The mitochondrial genome(mitogenome)of hybrid grouper Epinephelus moara(♀)×Epinephelus tukula(♂),a new hybrid progeny,can provide valuable information for analyzing phylogeny and molecular evolution.In this stu...The mitochondrial genome(mitogenome)of hybrid grouper Epinephelus moara(♀)×Epinephelus tukula(♂),a new hybrid progeny,can provide valuable information for analyzing phylogeny and molecular evolution.In this study,the mitogenome was analyzed using PCR amplification and sequenced,then the phylogenetic relationship of E.moara(♀)×E.tukula(♂)and 35 other species were constructed using Maximum Likelihood and NeighborJoining methods with the nucleotide sequences of 13 conserved protein-coding genes(PCGs).The complete mitogenome of E.moara(♀)×E.tukula(♂)was 16695 bp in length,which contained 13 PCGs,2 rRNA genes,22 tRNA genes,a replication origin and a control region.The composition and order of these genes were consistent with most other vertebrates.Of the 13 PCGs,12 PCGs were encoded on the heavy strand,and ND6 was encoded on the light strand.The mitogenome of the E.moara(♀)×E.tukula(♂)had a higher AT nucleotide content,a positive AT-skew and a negative GC-skew.All protein initiation codons were ATG,except for COX and ND4(GTG),ATP6(CTG),and ND3(ATA).ND2,COXII,ND3,ND4 and Cytb had T as the terminating codon,COXIII’s termination codon was TA,and the remaining PCGs of that were TAA.All tRNA genes,except for the lacking DHU-arm of tRNASer(AGN),were predicted to form a typical cloverleaf secondary structure.In addition,sequence similarity analysis(99%identity)and phylogenetic analysis(100%bootstrap value)indicated that the mitochondrial genome was maternally inherited.This study provides mitogenome data for studying genetic,phylogenetic relationships and breeding of grouper.展开更多
Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years These molecules have attracted much research interest because of their biochemical diversity, broad specificity...Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years These molecules have attracted much research interest because of their biochemical diversity, broad specificity on anti-viral, anti-bacterial, anti-fungi, anti-protozoan parasites, anti-tumoural, and wound-healing effects. Antimicrobial proteins and peptides play key roles in innate immunity. They interact directly with bacteria and kill them. The brown-spotted grouper, Epinephelusfario, is an important marine fish cultured in southem China. Recently, bacteria and virus have caused high mortality in E. fario cultures, but its endogenous antimicrobial peptides and proteins have not been explored. An antimicrobial component was found from the skin homogenate of E. fario. After the skin homogenate was digested with trypsin, its antimicrobial activity was lost, which showed that the antimicrobial component is a protein. The antimicrobial protein （Efap） was purified from the skin homogenate of E. fario by successive ion-exchange and gel filtration chromatography. Efap was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 41 kD. Efap exhibited antimicrobial activity both for the Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis, and for the Gram-negative bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Aeromonas hydrophila, Eschrrichiu coli, and Pseudomonas aeruginosa. Except A. hydrophila, P. aeruginosa, and E. coli （MIC〉20 mol/L）, most of the tested Gram-negative bacteria were sensitive to Efap （MIC〈20 mol/L）. Interestingly, Efap showed potent antimicrobial activity against Gram-positive bacteria S. aureus （MIC 5-10 mol/L） but comparatively weak antimicrobial activity against M. luteus and B. subtilis. The broad antimicrobial activities of Efap suggest that it contributes to the innate host defence of E. fario.展开更多
[Objective] The study aimed to evaluate the genetic variability and interspecific relationships among four species of groupers from South China Sea, including E. fario, E. merra, E. malabaricus and E. coioides. [Metho...[Objective] The study aimed to evaluate the genetic variability and interspecific relationships among four species of groupers from South China Sea, including E. fario, E. merra, E. malabaricus and E. coioides. [Method] Twenty one mircosatellite loci of groupers were selected from GenBank and eight high polymorphic loci were used to further genetic analysis. [Result] The mean number of alleles per locus （A）, effective number of alleles （Ne）, mean polymorphism information content （PIC）, observed heterozygosity （Ho） and expected heterozygosity （He） were 4.38±1.60, 3.69±0.86, 0.69±0.08, 0.67±0.08, 0.72±0.06 in E. malabaricus; 3.88±1.13, 3.55±1.04, 0.66±0.10, 0.68±0.21, 0.70±0.08 in E.coioides; 6.00±1.07, 4.68±0.65, 0.78±0.03, 0.73±0.25, 0.79±0.03 in E. fario; 5.50±1.07, 4.58±0.80, 0.76±0.05, 0.75±0.18, 0.78±0.04 in E. merra, respectively. [Conclusion] We compared the values above, the order of the genetic variability among these grouper species was E. fario E. merra E. malabaricus E. coioides. We found that the level of genetic variability of these groupers species was relatively higher than that of other marine fish, so their genetic status was good. In addition, the analyisis of genetic relationship showed that E. malabaricus and E. coioides was the closest and it was the farthest between E. merra and E. coioides.展开更多
