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
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 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
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
Two methods, tagging and ink injection, are used in the grouper releasing experiment. The studied fishes are the juvenile of Epinephelus akaara and Epinephelus awoara both artificially cultivated and caught from natur...Two methods, tagging and ink injection, are used in the grouper releasing experiment. The studied fishes are the juvenile of Epinephelus akaara and Epinephelus awoara both artificially cultivated and caught from natural waters, and their wild adults. Experimental results show that the inhabiting behaviours for both juvenile and adult fishes show distinct regionality, which move within an area of 2 n mile diameter 651 days and 48 days after being released, respectively. With the tagging method, the tagged fishes could only be recaptured in the year of release while, with the injection method, they could be caught in the lst, 2nd and 3rd years. It is confirmed that in the cement tank, the tagged fishes lose their 1/3 tags. That means that the tagging method is not fit for the release research while the injection method is. Generally, the recaptured rates of injected fishes are 1.4 ~ 4.5 %, 3.1 ~ 13.4 % and 2.7 % for the 1st, 2nd and 3rd years, respectively.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
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
Interferon-induced protein with tetratricopeptide repeats 1(IFIT1), also known as interferon-induced protein 56(IFI56) or Interferon-stimulated protein 56(ISG56), was originally identified as a protein induced upon tr...Interferon-induced protein with tetratricopeptide repeats 1(IFIT1), also known as interferon-induced protein 56(IFI56) or Interferon-stimulated protein 56(ISG56), was originally identified as a protein induced upon treatment with interferon and inhibited by viral replication and translational initiation. In this study, Epinephelus lanceolatus IFIT1(ELIFIT1) gene was cloned for the first time. The complete cDNA of El IFIT1 gene includes 2921 nucleotides, and encodes a 437-amino acid(AA) protein. The putative ELIFIT1 protein has 9 TRP domains and is highly similar with IFIT1 proteins in other teleosts. In healthy fish, ELIFIT1 gene was highly expressed in the blood, which indicate its specific function in the peripheral immune system. Its expression was also observed in various immunity-related tissues including spleen, intestine, and kidney, Inducted with spotted knifejaw iridovirus(SKIV), ELIFIT1 gene expression was upregulated in the spleen, kidney, and liver 24 h after induction and reached its peak at 72 h, indicating that ELIFIT1 may play an important role in antivirus. These findings contribute to the understanding of the antiviral regulation of ELIFIT1 gene in teleost.展开更多
In order to clarify the mechanism of retinomotor movements in banded grouper, this paper utilizes inhibition factors of microfilaments and microtubules cytochalasin-B and colchicine to study the roles of actin microfi...In order to clarify the mechanism of retinomotor movements in banded grouper, this paper utilizes inhibition factors of microfilaments and microtubules cytochalasin-B and colchicine to study the roles of actin microfilaments and microtubules in the movements of the REP pigment granles and cones. It provides evidences to explain the mechanism of retinomotor movements in fish.展开更多
The present study was conducted as a continuation of our previous study on the effects of fish meal replacement with soy bean meal, SBM in the diets of tiger grouper, Epinephelus fuscoguttatus which revealed the possi...The present study was conducted as a continuation of our previous study on the effects of fish meal replacement with soy bean meal, SBM in the diets of tiger grouper, Epinephelus fuscoguttatus which revealed the possible level of fish meal protein replacement of 30% with positive effect of phytase supplementation in one of the test diet. In the present study, all experimental diets with SBM inclusion (10%, 20%, 30%, 40% replacement levels) were supplemented with phytase and fed to juvenile tiger grouper with initial body weight of 44.57 ± 0.28 g for eight weeks. Apart from the effects on growth performance, diet utilization and fish whole-body composition, apparent digestibility coefficient of diets was also measured. Similar findings to our previous experiment were observed in the present study. Diet SM40P (40% replacement level) had produced the poorest growth performance and feed conversion ratio (FCR). However, it was observed that FCR of SM40P was improved when compared to SM40 (without phytase) in our previous experiment. Other than that, SBM-based diets were performed as good as the control diet in terms of growth, feed utilization and survival rate. Replacement of fish meal did not significantly influence body composition, except for whole body lipid content in SM40P. In general, dry matter and protein apparent digestibility coefficients (ADC) of the test diets were influenced by SBM inclusion in the diets. However, crude lipid ADC was very high in all dietary treatments indicating efficient utilization of dietary lipid by juvenile tiger grouper. The findings of the present study confirmed that SBM can be used to replace FM at a level of up to 30% and supplementation of phytase in SBM-based diets is recommended to improve the diet performance.展开更多
