Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,i...Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.展开更多
Aquatic vertebrates are very abundant in the world,and they are of tremendous importance in providing global food security and nutrition.However,emergent and resurgent viruses,such as ranavirus(e.g.,Rana grylio virus,...Aquatic vertebrates are very abundant in the world,and they are of tremendous importance in providing global food security and nutrition.However,emergent and resurgent viruses,such as ranavirus(e.g.,Rana grylio virus,RGV and Andriasd avidianus ranavirus,ADRV),herpesvirus(e.g.,Carassius carassius herpesvirus,CaHV),reovirus(e.g.,grass carp reovirus 109,GCRV-109,Scophthal musmaximus reovirus,SMReV and Micropterus salmoides reovirus,MsReV),and rhabdovirus(e.g.,Siniper cachuatsi rhabdovirus,SCRV and Scophthal musmaximus rhabdovirus,SMRV)can cause severe diseases in aquaculture animals and wild lower vertebrates,such as frogs,giant salamanders,fish,and so on.Here,we will briefly describe the symptoms produced by the aforementioned viruses and the molecular basis of the virus ehost interactions.This manuscript aims to provide an overview of viral diseases in lower vertebrates with an emphasis on visible symptomatic manifestations and pathogenesis.展开更多
The gonad specific expression of insulin-like growth factor 3(igf3)has suggested an important role of igf3 in fish reproduction.In this study,medaka igf3 was isolated and its expression patterns were compared with igf...The gonad specific expression of insulin-like growth factor 3(igf3)has suggested an important role of igf3 in fish reproduction.In this study,medaka igf3 was isolated and its expression patterns were compared with igf1,and germ cell gene vasa in adult medaka ovary.Molecular cloning and sequencing showed that the open reading frame(ORF)of medaka igf3 was comprised of 501 nucleotides and encoded 166 amino acid residues.Reverse transcription polymerase chain reaction(RT-PCR)analysis showed that igf3 RNA was specifically expressed in developing embryos and adult gonads of both sexes.Real time quantitative PCR(q-PCR)indicated that igf3 expression was gradually increased during oogenesis,and reached its highest level at stage V.Using Chromogenic and fluorescent in situ hybridization,igf3 was shown to be present in the germ cells of oogonia and in oocytes at stages I-III,as well as abundant in granulosa cells and theca cells of oocytes at stages IV-V.In addition,igf3 and igf1 were expressed mutually in the outer theca cells of stage V oocytes in ovary.Collectively,we demonstrated that gonad specific igf3 could mark medaka ovarian somatic cells and germ cells.These findings highlight the importance of igf3 during ovarian development.展开更多
Oryzias celebensis is determined by an XX-XY system,however,its sex-related genes have not been confirmed.The genes foxl2 and sox9b are important for gonadal development in vertebrates.In the present study,the foxl2 a...Oryzias celebensis is determined by an XX-XY system,however,its sex-related genes have not been confirmed.The genes foxl2 and sox9b are important for gonadal development in vertebrates.In the present study,the foxl2 and sox9b cDNAs were cloned and their expression patterns were analyzed.The cDNA sequence of foxl2 and sox9b were obtained by rapid amplification of cDNA ends(RACEs).Reverse transcription polymerase chain reaction(RT-PCR)showed that O.celebensis foxl2(Ocfoxl2)transcript was predominately expressed in the ovary while O.celebensis sox9b(Ocsox9b)transcript was mainly expressed in the testis.In embryos,Ocfoxl2 transcript was first detected in gastrula.In contrast,Ocsox9b transcript was of maternal origin as expression was detected at the two cell stage.By chromogenic and fluorescent in situ hybridization(ISH),Ocfoxl2 mRNA in the ovary was highly expressed in early oocytes and weakly expressed in the granulosa cells and thecal cells in the later oocytes.In contrast,Ocsox9b mRNA was mainly found in Sertoli cells surrounding spermatogonia and in spermatids and sperm in the testis.In summary,Ocfoxl2 identified somatic cells and differentiating oocytes in the ovary,indicating it is involved in ovarian development.Ocsox9b is identified in Sertoli cells and late developing male germ cells indicating it is involved in testicular development.展开更多