Crossbreeding between Epinephelas coioides （ ♀ ） and Epinephelus lanceolatus （♂） was conducted by artificial insemination. Fertilized eggs were col- lected at 0, 30, 90 s, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,...Crossbreeding between Epinephelas coioides （ ♀ ） and Epinephelus lanceolatus （♂） was conducted by artificial insemination. Fertilized eggs were col- lected at 0, 30, 90 s, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min post-insemination respectively, which were fixed by Smith＇s solution, embedded by paraffins and stained by H.E. According to the characteristics of Epinephelus coioides eggs, tissue section method was modified. The results showed that the sperms of Epinephelus lanceolatus rapidly entered the eggs of Epinephelus coioides at 30 s - 1 rain post-insemination. Observation results of tissue sections showed that mature eggs of Epinephelus coioides remained at metaphase of secondary maturation division. The eggs were activated after sperm penetration into the egg. With the develop- ment of secondary maturation division, at 2 rain post-insemination, eggs reached the metaphase of secondary maturation division ; at 3 - 6 rain post-insemination, sperm asters appeared; at 5 min post-insemination, eggs extruded secondary polar body; at 7 -15 rain post-insemination, male pronucleus and female pronucleus moved closer to each other and fused finally, forming a clear junction line; subsequently, zygote nucleus formed and karyotheca became faint; at 15 min pest-insem- ination, first karvokinetic division was develoued.展开更多
Using multi-molecular marker technologies and based on morphological criteria, the genetic relationship between Epinepheelus malabaricus and E, coioides was examined in the hope of resolving the long-standing issue of...Using multi-molecular marker technologies and based on morphological criteria, the genetic relationship between Epinepheelus malabaricus and E, coioides was examined in the hope of resolving the long-standing issue of identifying these two species. Results showed that： （ 1 ） E. coioides and E. malabaricus should be identified as two species, the consistency of mitochondrial DNA cytochrome b gene sequence between E. malabaricus and E. coioides is 94.4%, and the genetic similarity by AFLP was 0.753 9 ; （2） Hybridization exists between E. malabaricus and E. coioides, the specific RAPD and AFLP fragments are found to be useful in the identification of these two species, and the genetic properties （both with exterior and inheritance） of hybrid is significantly biased to the male parents ; and （3） AFLP was a potentially powerful tool in constructing the genetic linkage map for these two groupers.展开更多
Viral nervous necrosis （VNN） causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is i...Viral nervous necrosis （VNN） causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction （RT-PCR） was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro- sis virus （NNV）, in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus （IPNV）, infectious hematopoietic necrosis virus （IHNV）, spring viraemia of carp virus （SVCV）, epizootic haematopoietic necrosis virus （EHNV）, and large yellow croaker iridovirus （LYC1V）. With this method, the orange-spotted grouper （Epinephelus coioides） fry from hatcheries with or without incidence of the VN- N epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the t- wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.展开更多
This study aimed to study the development characteristics of Epinephelus fuscoguttatus at the embryo, larval, juvenile and young fish stages, thereby improving the breeding efficiency of E. fuscoguttatus. [Method] The...This study aimed to study the development characteristics of Epinephelus fuscoguttatus at the embryo, larval, juvenile and young fish stages, thereby improving the breeding efficiency of E. fuscoguttatus. [Method] The morphological characteristics and embryonic and post embryonic development of E. fuscoguttatus, including the occurrence and development of nervous system, digestive system and circulatory system, were observed and analyzed using eggs self-fertilized with artificially induced spawning in the breeding base of Hainan Province. [Result] The fertilized egg of E. fuscoguttatus is spherical, colorless transparent with single oil ball, and its diameter ranges from 0.83 to 0.94 mm. Based on the development process, the embryonic development of E. fuscoguttatus was divided into six stages, namely fertilized egg stage, cleavage stage, blastula stage, gastrula stage, idiosome formation stage and hatching stage. The entire embryonic development of E. fuscoguttatus required 20 h 47 min in the sea water with temperature of 28.4-31.9℃ salinity of 31-32 and pH of 8.4, and the total cumulative temperature was 455.361 ℃·h. The post embryonic development of E. fuscoguttatus was divided into larval stage, juvenile stage and young fish stage based on the features of the yolk-sac, second dorsal fin spine, pelvic fin spine, and body color. Accordingly, the larval stage was further classified into pre-larva and post-larva, with or without yolk-sac, respectively. The extension and shrinkage of the first pelvic fin spine and the second dorsal fin spine as well as the spinules on aforementioned spines were the most significant changes during the post embryonic development of E. fuscoguttatus. [Conclusion] This study will provide some basic data for industrial breeding of E. fuscoguttatus.展开更多
This study presents the complete mitochondrial genome of the hybrid Epinephelus moara♀× Epinephelus lanceolatus♂. The genome is 16886 bp in length, and contains 13 protein-coding genes, 2 r RNA genes, 22 t RNA ...This study presents the complete mitochondrial genome of the hybrid Epinephelus moara♀× Epinephelus lanceolatus♂. The genome is 16886 bp in length, and contains 13 protein-coding genes, 2 r RNA genes, 22 t RNA genes, a light-strand replication origin and a control region. Additionally, phylogenetic analysis based on the nucleotide sequences of 13 conserved protein-coding genes using the maximum likelihood method indicated that the mitochondrial genome is maternally inherited. This study presents genomic data for studying phylogenetic relationships and breeding of hybrid Epinephelinae.展开更多
