大菱鲆(Scophthalmus maximus)仔稚鱼发育期消化道可培养细菌的菌群特征分析

采用传统的细菌培养方法,对大菱鲆(Scophthalmus maximus)育苗生产过程中不同发育时期仔稚鱼的消化道、投喂饵料和养殖水源中的可培养细菌进行了菌群结构分析和优势菌株的16S rDNA同源性比较,揭示其形成过程和演替规律。结果显示,在大菱鲆仔稚鱼5?36日龄的不同发育时期,消化道中的细菌数量呈现了先升高后降低的变化趋势,在17?26日龄期间,仔稚鱼消化道可培养细菌数量级在105?106 CFU/g以上,并且与其他时期存在极显著差异(P<0.01)。弧菌总量呈现先升高后稳定的变化趋势,17日龄之前与之后存在显著差异(P<0.05)。至投喂颗粒饵料期,细菌总量和弧菌总量均稳定在104 CFU/g数量级,弧菌成为大菱鲆仔稚鱼消化道中的优势菌种。本研究发现,大菱鲆仔稚鱼发育早期消化道中的优势菌群变化明显,并且生物饵料中的细菌对消化道中的菌群结构影响较大,其中的Vibrio ichthyoenteri最终成为仔稚鱼消化道中的优势菌种。     

养殖水体添加嗜酸小球菌对鳜发育过程肠道微生物构成的影响

从鳜(Siniperca chuatsi)出膜仔鱼阶段开始向养殖水体添加5×10~7CFU/L的嗜酸小球菌(Pediococcus acidilactici),利用PCR-DGGE技术对鳜受精卵、出膜仔鱼、仔稚鱼和其开口饵料白鲢仔稚鱼的肠道微生物群落结构组成和多样性进行了分析。DGGE图谱的条带克隆测序结果显示,组成鳜鱼苗各阶段的细菌主要隶属于α-变形菌纲(Alphaproteobacteria)、β-变形菌纲(Betaproteobacteria)、γ-变形菌纲(Gammaproteobacteria)、异常球菌纲(Deinococci)、黄杆菌纲(Flavobacteria)。其中黄杆菌属(Flavobacterium)为鳜受精卵特有细菌;异常球菌属(Deinococcus)、红杆菌属(Rhodobacter)和菊苣假单胞菌(Pseudomonas cichorii)是受精卵和仔鱼共有优势细菌,但在仔稚鱼中没有或只有极少分布;肠杆菌属(Enterobacter)是鳜仔稚鱼和开口饵料白鲢仔稚鱼的共有优势细菌;类志贺邻单胞菌(Plesiomonas shigelloides)和豚鼠气单胞菌(Aeromonas caviae)在对照组仔稚鱼的分布明显比试验组丰富。     

应用高通量测序技术分析大菱鲆幼鱼肠道及其养殖环境的微生物群落结构

采用基于Illumina测序平台的高通量测序技术,对大菱鲆(Scophthalmus maximus)幼鱼肠道及其养殖水体、生物饵料中细菌种类及丰度进行研究。测序结果显示,养殖水体、生物饵料和大菱鲆幼鱼肠道等19个样品共获得有效序列547621条,可聚类于3771个可分类操作单元(OTUs),归属于养殖水体、生物饵料、健康幼鱼和发病幼鱼的操作分类单元(OTU)个数分别为3038、1090、87和777,其中,健康幼鱼与生物饵料、健康幼鱼与养殖水体特有的OTU个数分别为57和0,发病幼鱼与生物饵料、发病幼鱼与养殖水体特有的OTU个数分别为481和31。表明幼鱼肠道微生物多样性与生物饵料密切相关。根据细菌注释结果,拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)在大菱鲆幼鱼肠道中占优势地位,其中,健康幼鱼肠道微生物共聚类为8个门,发病幼鱼的肠道微生物可聚类为19个门。与健康幼鱼相比,发病幼鱼肠道门水平上的3种主要优势菌群落结构出现失衡。此外,对各样品中丰度最高的100位OTU分析显示,幼鱼肠道优势菌种类与生物饵料中的优势菌种类密切相关,而每个发病幼鱼肠道优势菌种类具有一定的独立性。本研究旨在为大菱鲆健康养殖和微生态调控提供实验依据。     