Aquatic viruses are naturally present in the aquatic environment and the number of viruses is staggering.Various multicellular organisms in aquatic ecosystems may be infected,cross-species transmitted,manipulated,and ...Aquatic viruses are naturally present in the aquatic environment and the number of viruses is staggering.Various multicellular organisms in aquatic ecosystems may be infected,cross-species transmitted,manipulated,and killed by aquatic viruses,which can lead to cascading ecological effects.The viruses in unicellular aquatic organisms can alter interactions between host individuals,and are essential in effecting or maintaining the dynamics of aquatic microbial communities,horizontal gene transfer,biodiversity,and modulating ecological processes globally.Meanwhile,hosts also impact viral abundance and diversity.Microbial diversity drives multifunctionality in ecosystems,while viruses shape complex microbial communities and are crucial for ecosystem functioning.This review focuses on molecular,genetic,evolutionary,and ecosystemic advances related to emerging and reemerging aquatic viruses,presents the contexts,novel tools,and investigative approaches pertaining to the study of aquatic virology,and discusses the mechanisms by which viruses affect aquatic ecosystems.The paper provides an efficient and broadly-based blueprint for improving understanding of aquatic viruses.展开更多
Diseases of grass carps often occur in juveniles but not in adults that may have established disease resistance during development.We performed both DNA bisulfite and transcriptome sequencing on liver libraries of 1-a...Diseases of grass carps often occur in juveniles but not in adults that may have established disease resistance during development.We performed both DNA bisulfite and transcriptome sequencing on liver libraries of 1-and 3-year-old grass carps.Differential DNA-methylation analysis exhibited a declined methylation level through development.Functional annotation revealed that identified differentially methylated genes(DMGs)and differentially expressed genes were enriched in immune-related pathways such as PI3K-Akt signaling pathway and its related pathways.Both differentially methylated and differentially expressed genes were clustered into the growth-and immune-related function networks.Subcellular localization analysis indicated that the DMGs localized on cell membrane were significantly enriched in calcium signaling pathway and neuroactive ligand-receptor interaction pathway,implying the importance of G protein-coupled receptors to development.These findings will broaden our understanding of the key genes and pathways that affect the immune system at different development stages and the developing protective strategies in grass carp.展开更多
Numerous studies have showed that sox3 is involved in neurogenesis and sex differentiation in vertebrates.However,the accurate expression pattern is still unclear in fish.In this study,medaka sox3 was isolated and its...Numerous studies have showed that sox3 is involved in neurogenesis and sex differentiation in vertebrates.However,the accurate expression pattern is still unclear in fish.In this study,medaka sox3 was isolated and its expression patterns were compared with germ cell gene vasa in adult gonads.By reverse transcription-polymerase chain reaction(RT-PCR)analysis,sox3 RNA expression was detected in the brain,eyes and gonads.By chromogenic and fluorescent in situ hybridization,sox3 was present in the oogonia and early stages of oocytes as well as granulosa cells and theca cells in the later stages;in the testis,sox3 was occurred in the Sertoli cells and sperm with a specific signal in the location of chromatid body of sperm;in the embryos,sox3 was expressed in the central nervous.These results suggest that medaka sox3 gene was occurred in somatic and germ cells in adult gonads of both sexes,involved in the process of spermatogenesis,as well as the development of the nervous system.This study provides a precise expression pattern of sox3 and suggests that sox3 may be involved in gonadal and nervous system development of fish.展开更多