The effects of fish meal (FM) replacement with graded level of soybean meal (SBM) on growth performance, feed utilization, survival rate and body composition were investigated in juvenile tiger grouper, Epinephelus fu...The effects of fish meal (FM) replacement with graded level of soybean meal (SBM) on growth performance, feed utilization, survival rate and body composition were investigated in juvenile tiger grouper, Epinephelus fuscoguttatus (initial body weight 13.9 ±0.65 g). Six experimental diets were formulated to contain 50% crude protein, 16% crude lipid and 365.8 kcal/100g feed with SBM replacing FM protein at 0% (SM 0), 10% (SM 0), 20% (SM 20), 30% (SM 30), 40% (SM 40) and 20% with phytase (SM 20P) replacement levels. At the end of the ten-week feeding trial, there were no significant differences detected in terms of growth performances (weight gain and specific growth rate), feed conversion ratio (FCR) and survival rates of fish fed with the control diet (SM 0), SM 20, SM 30 and SM 20P. Net protein utilization of fish fed SM 20P was higher than those fed with other diets suggesting an improved utilization of nutrients with phytase addition in the diet. Replacements of FM protein with SBM at 10% and 40% have resulted in significantly lower growth and poorer FCR than other replacement levels. Survival rates remain high (≥90%) throughout the trial. Whole-body proximate composition of the fish was significantly affected by the inclusion of SBM in the diets. It can be concluded that 20%-30% of FM protein replacement with SBM is recommended and addition of phytase in the SBM-based diet should be considered to improve nutrient utilization of tiger grouper juvenile.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.
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 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Two methods, tagging and ink injection, are used in the grouper releasing experiment. The studied fishes are the juvenile of Epinephelus akaara and Epinephelus awoara both artificially cultivated and caught from natural waters, and their wild adults. Experimental results show that the inhabiting behaviours for both juvenile and adult fishes show distinct regionality, which move within an area of 2 n mile diameter 651 days and 48 days after being released, respectively. With the tagging method, the tagged fishes could only be recaptured in the year of release while, with the injection method, they could be caught in the lst, 2nd and 3rd years. It is confirmed that in the cement tank, the tagged fishes lose their 1/3 tags. That means that the tagging method is not fit for the release research while the injection method is. Generally, the recaptured rates of injected fishes are 1.4 ~ 4.5 %, 3.1 ~ 13.4 % and 2.7 % for the 1st, 2nd and 3rd years, respectively.
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.
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 the Shandong Breeding Project (No. 2016LZGC009)the Projects from Laboratory for Marine Fisheries Science and Food Production Processes+2 种基金Pilot National Laboratory for Marine Science and Technology (Qingdao)(Nos. 2018-MFS-T08, 2017A STCP-OS15)the Central Public-interest Scientific Institution Basal Research Fund,CAFS (No. 2020TD20)the Central Public-Interest Scientific Institution Basal Re-search Fund,YSFRI,CAFS (No. 20603022018026)。
文摘Interferon-induced protein with tetratricopeptide repeats 1(IFIT1), also known as interferon-induced protein 56(IFI56) or Interferon-stimulated protein 56(ISG56), was originally identified as a protein induced upon treatment with interferon and inhibited by viral replication and translational initiation. In this study, Epinephelus lanceolatus IFIT1(ELIFIT1) gene was cloned for the first time. The complete cDNA of El IFIT1 gene includes 2921 nucleotides, and encodes a 437-amino acid(AA) protein. The putative ELIFIT1 protein has 9 TRP domains and is highly similar with IFIT1 proteins in other teleosts. In healthy fish, ELIFIT1 gene was highly expressed in the blood, which indicate its specific function in the peripheral immune system. Its expression was also observed in various immunity-related tissues including spleen, intestine, and kidney, Inducted with spotted knifejaw iridovirus(SKIV), ELIFIT1 gene expression was upregulated in the spleen, kidney, and liver 24 h after induction and reached its peak at 72 h, indicating that ELIFIT1 may play an important role in antivirus. These findings contribute to the understanding of the antiviral regulation of ELIFIT1 gene in teleost.
