异育银鲫(Carassius auratus gibelio)脊髓组织细胞系的建立及对CyHV-2的敏感性

鲫造血器官坏死病是近些年来危害养殖鲫鱼最为严重的传染性疾病,其病原为鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2, CyHV-2)。本研究以异育银鲫(Carassius auratus gibelio)脊髓组织为材料,进行组织细胞体外培养,构建对鲤疱疹病毒Ⅱ型敏感的异育银鲫脊髓细胞系(Spinalcordtissuecell lines of Carassius auratus gibelio, CSC)。采用组织块移植法,进行异育银鲫脊髓细胞的原代培养,并进行传代,已传代至第128代次,获得稳定传代的细胞系后,采用低渗法进行细胞染色体核型分析,并进行鲤疱疹病毒Ⅱ型的敏感性测试与传代。结果表明,以含10%胎牛血清的L-15培养基,在24℃恒温条件下,可稳定传代培养异育银鲫脊髓细胞,传代细胞呈成纤维样或纺锤状;核型分析显示,染色体数为156±2条,该细胞为三倍体细胞。通过对CyHV-2敏感性的测试,CSC细胞感染CyHV-2后产生典型的细胞病变,感染第7天的病变细胞液的TCID50达到109.33/mL,CyHV-2病毒可在CSC细胞上稳定传代;通过对病变细胞的电镜观察,可见大量直径为128-134nm大小的疱疹样病毒颗粒。以上研究结果表明,本研究构建的异育银鲫脊髓细胞系对CyHV-2敏感,并在细胞上能进行稳定传代,传代病毒具有较高病毒滴度,这为鲤疱疹病毒Ⅱ型的深入研究及疫苗开发奠定了重要的基础。

两种池养模式下异育银鲫(Carassius auratus gibelio)生物学表型对体质量影响效果的差异分析

水产养殖动物的增重机制既是其生存对策的重要组成部分,也是影响其生长性能和养殖产量的重要内因。异育银鲫(Carassius auratus gibelio)是我国的重要养殖鱼类,随机选取池塘生态主养模式(M_(1))和池塘生态套养模式(M_(2))下经7个月养殖的异育银鲫夏花苗种各70尾为研究对象,以全长(X_(1))、体高(X_(2))、体宽(X_(3))、头长(X_(4))、侧线长(X_(5))、尾柄高(X_(6))、腹鳍间距(X_(7))、胸鳍间距(X_(8))为体尺性状,净体质量(NW)、肠质量(W_(1))、肝质量(W_(2))、胃质量(W_(3))、鳃质量(W_(4))、心质量(W_(5))和鳔质量(W_(6))为称量性状,采用多元分析方法分别研究了M_(1)、M_(2)实验群体尺寸性状和称量性状对其体质量(BW)的影响效应。结果表明:(1)生物学测定结果显示,CV值大于10%的均为质量性状,小于10%的均为体尺性状,除NW、X_(1)、X_(5)和X_(6)均呈M_(1)<M_(2)(P<0.05),X_(4)和各脏器质量均呈M_(1)>M_(2)(P<0.05)外,其余性状间均无显著差异(P>0.05);(2)相关分析显示,除W_(5)与BW间相关系数均未达到显著水平(P>0.05)外,其余性状与BW间的相关系数均达到极显著水平(P<0.01);(3)经通径分析和复相关分析,M_(1)、M_(2)被保留体尺性状组合X_(5)-X_(1)-X_(2)和X_(2)-X_(3)-X_(1)-X_(5)的总决定系数和复相关指数均分别为0.889和0.927,称量性状组合NW-W_(2)-W_(1)-W_(3)和NW-W_(2)的总决定系数和复相关指数均分别为0.992和0.971;(4)经偏回归分析,得到了可用于估算BW的M_(1)最优方程组和M_(2)最优方程组。研究结果可为我国异育银鲫科学养殖提供有效参考。

