珠江口海域双胞旋沟藻赤潮对卤虫幼体、鱼苗和虾苗的急性毒性

为研究2009年10月下旬在广东珠海海域爆发的双胞旋沟藻赤潮对养殖鱼类及水体中其它生物的影响,实验以卤虫幼体、金鼓鱼苗和凡纳滨对虾苗作为受试生物,在赤潮现场测试双胞旋沟藻对卤虫幼体、鱼苗和虾苗的急性毒性效应。结果显示,24 h双胞旋沟藻对卤虫幼体的LC₅₀(半致死浓度)为9.55×10⁴/mL,藻密度为2.5×10³/mL的双胞旋沟藻对卤虫幼体的LT₅₀(半致死时间)为48.5 h。60 h内该赤潮水体对鱼苗和虾苗的存活无不利影响,卤虫幼体和金鼓鱼苗均可摄食双胞旋沟藻,卤虫幼体对双胞旋沟藻的摄食率低。研究表明,双胞旋沟藻赤潮水体对卤虫幼体有一定的毒性作用,但在低藻密度条件下,卤虫幼体能以该藻为食并维持其生命,双胞旋沟藻对金鼓鱼苗和凡纳滨对虾苗无急性毒性作用。     

黑点青鳉的生长和摄食行为发育

针对黑点青鳉实验室养殖成活率低的问题,对其摄食行为发育进行研究,了解其早期阶段的摄食行为发育状况,为人工养殖提供理论依据。通过单摄像机结合镜面成像的方法,对黑点青鳉胚后发育阶段90 d摄食行为的观察与分析,研究了投喂卤虫无节幼体条件下黑点青鳉的生长特征和摄食行为发育。结果显示,幼体体长平均生长率为2.579%/d,体长与日龄间关系为y=3.132+0.383x-0.004x²+0.000 03x³,R²=0.98。通过各项摄食行为指标将其幼体生长发育分为3期6个阶段:仔鱼期(0～10 d)分为前仔鱼期(0～4 d)和后仔鱼期(5～10 d),稚鱼期(11～30 d)分为前稚鱼期(11～18 d)、中稚鱼期(19～24 d)和后稚鱼期(25～30 d),幼鱼期(31～65 d)。黑点青鳉幼体出膜4 d后开始投喂初孵卤虫,至10 d所有鱼苗均能捕食。黑点青鳉仔鱼期幼体死亡率较高,但摄食能力稳步提升,至该期末,摄食成功率提升至45%～55%,摄食效率达0.5～0.6个/min;稚鱼期幼体的各项摄食指标均有大幅度提升,对饵料的响...     

食蚊鱼CYP19a基因的克隆与序列分析

硬骨鱼类CYP19基因与生物的性别分化和激素调节相关,因此可开发用来探究环境激素污染与基因表达的关系。本研究首次克隆和分析了食蚊鱼CYP19a cDNA的全系列,为将CYP19基因作为监测环境激素生物标志物的研究提供了全面的实验数据。根据CYP19a基因c DNA保守区域设计引物,扩增保守区域并测序。采用RACE法扩增食蚊鱼CYP19a基因c DNA序列全长,对其蛋白序列进行同源性分析,并将序列应用于CYP19a mRNA转录水平的RT-PCR法检测中。成功克隆食蚊鱼CYP19a基因全长,获得CYP19a基因总长为2020 bp,ORF为238~1791 bp,共编码518个氨基酸,对其编码的蛋白质进行有关信号肽、跨膜螺旋、亲水性/疏水性、一级结构、二级结构和三级结构分析,与其他硬骨鱼类底鰆、青鰆、平鲷、鲫、鲤和斑马鱼的性腺CYP19a基因作同源性比较,其基因相似度分别为93%、84%、84%、71%、71%和66%。用MEGA6.0软件对19个物种的CYP19a基因进行聚类分析,食蚊鱼CYP19a基因与底鰆、青鰆同源性最高,说明芳香化酶在进化上相对保守。确定从食蚊鱼性腺所克隆的CYP19a基因是芳香化酶基因,证明食蚊鱼的芳香化酶是由CYP19a和CYP19b两种基因编码的。食蚊鱼卵巢芳香化酶具有3个高度保守的片段,并具有催化活性。     