Germ cells of many organisms exhibit a unique perinuclear cytoplasmic cloud-like structure called the chromatoid body(CB).Specific components of the CB,such as vasa,are crucial for germline development.Herein we ident...Germ cells of many organisms exhibit a unique perinuclear cytoplasmic cloud-like structure called the chromatoid body(CB).Specific components of the CB,such as vasa,are crucial for germline development.Herein we identify another component of the CB,the Oryzias latipes gcnf homolog(Olgcnf).By reverse transcription polymerase chain reaction(RT-PCR),Olgcnf transcript was restricted to gonads in the adult tissues and was maternally provided and persisted throughout embryogenesis.By in situ hybridization on gonadal sections,Olgcnf transcript was restricted to germ cells.In the ovary,Olgcnf was predominantly expressed in the early stages of oocytes.In the testis,Olgcnf showed dynamic expression in round spermatids and sperm,accumulated in the CB during spermatid morphogenesis and concentrated in elongated spermatids of acrosome cap region and flagellar manchette.Moreover,Olgcnf was colocalized with vasa,which is the best-characterized component of the CB.Therefore,Olgcnf is specific to germ cells of both sexes and is a component of the CB during spermiogenesis.展开更多
In contrast to fish,amphibians inhabit both aquatic and terrestrial environments.To better understand osmoregulation in fish and amphibian,we have investigated the morphological changes in kidney cells to osmotic stre...In contrast to fish,amphibians inhabit both aquatic and terrestrial environments.To better understand osmoregulation in fish and amphibian,we have investigated the morphological changes in kidney cells to osmotic stress.To address this,kidney cell line isolated from the freshwater grass carp(CIK)and Chinese giant salamander(GSK)were challenged to different mediums with distinct osmotic pressures(100,300 and 700 mOsm).Morphological alterations of the fish and amphibian cells were compared by optical and electron microscopy.Following hyposmotic treatment(100 mOsm),both CIK and GSK cells became unhealthy and show condensed chromatin,swollen mitochondria and cytoplasmic vacuole.Meanwhile,after hyperosmotic treatment(700 mOsm),shrunken CIK cells with multipolar shape,pale or lightly stained cytoplasm,condensed chromatin,vacuoles and swollen mitochondria were detected.GSK cells were seriously damaged and most were completely lysed.The results suggest that fish kidney cells show a higher degree of tolerance to hyperosmoticity by comparing to amphibians and provide novel insights on the osmoregulatory capacity and adaptability of kidney cells between the two animal groups.展开更多
基金financially supported by the Science and Technology Commission of Shanghai Municipality(21ZR1427200).
文摘Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.
基金This work was partially supported by the following grants:the National Natural Science Foundation of China(31430091,31302214,31772890)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA08030202)the Project of State Key Laboratory of Freshwater Ecology and Biotechnology(2016FBZ01).
文摘Aquatic vertebrates are very abundant in the world,and they are of tremendous importance in providing global food security and nutrition.However,emergent and resurgent viruses,such as ranavirus(e.g.,Rana grylio virus,RGV and Andriasd avidianus ranavirus,ADRV),herpesvirus(e.g.,Carassius carassius herpesvirus,CaHV),reovirus(e.g.,grass carp reovirus 109,GCRV-109,Scophthal musmaximus reovirus,SMReV and Micropterus salmoides reovirus,MsReV),and rhabdovirus(e.g.,Siniper cachuatsi rhabdovirus,SCRV and Scophthal musmaximus rhabdovirus,SMRV)can cause severe diseases in aquaculture animals and wild lower vertebrates,such as frogs,giant salamanders,fish,and so on.Here,we will briefly describe the symptoms produced by the aforementioned viruses and the molecular basis of the virus ehost interactions.This manuscript aims to provide an overview of viral diseases in lower vertebrates with an emphasis on visible symptomatic manifestations and pathogenesis.
基金This work was supported by the National Natural Science Foundation of China(31672700)National Key R&D Program of China(2018YFD0901205).