Vibriofluvialis incubated in trypticase soy broth （TSB） showed stronger adhesion to the skin mucus of Epinephelus awoara than V. fluvialis grown on trypticase soy agar （TSA）, and this bacterial adhesion was assess...Vibriofluvialis incubated in trypticase soy broth （TSB） showed stronger adhesion to the skin mucus of Epinephelus awoara than V. fluvialis grown on trypticase soy agar （TSA）, and this bacterial adhesion was assessed in terms of saturation kinetics. Treating bacteria with antibody against O-antigens resulted in significantly reduced bacterial adhesion. In the early growth stage, the adhering bacteria numbers increased with incubation time, peaked at 24 h, and then dropped sharply. Prior heat treatment of the mucus at various temperatures resulted in different effects on bacterial adhesion. No remarkable effect on bacterial adhesion was detected after treating mucus with protease K and trypsin, whereas periodic acid treatment significantly enhanced bacterial adhesion. Extracts of K fluvialis surface structures at high concentrations significantly inhibited bacterial adhesion, while relatively low concentrations enhanced adhesion. V. fluvialis had strong affinity for a high molecular weight mucus component. These results indicate that V.fluvialis adhered strongly to E. awoara mucus by specific adhesion and that this adhesion was influenced by many factors, including bacterial conditions, mucus, and the environment.展开更多
Allograft inflammatory factor-1 （ AIF-1 ） is a cytoplasmic calcium-binding protein involved in inflammatory response-related diseases in mammals. Previously an identified AIF-1 gene was simply reported in yellow gro...Allograft inflammatory factor-1 （ AIF-1 ） is a cytoplasmic calcium-binding protein involved in inflammatory response-related diseases in mammals. Previously an identified AIF-1 gene was simply reported in yellow grouper. The characterization of AIF-1 gene and its expression at the gene and protein level are further described. Yellow grouper AIF-1 is composed of 147 amino acids, and 64% ~ 84% identical to other homologues. Basal level AIF-1 mRNA expression was noted in spleen, anterior kidney and kidney, using reverse-transcription polymerase chain reaction （RT-PCR）. After stimulation of LPS, the AIF-1 mRNA expression was up-regulated in tissues examined： spleen, anterior kidney, kidney, heart and liver, but not in muscle. The recombinant AIF-1 protein was expressed in Escherichia coli, and then purified for the development of antiserum. Western blotting analysis revealed a band with a molecular mass of about 17 ku.展开更多
On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OG...On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein （ MCP）gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction （RT-PCR） and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 （DE3）. Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers （8 cm in average length） were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group （ A1,30% ） was a little higher than the unvaccined group （ B2, 27.8% ）. Group B was challenged after three vaccine injections. The mortality in the vaccined group （B1, 16.7% ） was lower than the unvaccined group （132, 27.8% ）, And the relative percentage survival （RPS） value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 ： 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish （ Group C1, mortality of 18.18% ）, compared with the fish （ Group C2, mortality of 40% ） which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.展开更多
The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper, Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/k...The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper, Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/kg were investigated. After 20 weeks, the growth rates of the groups with high level of vitamin C apparently increased. The untreated fish had symptoms of vitamin C deficiency. The endogenous liver tissue vitamin C levels were found to reflect well the dietary treatments. After intraperitoneal injection or bath challenge with a virulent strain of Vibrio vulnificus , fish fed with high level vitamin C showed significantly higher survival rate compared with the normal control group. Vaccination with formalin inactivated V. vulnificus significantly enhanced the specific antibody production in fish treated with vitamin C, and completely protected from strong bacterial challenge the groups fed on fish with vitamin C 1500 and 2000 mg/kg diet.展开更多
The impact of water temperature(24, 27, 30 and 33℃) and salinity(15, 20, 25, 30 and 33) on oxygen consumption(OCR) and ammonium excretion rate(AER) of ♀ Epinephelus fuscoguttatus ×♂ E. lanceolatus hybrid group...The impact of water temperature(24, 27, 30 and 33℃) and salinity(15, 20, 25, 30 and 33) on oxygen consumption(OCR) and ammonium excretion rate(AER) of ♀ Epinephelus fuscoguttatus ×♂ E. lanceolatus hybrid grouper juveniles(9.39 ± 0.07 g) were investigated under the fed and un-fed conditions. The results showed that the OCR and AER were significantly(P < 0.05) affected by temperature and salinity under both fed and un-fed conditions. When temperature was 24–33℃, the OCR and AER of fed hybrid grouper juveniles were 85.68%–129.52% and 125.78%–287.63%, respectively, higher than those of un-fed hybrid grouper juveniles. The O/N ratio, protein use(P_u), Q_(10)(respiration) and Q_(10)(excretion) of fed hybrid grouper juveniles were 14.43–24.01, 28.35% – 48.48%, 1.69 and 3.01, respectively. The O/N ratio, P_u, Q_(10)(respiration), Q_(10)(excretion) of un-fed hybrid grouper juveniles were 20.39 – 31.79, 22.16% – 34.34%, 1.23 and 1.17, respectively. When salinity was 15–33, the OCR and AER of fed hybrid grouper juveniles increased by 87.42% – 116.85% and 215.38% – 353.57%, respectively, over those of un-fed hybrid grouper juveniles. The O/N ratio and P_u of fed hybrid grouper juveniles were 14.48 – 17.78, 39.36% – 49.43%, respectively. The O/N ratio and Pu of un-fed hybrid grouper juveniles were 20.39 – 31.79 and 22.16% – 34.34%, respectively. The specific dynamic action(SDA) of hybrid grouper juveniles was mainly related to protein metabolism. The results had a guiding significance to the large-scale intensive aquaculture of hybrid grouper juveniles.展开更多