罗氏沼虾细胞色素P450家族CYP302a1基因克隆及其在蜕皮周期中的表达

蜕皮是甲壳动物重要的生理活动,与其蜕皮激素的合成密切相关,细胞色素P450(CYP)302a1是甲壳动物蜕皮激素合成通路中的关键酶之一。本研究克隆了罗氏沼虾CYP302a1基因(Mr-CYP302a1),cDNA全长1 859 bp,开放阅读框(ORF)为1 629 bp,编码543个氨基酸(aa),分子量大小为61.09 ku,等电点为8.42。氨基酸序列分析显示CYP302a1基因的保守结构域含有5个P450基因家族特征保守区域:heme-binding、helix-K、helixC、helix-I及PERF。系统进化分析结果显示Mr-CYP302a1首先与绿虾CYP302a1聚为一支,然后与凡纳滨对虾及三疣梭子蟹等十足目甲壳动物的CYP302a1聚为一支,与甲壳动物的亲缘关系最近。实时荧光定量PCR(qRT-PCR)检测表明Mr-CYP302a1在罗氏沼虾的多个组织中均有表达,其中在Y器官中的表达量最高,性腺中次之。同时研究发现,MrCYP302a1基因在罗氏沼虾的蜕皮后期(A期和B期)表达量很低,蜕皮间期(C期)表达量开始上升,在蜕皮前期D1亚期达到峰值。对Mr-CYP302a...     

Comparison of age estimates from scale, opercular bone, otolith, vertebrae and dorsal fin ray in Labeo rohita (Hamilton), Catla catla (Hamilton) and Channa marulius (Hamilton)

The present study was undertaken with a view to compare the precision and reliability of the age readings obtained from different bony structures of some important freshwater teleosts viz., Labeo rohita (Hamilton), Catla catla (Hamilton) and Channa marulius (Hamilton). Standard procedures were followed to prepare and study the age structures. In L. rohita and C. marulius percent agreement between reader's age estimates was highest for scales, i.e. 96.3% and 90.5%, respectively and in C. catla percent agreement was highest (93.3%) for opercular bone. When scale ages were compared with other alternative structures viz., otoliths, opercular bone, vertebral centra and dorsal fin rays, percent agreement was found highest between scale and opercular bone age estimates (77.8%) in L. rohita and between scale and otoliths (94.8%) in C. marulius. In case of C. catla highest percent agreement was found between opercular bone and scale age estimates. In L. rohita each of the ageing structure showed significant (P < 0.05) underestimation of age in comparison to scales. In C. catla mean age estimates from opercular bone were comparable (P > 0.05) to the values obtained from all other structures except dorsal fin rays. In C. marulius mean age estimates from scales were comparable (P > 0.05) to those from all other structures except from dorsal fin rays. Results indicated scales to be the most suitable structure for ageing L. rohita and C. marulius and opercular bone for C. catla. However, in C. catla also scales may be used as a non-destructive method of age estimation with satisfactory results.     

基于RAG2基因序列分析仿石鲈科鱼类及其相关种类系统进化关系

为探讨仿石鲈科鱼类目前存在的分类争议问题,测定了仿石鲈科(Haemulidae),金线鱼科(Nemipteridae),眶棘鲈属(Scolopsis)等相关科属共33种鱼类RAG2基因部分序列,利用最大简约法(MP法)和贝叶斯分析法(BI法),并以黄鹦嘴鱼作为外类群构建分子系统进化树.结果显示,33种鱼类共形成三大类群,其中仿石鲈科8个属聚成一类群,金线鱼属和眶棘鲈属的种类聚成另一类群,眶棘鲈属未能与仿石鲈科形成单系.同时,基于属间遗传距离比较,眶棘鲈属与金线鱼科的遗传距离比其与仿石鲈科各属的遗传距离要小得多,表明眶棘鲈属与金线鱼科有更近的亲缘关系,支持眶棘鲈属隶属于金线鱼科的观点.此外,传统分类资料中归类于仿石鲈科的髭鲷属(Hapalogenys)也没有与仿石鲈科聚成单系,而是独自形成一支,位于进化树基部,显示出髭鲷属与仿石鲈科关系较远,与近年来认为髭鲷属应该从仿石鲈科中划分出去的结果一致.在仿石鲈科内部,少棘胡椒鲷属(Diagramma)中的少棘胡椒鲷与胡椒鲷属(Plectorhinchus)的种类关系很近.在进化树上,少棘胡椒鲷位于胡椒鲷属的内部,花尾胡椒鲷最先分化出来,位于该分支基部,支持少棘胡椒鲷归为胡椒鲷属,名称沿用原来的学名胡椒鲷的观点.     

