Effects of p,p'-DDE exposure on gonadal development and gene expression in Japanese medaka(Oryzias latipes)

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）.

Oryzias latipes鱼体对不同形态镧的积累特征

在试验室条件下研究了Oryziaslatipes鱼体对不同形态轻稀土La的积累特征及它对游离La吸收的动力学过程 ,并初步探讨了鱼对La的吸收机制。结果表明 ,试验鱼对La的积累量随水相游离La浓度增高、暴露时间增长而增加 ;水相富里酸的存在明显降低了La对鱼的生物有效性。此外 ,试验鱼也可以通过摄食获取水相中的La。

氯化镉、灭多威和硝基苯对日本青鳉(Oryzias latipes)行为抑制的生物应急监测

研究了氯化镉、灭多威和硝基苯对孵化以后7d左右日本青鳉(Oryzias latipes)行为毒性。结果表明,在不同污染物中,随暴露浓度的增加,第1尾日本青鳉产生行为抑制的时间逐渐缩短,并且半数行为抑制时间(time ofhalf behavior inhibition,THBI,Y)随暴露浓度(X)呈现幂指数关系Y=aXb,其中20<a<28,-1.2<b<-0.8。实验结果表明基于日本青鳉THBI分析,能初步实现水体突发性污染事故污染水平的应急监测,从而为污染事故应急预案提供支持。

Cu2＋和Cd2＋对日本青鳉(Oryzias latipes)早期发育阶段的急性毒性效应研究

为探究并比较淡水鱼种日本青鳉早期发育阶段对Cu^(2+)和Cd^(2+)等重金属胁迫的响应,在实验室通过半静态方式,对日本青鳉受精卵和仔稚鱼分别进行了48 h和96 h急性毒性实验。结果表明:Cu^(2+)对日本青鳉胚胎24、48 h-LC_(50)分别为8.164 mg·L^(-1)和6.965 mg·L^(-1);Cd^(2+)对日本青鳉胚胎24、48 h-LC_(50)分别为63.084 mg·L^(-1)和53.093 mg·L^(-1);较低浓度组Cu^(2+)(≤1.97 mg·L^(-1))时日本青鳉胚胎的发育速率快于对照组,而较高浓度组(≥3.87 mg·L^(-1))胚胎的发育速率则慢于对照组;与Cu^(2+)略有不同,无论浓度高低Cd^(2+)对胚胎的孵化速率均产生抑制作用;Cu^(2+)和Cd^(2+)质量浓度分别高于1.97 mg·L^(-1)和19.68 mg·L^(-1)时,两种重金属离子均显著降低胚胎的孵化率(P<0.05)。Cu^(2+)对日本青鳉初孵仔鱼24、48、72和96 h-LC_(50)分别为5.361 mg·L^(-1)、2.844 mg·L^(-1)、2.020 mg·L^(-1)和1.352 mg·L^(-1);Cd^(2+)对日本青鳉初孵仔鱼24、48、72和96 h-LC_(50)分别为15.907 mg·L^(-1)、10.550 mg·L^(-1)、7.986 mg·L^(-1)和6.346 mg·L^(-1);Cu^(2+)对日本青鳉稚鱼24、48、72和96 h-LC_(50)分别为5.732 mg·L^(-1)、4.037 mg·L^(-1)、2.498 mg·L^(-1)和1.955 mg·L^(-1);Cd^(2+)对日本青鳉稚鱼的24、48、72和96 h-LC_(50)分别为16.419 mg·L^(-1)、11.745 mg·L^(-1)、8.516 mg·L^(-1)和6.776 mg·L^(-1)。与其它淡水水生生物相比,日本青鳉仔稚鱼对铜和镉离子较为敏感。

Accumulation of Lathenum with Various Species in Oryzias Latipes Body

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.

Acute toxicity of DMAH to Oryzias latipes as modified by some influential factors

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.

Toxicity Effects of 1,4-Dinitrobenzene on Oryzias Latipes

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.

Pheomelanin Formation and Low Tyrosinase Activity in Fading Body Color Variant BdlR Strain Oryzias latipes

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.

Molecular Evolution of the Cytochrome b Gene in Geographically Isolated Populations of Oryzias Latipes

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）.