The paper reviews the recent advances in studying grouper nutrition requirement for the development of cost-effective and environmentally friendly artificial diets. It consists of seven parts: protein and amino acid, ...The paper reviews the recent advances in studying grouper nutrition requirement for the development of cost-effective and environmentally friendly artificial diets. It consists of seven parts: protein and amino acid, lipid and essential fatty acid, carbohydrate, vitamin, mineral, alternative protein source, broodstock and larval nutrition. The review provides some basic information for further investigation of nutrient requirements of groupers.展开更多
Aquaculture production efficiency may increase by using feed additives. This study investigated the effects of different dietary additives [w/w: 2% small peptides, 0.01% probiotics( Bacillus licheniformis) and 0.2% pr...Aquaculture production efficiency may increase by using feed additives. This study investigated the effects of different dietary additives [w/w: 2% small peptides, 0.01% probiotics( Bacillus licheniformis) and 0.2% prebiotics(inulin)] on growth performance, digestive enzyme activities, and oxidative stress in juvenile Epinephelus coioides reared in artificial seawater of two salt concentrations(13.5 vs. 28.5). Weight gain rate was significantly higher in fish fed the diet supplemented with small peptides, B. licheniformis, inulin, or synbiotics than that in fish fed the basal diet; the greatest weight gain rate was found in fish fed the small peptide treatment [56.0% higher than basal diet]. Higher feed efficiency was detected in fish fed the diet supplemented with small peptides than that of fish in the other dietary treatments. Total protease activity in the stomach and intestines was highest in fish fed the small peptide-treated diet, whereas lipase activity was highest in those fed synbiotics(combination of Bacillus licheniformis and inulin) than that in fish fed the other treatments. Antioxidant enzyme(total superoxide dismutase and catalase) activities and hepatic malondialdehyde content were higher in fish receiving the dietary supplements and maintained in artificial seawater containing 13.5 salinity compared with those in the control(28.5). Hepatic catalase activity in grouper fed the diets with small peptides or synbiotics decreased significantly compared with that in control fish. Overall, the three types of additives improved growth rate of juvenile grouper and digestive enzymes activities to varying degrees but did not effectively improve antioxidant capacity under low-salinity stress conditions.展开更多
The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. T...The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).展开更多
The effects of temperature and pH on embryonic development and survival rate of newly hatched larvae of Epinephelus fuscoguttatus were discussed by single factor experiment method. Under a salinity of 31‰, fertilized...The effects of temperature and pH on embryonic development and survival rate of newly hatched larvae of Epinephelus fuscoguttatus were discussed by single factor experiment method. Under a salinity of 31‰, fertilized eggs were incubated at different temperatures （17, 20, 23, 26, 29 （eontrol）, 32, 35 ℃ ） and different pH values （5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.4 （control）, 8.5, 9.0, 9.5 and 10.0）, the development rate of fertilized eggs, hatching rate, deformity rate, survival rate of newly hatched larvae, and optimum temperature and pH ranges for embryonic development were recorded and analyzed. The results showed that the incubation period y required by fertilized eggs was in remarkably negative correlation with temperature x, y =4 017. 3x-15277 , R2 =0. 971 3. At 29 ℃, the hatching rate was the highest, up to 92.00% ; this temperature was corresponding to the lowest deformity rate of newly hatched larvae （ 1.09% ） ; corresponding effective accumulative temperature was also the lowest （437. 410 ℃ . h）, and significantly different from other experimental groups （P 〈 0.05 ） ; but on the contrary, corresponding survival rates at 24 and 48 h reached, respectively, 88.03% and 82. 97% which were significantly different from other experimental groups （ P 〈 0.05）. At the pH of 8.4 in the CK group and the pH of 8.5 of one experimental group, the embryonic development rates were the highest; con-esponding hatching rates of fertilized eggs were the highest, respectively, of 88.33% and 83.33% which were significantly different from other groups （P 〈 0.05） ; and corresponding survival rates at 24 and 48 h were the highest, and reached 90.84% and 85.03%, respectively. In the pH range of 8.0 - 8.5, the deformity rate of newly hatched larvae was 0. It was indicating that for embryonic development of E. fuscoguttatus, the suitable water temperature value was in the range of 17 - 29 ℃, the optimum value was in the range of 23 -29℃; and the suitable pH value was in the range of 6.5 -9.5, and the optimum value was in the range of 8.0- 8.5.展开更多