香鱼白细胞介素17C基因克隆及鳗利斯顿氏菌侵染后的时空表达

为了解香鱼白细胞介素17(interleukin 17,IL-17)的序列特征、系统发育及其与鳗利斯顿氏菌侵染的相关性,实验采用RACE-PCR方法扩增出香鱼IL-17C基因c DNA序列,并通过实时荧光定量PCR(q RT-PCR)技术检测了鳗利斯顿氏菌侵染后香鱼各组织中IL-17C基因的表达变化。结果显示,香鱼IL-17C基因c DNA序列全长1 087 bp,含534 bp的开放阅读框,在3′端非编码区存在7个m RNA不稳定信号(ATTTA)。预测该基因编码177个氨基酸,N端19个氨基酸为信号肽序列,成熟蛋白中含有8个半胱氨酸残基。系统进化树分析显示不同物种的相同IL-17亚型聚为一支,香鱼IL-17C与其他脊椎动物IL-17C聚为一支;同源比对结果显示香鱼IL-17C与虹鳟的IL-17C1和IL-17C2亲缘关系最近,相似性分别为40.72%和33.51%。q RT-PCR检测显示IL-17C基因在健康香鱼的心、肝、脾、头肾、鳃、脑、肌肉和肠等组织中均有表达,鳃和头肾中表达量较高。经鳗利斯顿氏菌侵染后,香鱼肝脏中IL-17C基因表达变化最大,菌侵染8 h时的表达量为对照组的9.17倍;其次为脾,菌侵染4 h时的表达量为对照组的6.69倍。研究表明,香鱼IL-17C基因的表达与病原菌侵染密切相关,可能在免疫功能中具有重要作用。     

尼罗罗非鱼无乳链球菌Sip蛋白乳酸菌活载体口服疫苗的研制及其免疫效果

链球菌病是威胁我国罗非鱼养殖产业健康发展的重要病害之一。为研制出免疫效果好、操作简便的罗非鱼链球菌病疫苗,本研究构建重组表达无乳链球菌Sip蛋白的穿梭质粒pNZ8124-Sip,通过酶切和测序验证后电转化乳酸乳球菌NZ9000,获得能够诱导重组表达无乳链球菌Sip蛋白的乳酸菌活菌载体疫苗。采用SPS-PAGE电泳摸索最佳诱导浓度和诱导时间以获得最大表达量,通过镍柱纯化目的蛋白并进行Western blot检测;利用不同浓度的重组乳酸菌活载体疫苗灌胃口服免疫尼罗罗非鱼,采用间接ELISA法测定免疫后血清抗体水平变化,通过人工腹腔注射感染无乳链球菌获得相对免疫保护率。研究结果显示,构建的重组乳酸乳球菌可通过nisin诱导表达大小为48 ku特异性蛋白,与目的蛋白大小一致;PAGE电泳显示,重组蛋白主要以可溶蛋白和包涵体2种形式存在,其中胞内可溶性蛋白浓度达7.65 mg/mL;诱导表达的最佳条件为100 ng/mL nisin诱导6 h;Western blot检测结果显示,诱导蛋白可与鼠抗His标签抗体特异性结合。口服免疫结果显示,中浓度组(2.24×10~(10) CFU/mL)和低浓度组(2.24×10~9 CFU/mL)免疫2次能够显著提高尼罗罗非鱼的血清抗体水平和抗无乳链球菌感染能力,中浓度免疫组的相对免疫保护率最高为41.0%。本研究可为罗非鱼链球菌病口服疫苗的研究奠定基础,具有广阔的应用前景。     

