In the coastal environment,the co-occurrence of antibiotic and nanoplastic pollution is common.Investigating their individual and combined toxicity to marine organisms is of great necessity.In the present study,the re...In the coastal environment,the co-occurrence of antibiotic and nanoplastic pollution is common.Investigating their individual and combined toxicity to marine organisms is of great necessity.In the present study,the reproductive toxicity of sulfamethazine(SMZ)and nanoplastics(polystyrene,PS)via the dietary route on the spermatogenesis of marine medaka(Oryzias melastigma)was examined.After 30 d of dietary exposure,SMZ alone decreased the gonadosomatic index(GSI)value(~35%)and the proportion of undifferentiated type A spermatogonia(A_(und))(~40%),probably by disrupting the testicular sex hormone production,the spermatogenesis-related growth factor network and the balance of apoptosis.Individual exposure to PS did not affect the GSI value or the proportions of germ cells at different developmental stages,but dysregulated the expression of several spermatogenesis-related genes.Interestingly,the presence of PS alleviated the decreased GSI value caused by SMZ.This alleviation effect was achieved by enhancing the spermatogonia differentiation instead of reversing the suppressed self-renewal of A_(und),suggesting that the mixture of PS and SMZ could cause reproductive effects in a different way.These findings expand our knowledge of threats of ubiquitous antibiotic and nanoplastic pollution to fish reproduction and population.展开更多
Although 1,1-dichloro-2,2-bis（p-chlorophenyl）-ethylene （p,p′-DDE）, the major and most persistent metabolite of dichlorodiphenyltrichloroethane （DDT）, was continually detected in wild fishes that showed abnormal...Although 1,1-dichloro-2,2-bis（p-chlorophenyl）-ethylene （p,p′-DDE）, the major and most persistent metabolite of dichlorodiphenyltrichloroethane （DDT）, was continually detected in wild fishes that showed abnormal gonad development such as intersex, little is known about the impact of p,p′-DDE exposure on gonad development in fishes. To survey the effects of p,p′-DDE on gonadal development and gene expressions, male juvenile （20-d post hatch） Japanese medaka （Oryzias latipes） was exposed to 1, 5, 20, and 100 μg/L p,p′-DDE for two months. Increased hepatosomatic index （HSI） and decreased gonadosomatic index （GSI） were found in the p,p′-DDE-treated groups. Intersex was found in 100 μg/L p,p′-DDE exposure group, as well as 100 ng/L 17α-ethynylestradiol （EE2） group. By quantitative real-time RT-PCR, it was found that gene expressions of vitellogenins （VTG-1, VTG-2）, choriogenins （CHG-H, CHG-L）, and estrogen receptor α （ER-α） in the liver of the fish were significantly up-regulated by p,p′-DDE exposure. VTG-1 and VTG- 2 were recommended as the preferred biomarker for assessing anti-androgenic p,p′-DDE because they were the highest up-regulated among the genes and showed good dose-response relationship. The up-regulated ER-α suggested that a potential synergetic effect would occur when p,p′-DDE coexists with other ER-α-binding endocrine-disrupting chemicals （EDCs）.展开更多
The accumulation and absorption kinetics of La by fish (Oryzias latipes) from both water and food were tested in fresh water. The effects of La concentration in water and food and the existence of fulvic acid on t...The accumulation and absorption kinetics of La by fish (Oryzias latipes) from both water and food were tested in fresh water. The effects of La concentration in water and food and the existence of fulvic acid on the absorption were investigated. It was demonstrated that the amount of La accumulated in fish increases with the increase in La concentration in water and food and exposure duration, which the normal physiological activity of fish was inhibited. With fulvic in the system, the absorption rate decreases significantly indicating that the fulvic combined La is not available to the fish uptake.展开更多
As a new insecticide,less information about the aquatic biological toxicity of DMAH [N-(2,4-dimethylphenyl)-N'-methylamidine hydrochloride]is known.Acute 96h static bioassays were conducted with Oryzias latipes to...As a new insecticide,less information about the aquatic biological toxicity of DMAH [N-(2,4-dimethylphenyl)-N'-methylamidine hydrochloride]is known.Acute 96h static bioassays were conducted with Oryzias latipes to assess the toxicity of DMAH and the influence of environ- mental factors.The results indicated that,at 23℃,the 24h,48h and 96h LC_(50)are 14.6 mg/L,13. 06 mg/L and 12.39 mg/L,respectively,the safe concentration(SC)of DMAH to Oryzias latipes is 1.16-1.32 mg/L.The changes of temperature,pH value and hardness of water will significantly influence the toxicity of DMAH to fish.展开更多