基金The Key Research and Development Program of Shandong Province under contact No.2019GHY112063the Breeding Project of Shandong Province under contract No.2019LZGC020+1 种基金the Central Public-interest Scientific Institution Basal Research Fund Chinese Academy of Fishery Sciences under contract Nos 2020XT0601,2020TD19 and 2020TD25the Yellow Sea Fisheries Research Institute Research Fees under contract Nos 20603022019002 and 20603022020015
文摘The mitochondrial genome(mitogenome)of hybrid grouper Epinephelus moara(♀)×Epinephelus tukula(♂),a new hybrid progeny,can provide valuable information for analyzing phylogeny and molecular evolution.In this study,the mitogenome was analyzed using PCR amplification and sequenced,then the phylogenetic relationship of E.moara(♀)×E.tukula(♂)and 35 other species were constructed using Maximum Likelihood and NeighborJoining methods with the nucleotide sequences of 13 conserved protein-coding genes(PCGs).The complete mitogenome of E.moara(♀)×E.tukula(♂)was 16695 bp in length,which contained 13 PCGs,2 rRNA genes,22 tRNA genes,a replication origin and a control region.The composition and order of these genes were consistent with most other vertebrates.Of the 13 PCGs,12 PCGs were encoded on the heavy strand,and ND6 was encoded on the light strand.The mitogenome of the E.moara(♀)×E.tukula(♂)had a higher AT nucleotide content,a positive AT-skew and a negative GC-skew.All protein initiation codons were ATG,except for COX and ND4(GTG),ATP6(CTG),and ND3(ATA).ND2,COXII,ND3,ND4 and Cytb had T as the terminating codon,COXIII’s termination codon was TA,and the remaining PCGs of that were TAA.All tRNA genes,except for the lacking DHU-arm of tRNASer(AGN),were predicted to form a typical cloverleaf secondary structure.In addition,sequence similarity analysis(99%identity)and phylogenetic analysis(100%bootstrap value)indicated that the mitochondrial genome was maternally inherited.This study provides mitogenome data for studying genetic,phylogenetic relationships and breeding of grouper.
基金Key Research Program for International Cooperation(2005DFA30610)Program for New Century Excellent Talents in University(NCET-05-0755)+2 种基金National Natural Science Foundation(30700128)Natural Science Foundation of Hainan Province(80623)Research Foundation of Education Department of Hainan Province(Hj200731)
文摘Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years These molecules have attracted much research interest because of their biochemical diversity, broad specificity on anti-viral, anti-bacterial, anti-fungi, anti-protozoan parasites, anti-tumoural, and wound-healing effects. Antimicrobial proteins and peptides play key roles in innate immunity. They interact directly with bacteria and kill them. The brown-spotted grouper, Epinephelusfario, is an important marine fish cultured in southem China. Recently, bacteria and virus have caused high mortality in E. fario cultures, but its endogenous antimicrobial peptides and proteins have not been explored. An antimicrobial component was found from the skin homogenate of E. fario. After the skin homogenate was digested with trypsin, its antimicrobial activity was lost, which showed that the antimicrobial component is a protein. The antimicrobial protein （Efap） was purified from the skin homogenate of E. fario by successive ion-exchange and gel filtration chromatography. Efap was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 41 kD. Efap exhibited antimicrobial activity both for the Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis, and for the Gram-negative bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Aeromonas hydrophila, Eschrrichiu coli, and Pseudomonas aeruginosa. Except A. hydrophila, P. aeruginosa, and E. coli （MIC〉20 mol/L）, most of the tested Gram-negative bacteria were sensitive to Efap （MIC〈20 mol/L）. Interestingly, Efap showed potent antimicrobial activity against Gram-positive bacteria S. aureus （MIC 5-10 mol/L） but comparatively weak antimicrobial activity against M. luteus and B. subtilis. The broad antimicrobial activities of Efap suggest that it contributes to the innate host defence of E. fario.
基金Supported by the National High Technology Research and Development Program of China(863Program,No.2007AA091404)the National Key Technology Research and Development Program(2007BAD29B03)A Project Funded by the Academic Program Development of Jiangsu Higher Education Institutions(PAPD)~~
文摘[Objective] The study aimed to evaluate the genetic variability and interspecific relationships among four species of groupers from South China Sea, including E. fario, E. merra, E. malabaricus and E. coioides. [Method] Twenty one mircosatellite loci of groupers were selected from GenBank and eight high polymorphic loci were used to further genetic analysis. [Result] The mean number of alleles per locus （A）, effective number of alleles （Ne）, mean polymorphism information content （PIC）, observed heterozygosity （Ho） and expected heterozygosity （He） were 4.38±1.60, 3.69±0.86, 0.69±0.08, 0.67±0.08, 0.72±0.06 in E. malabaricus; 3.88±1.13, 3.55±1.04, 0.66±0.10, 0.68±0.21, 0.70±0.08 in E.coioides; 6.00±1.07, 4.68±0.65, 0.78±0.03, 0.73±0.25, 0.79±0.03 in E. fario; 5.50±1.07, 4.58±0.80, 0.76±0.05, 0.75±0.18, 0.78±0.04 in E. merra, respectively. [Conclusion] We compared the values above, the order of the genetic variability among these grouper species was E. fario E. merra E. malabaricus E. coioides. We found that the level of genetic variability of these groupers species was relatively higher than that of other marine fish, so their genetic status was good. In addition, the analyisis of genetic relationship showed that E. malabaricus and E. coioides was the closest and it was the farthest between E. merra and E. coioides.