SOST基因在淇河鲫肌间骨骨化过程中的表达研究

为探索硬化蛋白(sclerostin,SOST)基因在淇河鲫肌间骨骨化过程中的调控作用,本研究以淇河鲫仔稚鱼为研究对象,利用整体骨骼染色法对肌间骨的形态发生进行观察,并采用q RT-PCR、Western Blot和免疫组织化学技术检测SOST基因在肌间骨不同骨化阶段的m RNA和蛋白表达变化情况。结果显示,淇河鲫发育至24 dpf(day post fertilization),肌膈中出现纤维束;25 dpf髓弓小骨出现,28 dpf脉弓小骨出现,33 dpf髓弓小骨出现分叉,45 dpf肌间小骨发育完全。SOST基因在肌间骨骨化发生各阶段均有表达,23 dpf的表达量最低(P0.05),随着肌间小骨的骨化发育,SOST基因呈递增趋势,45 dpf表达量显著高于其他骨化阶段(P0.05);SOST蛋白在45 dpf时显著高于23和27 dpf(P0.05),与其他发育阶段无显著差异(P0.05)。SOST m RNA和蛋白变化趋势基本相同,都在23dpf处于最低,肌间骨全部出现后表达量达到最高,与淇河鲫肌间骨的形态发生趋势一致。因此,推测SOST与淇河鲫肌间骨骨化具有一定的相关性,可调控肌间骨的分化和形成。     

罗氏沼虾不同养殖模式对水体浮游生物的影响

通过围隔实验比较罗氏沼虾不同养殖模式对水体浮游生物的影响。实验设置6种养殖模式:罗氏沼虾单养(MP)、罗氏沼虾+浮萍(水面覆盖率5%)(PP)、罗氏沼虾+鲢(PF)、罗氏沼虾+背角无齿蚌+鲢(PMF)、罗氏沼虾+背角无齿蚌+浮萍(PMP)、罗氏沼虾+背角无齿蚌+浮萍+鲢(PMPF)。养殖64 d后,测定不同模式中浮游植物和三大类浮游动物(轮虫、枝角类及桡足类)的种类和数量。结果显示,上述6种模式中浮游植物共同优势种有4种,但优势度指数最大的浮游植物不同,MP组是锥囊藻属,有浮萍的PP和PMP组均为细小平裂藻,混养鲢的PF、PMF和PMPF组均为针杆藻。不同养殖模式无共同的浮游动物优势种。养殖模式对浮游生物密度具有显著影响,PF组浮游植物密度最高,MP组浮游植物密度最低,PF组浮游植物密度比MP、PP和PMP组分别高78%、53%和61%。相反,浮游动物密度MP组最高,PF组最低。混养鲢的PF、PMF和PMPF组浮游动物密度显著低于其他3组。研究表明,罗氏沼虾养殖中混养鲢可增加浮游植物密度而降低浮游动物密度,浮萍和鲢影响池塘优势种。     

淇河鲫cyp19a1b基因的克隆表达及芳香化酶抑制剂对其表达的影响

为探索脑型芳香化酶基因(cyp19a1b)在雌核发育三倍体鱼淇河鲫性别决定与分化过程中的作用,利用RACE方法克隆淇河鲫cyp19a1b基因cDNA全长序列,采用Real-time PCR分析其在不同组织、胚胎及胚后不同发育时期的表达情况,同时检测芳香化酶抑制剂Letrozole诱导性逆转及腹腔注射人绒毛膜促性腺激素(hCG)后cyp19a1b在脑中的表达情况。结果显示,淇河鲫cyp19a1b cDNA全长2 984 bp(GenBank ID:MF926270),包含132 bp5′非编码区,1 319 bp 3′非编码区,1 533 bp开放读码框,编码510个氨基酸残基;氨基酸序列比对及系统进化分析结果显示,淇河鲫Cyp19a1b与其他鲤科鱼类同源性较高,与哺乳类、爬行类等脊椎动物同源性较低,这与其分类地位一致;组织分布检测结果显示,cyp19a1b基因在淇河鲫脑中表达量最高,在卵巢等其他组织中表达量较低;Realtime PCR结果表明,在胚胎发育过程中cyp19a1b在外源精子刺激后从囊胚期开始上调,神经胚期达到最高,随后降低;出膜后随着发育的进行,该基因在脑中表达量逐步上调,在躯体中一直维持在较低水平;除此之外,伴随着Letrozole诱导的性逆转,cyp19a1b在脑中表达量降低;腹腔注射hCG可以促进cyp19a1b在脑中表达。研究表明,淇河鲫cyp19a1b基因可能通过参与神经系统的形成及神经内分泌活动,在性别决定与分化过程中起到一定的作用。     