文摘In order to clarify the mechanism of retinomotor movements in banded grouper, this paper utilizes inhibition factors of microfilaments and microtubules cytochalasin-B and colchicine to study the roles of actin microfilaments and microtubules in the movements of the REP pigment granles and cones. It provides evidences to explain the mechanism of retinomotor movements in fish.
文摘The present study was conducted as a continuation of our previous study on the effects of fish meal replacement with soy bean meal, SBM in the diets of tiger grouper, Epinephelus fuscoguttatus which revealed the possible level of fish meal protein replacement of 30% with positive effect of phytase supplementation in one of the test diet. In the present study, all experimental diets with SBM inclusion (10%, 20%, 30%, 40% replacement levels) were supplemented with phytase and fed to juvenile tiger grouper with initial body weight of 44.57 ± 0.28 g for eight weeks. Apart from the effects on growth performance, diet utilization and fish whole-body composition, apparent digestibility coefficient of diets was also measured. Similar findings to our previous experiment were observed in the present study. Diet SM40P (40% replacement level) had produced the poorest growth performance and feed conversion ratio (FCR). However, it was observed that FCR of SM40P was improved when compared to SM40 (without phytase) in our previous experiment. Other than that, SBM-based diets were performed as good as the control diet in terms of growth, feed utilization and survival rate. Replacement of fish meal did not significantly influence body composition, except for whole body lipid content in SM40P. In general, dry matter and protein apparent digestibility coefficients (ADC) of the test diets were influenced by SBM inclusion in the diets. However, crude lipid ADC was very high in all dietary treatments indicating efficient utilization of dietary lipid by juvenile tiger grouper. The findings of the present study confirmed that SBM can be used to replace FM at a level of up to 30% and supplementation of phytase in SBM-based diets is recommended to improve the diet performance.
文摘The effects of fish meal (FM) replacement with graded level of soybean meal (SBM) on growth performance, feed utilization, survival rate and body composition were investigated in juvenile tiger grouper, Epinephelus fuscoguttatus (initial body weight 13.9 ±0.65 g). Six experimental diets were formulated to contain 50% crude protein, 16% crude lipid and 365.8 kcal/100g feed with SBM replacing FM protein at 0% (SM 0), 10% (SM 0), 20% (SM 20), 30% (SM 30), 40% (SM 40) and 20% with phytase (SM 20P) replacement levels. At the end of the ten-week feeding trial, there were no significant differences detected in terms of growth performances (weight gain and specific growth rate), feed conversion ratio (FCR) and survival rates of fish fed with the control diet (SM 0), SM 20, SM 30 and SM 20P. Net protein utilization of fish fed SM 20P was higher than those fed with other diets suggesting an improved utilization of nutrients with phytase addition in the diet. Replacements of FM protein with SBM at 10% and 40% have resulted in significantly lower growth and poorer FCR than other replacement levels. Survival rates remain high (≥90%) throughout the trial. Whole-body proximate composition of the fish was significantly affected by the inclusion of SBM in the diets. It can be concluded that 20%-30% of FM protein replacement with SBM is recommended and addition of phytase in the SBM-based diet should be considered to improve nutrient utilization of tiger grouper juvenile.