两种池塘养殖模式下异育银鲫(Carassius auratus gibelio)的形质特征差异分析

探究引起银鲫在不同养殖模式下的形质差异原因及形质饰变途径和机制,进而揭示养殖模式致其改变生存对策的内在逻辑,对于指导银鲫养殖模式的改进与优化具重要现实意义。随机选取池塘生态主养模式(M_(1))和池塘生态套养模式(M_(2))下经7个月养殖的异育银鲫夏花苗种各70尾为研究对象,采用主成分分析和判别分析方法,系统开展了两种池养模式下异育银鲫形质差异研究,结果表明:(1)池养期间两实验群体的生长速度总体上呈M_(2)>M_(1),在21项生物学表型性状中,M_(2)实验群体显著大于M_(1)实验群体的为L1(体长)、L4(头宽)、L11(肛后体长)和NM (净体质量)(P<0.05);(2)所涉20项形质评价性状中, M_(1)、M_(2)实验群体间具显著差异(P<0.05)的高达16项,聚类分析也指示两者间的欧式距离已达到显著水平(P<0.05),即M_(1)、M_(2)实验群体已在形质特征上出现显著分化;(3)经主成分分析,提取到的5个特征值均大于1的主成分,其累积贡献率达80.844%,其中PC1可归纳为表征机体消化代谢水平的公共因子, PC_(2)可归纳为表征脏器可容纳空间几何比例的公共因子, PC_(3)、PC_(4)和PC5可统归为表征食物获取方式的公共因子;(4)采用逐步判别法,以判别贡献率较大的眼间距/侧线长、净重系数、肝系数、胃系数、肠系数、鳃系数和Fulton指数为自变量,所建Fisher分类函数方程组可较清晰区分M_(1)和M_(2)实验个体,其中M_(1)实验群体的判别准确率P1、P2分别为98.6%和94.5%, M_(2)实验群体则分别为94.3%和98.5%,两实验群体的综合判别准确率为96.4%。所得结果可为异育银鲫池塘生态主养模式和生态套养模式的改良与优化提供科学依据。

Protection of Carassius auratus Gibelio against infection by Aeromonas hydrophila using specific immunoglobulins from hen egg yolk

Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.

Immunotoxicity of bisphenol A to Carassius auratus lymphocytes and macrophages following in vitro exposure

Bisphenol A （BPA） is the monomer component of polycarbonate plastics and classified as an endocrine disrupting chemical （EDC）. The reproductive toxicity of BPA has been extensively studied in mammals; however, relatively little information is available on the immunotoxic responses of fish to BPA. In this study, we investigated the effects of BPA on the immune functions of lymphocytes and macrophages in Carassius auratus. The effects of BPA were compared with those of two natural steroid hormones, estradiol and hydrocortisone. Proliferation of the two types of cells in response to PHA was measured using colorimetric MTT assay. Macrophage respiratory burst stimulated by Con A was measured using chemiluminescence assay. Results showed that BPA （0.054-5.4 mg/L）, estradiol （0.0002-2.0 mg/L） and hydrocortisone （5-50 mg/L） significantly induced Carassius auratus lymphocyte proliferation while higher doses of hydrocortisone （500-5000 mg/L） appeared to be inhibitory. BPA （0.005-50 mg/L）, estradiol （0.005-800 mg/L） and hydrocortisone （0.005-500 mg/L） markedly enhanced macrophage proliferation, whereas higher doses of BPA （500-1000 mg/L） appeared to inhibit cell proliferation. Furthermore, higher dosage of BPA （50 mg/L） and hydrocortisone （50 and 500 mg/L） suppressed the macrophages respiratory burst while estradiol is stimulative all the doses tested （0.05-500 mg/L）. In conclusion, BPA could have immunotoxicity to Carassius auratus and functional changes of lymphocyte and macrophage in Carassius auratus may be different between low and high dosages.