The risk of parasite transfer to juvenile fishes by live copepod food with the example Triaenophorus crassus and Triaenophorus nodulosus

Natural zooplankton is a potential food resource for juvenile fish in fish farms as it is a good source of fats, carbohydrates, and protein. However, it is also a potential source of parasites and pathogens. The present study was conducted (1) to estimate the risk of parasite transfer by live copepod food under intensive farming conditions using the parasites Triaenophorus crassus and Triaenophorus nodulosus as example and (2) to look for strategies to avoid parasite infestation of juvenile fish.An easy and routinely applicable aniline blue staining method was developed to check the infestation levels of copepods with procercoids during the on-growing season of juvenile fish. From the copepod species occurring in zooplankton Cyclops spp. was infested frequently (9.4 ± 12.0%, maximum 38.7%), Diaptomus spp. infrequently (1.5 ± 1.6%, maximum 3.2%), Daphnia spp. never. Juvenile grayling, Thymallus thymallus, and corgonids, Coregonus sp., which had been fed with natural zooplankton revealed infestations rates with T. crassus of circa 5%, with T. nodulosus of circa 10%. As the occurrence of procercoids in the zooplankton was temporary limited, Triaenophorus infestation can be avoided by using artificial food instead of live copepods during the risky season.To prevent parasite infestation of juvenile fish methods were investigated to eradicate procercoids from copepods by chemical treatment (sodium chloride, hydrogen peroxide, citric acid treatment) and by freeze-thawing methods. Chemical methods failed to remove procercoids. In fish, which had been fed with frozen thawed zooplankton, no Triaenophorus spp. infestation was observed. Their survival rates were similar as in the control fed with live zooplankton, however the weight of the fish was significantly lower.     

草鱼NK-lysin基因的克隆、原核表达与活性分析

为研究草鱼抗菌肽NK-lysin基因的组成结构、在组织中的表达差异及其抑菌活性,实验根据已知鱼类NK-lysin基因保守区序列设计上下游引物,采用RT-PCR和RACEPCR技术克隆草鱼NK-lysin基因(Cinkl),RT-PCR进行各组织间的表达分析,并构建Cinkl成熟肽的原核表达载体,采用琼脂糖孔穴扩散法检测重组蛋白的抑菌活性。结果显示,Cinkl c DNA全长768 bp,编码121个氨基酸;基因组DNA全长3 361 bp,包含4个外显子和3个内含子。经多序列比对分析和结构域预测表明,Ci Nkl氨基酸序列具有SAPLIP家族的特征:含有6个保守的半胱氨酸和saposin B蛋白结构域,与斑马鱼Nklc、Nkld的相似性分别为57.98%和63.03%。构建NK-lysin氨基酸序列系统进化树,发现Ci Nkl与斑马鱼Nklc、Nkld聚为一支。RT-PCR检测结果显示,Cinkl m RNA在所有检测的草鱼组织中均有表达,在脾脏中表达量最高,心脏、鳃、中肾和头肾的表达量次之,皮肤、脑、肝脏和肠有微量表达。p ET-28b-MBP与Ci Nkl成熟肽序列构建的原核表达质粒在宿主菌Rosetta(DE3)表达出约57 ku的可溶性融合蛋白,利用Ni-NTA His·Bind树脂进行纯化,Ci Nkl重组蛋白对G–细菌如大肠杆菌M15株、嗜水气单胞菌,以及G+细菌如金黄色葡萄球菌均具有抑菌活性。基于草鱼NK-lysin的组织表达特征和重组蛋白的抑菌活性,推测NK-lysin在草鱼的先天性免疫防御中发挥调节作用。     

