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
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
In this study,we conducted a 56-d feeding trial to investigate the effects of replacing the fish oil(FO)with palm oil(PO)on the performance,tissue fatty acid(FA)composition,and mRNA levels of genes related to hepatic ...In this study,we conducted a 56-d feeding trial to investigate the effects of replacing the fish oil(FO)with palm oil(PO)on the performance,tissue fatty acid(FA)composition,and mRNA levels of genes related to hepatic lipid metabolism in grouper(Epinephelus coioides).Five isolipidic(13%crude lipid)and isonitrogenous(48%CP)diets were formulated by incrementally adding PO to the control diet(25%fish meal and 9%added FO)to replace FO in the control diets.Triplicate groups of 30 groupers(initial weight:12.6±0.1 g)were fed one of the diets twice daily,to apparent satiety.The replacement of FO with 50%PO revealed maximum growth without affecting the performance and whole-body proximate compositions,and replacing FO with 100%PO revealed a comparable(P>0.05)growth with that of the control diet,suggesting PO as a suitable alternative to FO.The analysis of FA profiles in the dorsal muscle and liver though reflected the FA profile of the diet,PO substitutions above 50%could compromise(P<0.05)the FA profile in the liver and flesh of the fish species in comparison with the control diet.Furthermore,the mRNA levels of FAS,G6PD,LPL,PPARА,and D6FAD genes in the liver had positive linear and/or quadratic responses,but the SCD,HSL,ATGL,FABP,SREBP-1C and ELOVL5 had the opposite trend,with increasing dietary PO inclusion levels,whereas the mRNA level of ACC was not affected by dietary treatments.The optimal level of PO substitution for FO was estimated to be 47.1%of the feed,based on the regression analysis of percent weight gains against dietary PO inclusion levels;however,it might affect the FA profile in the liver and flesh of the fish species,and further study is required to investigate whether the changes in tissue FA composition will affect the welfare and market value over a production cycle of grouper.展开更多
Groupers are dispersed worldwide in the tropical and subtropical waters.They are prized in the live reef fish trade,making them candidates with high market value and consumer demand.In the Asian-Pacific region,the bro...Groupers are dispersed worldwide in the tropical and subtropical waters.They are prized in the live reef fish trade,making them candidates with high market value and consumer demand.In the Asian-Pacific region,the brown-marbled grouper(Epinephelus fuscoguttatus)is widely adapted as an aquaculture species.However,health management remains a major concern in the stressful intensive culture process.The present review discusses techniques and current knowledge in the cultivation of E.fuscoguttatus towards healthy fish growth focusing on aspects of immunity,feed,and husbandry.Understanding how the fish immune system responds during infections provides insights into the intricate ways fish resist pathogens.This information is helpful when developing vaccination strategies or immunostimulant compounds to strengthen fish immunity.Feeds that are formulated according to the needs of the fish ensure optimal growth and using suitable alternative ingredients may lower production cost without compromising fish health.Good husbandry practices contribute to a favourable environment for the fish to grow,while interspecific hybridization may be a convenient approach to generate hardier species.Nevertheless,it has been noted that research in E.fuscoguttatus is relatively scarce in contrast to the closely-related orange-spotted grouper(E.coioides),which is used in the present article as a reference.Ultimately,the identified gaps in knowledge between the two species warrant species-specific research in E.fuscoguttatus to promote fish health and ensure continued success in aquaculture.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
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.
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This study was supported by funding from the National Natural Science Foundation of China(Grant No.31772861)the Science and Technology Project of Fujian Province of China(No.2020N0012).
文摘In this study,we conducted a 56-d feeding trial to investigate the effects of replacing the fish oil(FO)with palm oil(PO)on the performance,tissue fatty acid(FA)composition,and mRNA levels of genes related to hepatic lipid metabolism in grouper(Epinephelus coioides).Five isolipidic(13%crude lipid)and isonitrogenous(48%CP)diets were formulated by incrementally adding PO to the control diet(25%fish meal and 9%added FO)to replace FO in the control diets.Triplicate groups of 30 groupers(initial weight:12.6±0.1 g)were fed one of the diets twice daily,to apparent satiety.The replacement of FO with 50%PO revealed maximum growth without affecting the performance and whole-body proximate compositions,and replacing FO with 100%PO revealed a comparable(P>0.05)growth with that of the control diet,suggesting PO as a suitable alternative to FO.The analysis of FA profiles in the dorsal muscle and liver though reflected the FA profile of the diet,PO substitutions above 50%could compromise(P<0.05)the FA profile in the liver and flesh of the fish species in comparison with the control diet.Furthermore,the mRNA levels of FAS,G6PD,LPL,PPARА,and D6FAD genes in the liver had positive linear and/or quadratic responses,but the SCD,HSL,ATGL,FABP,SREBP-1C and ELOVL5 had the opposite trend,with increasing dietary PO inclusion levels,whereas the mRNA level of ACC was not affected by dietary treatments.The optimal level of PO substitution for FO was estimated to be 47.1%of the feed,based on the regression analysis of percent weight gains against dietary PO inclusion levels;however,it might affect the FA profile in the liver and flesh of the fish species,and further study is required to investigate whether the changes in tissue FA composition will affect the welfare and market value over a production cycle of grouper.
基金Our research was supported by Malaysian Ministry of Education(MOE)Higher Institution Centre of Excellence(HICoE)Grant No.6369100Malaysian Ministry of Science,Technology and Innovation(MOSTI)EScience Fund No.5450576.
文摘Groupers are dispersed worldwide in the tropical and subtropical waters.They are prized in the live reef fish trade,making them candidates with high market value and consumer demand.In the Asian-Pacific region,the brown-marbled grouper(Epinephelus fuscoguttatus)is widely adapted as an aquaculture species.However,health management remains a major concern in the stressful intensive culture process.The present review discusses techniques and current knowledge in the cultivation of E.fuscoguttatus towards healthy fish growth focusing on aspects of immunity,feed,and husbandry.Understanding how the fish immune system responds during infections provides insights into the intricate ways fish resist pathogens.This information is helpful when developing vaccination strategies or immunostimulant compounds to strengthen fish immunity.Feeds that are formulated according to the needs of the fish ensure optimal growth and using suitable alternative ingredients may lower production cost without compromising fish health.Good husbandry practices contribute to a favourable environment for the fish to grow,while interspecific hybridization may be a convenient approach to generate hardier species.Nevertheless,it has been noted that research in E.fuscoguttatus is relatively scarce in contrast to the closely-related orange-spotted grouper(E.coioides),which is used in the present article as a reference.Ultimately,the identified gaps in knowledge between the two species warrant species-specific research in E.fuscoguttatus to promote fish health and ensure continued success in aquaculture.
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.