生物絮团中异养亚硝化菌的分离鉴定及其特性

采用富集培养、分离纯化等微生物学手段,从对虾养殖池的生物絮团中筛选出两株对氨氮具有高转化率的菌株.16S rRNA测序及系统发育分析结果表明,两株菌均属于盐单胞菌属,菌株2011072708与食物盐单胞菌Halomonas alimentaria有99％的同源性,而菌株2011072709与胜利盐单胞菌H.shengliensis有100％的同源性.比较研究了两株菌在不同温度、盐度、pH、碳氮比条件下对氨氮的转化率,菌株2011072708在温度37℃、盐度30～40、pH 8、碳氮比28的条件下对氨氮的转化率最高;菌株2011072709在温度27～42℃、盐度40～50、pH 6、碳氮比21的条件下对氨氮的转化率最高.研究结果表明,胜利盐单胞菌（2011072709）对温度、盐度、pH、碳氮比等各方面的适应性优于食物盐单胞菌（2011072708）,更适合在生物絮团技术中得到应用.     

一株高效氨氮及亚硝态氮去除功能菌株的分离鉴定及在生物絮团对虾养殖中的应用

从凡纳滨对虾(Litopenaeus vannamei)养殖水体中分离出具有高效氨氮及亚硝态氮去除功能的菌株Y2,生理生化和16SrRNA基因序列比对分析结果显示该菌株为麦氏交替单胞菌(Alteromonas macleodii)。进一步通过生长实验进行温度、酸碱度、盐度的培养条件优化,利用抗生素药敏实验筛选菌株特定抗性;通过卤虫浸泡感染的方法检测麦氏交替单胞菌Y2的安全性,并测定海水培养液OD_(600)及含氮无机污染物的浓度,探究菌株Y2生长与水体中氨氮、亚硝态氮、硝态氮之间的动态变化关系;通过28d对虾养殖试验,监测水质、生物絮团形成量、致病菌数量及对虾成活率生长速率,进一步阐明菌株在实际养殖中的功效。结果表明,该菌株Y2对苯唑西林、克林霉素有抗性;对卤虫的48 h半致死浓度高于1.9×10~8 cfu/mL,显著高于哈氏弧菌(10~2 cfu/mL)。此外,该菌具有持续去除水体中氨氮、亚硝态氮的功能。在养殖实验中能抑制潜在病原菌弧菌生长、提高对虾存活率及生长率,并且能在水体中稳定存活较长时间。综上所述,菌株Y2是养殖用益生菌制剂的优良备选菌株,可作为生物絮团养殖系统中调节水质的关键菌株。     

不同C/N对草鱼池生物絮团的形成及水质的影响研究

为了研究草鱼池生物絮团形成所需的适合C/N,实验分析不同C/N水平对水泥池中生物絮团的形成、水质及草鱼生长的影响。对照组投喂基础饲料(C/N为10.8∶1),实验组在基础饲料上添加葡萄糖,控制C/N分别为15∶1、20∶1和25∶1。结果显示,当C/N≥15时,形成的生物絮团可以有效的调节水质,降低水体中的氨氮、亚硝酸盐氮水平;各组的生物絮团体积指数(FVI)随养殖时间逐步增加,在第14天趋于稳定;随着C/N增高,尽管实验组水体中形成的生物絮团粗蛋白含量显著高于对照组(P<0.05),但是草鱼生长却呈下降趋势。综合而言,生物絮团技术应用于草鱼养殖适宜的C/N为15,该比值能促进生物絮团的形成,并能有效降低水中的氨氮、亚硝酸盐氮水平。     

凡纳滨对虾养殖系统中异养和自养型生物絮团的微生物特性及其与养殖水环境的关系

生物絮团的群落结构特征与其营养类型密切相关, 并与系统水质相互影响。本研究应用高通量测序技术研究了凡纳滨对虾(Litopenaeus vannamei)养殖系统中异养、自养型生物絮团的微生物群落结构特征, 讨论了絮团微生物与养殖水环境的相互作用。群落结构分析表明, 异养、自养型生物絮团的优势门类均为变形菌门(Proteobacteria, 相对丰度占比 24.2%~70.45%)、拟杆菌门(Bacteroldota, 相对丰度占比 8.45%~28.09%); 属水平上, 对构建生物絮团骨架起重要作用的亮发菌属(Leucothrix)相对丰度在两种生物絮团间无显著差异(P＞0.05); 此外, 注释为硝化螺旋菌门(Nitrospirota)的 OTU 仅存在于自养絮团。功能基因预测分析表明, 自养型生物絮团 amoA、amoB 等硝化基因的丰度(0.17%, 0.20%)明显高于异养型生物絮团(0.10%, 0.09%)。絮团微生物组成的变化改变了水体氮循环路径, 造成氨氮、亚硝酸盐、硝酸盐浓度的不同, 并受到水质差异的反作用。生物絮团的营养类型对对虾特定生长率无显著性影响。结论认为: 与异养型生物絮团相比, 自养型生物絮团硝化细菌和硝化基因的丰度、多样性明显升高, 微生物组成与功能更加合理, 能有效控制养殖水质, 维持养殖系统的平衡与良性发展。     

南美白对虾淡水生物絮团循环水养殖试验

<正>生物絮团通过异养细菌分解水体中的有机污染物,转化为无机氮与菌体蛋白,消除水体中的氨氮、亚硝酸盐等有害物质;养殖水体中保持一定量的生物絮团,对净化养殖水体水质、降低饵料系数、减少病害等作用显著;并且可操作性强,可以人为向水体中添加碳源和有益微生物培养生物絮团,是淡水高效养殖南美白对虾的新模式。     

