虾夷扇贝脓胞病病原的分离、鉴定与致病性

开展对大连市长海县附近养殖海域近几年夏季出现的大规模虾夷扇贝死亡现象的研究,能为虾夷扇贝养殖过程中的疾病防治提供基础理论支持。以患病虾夷扇贝为研究对象,通过对病变组织进行细菌分离、提纯培养,并采用人工感染试验确定虾夷扇贝脓胞病的病原性质;通过生理生化特性、16SrRNA基因部分序列和药物敏感性测定,对虾夷扇贝脓胞病进行了鉴定。结果表明,从患病组织中分离出的其中一株细菌经人工感染试验证实为虾夷扇贝脓胞病的病原菌,在水温19℃、注射浓度为1.09×105CFU/mL的条件下,该菌有较强的致病性。通过对该病原菌的生理生化特性测定和16SrRNA基因部分序列分析,确定虾夷扇贝脓胞病的病原为查氏弧菌。     

微酸性电解水对活品虾夷扇贝存活率的影响及杀菌效果

以活品虾夷扇贝(Patinopecten yessoensis)为实验对象,用不同理化性质的微酸性电解水(slightly acidic electrolyzed water,SAEW)处理受试扇贝,并用副溶血性弧菌人工浸染经微酸性电解水处理过的扇贝,检测其存活率、微酸性电解水杀菌效果、副溶血性弧菌的变化规律,以及虾夷扇贝在不同电解水处理阶段的菌相。结果显示,微酸性电解水处理1 min、2 min、4 min,扇贝存活率均为80%,处理8 min存活率为82%,均高于染菌组和对照组;电解水处理时间与细菌总数和大肠菌群呈显著负相关(P0.05),与处理时采用的电解水有效氯浓度没有显著性关系(P0.05)。采用电解水处理8 min后,活品虾夷扇贝体内染上的副溶血性弧菌数量从1 100 MPN/g降至28 MPN/g。扇贝初期主要菌群为假单胞菌和弧菌,在经过微酸性电解水处理后,其体内的菌群趋于复杂化,优势菌群有所改变,细菌总数有所下降。24 h后经电解水处理或者未处理的虾夷扇贝体内优势腐败菌均为假单胞菌。研究表明,SAEW在虾夷扇贝净化中具有一定的应用潜力。     

虾夷扇贝C型凝集素母源传递与抑菌作用的初步研究

为研究虾夷扇贝C型凝集素的母源传递及其抑菌作用,实验运用qRT-PCR技术检测了经鳗弧菌刺激后虾夷扇贝卵巢中C型凝集素的表达模式,比较分析了C型凝集素基因在正常虾夷扇贝和鳗弧菌刺激的虾夷扇贝所产的卵及其胚胎发育前期的存在与变化;通过抑菌实验研究了卵胞浆中C型凝集素抑制细菌存活的作用.结果表明,鳗弧菌刺激能够诱导虾夷扇贝卵巢中的C型凝集素mRNA表达量显著变化,最高表达量出现在刺激8h后,为对照组的6.2倍;母体中C型凝集素可以传递给卵和胚胎,刺激组表达量极显著高于正常组,且表达量都随胚胎发育逐渐降低,至受精36 h时分别为正常对照卵的0.3和0.2倍;蛋白终浓度为200和400 μg/mL的卵无细胞体系都具有一定的抑菌作用,与C型凝集素家族抗体反应后,细菌存活率显著上升,说明母源C型凝集素在抑制细菌存活方面发挥了重要的作用.     

3种扇贝遗传结构的RAPD分析

用RAPD方法对扇贝科中的虾夷扇贝、海湾扇贝和栉孔扇贝的基因组DNA的特异性遗传标记进行分析,筛选了43个随机引物,有13个引物获得了片段长度在250～2000 bp之间且重复性良好的谱带,每个引物分别获得了4～17个大小不等的片段,共获得了170个扩增片段,其中12个引物得到特异性片段58个,可以将两种或者3种扇贝区别开来。通过Popgene 3.2分析软件包统计表明,虾夷扇贝与海湾扇贝的遗传距离最大,为0.2481;栉孔扇贝与海湾扇贝的遗传距离次之,为0.2161;虾夷扇贝和栉孔扇贝遗传距离最小,其值为0.2133。根据遗传距离指数,用MEGA3统计软件中的UPGMA方法进行聚类分析,构建系统树。     