Use of Microbacterium sp. and Exiguobacterium mexicanum to improve the survival and development of Artemia under xenic conditions

The effect of Microbacterium sp. strain 8L and Exiguobacterium mexicanum strain 8N was evaluated in the diet of Artemia under xenic conditions. Viable cultures of bacteria were provided to xenic cultures of Artemia in combination with Sacharomyces cerevisiae, cornflour or Spirulina, and the effect on the survival and growth was recorded. The use of these bacterial strains improves significantly the survival of Artemia independently of the used food (P < 0.05), and variable results were observed in the growth.     

引起混养塘中异育银鲫和鲢发病死亡的病原及组织病理

混养的异育银鲫和鲢鱼种大批死亡,为明确发病死亡的病原和组织损伤并提供相关的疾病防控措施,进行了病鱼肉眼和显微镜检查、细菌学检测、病毒学检测、组织病理和药敏试验研究。结果发现,除在患病鱼体表偶然发现有少量不会引起充血等症状的杯体虫和车轮虫外,未在体内外发现其他寄生虫和真菌类病原;通过细菌分离、人工回感试验、生理生化特性和16S r RNA基因序列分析,从患病异育银鲫和鲢分离到的致病菌株均为嗜水气单胞菌;根据异育银鲫和鲢病毒性疾病的现状,使用鲤疱疹病毒2型(Cyprinid herpesvirus 2,Cy HV-2)DNA聚合酶基因的特异性引物分别对自然发病的异育银鲫和鲢进行PCR检测,只有异育银鲫检测到Cy HV-2,分别用它们的除菌组织上清液进行人工感染试验,只有异育银鲫出现充血症状和死亡现象;由此得出嗜水气单胞菌是异育银鲫和鲢发病死亡的主要病原,Cy HV-2是异育银鲫混合感染的次要病原。患病鲢与患病异育银鲫呈现出类似的组织病理现象,又有一些各自特有的组织病理表现,单纯细菌感染的鲢轻度病变以细胞颗粒变性为主,坏死细胞以核溶解为主,细菌和病毒混合感染的异育银鲫肝脏轻度病变以细胞滴状玻璃样变的变性为主,坏死组织细胞以核固缩和核碎裂为主,在肾脏和脾脏出现染色质边集于核膜的肿大细胞核,主要组织器官出现从变性到坏死的病理变化过程,最终失去应有的功能而死亡。依据药敏试验结果,建议内服诺氟沙星和氟苯尼考等抗生素防治本病的嗜水气单胞菌感染,混合感染Cy HV-2的异育银鲫可以通过注射Cy HV-2疫苗和生态养殖的方法控制和减少该病毒病感染和发展。     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Susceptibility of corkwing wrasse Symphodus melops, goldsinny wrasse Ctenolabrus rupestis, and Atlantic salmon Salmo salar smolt, to experimental challenge with Vibrio tapetis and Vibrio splendidus isolated from corkwing wrasse

The virulence of two Vibrio strains, previously isolated from diseased corkwing wrasse Symphodus melops and identified as V. tapetis and V. splendidus, to corkwing and goldsinny wrasse Ctenolabrus rupestris and to Atlantic salmon Salmo salar, was studied under laboratory conditions. Both bacteria were shown to be opportunistically pathogenic to corkwing wrasse, causing significantly higher mortality in the challenged groups than in the controls. Bacterial cultivation of kidney samples and re-isolation of V. tapetis and V. splendidus from most mortalities confirmed the two strains as the probable cause of mortality in the challenged groups. The control group also suffered relatively high mortality, but no specific pathogens that were suspected to be the main cause of death were isolated, other than a mixture of Vibrio spp. and, in the case of one individual, atypical Aeromonas salmonicida. Following injection challenge with both bacterial strains, no mortality was recorded in Atlantic salmon. In bath challenge trials with goldsinny wrasse, only slight mortality was observed in the challenged groups and the unchallenged control group. Bacterial examination showed that atypical Aeromonas salmonicida was the probable cause of death in both bath challenged and control groups of goldsinny wrasse, and no indication of infection by any Vibrio sp. was found.     