文摘Crossbreeding between Epinephelas coioides （ ♀ ） and Epinephelus lanceolatus （♂） was conducted by artificial insemination. Fertilized eggs were col- lected at 0, 30, 90 s, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min post-insemination respectively, which were fixed by Smith＇s solution, embedded by paraffins and stained by H.E. According to the characteristics of Epinephelus coioides eggs, tissue section method was modified. The results showed that the sperms of Epinephelus lanceolatus rapidly entered the eggs of Epinephelus coioides at 30 s - 1 rain post-insemination. Observation results of tissue sections showed that mature eggs of Epinephelus coioides remained at metaphase of secondary maturation division. The eggs were activated after sperm penetration into the egg. With the develop- ment of secondary maturation division, at 2 rain post-insemination, eggs reached the metaphase of secondary maturation division ; at 3 - 6 rain post-insemination, sperm asters appeared; at 5 min post-insemination, eggs extruded secondary polar body; at 7 -15 rain post-insemination, male pronucleus and female pronucleus moved closer to each other and fused finally, forming a clear junction line; subsequently, zygote nucleus formed and karyotheca became faint; at 15 min pest-insem- ination, first karvokinetic division was develoued.
基金This study was supported by the National Natural Science Foundation of China under contract Nos 40306023 and 40576064the Important Program of Science and Technology of Fujian Province in China under contract No.2004NZ03.
文摘Using multi-molecular marker technologies and based on morphological criteria, the genetic relationship between Epinepheelus malabaricus and E, coioides was examined in the hope of resolving the long-standing issue of identifying these two species. Results showed that： （ 1 ） E. coioides and E. malabaricus should be identified as two species, the consistency of mitochondrial DNA cytochrome b gene sequence between E. malabaricus and E. coioides is 94.4%, and the genetic similarity by AFLP was 0.753 9 ; （2） Hybridization exists between E. malabaricus and E. coioides, the specific RAPD and AFLP fragments are found to be useful in the identification of these two species, and the genetic properties （both with exterior and inheritance） of hybrid is significantly biased to the male parents ; and （3） AFLP was a potentially powerful tool in constructing the genetic linkage map for these two groupers.
基金The National Basic Research Program under contract No.2012CB114402the National High Technology Research and Development Program of China (863 Program) under contract No.2012AA092202Scientific Research Project of Marine Public Welfare Industry of China under contract No.200905020-07
文摘Viral nervous necrosis （VNN） causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction （RT-PCR） was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro- sis virus （NNV）, in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus （IPNV）, infectious hematopoietic necrosis virus （IHNV）, spring viraemia of carp virus （SVCV）, epizootic haematopoietic necrosis virus （EHNV）, and large yellow croaker iridovirus （LYC1V）. With this method, the orange-spotted grouper （Epinephelus coioides） fry from hatcheries with or without incidence of the VN- N epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the t- wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.
基金Supported by Special Project of Hainan Provincial Engineering Research Center(GCZX2013005)Technology Research and Development Project of Hainan Provincial Research Institutes(KYYS-2014-59,KYYS-2013-24,11-20410-0010)Applied Technology Research and Development and Demonstration Project of Hainan Province(ZDXM2015025)~~
文摘This study aimed to study the development characteristics of Epinephelus fuscoguttatus at the embryo, larval, juvenile and young fish stages, thereby improving the breeding efficiency of E. fuscoguttatus. [Method] The morphological characteristics and embryonic and post embryonic development of E. fuscoguttatus, including the occurrence and development of nervous system, digestive system and circulatory system, were observed and analyzed using eggs self-fertilized with artificially induced spawning in the breeding base of Hainan Province. [Result] The fertilized egg of E. fuscoguttatus is spherical, colorless transparent with single oil ball, and its diameter ranges from 0.83 to 0.94 mm. Based on the development process, the embryonic development of E. fuscoguttatus was divided into six stages, namely fertilized egg stage, cleavage stage, blastula stage, gastrula stage, idiosome formation stage and hatching stage. The entire embryonic development of E. fuscoguttatus required 20 h 47 min in the sea water with temperature of 28.4-31.9℃ salinity of 31-32 and pH of 8.4, and the total cumulative temperature was 455.361 ℃·h. The post embryonic development of E. fuscoguttatus was divided into larval stage, juvenile stage and young fish stage based on the features of the yolk-sac, second dorsal fin spine, pelvic fin spine, and body color. Accordingly, the larval stage was further classified into pre-larva and post-larva, with or without yolk-sac, respectively. The extension and shrinkage of the first pelvic fin spine and the second dorsal fin spine as well as the spinules on aforementioned spines were the most significant changes during the post embryonic development of E. fuscoguttatus. [Conclusion] This study will provide some basic data for industrial breeding of E. fuscoguttatus.