Dinitrobenzene(1,4-DNB) is one of the preferential toxicants stipulated by OECD and EPA of USA. With Oryzias latipes, which were chosen as the acceptors according to the international standard, the acute, subacute and...Dinitrobenzene(1,4-DNB) is one of the preferential toxicants stipulated by OECD and EPA of USA. With Oryzias latipes, which were chosen as the acceptors according to the international standard, the acute, subacute and accumulative toxicity experiments were conducted. The threshold mass concentration of O.latipes was 1.360 mg/L. The subacute experiment showed that O.latipes were hardly affected when the mass concentration of 1,4-DNB was 0.0292 mg/L, but were significantly affected when the mass concentration of 1,4-DNB was 0.0420, 0.0608 or 0.0875 mg/L in contrast to the control sample(P<0.05). The accumulative rate(K) was 1.21. This shows that there are significant accumulative actions of 1,4-DNB in fish. When the mass concentration of 1,4-DNB is in the range of 0.032_0.320 mg/L, the higher the 1,4-DNB concentration, the more strongly the Na +/K +-ATPase is inhibited. The calculated IC_ 50 values in gill, kidney and liver of O. latipes are 0.128, 0.132 and 0.428 mg/L, respectively.展开更多
BdlR strain killifish Oryzias latipes, which can be found in natural populations in Kochi Prefecture, Japan, exhibit a fading body-color. The aim of this article is trace of body fade by enzymatic analysis especially ...BdlR strain killifish Oryzias latipes, which can be found in natural populations in Kochi Prefecture, Japan, exhibit a fading body-color. The aim of this article is trace of body fade by enzymatic analysis especially blacking key enzyme tyrosinase. A single scale was collected from a single specimen of this strain at the dorsal axis, and the supernatant fi-action developed from this sample served as a tyrosinase extract. The extract fraction showed low tyrosinase activity when L-tyrosine, L-dopa and L-dopamine were used as enzyme substrates. After addition of L-cysteine to the sample scale, surface color gradually faded as pheomelanin formed. Tyrosinase was believed to have been in the folded phase and especially active arotmd the catalytic site and copper was detected in the scale. In hereafter, molecular analysis can attain above purpose.展开更多
The mitochondrial cytochrome b gene was isolated from the caudal fin of Oryzias latipes and PCR was undertaken to determine phylogeny. The nucleotide sequence of the complete cytochrome b gene including the 5＇ and 3...The mitochondrial cytochrome b gene was isolated from the caudal fin of Oryzias latipes and PCR was undertaken to determine phylogeny. The nucleotide sequence of the complete cytochrome b gene including the 5＇ and 3＇ ends was 1,143 base pairs （bp） and 1,137 bp encoding 380 and 378 amino acids in the K-11 strain and HS strain, respectively. In addition, higher substitutions both in base and amino acid residues occurred more frequently between the former control （Hd-rR strain） and HS strains than the K-I 1 strain. Approximately similar values in polymorphism, as assessed by restriction endonuclease digestion, were detected when utilizing 20 different enzymes. Therefore, the evolutionary processes were unlikely to involve common ancestors, especially between the K-11 strain and HS strains, in O. latipes populations in Japan. The sequence had been deposited in GenBank Data Base under accession number AB480878 （K-I I strain） and AB480879 （HS strain）.展开更多
Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and NPY8BR.Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest th...Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and NPY8BR.Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation;this has been found in only a few fish,at present.In order to better understand the physiological function of npy8br,especially in digestion,we used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology to generate npy8br-/-japanese medaka(Oryzias latipes).We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability,ultimately affecting their growth.Specifically,npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes(npy and agrp).npy8br-/-medaka larvae fed for 10 d(10th day of feeding)still had incompletely digested brine shrimp(Artemia nauplii)in the digestive tract 8 h after feeding,the messenger RNA(mRNA)expression levels of digestion-related genes(amy,lpl,ctra,and ctrb)were significantly decreased,and the activity of amylase,trypsin,and lipase also significantly decreased.The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes(gh and igf1).Hematoxylin and eosin(H&E)sections of intestinal tissue showed that npy8br-/-medaka larvae had damaged intestine,thinned intestinal wall,and shortened intestinal villi.So far,this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.展开更多