文摘The gonad specific expression of insulin-like growth factor 3(igf3)has suggested an important role of igf3 in fish reproduction.In this study,medaka igf3 was isolated and its expression patterns were compared with igf1,and germ cell gene vasa in adult medaka ovary.Molecular cloning and sequencing showed that the open reading frame(ORF)of medaka igf3 was comprised of 501 nucleotides and encoded 166 amino acid residues.Reverse transcription polymerase chain reaction(RT-PCR)analysis showed that igf3 RNA was specifically expressed in developing embryos and adult gonads of both sexes.Real time quantitative PCR(q-PCR)indicated that igf3 expression was gradually increased during oogenesis,and reached its highest level at stage V.Using Chromogenic and fluorescent in situ hybridization,igf3 was shown to be present in the germ cells of oogonia and in oocytes at stages I-III,as well as abundant in granulosa cells and theca cells of oocytes at stages IV-V.In addition,igf3 and igf1 were expressed mutually in the outer theca cells of stage V oocytes in ovary.Collectively,we demonstrated that gonad specific igf3 could mark medaka ovarian somatic cells and germ cells.These findings highlight the importance of igf3 during ovarian development.
基金This work was supported by National Key R&D Program of China(2018YFD0901205)National Natural Science Foundation of China(31672700,31372520).
文摘Oryzias celebensis is determined by an XX-XY system,however,its sex-related genes have not been confirmed.The genes foxl2 and sox9b are important for gonadal development in vertebrates.In the present study,the foxl2 and sox9b cDNAs were cloned and their expression patterns were analyzed.The cDNA sequence of foxl2 and sox9b were obtained by rapid amplification of cDNA ends(RACEs).Reverse transcription polymerase chain reaction(RT-PCR)showed that O.celebensis foxl2(Ocfoxl2)transcript was predominately expressed in the ovary while O.celebensis sox9b(Ocsox9b)transcript was mainly expressed in the testis.In embryos,Ocfoxl2 transcript was first detected in gastrula.In contrast,Ocsox9b transcript was of maternal origin as expression was detected at the two cell stage.By chromogenic and fluorescent in situ hybridization(ISH),Ocfoxl2 mRNA in the ovary was highly expressed in early oocytes and weakly expressed in the granulosa cells and thecal cells in the later oocytes.In contrast,Ocsox9b mRNA was mainly found in Sertoli cells surrounding spermatogonia and in spermatids and sperm in the testis.In summary,Ocfoxl2 identified somatic cells and differentiating oocytes in the ovary,indicating it is involved in ovarian development.Ocsox9b is identified in Sertoli cells and late developing male germ cells indicating it is involved in testicular development.
基金supported by the National Key R&D Plan of the Ministry of Science and Technology,China(2018YFA0903101,2018YFD0900302)the Key Program of Frontier Sciences of the Chinese Academy of Sciences(KJZD-SW-L11).
文摘Aquatic viruses are naturally present in the aquatic environment and the number of viruses is staggering.Various multicellular organisms in aquatic ecosystems may be infected,cross-species transmitted,manipulated,and killed by aquatic viruses,which can lead to cascading ecological effects.The viruses in unicellular aquatic organisms can alter interactions between host individuals,and are essential in effecting or maintaining the dynamics of aquatic microbial communities,horizontal gene transfer,biodiversity,and modulating ecological processes globally.Meanwhile,hosts also impact viral abundance and diversity.Microbial diversity drives multifunctionality in ecosystems,while viruses shape complex microbial communities and are crucial for ecosystem functioning.This review focuses on molecular,genetic,evolutionary,and ecosystemic advances related to emerging and reemerging aquatic viruses,presents the contexts,novel tools,and investigative approaches pertaining to the study of aquatic virology,and discusses the mechanisms by which viruses affect aquatic ecosystems.The paper provides an efficient and broadly-based blueprint for improving understanding of aquatic viruses.
基金This work is supported by the National Key Research and Development Program of China(2018YFD0900601,2018YFD0900101)Doctor Fund of Shanghai Ocean University(A2-2006-00-200301)Science and Technology Development Fund of Shanghai Ocean University(A2-2006-00-200205).