基金supported by grants from Dahuanong Aquatic Company, Guangdong, and Taishan Scholar Project, Shandong, China
文摘This study presents the complete mitochondrial genome of the hybrid Epinephelus moara♀× Epinephelus lanceolatus♂. The genome is 16886 bp in length, and contains 13 protein-coding genes, 2 r RNA genes, 22 t RNA genes, a light-strand replication origin and a control region. Additionally, phylogenetic analysis based on the nucleotide sequences of 13 conserved protein-coding genes using the maximum likelihood method indicated that the mitochondrial genome is maternally inherited. This study presents genomic data for studying phylogenetic relationships and breeding of hybrid Epinephelinae.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2007AA09Z115)Technology Program of Xiamen(No.3502Z20073019)
文摘Vibriofluvialis incubated in trypticase soy broth （TSB） showed stronger adhesion to the skin mucus of Epinephelus awoara than V. fluvialis grown on trypticase soy agar （TSA）, and this bacterial adhesion was assessed in terms of saturation kinetics. Treating bacteria with antibody against O-antigens resulted in significantly reduced bacterial adhesion. In the early growth stage, the adhering bacteria numbers increased with incubation time, peaked at 24 h, and then dropped sharply. Prior heat treatment of the mucus at various temperatures resulted in different effects on bacterial adhesion. No remarkable effect on bacterial adhesion was detected after treating mucus with protease K and trypsin, whereas periodic acid treatment significantly enhanced bacterial adhesion. Extracts of K fluvialis surface structures at high concentrations significantly inhibited bacterial adhesion, while relatively low concentrations enhanced adhesion. V. fluvialis had strong affinity for a high molecular weight mucus component. These results indicate that V.fluvialis adhered strongly to E. awoara mucus by specific adhesion and that this adhesion was influenced by many factors, including bacterial conditions, mucus, and the environment.
基金The National Natural Science Foundation of China under contract No. 30671619
文摘Allograft inflammatory factor-1 （ AIF-1 ） is a cytoplasmic calcium-binding protein involved in inflammatory response-related diseases in mammals. Previously an identified AIF-1 gene was simply reported in yellow grouper. The characterization of AIF-1 gene and its expression at the gene and protein level are further described. Yellow grouper AIF-1 is composed of 147 amino acids, and 64% ~ 84% identical to other homologues. Basal level AIF-1 mRNA expression was noted in spleen, anterior kidney and kidney, using reverse-transcription polymerase chain reaction （RT-PCR）. After stimulation of LPS, the AIF-1 mRNA expression was up-regulated in tissues examined： spleen, anterior kidney, kidney, heart and liver, but not in muscle. The recombinant AIF-1 protein was expressed in Escherichia coli, and then purified for the development of antiserum. Western blotting analysis revealed a band with a molecular mass of about 17 ku.
基金This work was supported by the National"863"Projects of China under contract No.2001AA601010the Natural Science Foundation of Jinan University,China under contract No.51204062.
文摘On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein （ MCP）gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction （RT-PCR） and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 （DE3）. Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers （8 cm in average length） were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group （ A1,30% ） was a little higher than the unvaccined group （ B2, 27.8% ）. Group B was challenged after three vaccine injections. The mortality in the vaccined group （B1, 16.7% ） was lower than the unvaccined group （132, 27.8% ）, And the relative percentage survival （RPS） value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 ： 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish （ Group C1, mortality of 18.18% ）, compared with the fish （ Group C2, mortality of 40% ） which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.
文摘The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper, Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/kg were investigated. After 20 weeks, the growth rates of the groups with high level of vitamin C apparently increased. The untreated fish had symptoms of vitamin C deficiency. The endogenous liver tissue vitamin C levels were found to reflect well the dietary treatments. After intraperitoneal injection or bath challenge with a virulent strain of Vibrio vulnificus , fish fed with high level vitamin C showed significantly higher survival rate compared with the normal control group. Vaccination with formalin inactivated V. vulnificus significantly enhanced the specific antibody production in fish treated with vitamin C, and completely protected from strong bacterial challenge the groups fed on fish with vitamin C 1500 and 2000 mg/kg diet.
基金supported by the project of National Science and Technology Supporting Plan (No. 2011BAD13B04)
文摘The impact of water temperature(24, 27, 30 and 33℃) and salinity(15, 20, 25, 30 and 33) on oxygen consumption(OCR) and ammonium excretion rate(AER) of ♀ Epinephelus fuscoguttatus ×♂ E. lanceolatus hybrid grouper juveniles(9.39 ± 0.07 g) were investigated under the fed and un-fed conditions. The results showed that the OCR and AER were significantly(P < 0.05) affected by temperature and salinity under both fed and un-fed conditions. When temperature was 24–33℃, the OCR and AER of fed hybrid grouper juveniles were 85.68%–129.52% and 125.78%–287.63%, respectively, higher than those of un-fed hybrid grouper juveniles. The O/N ratio, protein use(P_u), Q_(10)(respiration) and Q_(10)(excretion) of fed hybrid grouper juveniles were 14.43–24.01, 28.35% – 48.48%, 1.69 and 3.01, respectively. The O/N ratio, P_u, Q_(10)(respiration), Q_(10)(excretion) of un-fed hybrid grouper juveniles were 20.39 – 31.79, 22.16% – 34.34%, 1.23 and 1.17, respectively. When salinity was 15–33, the OCR and AER of fed hybrid grouper juveniles increased by 87.42% – 116.85% and 215.38% – 353.57%, respectively, over those of un-fed hybrid grouper juveniles. The O/N ratio and P_u of fed hybrid grouper juveniles were 14.48 – 17.78, 39.36% – 49.43%, respectively. The O/N ratio and Pu of un-fed hybrid grouper juveniles were 20.39 – 31.79 and 22.16% – 34.34%, respectively. The specific dynamic action(SDA) of hybrid grouper juveniles was mainly related to protein metabolism. The results had a guiding significance to the large-scale intensive aquaculture of hybrid grouper juveniles.