Benzo[a]pyrene(BaP),a ubiquitous pollutant,raises environmental health concerns due to induction of bone toxicity in the unexposed offspring.Exposure of F0 ancestor medaka(Oryzias latipes)to 1μg/L BaP for 21 days cau...Benzo[a]pyrene(BaP),a ubiquitous pollutant,raises environmental health concerns due to induction of bone toxicity in the unexposed offspring.Exposure of F0 ancestor medaka(Oryzias latipes)to 1μg/L BaP for 21 days causes reduced vertebral bone thickness in the unexposed F3 male offspring.To reveal the inherited modifications,osteoblast(OB)abundance and molecular signaling pathways of transgenerational BaP-induced bone thinning were assessed.Histomorphometric analysis showed a reduction in OB abundance.Analyses of the miRNA and mRNA transcriptomes revealed the dysregulation of Wnt signaling(frzb/ola-miR-1–3p,sfrp5/ola-miR-96–5p/miR-455–5p)and bone morphogenetic protein(Bmp)signaling(bmp3/ola-miR-96–5p/miR-181b-5p/miR-199a-5p/miR-205–5p/miR-455–5p).Both pathways are major indicators of impaired bone formation,while the altered Rank signaling in osteoclasts(c-fos/miR-205–5p)suggests a potentially augmented bone resorption.Interestingly,a typical BaP-responsive pathway,the Nrf2-mediated oxidative stress response(gst/ola-miR-181b-5p/miR-199a-5p/miR-205),was also affected.Moreover,mRNA levels of epigenetic modification enzymes(e.g.,hdac6,hdac7,kdm5b)were found dysregulated.The findings indicated that epigenetic factors(e.g.,miRNAs,histone modifications)may directly regulate the expression of genes associated with transgenerational BaP bone toxicity and warrants further studies.The identified candidate genes and miRNAs may serve as potential biomarkers for BaP-induced bone disease and as indicators of historic exposures in wild fish for conservation purposes.展开更多
Chinese medaka(Oryzias sinensis),widely distributed in the plateau lakes,Yunnan Province,which was the indicative specie for the habitat quality of riparian zone in the wetland ecosystems and also was the key specie i...Chinese medaka(Oryzias sinensis),widely distributed in the plateau lakes,Yunnan Province,which was the indicative specie for the habitat quality of riparian zone in the wetland ecosystems and also was the key specie in the lake ecosystems,and also was one of the fishes who suffered the severe invasion of alien species.There,we investigated that the genetic diversity and population structure of the wild Chinese medaka by using inter-simple sequence repeat(ISSR)analysis.The samples(20 individuals in total)of Chinese medaka in three populations in the drainage area of Dian Lake were obtained.The 7 ISSR primers were picked out to assess the genetic diversity.The ISSR primers combinations revealed 82.69%polymorphism,among 52 genetic loci amplified.The sum of effective number of alleles and observed number of alleles were 1.492 4 and 1.826 9,respectively.The Shannon genetic diversity and Nei genetic diversity indexes of all the populations were 0.434 5 and 0.289 7,respectively.The gene differentiation(GST)was 0.198 9,and the gene flow(NM)was 2.013 2.The AMOVA demonstrated that only 6.65%of genetic variance came from inter-population,and 93.35%of genetic variance came from intra-population.Phylogenetic tree showed that 20 individuals from 3 populations were clustering mixed each other.Also,the principal coordinate analysis(PCA)supported the cluster analysis results.These results indicated that the genetic diversity of Oryzias sinensis was at the middle level,and there was no obvious genetic differentiation among the three populations of Oryzias sinensis.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
Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRN...Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.展开更多
In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand deposition.LPL deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause hypertriglyceridemia.The lpl gene ...In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand deposition.LPL deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause hypertriglyceridemia.The lpl gene is not expressed in adult mammalian liver,but is in adult fish liver.The functions provided by the lpl gene are diverse in vertebrates.Here,we knocked out the lpl gene in Japanese medaka(Oryzias latipes)with the CRISPR/Cas9 system.The lpl-knockout(KO)homozygous individuals showed severe developmental defects with an extremely emaciated and deformedbody and onlyaccounted for about5%of the F2fish.This is consistentwiththefindings inmicebut disaccords with the results for zebrafish(Danio rerio).Compared with wild-type(WT)madaka,the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle,showed no significant difference in the liver,and was significantly lower in the heart.Under lpl heterozygous deficiency,the relative area of Oil RedO and triglycerides(TG)level in the liver,heart and muscle tissue covaried with levels of lpl mRNA in medaka.The lpl heterozygous deficiency did not affect the levelsofTG,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol(TC)in the plasma of medaka,which is inconsistent with the findings in mammals.In general,the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.展开更多
基金The National Natural Science Foundation of China under contract No.42106119the Department of Science and Technology of Fujian Province under contract Nos 2022J02052,2020J05175 and 2020J05178+1 种基金the Fujian Provincial Department of Ocean and Fisheries under contract No.FJHJF-L-2022-12the Yancheng Fishery High Quality Development Project under contract No.YCSCYJ2021023.