Neuropeptide Y receptor Y8b(npy8br)regulates feeding and digestion in Japanese medaka(Oryzias latipes)larvae:evidence from gene knockout

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.

活动范围对青鳉鱼(Oryzias latipes)运动模式的影响

重尾分布现象广泛存在于自然界的各种生物运动模式之中,重尾分布现象会随环境的变化而变化。鱼类为适应生存环境,往往会选择更加高效的个体运动模式。以青鳉鱼(Oryzias latipes)为研究对象,在相同浅水条件(5 cm)下,控制其活动范围,探究青鳉鱼在不同水域面积(1.0 m×1.0 m、0.6 m×0.6 m、0.4 m×0.4 m、0.2 m×0.2 m以及0.1 m×0.1 m)条件下的运动模式。利用MATLAB软件将青鳉鱼的运动轨迹转换为具体数据,通过移动速度、移动加速度、单步步长和曲率半径4个方向对青鳉鱼的运动模式进行分析,并采用对数正态分布函数对实验数据进行拟合。研究结果显示,在较小活动范围中(0.1 m×0.1 m),青鳉鱼运动模式中重尾分布现象会被抑制,并且抑制程度与活动范围的大小有直接关系,具体表现在移动速度、移动加速度、单步步长和曲率半径取值的随机性会随活动范围的缩小而降低。该研究将为解释鱼类行为提供新视角,为探究活动范围对鱼类运动模式的影响奠定基础。

Efficient gene editing in a medaka(Oryzias latipes)cell line and embryos by SpCas9/tRNA-gRNA

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.

Knockout of lipoprotein lipase with CRISPR/Cas9 causes severe developmental defects and affects lipid deposition in Japanese medaka(Oryzias latipes)

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.

Transgenerational bone toxicity in F3 medaka (Oryzias latipes) induced by ancestral benzo[a]pyrene exposure: Cellular and transcriptomic insights

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.

含吡啶类腈类医药中间体废盐对海洋青鳉鱼胚胎的毒性效应

工业废盐是化工生产过程中产生的副产结晶盐。农药、医药等精细化工行业的生产中往往伴随着大量废盐的产生,《国家危险废物名录》中明确将多种化学制药、农药生产过程中产生的废盐列为危险废物。然而,由于处理成本高昂和处置能力不足,废盐限制了企业的健康发展。在《危险废物环境管理指南化工废盐》中明确了工业废盐无害化排海的相关要求,但目前仍缺少相关领域的实验研究。海洋青鳉鱼(Oryzias melastigma)具有与斑马鱼类似的研究特征优势,是国际生命科学学会(ILSI)与健康和环境科学研究所(HESI)认定的海洋生态毒理学的重要研究工具。研究了含吡啶类腈类医药中间体废盐和经过实验室处理后的除杂盐对海洋青鳉鱼胚胎的急性毒性、发育毒性以及对抗氧化系统、免疫系统的影响。结果表明,废盐暴露对海洋青鳉鱼胚胎的生存和发育有显著影响并造成系统性损伤,而除杂盐对海洋青鳉鱼胚胎没有较大影响。本研究为废盐排海提供了基础的理论支持,也为后续实验开展提供了良好的思路引导。

Genetic Diversity of Chinese Medaka(Oryzias sinensis)in Dian Lake Drainage Area by ISSR Analysis

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.