文摘The paper reviews the recent advances in studying grouper nutrition requirement for the development of cost-effective and environmentally friendly artificial diets. It consists of seven parts: protein and amino acid, lipid and essential fatty acid, carbohydrate, vitamin, mineral, alternative protein source, broodstock and larval nutrition. The review provides some basic information for further investigation of nutrient requirements of groupers.
基金Supported by the Ministry of Science and Technology of China(No.2011BAD13B09)the Project of a Special Fund for Public Welfare Industrial(Agriculture)Research of China(No.200903001-5)
文摘Aquaculture production efficiency may increase by using feed additives. This study investigated the effects of different dietary additives [w/w: 2% small peptides, 0.01% probiotics( Bacillus licheniformis) and 0.2% prebiotics(inulin)] on growth performance, digestive enzyme activities, and oxidative stress in juvenile Epinephelus coioides reared in artificial seawater of two salt concentrations(13.5 vs. 28.5). Weight gain rate was significantly higher in fish fed the diet supplemented with small peptides, B. licheniformis, inulin, or synbiotics than that in fish fed the basal diet; the greatest weight gain rate was found in fish fed the small peptide treatment [56.0% higher than basal diet]. Higher feed efficiency was detected in fish fed the diet supplemented with small peptides than that of fish in the other dietary treatments. Total protease activity in the stomach and intestines was highest in fish fed the small peptide-treated diet, whereas lipase activity was highest in those fed synbiotics(combination of Bacillus licheniformis and inulin) than that in fish fed the other treatments. Antioxidant enzyme(total superoxide dismutase and catalase) activities and hepatic malondialdehyde content were higher in fish receiving the dietary supplements and maintained in artificial seawater containing 13.5 salinity compared with those in the control(28.5). Hepatic catalase activity in grouper fed the diets with small peptides or synbiotics decreased significantly compared with that in control fish. Overall, the three types of additives improved growth rate of juvenile grouper and digestive enzymes activities to varying degrees but did not effectively improve antioxidant capacity under low-salinity stress conditions.
文摘The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).
基金Supported by Special Fund for Construction of Hainan Key Laboratory and Engineering Technical Research Center(gczx2013005)Special Fund for Technology Development by Scientific Research Institutes of Hainan Province(KYYS-2014-59)+1 种基金Special Fund for Application Technology Development and Demonstration(ZDXM2015025)Hainan Provincial-level Department Budget Project
文摘The effects of temperature and pH on embryonic development and survival rate of newly hatched larvae of Epinephelus fuscoguttatus were discussed by single factor experiment method. Under a salinity of 31‰, fertilized eggs were incubated at different temperatures （17, 20, 23, 26, 29 （eontrol）, 32, 35 ℃ ） and different pH values （5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.4 （control）, 8.5, 9.0, 9.5 and 10.0）, the development rate of fertilized eggs, hatching rate, deformity rate, survival rate of newly hatched larvae, and optimum temperature and pH ranges for embryonic development were recorded and analyzed. The results showed that the incubation period y required by fertilized eggs was in remarkably negative correlation with temperature x, y =4 017. 3x-15277 , R2 =0. 971 3. At 29 ℃, the hatching rate was the highest, up to 92.00% ; this temperature was corresponding to the lowest deformity rate of newly hatched larvae （ 1.09% ） ; corresponding effective accumulative temperature was also the lowest （437. 410 ℃ . h）, and significantly different from other experimental groups （P 〈 0.05 ） ; but on the contrary, corresponding survival rates at 24 and 48 h reached, respectively, 88.03% and 82. 97% which were significantly different from other experimental groups （ P 〈 0.05）. At the pH of 8.4 in the CK group and the pH of 8.5 of one experimental group, the embryonic development rates were the highest; con-esponding hatching rates of fertilized eggs were the highest, respectively, of 88.33% and 83.33% which were significantly different from other groups （P 〈 0.05） ; and corresponding survival rates at 24 and 48 h were the highest, and reached 90.84% and 85.03%, respectively. In the pH range of 8.0 - 8.5, the deformity rate of newly hatched larvae was 0. It was indicating that for embryonic development of E. fuscoguttatus, the suitable water temperature value was in the range of 17 - 29 ℃, the optimum value was in the range of 23 -29℃; and the suitable pH value was in the range of 6.5 -9.5, and the optimum value was in the range of 8.0- 8.5.