文摘In the coastal environment,the co-occurrence of antibiotic and nanoplastic pollution is common.Investigating their individual and combined toxicity to marine organisms is of great necessity.In the present study,the reproductive toxicity of sulfamethazine(SMZ)and nanoplastics(polystyrene,PS)via the dietary route on the spermatogenesis of marine medaka(Oryzias melastigma)was examined.After 30 d of dietary exposure,SMZ alone decreased the gonadosomatic index(GSI)value(~35%)and the proportion of undifferentiated type A spermatogonia(A_(und))(~40%),probably by disrupting the testicular sex hormone production,the spermatogenesis-related growth factor network and the balance of apoptosis.Individual exposure to PS did not affect the GSI value or the proportions of germ cells at different developmental stages,but dysregulated the expression of several spermatogenesis-related genes.Interestingly,the presence of PS alleviated the decreased GSI value caused by SMZ.This alleviation effect was achieved by enhancing the spermatogonia differentiation instead of reversing the suppressed self-renewal of A_(und),suggesting that the mixture of PS and SMZ could cause reproductive effects in a different way.These findings expand our knowledge of threats of ubiquitous antibiotic and nanoplastic pollution to fish reproduction and population.
基金supported by the National Natural Science Foundation of China(No.40632009,20607002)the National Basic Research Program(973)of China(No.2007CB407304).
文摘Although 1,1-dichloro-2,2-bis（p-chlorophenyl）-ethylene （p,p′-DDE）, the major and most persistent metabolite of dichlorodiphenyltrichloroethane （DDT）, was continually detected in wild fishes that showed abnormal gonad development such as intersex, little is known about the impact of p,p′-DDE exposure on gonad development in fishes. To survey the effects of p,p′-DDE on gonadal development and gene expressions, male juvenile （20-d post hatch） Japanese medaka （Oryzias latipes） was exposed to 1, 5, 20, and 100 μg/L p,p′-DDE for two months. Increased hepatosomatic index （HSI） and decreased gonadosomatic index （GSI） were found in the p,p′-DDE-treated groups. Intersex was found in 100 μg/L p,p′-DDE exposure group, as well as 100 ng/L 17α-ethynylestradiol （EE2） group. By quantitative real-time RT-PCR, it was found that gene expressions of vitellogenins （VTG-1, VTG-2）, choriogenins （CHG-H, CHG-L）, and estrogen receptor α （ER-α） in the liver of the fish were significantly up-regulated by p,p′-DDE exposure. VTG-1 and VTG- 2 were recommended as the preferred biomarker for assessing anti-androgenic p,p′-DDE because they were the highest up-regulated among the genes and showed good dose-response relationship. The up-regulated ER-α suggested that a potential synergetic effect would occur when p,p′-DDE coexists with other ER-α-binding endocrine-disrupting chemicals （EDCs）.
文摘The accumulation and absorption kinetics of La by fish (Oryzias latipes) from both water and food were tested in fresh water. The effects of La concentration in water and food and the existence of fulvic acid on the absorption were investigated. It was demonstrated that the amount of La accumulated in fish increases with the increase in La concentration in water and food and exposure duration, which the normal physiological activity of fish was inhibited. With fulvic in the system, the absorption rate decreases significantly indicating that the fulvic combined La is not available to the fish uptake.
基金This research was supported by the National Natural Science Foundation of China.
文摘As a new insecticide,less information about the aquatic biological toxicity of DMAH [N-(2,4-dimethylphenyl)-N'-methylamidine hydrochloride]is known.Acute 96h static bioassays were conducted with Oryzias latipes to assess the toxicity of DMAH and the influence of environ- mental factors.The results indicated that,at 23℃,the 24h,48h and 96h LC_(50)are 14.6 mg/L,13. 06 mg/L and 12.39 mg/L,respectively,the safe concentration(SC)of DMAH to Oryzias latipes is 1.16-1.32 mg/L.The changes of temperature,pH value and hardness of water will significantly influence the toxicity of DMAH to fish.