添加外源益生菌对大菱鲆育苗生物饵料菌群结构的影响

为评价在大菱鲆育苗生产中添加外源益生菌对生物饵料轮虫和卤虫微生物菌群结构的影响,运用基于illumina HiSeq平台的高通量测序技术对添加益生菌和按照生产流程正常强化的轮虫、卤虫进行了菌群结构的分析和对比。添加外源益生菌的实验组轮虫和卤虫中的菌群物种多样性均明显高于对照组。在轮虫强化过程中,不同时期的对照组样品中菌群结构差异较明显,优势细菌种类变化较大。而添加外源益生菌后的各时期实验组轮虫菌群结构很相似,优势菌群的种类更为丰富,Lactococcus sp.、Pseudoalteromonas sp.和Alteromonas sp.等一直是各实验组中的优势细菌。在卤虫强化过程中,各对照组样品的菌群结构高度相似,优势细菌Cobetia sp.的相对丰度高达54%~65.2%。而在添加益生菌后,各实验组中的菌群结构仍高度相似,但Cobetia sp.的比例下降至4.3%~25.3%,最优势的细菌为Pseudoalteromonas sp.和Alteromonas sp.等几种,菌群结构中的物种均匀度更好。研究表明,在轮虫和卤虫强化过程中添加外源益生菌,能够改变生物饵料的菌群结构,使生物饵料中的细菌种类均匀度更好,并使菌群结构趋于稳定。     

牙鲆(Paralichthys olivaceus)仔稚幼鱼肠道菌群结构比较分析

采用MiSeq 16S rRNA高通量测序技术和生物信息学分析方法,构建了牙鲆(Paralichthys olivaceus)工厂化人工育苗模式下仔稚幼鱼阶段6个不同发育时期18个样品的16SrRNA基因测序文库,共获得7462个OTU (Operational Taxonomic Unit),分类为42个菌门972个菌属.对肠道菌群的形成过程及结构多样性变化分析显示,牙鲆初孵仔鱼的菌群组成多样性丰富,体内的优势菌为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes);在9日龄和21日龄摄食轮虫(Rotifer)和卤虫(Artemia sp.)幼体样品中,肠道的优势菌群结构较单一,变形菌门成为此时期肠道的优势菌群;45日龄摄食配合饲料后,肠道中变形菌门的相对丰度显著降低,厚壁菌门和拟杆菌门的相对丰度明显增大,成为肠道菌群的优势菌群.在属水平的菌群结构中发现,牙鲆仔稚幼鱼肠道优势菌群的种类和数量都发生了较大变化,在9日龄和21日龄时期肠道中弧菌属(Vibrio)相对丰度最高,到45日龄后相对丰度锐减到最低水平;拟杆菌属(Bacteroides)和普氏菌属(Prevotella)在80日龄后达到较高水平,成为肠道优势菌属;厚壁菌门的8个菌属在80-115日龄时期均发展成为优势菌属,定植于牙鲆的肠道.本研究揭示了工厂化人工育苗模式下牙鲆仔稚幼鱼肠道菌群结构及演替规律.     

牙鲆(Paralichthys olivaceus)仔稚幼鱼肠道菌群结构比较分析

采用MiSeq 16S rRNA高通量测序技术和生物信息学分析方法,构建了牙鲆(Paralichthys olivaceus)工厂化人工育苗模式下仔稚幼鱼阶段6个不同发育时期18个样品的16S rRNA基因测序文库,共获得7462个OTU(Operational Taxonomic Unit),分类为42个菌门972个菌属。对肠道菌群的形成过程及结构多样性变化分析显示,牙鲆初孵仔鱼的菌群组成多样性丰富,体内的优势菌为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes);在9日龄和21日龄摄食轮虫(Rotifer)和卤虫(Artemia sp.)幼体样品中,肠道的优势菌群结构较单一,变形菌门成为此时期肠道的优势菌群;45日龄摄食配合饲料后,肠道中变形菌门的相对丰度显著降低,厚壁菌门和拟杆菌门的相对丰度明显增大,成为肠道菌群的优势菌群。在属水平的菌群结构中发现,牙鲆仔稚幼鱼肠道优势菌群的种类和数量都发生了较大变化,在9日龄和21日龄时期肠道中弧菌属(Vibrio)相对丰度最高,到45日龄后相对丰度锐减到最低水平;拟杆菌属(Bacteroides)和普氏菌属(Prevotella)在80日龄后达到较高水平,成为肠道优势菌属;厚壁菌门的8个菌属在80–115日龄时期均发展成为优势菌属,定植于牙鲆的肠道。本研究揭示了工厂化人工育苗模式下牙鲆仔稚幼鱼肠道菌群结构及演替规律。     