几种不同碳源对凡纳滨对虾生物絮团技术育苗效果的影响

为提高凡纳滨对虾种苗生产的生态化水平,分别以蔗糖、葡萄糖、淀粉为添加碳源,添加量为投饵量的50%,同时添加地衣芽孢杆菌,在1 000 L的水体中进行凡纳滨对虾生物絮团技术育苗实验。结果表明添加碳源组絮团含量明显高于对照组(不添加碳源和芽孢杆菌),且蔗糖组絮团的形成比淀粉组和葡萄糖组早;蔗糖组和葡萄糖组的氨氮、亚硝酸盐含量均显著低于对照组和淀粉组,其亚硝酸盐峰值浓度分别比对照组降低25.4%和31.4%,且未换水即自行下降;添加碳源各组絮团的粗蛋白、粗脂肪含量均显著高于对照组,粗蛋白含量最高的蔗糖组达到32.6%。仔虾幼体P14时,葡萄糖组和蔗糖组的仔虾体长分别比对照组增长22.8%和19.2%(P0.01),差异极显著。表明在凡纳滨对虾生物絮团技术育苗中,蔗糖和葡萄糖作为添加碳源是合适的。     

凡纳滨对虾室外生物絮团养殖池水体理化因子和细菌的变化

在室外凡纳滨对虾池塘构建生物絮团养殖池,研究生物絮团池中理化因子的变化和对对虾生长状况的影响。试验中选取在池塘取3个定点作为平行组。经过122 d的试验,结果表明,生物絮团可以降低氨氮、亚硝酸盐浓度,维持硝酸盐浓度在合理的范围。试验期间,氨氮浓度的变化范围是(0.02±0.01)～(8.53±0.60)mg/L;亚硝酸盐浓度的变化范围是0～(5.18±0.03)mg/L;硝酸盐的浓度范围是0～(11.11±0.39)mg/L。pH的变化范围是(6.61±0.03)～(8.31±0.02)。从微生物物种分类上分析,在门的水平中分布变形菌门(Proteobacteria)所占的比例最高为62.43%+1.26%;从优势科水平来看,红杆菌科(Rhodobacteraceae)所占比例最高为22.01%+2.25%;从优势属水平来看,Candidatus Aquiluna所占的比例最高为8.10%+0.39%,其次是聚球蓝细菌属3.32%+0.31%。此研究发现,室外凡纳滨对虾养殖池生物絮团细菌的组成和多样性都极其丰富,通过结合分析这些微生物的功能特点,为生物絮团技术在室外养殖生产中的进一步应用奠定基础。     

一株芽孢杆菌的分离鉴定及在生物絮团对虾养殖中的应用

从对虾养殖池中分离到1株细菌(编号2013042402,简称菌株02),分别用16S rDNA序列比对法和细菌全细胞脂肪酸气相色谱法对该菌进行鉴定.结果显示,菌株02为芽孢杆菌(Bacillus sp.).为探讨该芽孢杆菌在生物絮团对虾养殖中的使用效果,实验分别设置加菌加糖组(菌株02的量为2.0× 104 CFU/ml,蔗糖量为饵料的70％)、加菌组、加糖组(生物絮团组)及空白对照组,研究了菌株02对养殖水质(温度、盐度、溶氧、pH、氨氮及亚硝酸氮)、对虾存活率及水体中主要菌群组成等指标的影响.结果显示,加菌加糖组能显著降低养殖水体中的氨氮和亚硝酸氮浓度,提高对虾存活率.生物絮团对虾养殖系统中添加菌株02,能够改善菌群结构,抑制弧菌生长.研究结果可为生物絮团对虾养殖中定向培养有益微生物提供技术支持.     

不同碳源培养生物絮团对南美白对虾养殖影响试验

以红糖、糖蜜、豆粕作为碳源研究三种碳源培养的生物絮团对南美白对虾养殖的影响,结果表明:生物絮团含量:红糖组>糖蜜组>豆粕组,三种碳源培养的生物絮团能够有效降低水体中的氨氮和亚硝酸盐含量,对pH值影响不大,三个试验组的成活率均高于对照组,其中红糖组>糖蜜组>豆粕组。综上所述:红糖、糖蜜和豆粕作为碳源的生物絮团养殖均能降低水体中的氨氮和亚硝酸盐,提高南美白对虾的成活率,同时生物絮团养殖可以降低换水量,减少养殖废水排放,生物需团技术在今后的南美白对虾及其他经济鱼类养殖过程中值得推广。     

生物絮团对锦鲤生长及养殖水体水质的影响

为了研究生物絮团对观赏鱼类生长影响及对养殖水质净化效果,通过设置对照组和生物絮团组(碳氮比为20:1)进行了锦鲤养殖效果对比试验。30d的试验结果显示,生物絮团组锦鲤的特定生长率相比对照组显著提高(P0.05),饲料系数相比对照组显著降低(P0.05),成活率两者之间无显著差异(P0.05)。在池塘水质净化方面,生物絮团组的亚硝酸盐氮浓度和氨氮含量变化趋势一致,呈现先升高后逐渐下降的趋势,生物絮团系统达到稳定后,生物絮团组的二态氮含量显著低于对照组(P0.05)。研究表明,生物絮团技术应用在锦鲤养殖中能有效净化池塘水质,同时可促进锦鲤生长。生物絮团通过实现饲料中蛋白质的二次有效利用,提高了饲料利用效率,降低了养殖成本、减少了水体污染。     

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).     

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.     

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.