Toxic effects of crude-oil-contaminated soil in aquatic environment on Carassius auratus and their hepatic antioxidant defense system

Under the indoor simulant conditions, toxic effects of crude-oil-contaminated soil which was put into aquatic environment on the young fishes Carassius auratus and their hepatic antioxidant system after a 20-d exposure were investigated. Results showed that the relationship between the mortality of C. auratus and the exposed doses could be divided into 3 phases： fishes exposed to the low dose groups （0.5-5.0 g/L） were dead due to the ingestion of crude-oil-contaminated soils in aquatic environment; at the medium dose groups （5.0-25.0 g/L） fishes were dead due to the penetration of toxic substances; at the high dose groups （25.0-50.0 g/L） fishes were dead due to environmental stress. The highest mortality and death speed were found in the 1.0 g/L dose group, and the death speed was sharply increased in the 50.0 g/L dose group in the late phase of exposure. The activities of superoxide dismutase （SOD）, catalase （CAT） and glutathione S-transferase （GST） and the content of malaondialdehyde （MDA） in the hepatic tissues of C. auratus were induced significantly. The activity of SOD was increased and then decreased. It was significantly inhibited in the 50.0 g/L dose group. The activity of CAT was highly induced, and restored to a level which is little more than the control when the exposed doses exceeded 10.0 g/L. The activity of GST was the most sensitive, it was significantly induced in all dose groups, and the highest elevation was up to 6 times in the 0.5 g/L dose group comparing with the control. The MDA content was significantly elevated in the 50.0 g/L dose group, and the changes of the MDA content were opposite with the changes of GST activity.

Effects of Selected Metal Oxide Nanoparticles on Multiple Biomarkers in Carassius auratus

Objective To study the biological effects of nanoscale copper oxide （nCuO）, zinc oxide （nZnO）, cerium dioxide （nCeO2） and their mixtures on Carassius auratus. Methods Juvenile fish （Carossius auratus） were exposed to aqueous suspensions of nCuO, nZnO, and nCeO2 （alone and in mixtures） at concentrations of 20, 40, 80, 3.60, and 320 mg/L. The biomarkers-acetylcholinesterase （ACHE） in brain, sodium/potassium-activated ATPase （Na~/K＊-ATPase） in gill, and superoxide dismutase （SOD） and catalase （CAT） in liver-were determined after 4 days of exposure. Integrated biomarker response （IBR） was calculated by combining multiple biomarkers into a single value. Results AChE and SOD activities were significantly inhibited by all test metal oxide nanoparticles （NPs） at high concentrations （__.160 rag/L） with the exception of nCeO2. Na~/K＋-ATPase induction exhibited bell-shaped concentration-response curves. CAT activity was significantly inhibited at concentrations equal to or higher than 160 mg/L. The order of IBR values was nCeO2 = nZnO/nCeO2= nCuO/nCeO2 〈 nCuO/nZnO/nCeO2 〈 nZnO 〈 nCuO 〈 nCuO/nZnO. The joint effect seemed to be synergistic for nCuO/nZnO mixtures, additive for the ternary mixture and less than additive or antagonistic for the binary mixtures containing nCe02. Conclusion Concentration-dependent changes of enzymatic activities （ACHE, Na~/K＊-ATPase, SOD, and CAT） were observed in fish exposed to nanoscale metal oxides. IBR analysis allowed good discrimination between the different exposures and might be a useful tool for the quantification of integrated negative effects induced by NPs toward fish.