文摘Dinitrobenzene(1,4-DNB) is one of the preferential toxicants stipulated by OECD and EPA of USA. With Oryzias latipes, which were chosen as the acceptors according to the international standard, the acute, subacute and accumulative toxicity experiments were conducted. The threshold mass concentration of O.latipes was 1.360 mg/L. The subacute experiment showed that O.latipes were hardly affected when the mass concentration of 1,4-DNB was 0.0292 mg/L, but were significantly affected when the mass concentration of 1,4-DNB was 0.0420, 0.0608 or 0.0875 mg/L in contrast to the control sample(P<0.05). The accumulative rate(K) was 1.21. This shows that there are significant accumulative actions of 1,4-DNB in fish. When the mass concentration of 1,4-DNB is in the range of 0.032_0.320 mg/L, the higher the 1,4-DNB concentration, the more strongly the Na +/K +-ATPase is inhibited. The calculated IC_ 50 values in gill, kidney and liver of O. latipes are 0.128, 0.132 and 0.428 mg/L, respectively.
文摘BdlR strain killifish Oryzias latipes, which can be found in natural populations in Kochi Prefecture, Japan, exhibit a fading body-color. The aim of this article is trace of body fade by enzymatic analysis especially blacking key enzyme tyrosinase. A single scale was collected from a single specimen of this strain at the dorsal axis, and the supernatant fi-action developed from this sample served as a tyrosinase extract. The extract fraction showed low tyrosinase activity when L-tyrosine, L-dopa and L-dopamine were used as enzyme substrates. After addition of L-cysteine to the sample scale, surface color gradually faded as pheomelanin formed. Tyrosinase was believed to have been in the folded phase and especially active arotmd the catalytic site and copper was detected in the scale. In hereafter, molecular analysis can attain above purpose.
文摘The mitochondrial cytochrome b gene was isolated from the caudal fin of Oryzias latipes and PCR was undertaken to determine phylogeny. The nucleotide sequence of the complete cytochrome b gene including the 5＇ and 3＇ ends was 1,143 base pairs （bp） and 1,137 bp encoding 380 and 378 amino acids in the K-11 strain and HS strain, respectively. In addition, higher substitutions both in base and amino acid residues occurred more frequently between the former control （Hd-rR strain） and HS strains than the K-I 1 strain. Approximately similar values in polymorphism, as assessed by restriction endonuclease digestion, were detected when utilizing 20 different enzymes. Therefore, the evolutionary processes were unlikely to involve common ancestors, especially between the K-11 strain and HS strains, in O. latipes populations in Japan. The sequence had been deposited in GenBank Data Base under accession number AB480878 （K-I I strain） and AB480879 （HS strain）.
基金supported by the National Natural Science Foundation of China(No.31972809)the Key Research&Development Program of Hubei Province(No.2022BBA0051),China.
文摘Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and NPY8BR.Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation;this has been found in only a few fish,at present.In order to better understand the physiological function of npy8br,especially in digestion,we used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology to generate npy8br-/-japanese medaka(Oryzias latipes).We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability,ultimately affecting their growth.Specifically,npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes(npy and agrp).npy8br-/-medaka larvae fed for 10 d(10th day of feeding)still had incompletely digested brine shrimp(Artemia nauplii)in the digestive tract 8 h after feeding,the messenger RNA(mRNA)expression levels of digestion-related genes(amy,lpl,ctra,and ctrb)were significantly decreased,and the activity of amylase,trypsin,and lipase also significantly decreased.The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes(gh and igf1).Hematoxylin and eosin(H&E)sections of intestinal tissue showed that npy8br-/-medaka larvae had damaged intestine,thinned intestinal wall,and shortened intestinal villi.So far,this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.
基金supported by the Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(No.SMSEGL20SC02)the National Natural Science Foundation of China(No.41977371)+1 种基金a Project grant from the Shenzhen and Technology Innovation Commission(No.JCYJ20170818094137791)supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award number 1R15ES032936-01.