半滑舌鳎Cs-UBE2D4基因的克隆、表达模式及启动子活性

泛素-蛋白酶体系统作为重要的蛋白质修饰途径,参与多个生物学过程,其中泛素结合酶E2作为关键酶可以决定泛素化对靶蛋白的影响。为了探究泛素结合酶E2在半滑舌鳎雌性性腺发育中的作用,本研究克隆了半滑舌鳎Cs-UBE2D4基因的开放阅读框ORF,检测了该基因在不同组织和不同发育阶段性腺中的表达模式;构建了其启动子报告基因载体(pGL3-Cs-UBE2D4)并进行了启动子活性分析。Cs-UBE2D4开放阅读框为360 bp,编码119个氨基酸,含有保守的泛素结合酶E2结构域。荧光定量PCR结果显示,Cs-UBE2D4在雌鱼的各组织中广泛表达,其中在卵巢和肝脏中表达量较高,而在雄鱼中几乎检测不到表达;在卵巢各发育阶段中,该基因的表达量从90日龄开始上调,6月龄达到峰值,之后开始下调,在1.5龄时表达量最低,在3龄时表达量有所回升,其中6月龄表达量为90日龄表达量的1.97倍。双荧光素酶活性检测结果显示,Cs-UBE2D4启动子具有较强的转录活性。本实验将为进一步开展Cs-UBE2D4基因对半滑舌鳎卵巢发育和性别调控相关机制的研究提供理论依据。     

抑食金球藻对翡翠贻贝抗氧化酶系统的影响

本研究以单种抑食金球藻及其与亚心形四爿藻的混合藻为实验组,以亚心形四爿藻为对照,探究抑食金球藻对翡翠贻贝超氧化物歧化酶(SOD)活性、丙二醛(MDA)和谷胱甘肽(GSH)含量的影响。结果显示,抑食金球藻显著影响翡翠贻贝的SOD活性、MDA和GSH含量,各实验组均表现出诱导升高与抑制降低交替出现的规律。单种高、中浓度的抑食金球藻在短期(3 h)内即可对翡翠贻贝的SOD活性产生影响,混合藻组中的SOD活性上升较慢,说明亚心形四爿藻可以延滞氧化还原系统的反应时间。各实验组翡翠贻贝GSH含量在2 d内均显著低于对照组,且混合藻组与单种抑食金球藻组相差不大,提示亚心形四爿藻对抗氧化还原损伤并未起到明显的缓解作用。     

花鲈ncc、nkcc基因在海、淡水适应中鳃组织的表达与定位分析

为探究ncc、nkcc基因在花鲈渗透调节中发挥的作用,实验通过全基因组鉴定、多重序列比对、系统进化树构建以及蛋白结构预测对花鲈ncc进行了鉴定及序列分析,利用实时荧光定量PCR (qRT-PCR)检测ncc和nkcc在海水、淡水花鲈鳃组织中的表达水平,利用原位杂交技术确定ncc2和nkcc1a在海水及淡水花鲈鳃中的表达位置。结果显示,从花鲈中鉴定出2个ncc基因,即ncc1和ncc2,其编码序列(CDS)长度分别为2 691和3 120bp,编码896和1 039个氨基酸,在进化上具有保守性。ncc2在淡水花鲈鳃组织中的表达量显著高于海水,而nkcc1a在海水花鲈鳃组织中的表达量显著高于淡水,ncc1、nkcc1b、nkcc2在海淡水中的表达量则无显著差异。淡水适应过程中花鲈鳃组织中的ncc2的表达量逐渐上调,而nkcc1a的表达量逐渐下调;海水适应过程则呈现相反的表达趋势。此外,原位杂交结果显示,ncc2和nkcc1a基因分别位于淡水与海水中鳃组织的相邻鳃小片间的鳃丝上皮。以上结果表明,ncc2和nkcc1a基因分别编码淡水及海水花鲈鳃中重要的Na⁺及Cl