Biochemical responses of freshwater fish Carassius auratus to polycyclic aromatic hydrocarbons and pesticides

The freshwater fish Carassius auratus was chosen as an experimental subject, and their hepatic biochemical responses to the medium-term exposure of Benzo（k）fluoranthene （BkF） alone and in combination with PCB 118 and dichlorodiphenyltrichloroethane （DDT） were investigated by measuring the reduced glutathione （GSH）, glutathione S-transferase （GST）, and thiobarbituric acid reactive substances （TBARS）, to assess sublethal effects. The hepatic GSH content was significantly inhibited by organic pollutants, alone and in mixtures, while the TBARS content was significantly induced after three days of exposure. Bell-shaped concentration-response charts of GST activities were obtained. Significant dose-response relationships were found for hepatic GSH and TBARS contents of all concentrations and for the GST activity, except at the highest concentration. The GSH content, GST activity, and TBARS content in Carassius auratus were confirmed as useful biomarkers of exposure to organic pollutions.

Induction Effect of Cadmium on Peripheral Erythrocyte Micronuclei in Carassius auratus and Oriental weatherfish

[Objective] The study aimed to the effects of various Cd2＋ concentrations and exposure time on erythrocyte micronuclei in peripheral erythrocyte micronuclei in Carassius auratus and Oriental weatherfish. [ Method ] Using static water culture, Carassius auratus and Oriental weatherfish were exposed to solutions containing Cd2＋ with mass concentrations of 0.08, 1.72 and 6.84 mg/L, and blood was collected from tail veins to make smears which were observed under a oil lens afterwards. [ Result] In the initial exposure period, Carassius auratus and Onental weatherfish were unwell obviously, being afraid and unpeaceful and escaping everywhere. With the increase in Cd2＋ concentration, this phenomenon became more distinct. In addition, as the prolonging of exposure time, fishes moved slowly, powerlessly and less and reacted slowly, and moe and more excrement deposited at the bottom of aquariums. [ Conclusion] Cd2 ＋ could obviously induce Carassius auratus and Oriental weatherfish to generate micronuclei, showing certain dose-effect and time-effect relation under certain conditions.

Effect of lead on cytoskeletal protein stability in crucian carp Carassius auratus

Inorganic lead （Pb） is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb（NO3）2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level.

Effect of Atrazine on Antioxidant Enzyme and Its Bioaccumulation in Kidney of Crucian Carp,Carassius auratus

Etrazine is one of the most widely used herbicides in China and the world. Acute and chronic toxicity tests werc carried out to assess the possible toxicity effect of atrazine on crucian carp （Carassius auratus）. Results showed that 96 h LC,. of atrazine to Carassius auratus was 105.94 mg. L-1. The enzyme activities of superoxide dismutase （SOD）, catalase （CAT） and glutathione-S-transferases （GST） in kidney of Carassius auratus were all influenced by atraizine, and CAT was more sensitive to atrazine compared with SOD and GST. Atrazine residues in kidney of Carassius aura/us reached the stable state at day 19, and the bioaccumulation factors （BAF） of atrazine in kidney of Carassius auratus treated with 1.0 mg. L-1 and 10.0 mg. L-1 atrazine were 8.3 and 4.4, respectively. The research demonstrated that atrazine could cause oxidative stress to fish kidney, but atrazine was not easy to accumulate in Carassius auratus kidney, and the antioxidant enzymes could be used as biomarker to the early detection of pollution.

Growth and Development of Larvae and Juveniles of Carassius auratus gibelio

The growth and development of larvae and juveniles was morphologically investigated in Carassius auratus gibelio. The results showed that it took 52 h from fertilization to hatching at 22 - 25℃. The newly hatched larvae had transparent body, with the head fight near the yolk sac; the yolk sac accounted for 2/3 of larval volume, which was exhausted in the four-day-old larvae that started the exogenous nutrient period. The larvae were found to develop into juveniles from a few scales on the 20^th day after hatching to whole scales, fingerling, on the 28^th day after hatching. The relationship between total length（TL） and age in days（D） was described as ： TL = 0.001 2D3 - 0. 038 8/32 ＋ 1.043 2D ＋ 3. 945 8 （ r^2 = 0.998 ）. The relationship between body length （L） and body weight （W） of larvae and juveniles was expressed as： m = 1E -05L^3.3769（ r^2 = 0.982 7）. The formula between body length and body height during the development of the fish was described as ： H = 0.0（30 1L3 -0. 028 4L2 ＋ 1. 281 6L -7. 250 5 （ r^2 = 0. 988 6）. The findings provided the theoretical instruction for practical production of C. auratus gibelio.