虾夷扇贝EST-SSR标记在栉孔扇贝中的通用性研究

为研究虾夷扇贝(Patinopecten yessoensis)和栉孔扇贝(Chlamys farreri)的群体遗传多样性并分析其亲缘关系,对在虾夷扇贝中开发的60个EST-SSR位点在栉孔扇贝中的通用性进行了研究。结果显示,21个位点可在栉孔扇贝中扩增出特异性条带,通用性比例达35.00%,其中,17个位点具有多态性,多态性比例达28.33%。在虾夷扇贝和栉孔扇贝群体中,平均等位基因数(N_a)分别为2.7647和2.3529;平均有效等位基因数(N_e)分别为1.9487和1.6350;平均观测杂合度(H_o)为0.6314和0.3333;平均期望杂合度(H_e)为0.4569和0.3139;平均多态信息含量(PIC)为0.3726和0.2597;Nei's(1973)基因多样性指数(I)分别为0.4493和0.3087;Shannon信息指数分别为0.7176和0.5041;固定指数(F_(is))检测发现,7个位点偏离了Hardy-Weinberg平衡;遗传分化系数(F_(st))为0.2398。本研究开发的21对通用性EST-SSR标记,为进一步开展虾夷扇贝和栉孔扇贝遗传多样性分析、分子辅助育种、基因发掘和种质资源评价奠定了基础。     

急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组DNA甲基化的影响

为探讨急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组 DNA 甲基化水平的影响,本研究从表观遗传学角度,运用甲基化敏感扩增多态性(Methylation-sensitive amplification polymorphism,MSAP)技术比较分析了升温海水(17℃和24℃)急性胁迫虾夷扇贝(原暂养于9℃的海水中)9 h 和24 h 后基因组 DNA 甲基化的变化情况。结果显示,9对引物在各组的总扩增位点数为314–337,总甲基化位点为79–94,所占比例为23.45%–28.51%;所有处理组基因组 DNA 总甲基化率低于对照组,急性升温胁迫使虾夷扇贝基因组 DNA 发生去甲基化,而且去甲基化程度随胁迫温差增大和胁迫时间增长而增强,说明急性升温胁迫能够改变 DNA 甲基化水平和模式。本研究为进行虾夷扇贝抗逆基因的筛查提供了新思路和研究基础,丰富了表观遗传学在扇贝中的研究资料。     

急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组DNA甲基化的影响

为探讨急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组DNA甲基化水平的影响,本研究从表观遗传学角度,运用甲基化敏感扩增多态性(Methylation-sensitive amplification polymorphism,MSAP)技术比较分析了升温海水(17℃和24℃)急性胁迫虾夷扇贝(原暂养于9℃的海水中)9 h和24 h后基因组DNA甲基化的变化情况。结果显示,9对引物在各组的总扩增位点数为314–337,总甲基化位点为79–94,所占比例为23.45%–28.51%;所有处理组基因组DNA总甲基化率低于对照组,急性升温胁迫使虾夷扇贝基因组DNA发生去甲基化,而且去甲基化程度随胁迫温差增大和胁迫时间增长而增强,说明急性升温胁迫能够改变DNA甲基化水平和模式。本研究为进行虾夷扇贝抗逆基因的筛查提供了新思路和研究基础,丰富了表观遗传学在扇贝中的研究资料。     

扇贝血细胞胞内酶活性的细胞学观察

分别对海湾扇贝、虾夷扇贝、栉孔扇贝的3种血细胞胞内酶：碱性磷酸酶(ALP)、过氧化物酶(POD)、酚氧化酶(PO)进行了免疫细胞化学研究,并对血细胞酶反应阳性率作了统计。结果表明,3种扇贝血细胞ALP阳性率相差不大,海湾扇贝为46．6％,虾夷扇贝为42．4％,栉孔扇贝为36．8％;3种扇贝血细胞POD阳性率相差较大,栉孔扇贝阳性率高达50．2％,海湾扇贝为44．2％,虾夷扇贝仅为17．2％。3种扇贝血细胞PO孵育反应后,由于海湾扇贝、虾夷扇贝PO反应活性很低,瑞氏染液复染后,无法区分阴性、阳性细胞。实验中仅对栉孔扇贝PO阳性率进行了统计,栉孔扇贝血细胞PO阳性率为39．3％。     

虾夷扇贝Dmrt1基因的分子鉴定及表达模式分析

Dmrt1是Dmrt双性和mab-3相关转录因子基因家族的重要成员之一,在生殖细胞分化和性别调控中发挥着重要作用.为探究其在虾夷扇贝(Patinopecten yessoensis)性别分化中的表达调控模式,本研究利用生物信息学的方法分析了虾夷扇贝Dmrt1的序列特征;采用半定量PCR (RT-PCR)检测了Dmrt1...     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.