布洛芬对海洋青鳉鱼生命初期毒性效应

为探究布洛芬(Ibuprofen,IBP)对海洋青鳉鱼(Oryzias melastigma)早期生命阶段的毒性效应,将海洋青鳉鱼胚胎在不同浓度(0、1、10、100、1000μg·L^(-1))IBP溶液中暴露至孵化后的第30天(未孵化胚胎暴露20 d).观察统计胚胎的孵化率、孵化时间、心率、仔鱼30 d存活率以及体长体重等发育参数,并检测胚胎的抗氧化酶(超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽巯基转移酶(Glutathione S-transferase,GST))活性、抗氧化剂(还原型谷胱甘肽(glutathione,GSH))、氧化产物(丙二醛(malonic dialdehyde,MDA))浓度和神经递质降解酶(乙酰胆碱酯酶(acetylcholinesterase,AChE))活性变化,以评估IBP对海洋青鳉鱼早期生命阶段的发育毒性、抗氧化系统以及神经递质系统的影响.结果表明,1μg·L^(-1)IBP对海洋青鳉鱼胚胎及仔鱼不存在发育毒性,其余各浓度导致胚胎孵化率、孵化时间、心率和仔鱼存活率降低,所有浓度下仔鱼体长体重均未变化;在第5天时,随浓度升高,SOD活性和GSH含量呈下降趋势,CAT、GST活性和MDA含量先上升后下降,第8天时各指标趋于正常;IBP浓度的升高导致AChE活性先增加后降低.综上,环境相关浓度下的IBP对海洋青鳉鱼胚胎及仔鱼不存在发育毒性,各浓度IBP均会导致胚胎产生氧化应激和神经毒性效应.

染织排水对日本青鳉幼鱼和胚胎的毒性效应

采用日本青鳉幼鱼及胚胎暴露评估染织排水的毒性效应。4个测试水样采自染织工厂排水口。96h急性毒性试验表明:1和2号水样对幼鱼急性毒性效应不明显,3和4号水样对幼鱼具有急性毒性效应,毒性单位分别为0.47、0.53、9.93和1.68TUa,1和2号水样为微毒,3号水样为中毒,4号水样为低毒。14d胚胎幼鱼慢性毒性试验表明:1、3和4号水样引起胚胎死亡,胚胎孵化的无观察效应浓度(NOEC)分别为90%、20%和33%。4号水样52%和64%试验组延长了胚胎孵化的时间并与对照组产生了显著性差异(p<0.05)。2号水样严重抑制胚胎孵化,胚胎孵化率的NOEC为2.5%,其EC25为39.2%。水样的慢性毒性单位分别为:1.11、40、5和3.03TUc。鱼苗经3d暴露后,仅1和3号水样中的试验组对幼鱼产生致死效应。存活率NOEC分别为90%、20%、10%和64%。

城镇污水处理厂进出水对日本青鳉早期阶段亚慢性毒性效应

采用日本青鳉胚胎及幼鱼暴露评估污水处理厂进出水的综合毒性效应。水样采自广州市3个污水处理厂的进水口和出水口。96 h青鳉幼鱼急性毒性试验表明:6个水样均没有对青鳉幼鱼产生急性毒性效应。14 d胚胎暴露试验结果:3个污水处理厂的进水及1号和3号污水处理厂的出水均引起胚胎死亡,胚胎孵化率均低于70%且均与对照组产生了显著性差异(p<0.05)。3个厂的进水和1号、2号厂出水的鱼苗畸形率与对照组也有显著差异(p<0.05),其中3号厂进水鱼苗畸形率高达(33.73±8.94)%。而6个水样的胚胎孵化时间均与对照组无显著差异(p>0.05),均在9 d左右。7 d鱼苗暴露试验结果:1、2、3号3个水厂进水的鱼苗死亡率分别是(30.16±2.75)%,(24.07±1.60)%,(37.90±4.77)%,与对照组产生明显差异(p<0.05)。出水对鱼苗的致死效应明显低于进水,但出水对鱼苗的致死效应相对于对照组仍有显著差异(p<0.05)。

2种除草剂联合胁迫下日本青鳉的逐级行为响应

探讨了日本青鳉在两种除草剂阿特拉津和百草枯联合暴露下的逐级行为响应,采用水质安全在线生物预警系统(BEWs)记录行为强度数据,分析不同暴露浓度、不同暴露时间日本青鳉的行为响应.10、5、1和0.1TU的暴露浓度下行为反应时间分别为:0.74、7.7、29.4和42.2h,而每个浓度不同配比之间行为反应时间差异明显.结果表明:在两种除草剂的暴露下,日本青鳉的逐级行为响应既受化合物浓度高低的影响,又受暴露时间的影响,而且每个浓度两种药物不同配比暴露下的青鳉鱼的逐级行为响应基本一致,每个浓度不同配比之间行为反应时间差异明显,即两种作用机制不同的除草剂对日本青鳉的行为毒性是协同作用.