Effects of Potassium Permanganate on Respiratory Movement in Carassius auratus under Different Conditions

[ Objective] To observe the effects of potassium permanganate on respiratory rate and cough frequency of Carassius auratus in different conditions. [Method] According to the simple factor design of experiment, the respiratory rate and cough frequency of Carassius auratus were recorded. The factors included concentration of potassium permanganate, temperature, duration time and pH values. [ Result] The respiratory rate and cough frequency of Carassius auratus were increased and then decreased with the increasing concentrations of potassium permanganate, rising temperature, and duration of medicated bath. The Carassius auratus had the best respiratory function at pH 7.0. The strong acid and strong alkali caused lesions and inhibited the respiratory function of Carassius auratus. [ Conclusion ] The potassium permanganate at different concentrations may have an impact on the cough frequency and respiratory rate of Carassius auratus.

In vivo Pharm acodynam ic Effect of Thiam phenicol in Serum of Carassius auratus on Aerom onas hydrophila

This study was to investigate the in vitro pharmacodynamic effect of thiamphenicol( TAP) in serum of Carassius auratus on Aeromonas hydrophila. By combining the in vivo pharmacokinetics and in vitro pharmacodynamics,the pharmacodynamic effect of TAP on Aeromonas hydrophila was studied,and the data were processed and analyzed by software Excel 2007,Kinetica3P97 and Kinetica4. 4. The results showed that oral administration of singly 30 mg /kg TAP assumed a rapid assimilation-quickly peaking-slowly dispelling trend in Carassius auratus. The related parameters were measured as follows: time of peaked plasma concentration of TAP( Tpeak) of 1.5 h,peak concentration( Cmax) of 37.172 μg/mL and absorption rate( ka) of 1.523 h,half-life period T1/2( ka) of 0.455 h,lag time( TL)of 0. 02 h,elimination half life T1/2( ke) of 16.712 h. The half maximal effective concentration( EC50) was 14.28 h. The PK-PD parameters were 32.41 h in AUC0- 24/ MICserumand 23. 23 in Cmax/MICserum. Employing an inhibitory Sigmoid Emax model,the administration dosage of TAP for preventing Aeromonas hydrophila-caused bacterial septicemia was 8. 61- 46. 20 mg /kg in clinical application. Based on these,we proposed the optimal administration route for preventing and controlling the Aeromonas hydrophila-caused bacterial septicemia: delivering TAP at the ratio of 46. 20 mg /kg on diseased Carassius auratus by mixing with baits or oral administration,followed by delivering with baits at ratio of 8. 61 mg /kg for preventing the Aeromonas hydrophila-caused bacterial septicemia. The results provided references for applying thiamphenicol for preventing and controlling the bacterial septicemia in aquatic livestock.

Changes in Bacterial Populations in Aquatic Environment of Carassius auratus gibelio during an Outbreak of Diseases

During an outbreak of diseases caused by co-infection with both parasites and bacteria in silver crucian carp （ Carassius auratus gibelio）, aerobic anoxy- genic phototrophic （AAP） bacteria in the aquatic environment were identified by PCR using the universal primers of bacterial 16S rDNA. The bacterial populations in the ponds infected and non-infected by the diseases were measured with acridine orange direct count （AODC） method, and the data were analyzed using SPSS software. The results showed that nine dominant cuhurable bacterial species： Shewanella putrefaciens, Acinetobacter sp. , Aeromonas sp. , Proteus vulgaris, Xan- thomonas sp. , Citrobacterfieundii, Morganella morganii, Vagococcus lutrae and Providencia sp. were identified, some of which were common pathogens. The bac- terial population in infected ponds was 7. 462 x 10^6 ind./ml in July, and 1. 007 4 x 10^7 ind./ml in August, and that in non-infected ponds was 1. 460 x 10x6ind./ml in July, and 1. 911 x10^6 ind./ml in August, with significant differences between the infected and non-infected ponds. The results suggested that the outbreak of diseases in silver crucian carp was to some extent related to the bacterial population in water environment.