文摘Benzo[a]pyrene(BaP),a ubiquitous pollutant,raises environmental health concerns due to induction of bone toxicity in the unexposed offspring.Exposure of F0 ancestor medaka(Oryzias latipes)to 1μg/L BaP for 21 days causes reduced vertebral bone thickness in the unexposed F3 male offspring.To reveal the inherited modifications,osteoblast(OB)abundance and molecular signaling pathways of transgenerational BaP-induced bone thinning were assessed.Histomorphometric analysis showed a reduction in OB abundance.Analyses of the miRNA and mRNA transcriptomes revealed the dysregulation of Wnt signaling(frzb/ola-miR-1–3p,sfrp5/ola-miR-96–5p/miR-455–5p)and bone morphogenetic protein(Bmp)signaling(bmp3/ola-miR-96–5p/miR-181b-5p/miR-199a-5p/miR-205–5p/miR-455–5p).Both pathways are major indicators of impaired bone formation,while the altered Rank signaling in osteoclasts(c-fos/miR-205–5p)suggests a potentially augmented bone resorption.Interestingly,a typical BaP-responsive pathway,the Nrf2-mediated oxidative stress response(gst/ola-miR-181b-5p/miR-199a-5p/miR-205),was also affected.Moreover,mRNA levels of epigenetic modification enzymes(e.g.,hdac6,hdac7,kdm5b)were found dysregulated.The findings indicated that epigenetic factors(e.g.,miRNAs,histone modifications)may directly regulate the expression of genes associated with transgenerational BaP bone toxicity and warrants further studies.The identified candidate genes and miRNAs may serve as potential biomarkers for BaP-induced bone disease and as indicators of historic exposures in wild fish for conservation purposes.
基金Under the auspices of Special Program for Key Basic Research Project of the Ministry of Science and Technology,China(973 Program)(2012CB426509)General Project of Research Foundation of Yunnan Provincial Education Department(2014Y335)Research Staring Foundation of Southwest Forestry University(111041)
文摘Chinese medaka(Oryzias sinensis),widely distributed in the plateau lakes,Yunnan Province,which was the indicative specie for the habitat quality of riparian zone in the wetland ecosystems and also was the key specie in the lake ecosystems,and also was one of the fishes who suffered the severe invasion of alien species.There,we investigated that the genetic diversity and population structure of the wild Chinese medaka by using inter-simple sequence repeat(ISSR)analysis.The samples(20 individuals in total)of Chinese medaka in three populations in the drainage area of Dian Lake were obtained.The 7 ISSR primers were picked out to assess the genetic diversity.The ISSR primers combinations revealed 82.69%polymorphism,among 52 genetic loci amplified.The sum of effective number of alleles and observed number of alleles were 1.492 4 and 1.826 9,respectively.The Shannon genetic diversity and Nei genetic diversity indexes of all the populations were 0.434 5 and 0.289 7,respectively.The gene differentiation(GST)was 0.198 9,and the gene flow(NM)was 2.013 2.The AMOVA demonstrated that only 6.65%of genetic variance came from inter-population,and 93.35%of genetic variance came from intra-population.Phylogenetic tree showed that 20 individuals from 3 populations were clustering mixed each other.Also,the principal coordinate analysis(PCA)supported the cluster analysis results.These results indicated that the genetic diversity of Oryzias sinensis was at the middle level,and there was no obvious genetic differentiation among the three populations of Oryzias sinensis.
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This study was supported by the National Natural Science Foundation of China(Nos.31771648 and 31672653)the Scientific Research Foundation of Jimei University(No.ZQ2020003)the National Key Basic Research Program of China(No.2013CB967700).
文摘Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
基金the National Natural Science Foundation of China(31972809)China Agriculture Research System(CARS-46)the National Key R&D Program of China(2019YFD0900500).
文摘In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand deposition.LPL deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause hypertriglyceridemia.The lpl gene is not expressed in adult mammalian liver,but is in adult fish liver.The functions provided by the lpl gene are diverse in vertebrates.Here,we knocked out the lpl gene in Japanese medaka(Oryzias latipes)with the CRISPR/Cas9 system.The lpl-knockout(KO)homozygous individuals showed severe developmental defects with an extremely emaciated and deformedbody and onlyaccounted for about5%of the F2fish.This is consistentwiththefindings inmicebut disaccords with the results for zebrafish(Danio rerio).Compared with wild-type(WT)madaka,the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle,showed no significant difference in the liver,and was significantly lower in the heart.Under lpl heterozygous deficiency,the relative area of Oil RedO and triglycerides(TG)level in the liver,heart and muscle tissue covaried with levels of lpl mRNA in medaka.The lpl heterozygous deficiency did not affect the levelsofTG,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol(TC)in the plasma of medaka,which is inconsistent with the findings in mammals.In general,the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.