文摘Diseases of grass carps often occur in juveniles but not in adults that may have established disease resistance during development.We performed both DNA bisulfite and transcriptome sequencing on liver libraries of 1-and 3-year-old grass carps.Differential DNA-methylation analysis exhibited a declined methylation level through development.Functional annotation revealed that identified differentially methylated genes(DMGs)and differentially expressed genes were enriched in immune-related pathways such as PI3K-Akt signaling pathway and its related pathways.Both differentially methylated and differentially expressed genes were clustered into the growth-and immune-related function networks.Subcellular localization analysis indicated that the DMGs localized on cell membrane were significantly enriched in calcium signaling pathway and neuroactive ligand-receptor interaction pathway,implying the importance of G protein-coupled receptors to development.These findings will broaden our understanding of the key genes and pathways that affect the immune system at different development stages and the developing protective strategies in grass carp.
基金the National Key R&D Program of China(2018YFD0901205)National Natural Science Foundation of China(31672700).
文摘Numerous studies have showed that sox3 is involved in neurogenesis and sex differentiation in vertebrates.However,the accurate expression pattern is still unclear in fish.In this study,medaka sox3 was isolated and its expression patterns were compared with germ cell gene vasa in adult gonads.By reverse transcription-polymerase chain reaction(RT-PCR)analysis,sox3 RNA expression was detected in the brain,eyes and gonads.By chromogenic and fluorescent in situ hybridization,sox3 was present in the oogonia and early stages of oocytes as well as granulosa cells and theca cells in the later stages;in the testis,sox3 was occurred in the Sertoli cells and sperm with a specific signal in the location of chromatid body of sperm;in the embryos,sox3 was expressed in the central nervous.These results suggest that medaka sox3 gene was occurred in somatic and germ cells in adult gonads of both sexes,involved in the process of spermatogenesis,as well as the development of the nervous system.This study provides a precise expression pattern of sox3 and suggests that sox3 may be involved in gonadal and nervous system development of fish.
基金This work was supported by the National Natural Science Foundation of China(31672700)National Key R&D Program of China(2018YFD0901205).
文摘Germ cells of many organisms exhibit a unique perinuclear cytoplasmic cloud-like structure called the chromatoid body(CB).Specific components of the CB,such as vasa,are crucial for germline development.Herein we identify another component of the CB,the Oryzias latipes gcnf homolog(Olgcnf).By reverse transcription polymerase chain reaction(RT-PCR),Olgcnf transcript was restricted to gonads in the adult tissues and was maternally provided and persisted throughout embryogenesis.By in situ hybridization on gonadal sections,Olgcnf transcript was restricted to germ cells.In the ovary,Olgcnf was predominantly expressed in the early stages of oocytes.In the testis,Olgcnf showed dynamic expression in round spermatids and sperm,accumulated in the CB during spermatid morphogenesis and concentrated in elongated spermatids of acrosome cap region and flagellar manchette.Moreover,Olgcnf was colocalized with vasa,which is the best-characterized component of the CB.Therefore,Olgcnf is specific to germ cells of both sexes and is a component of the CB during spermiogenesis.
基金This work was supported by the National Natural Science Foundation of China(31302214).
文摘In contrast to fish,amphibians inhabit both aquatic and terrestrial environments.To better understand osmoregulation in fish and amphibian,we have investigated the morphological changes in kidney cells to osmotic stress.To address this,kidney cell line isolated from the freshwater grass carp(CIK)and Chinese giant salamander(GSK)were challenged to different mediums with distinct osmotic pressures(100,300 and 700 mOsm).Morphological alterations of the fish and amphibian cells were compared by optical and electron microscopy.Following hyposmotic treatment(100 mOsm),both CIK and GSK cells became unhealthy and show condensed chromatin,swollen mitochondria and cytoplasmic vacuole.Meanwhile,after hyperosmotic treatment(700 mOsm),shrunken CIK cells with multipolar shape,pale or lightly stained cytoplasm,condensed chromatin,vacuoles and swollen mitochondria were detected.GSK cells were seriously damaged and most were completely lysed.The results suggest that fish kidney cells show a higher degree of tolerance to hyperosmoticity by comparing to amphibians and provide novel insights on the osmoregulatory capacity and adaptability of kidney cells between the two animal groups.