Time-Effect Relationship of Toxicity Induced by Roundup and Its Main Constituents in Liver of Carassius Auratus

In order to evaluate the eco-toxicological effects of Roundup&reg on Carassius auratus (C. auratus), fish were exposed to 32 μg/L Roundup&reg, isopropylamine salt of glyphosate (G.I.S) and polyoxyethylene amine (POEA) over different periods (0.5, 1, 3, 7 and 14 d). Hydroxyl radical (·OH), malondialdehyde (MDA) and acetylcholinesterase (AChE) in liver were detected in this study. Results showed that the generation of ·OH increased before 7 d, but without significantly difference. ·OH was induced at 1 d for POEA group, 3 d for Roundup&reg group and 7 d for G.I.S group. At 14 d, ·OH generation returned to normal levels. MDA contents all increased significantly (p < 0.01) during 7 days and then reached a normal level at 14 d. AChE activity in all group tests revealed a significant inhibition (p < 0.01) after 7 days exposure and then rebounded a little, but remained below the control after 14 days exposure. The rate of AChE inhibition range from 13% - 42% in Roundup&reg, 6% - 40% in G.I.S, and 21% - 54% in POEA, suggesting that POEA was more toxic compared to Roundup&reg and G.I.S. 32 μg/L Roundup&reg exposure led to the change of physiological and biochemical indexes in C. auratus, which was a reversible process in the long run.

Studies on Physiological and Biochemical Properties of Female Specific Serum Protein and Its Immunocytochemical Localization in Carassius auratus cuvieri(Temminck & Schlegel)

Female-specific serum protein(FSSP)is normally present in the sera of female fish,but it is notfound in males.However,estradiol benzoate(EB)was found to induce the appearance of FSSP inmale fish and immature female fish.Massive doses of FSSP caused a rapid increase in FSSP.Ultrastructural examination of the liver indicated an extensive proliferation of the rough endoplasmicreticulum and a decrease in glycogen and lipid droplets after EB injection.A preparative PAGE for isolating highly purified FSSP from the serum of EB-treated fish,Carassiusauratus cuvieri(Temminck & Schlegel),is described.Purified FSSP obtained from EB-induced O-crucian fish has a molecular weight of 466,000±4,000(n=10),while that in mature female serumis 480,000±40,000(n=2).FSSP appears to be a dimer,with the size of the possible monomerbeing 240,000±8,000(n=6).SDS-PAGE on gradient gels indicated that sera from males given multiple(12)injections of EBcontain a main band with a molecular weight of 147,000±6,000(n=6).However,the same serumsamples provided three bands of protein on the PAGE gels.Antiserum was raised against the electrophoretically purified FSSP.The resulting antibody formeda single,continuous precipitation line with sera from EB-treated males and vitellogenic females,butnot with that from normal males.lmmunocytochemistry(PAP method)was used to locate FSSP in the liver and ovary of maturefemales and the liver of EB-treated males.Strongly positive particles were found clustered in groupsaround liver cell nuclei under light microscopy,and the yolk granules in the oocytes were also filledwith positive particles.

