 microg fish(-1), but no toxic response was observed in the fish even if Poly(I:C) was injected at a dose of 200 microg fish . In fish injected with Poly(I:C) at 200 pg fish(-1), a high level of antiviral state of > 90% RPS against RGNNV challenge lasted for at least 4 d after Poly(I:C) injection. However, no curative effect by Poly(I:C) injection was observed in fish already infected with RGNNV. It is considered that the present immunization method using Poly(I:C) followed by a live virus injection could offer protection against various viral infections in a broader range of fish species.

Variable region of betanodavirus RNA2 is sufficient to determine host specificity

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.

Role of Follicle‐Stimulating Hormone and Androgens on the Sexual Inversion of Sevenband Grouper Epinephelus septemfasciatus

AbstractThis study was designed to characterize the amount of androgen implant and the time period necessary to cause sexual inversion in sevenband grouper Epinephelus septemfasciatus and to determine whether administration of follicle‐stimulating hormone (FSH) with or without androgens had any effect on the sexual state of this species. Seventy‐two fish were treated with 10 μg of recombinant FSH/kg body weight (BW) or saline control in cocoa butter. Two days later, cocoa butter and α‐cellulose pellet implants of control, 0.1 mg of low‐androgen mixture (LoAN)/kg BW, or 5 mg of high‐androgen mixture (HiAN)/kg BW were injected into the same fish. Fish were sampled after 3 and 5 weeks. Gonadosomatic indices (GSIs) at 3 weeks for FSH, HiAN, and FSH+HiAN treatment groups were significantly lower than the control values. At 5 weeks, no significant difference was observed, although the GSIs for HiAN treatment groups were lowest at both sampling points. The highest GSI values recorded were in the FSH‐only treatment group at 5 weeks. All the controls remained in the immature female state throughout the study. High‐dose androgen levels caused sexual transition of gonadal tissue at 3 weeks with spermatocytes present in all individuals. At 5 weeks, all individuals in the HiAN treatment group had complete inversion to male gonad, and sperm sinus development occurred in four individuals. However, incomplete sexual inversion was observed in the FSH+HiAN treatment groups with some ovarian tissue persisting even at 5 weeks. Exogenous FSH treatment alone appears to stimulate ovarian growth in the postspawning period. The 3‐week period of sexual transition in this study is one of the shortest time periods recorded for grouper sexual inversion. This may be due to androgen treatment in the postspawning period, the proposed period of natural sex change in several grouper species.

Phylogenetic analysis of betanodaviruses isolated from cultured fish in Korea

Since the publication of the first report on fish nodaviruses in Korea in 1998, fish nodaviruses have caused widespread epizootic events among various fish species in Korea. However, the genotypes of fish nodaviruses in Korea have not yet been determined due to a lack of information about their nucleotide sequences. In this study, we isolated 5 fish nodaviruses from 4 fish species cultured in 4 different regions in Korea: rock bream Oplegnathus fasciatus, Japanese flounder Paralichthys olivaceus, sevenband grouper Epinephelus septemfasciatus, and grey mullet Mugil cophalus. The full open-reading frame (ORF) encoding the coat protein (1017 nt) was sequenced from each of the 5 fish nodaviruses and the nucleotide sequences were phylogenetically analyzed. Results showed that even though their sequences were not identical, all 5 Korean isolates were clustered in the RGNNV genotype. This is the first report on the phylogenetic analysis of fish nodaviruses from cultured fish in Korea.

マハタ仔魚のワムシサイズに対する摂餌選択性

マハタ仔魚のワムシサイズに対する摂餌選択性