Unveiling potential sex-determining genes and sex-specific markers in autotetraploid Carassius auratus

Autotetraploid Carassius auratus is a stable hereditary autotetraploid fish resulting from the hybridization of Carassius auratus red var.(RCC,♀)×Megalobrama amblycephala(BSB,♂),containing four sets of RCC chromosomes.However,the molecular mechanism underlying the determination of sex in this species remains largely unknown.Currently,there lacks a full understanding of the molecular mechanisms governing sex determination and specific molecular markers to differentiate sex in this species.In this study,25,801,677 SNPs(Singlenucleotide polymorphism)and 6,210,306 Indels(insertion-deletion)were obtained from whole-genome resequencing of 100 individuals(including 50 female and 50 male).Further identification confirmed the candidate chromosomes as Chr46B,with the sex-determining region located at Chr46B:22,500,000‒22,800,000 bp.Based on the male-specific insertion(26 bp)within the candidate sex-determining region,a pair of sex-specific molecular markers has been identified.In addition,based on the screening of candidate sex-determining region genes and RT-qPCR validation analysis,ADAM10,AQP9 and tc1a were identified as candidate sex-determining genes.These findings provide a robust foundation for investigating sex determination mechanisms in fish,the evolution of sex chromosomes,and the development of monosex populations.

Astragalus polysaccharide orβ-glucan combined with inactivated vaccine markedly prevent CyHV-2 infection in Carassius auratus gibelio

Herpesviral hematopoietic necrosis disease caused by cyprinid herpesvirus 2(CyHV-2)results in huge economic losses for the gibel carp(Carassius auratus gibelio)aquaculture industry.A vaccination strategy is a feasible method to prevent CyHV-2 infection and the resulting economic losses.In this study,inactivators(includingβ-propiolactone,formaldehyde and binary ethylenimine)were used to prepare an inactivated vaccine.The optimal inactivated CyHV-2 vaccine(0.2%β-propiolactone inactivates CyHV-2 for 48 h)mixed withβ-glucan,anisodamine,chitosan,and astragalus polysaccharide(APS)adjuvants were injected into gibel carp.Theβ-glucan and APS combined with inactivated vaccine significantly improved the relative immune protection rate of gibel carp against CyHV-2 infection.The highest specific antibody levels inβ-glucan and APS groups were found in serum by ELISA assay and antibody neutralization titer.Furthermore,adjuvant addition groups produced better results for lysozyme,total superoxide dismutase,and complement C3 in serum.β-glucan and APS combined with vaccine treatment effectively enhanced mRNA expressions of important immune genes(IL-1β,IgM,IL-2 and IFN-γ2).Minimal tissue lesions(gill,spleen,and head kidney)and virus replication were seen in theβ-glucan and APS groups by histopathological examination and expression analysis of CyHV-2 TK gene.These results indicated that the combination of inactivated vaccine and adjuvantβ-glucan or APS is an effective method against CyHV-2 infection and provided a case study reference for prevention of fish viral diseases using inactivated vaccines.

A novel cell line established from skin tissue of gibel carp(Carassius gibelio)is susceptible to Carassius auratus herpesvirus infection

Frequent outbreaks of emerging infectious diseases in fish,such as Carassius auratus herpesvirus(CaHV)infection has caused great economic losses in China.However,the lack of a sensitive cell culture system has limited studies of CaHV.In the present study,a new cell line(gibel carp skin cell,GiCS)derived from gibel carp(Carassius gibelio)skin tissue was established to create a valuable tool for research of the virus.The GiCS cells consisted mainly of epithelial-like cells,which grew well at 25℃in L-15 medium supplemented with 10–20%fetal bovine serum.Chromosomal analysis revealed that the skin cell line remained amphitriploid,with most chromosome counts being 156(54%).The GiCS cells can be efficiently transfected and expressed exogenous genes.In particular,the GiCS cells showed high susceptibility to CaHV infection,which was confirmed by virus infection tests,detection of viral gene expression,and ultrastructural observation.To our knowledge,it is the first cell line that is highly permissive to CaHV infection.In addition,the cells also showed susceptibility to several aquatic animal viruses from different families including Iridoviridae,Rhabdoviridae,and Reoviridae.In conclusion,these results indicated that the establishment of the GiCS cell line is a significant advance that will be beneficial to future studies of CaHV and other aquatic animal viruses.