高蛋白配合饲料替代饲喂对拟穴青蟹肠道菌群的影响

为探明蟹公寓养殖模式下,高蛋白配合饲料替代杂鱼饲喂对拟穴青蟹菌群的影响,采用16S rRNA高通量测序技术,比较分析养殖过程中不同饵料组拟穴青蟹的肠道菌群结构。结果显示,各饵料组中优势菌群均为变形菌门、厚壁菌门、梭杆菌门和拟杆菌门,占操作分类单元总数85.84%以上。不同饵料组共有细菌操作分类单元(695个)占总数的18.92%,其序列数占总序列数的95.39%。主坐标分析显示不同饵料组肠道菌群的群落结构未产生显著分化。共现性网络分析显示,随养殖过程推进,微生物互作网络复杂度降低。功能预测分析显示,细菌次生代谢产物的生物合成、转运和分解代谢等功能主要在养殖前期出现显著性差异,而养殖后期饵料组间无显著性差异。试验结果表明,蟹公寓养殖过程中,不同饵料会对拟穴青蟹的肠道菌群造成影响。与杂鱼组相比,高蛋白配合饲料在维持肠道菌群多样性方面有一定优势,同时未显著影响肠道细菌群落结构和功能。     

养殖刺参早期发育阶段体内可培养细菌的菌群特征及其与环境菌群相关性分析

采用传统细菌培养方法，对养殖刺参(Apostichopus japonicus)早期发育各阶段幼体体内及环境(投入饵料及培育用水)菌群的组成与结构展开研究，对分离的优势细菌进行分子鉴定，在此基础上，进行了刺参幼体体内菌群结构与环境菌群结构相关性分析。幼体各发育期的细菌培养结果显示，在幼体开口前的各发育时期(性腺、卵、受精卵、原肠胚)均无可培养细菌，在投饵以后，耳状幼体、樽形幼体体内可分离到可培养细菌，幼体发育到稚参以后，消化道可培养细菌总数急剧增加，并在4月龄时达到108 CFU/g数量级。在幼体体内可培养细菌中，弧菌(Vibrio)占比为2.2%~77.3%。对环境菌群的细菌培养结果显示，培育用水中细菌含量变化不显著，随着幼体发育期饵料的转变，不同时期饵料中细菌含量差异显著。整个养殖系统中共分离到65株优势细菌，16S rDNA鉴定结果显示，所分离的65株优势菌鉴定为14个属43种细菌。相关性分析结果显示，随着幼体的发育，生物饵料中的细菌对消化道中的菌群结构影响越来越大。本研究结果为解析刺参消化道菌群的形成过程和演替规律以及养殖用益生菌的筛选与应用奠定了基础。     

不同健康程度和抗生素氟苯尼考干预下斑石鲷肠道菌群的结构差异

为了研究不同健康程度和抗生素氟苯尼考干预下斑石鲷肠道菌群结构的差异及其与养殖环境中菌群结构的相关性,采用Illumina Hi Seq PE250高通量测序的方法对健康、亚健康、典型黑身病和口服氟苯尼考条件下的斑石鲷肠道、养殖水体和颗粒饵料中的细菌多样性及群落结构进行了分析比较。结果显示,养殖水体中细菌多样性高于肠道和颗粒饵料。不同健康程度及氟苯尼考干预下斑石鲷肠道中细菌均以变形菌门、厚壁菌门和软壁菌门为主,且对应的操作分类单元(OTU)占样品全部OTU的比例均达到85%以上。黑身病的发生可影响斑石鲷肠道中丰度最高的前20种优势细菌种类的排名次序,其中变形菌门中的弧菌属的相对丰度显著增加,且随着弧菌属丰度的增加,斑石鲷的黑身病症状也逐渐加重。饵料中添加氟苯尼考投喂斑石鲷能使患病鱼肠道弧菌属的丰度从60.33%下降到1.29%,较大程度改变了肠道的菌群结构,并证实氟苯尼考有效防治黑身病。其次,养殖水体和颗粒饵料对斑石鲷肠道菌群也有一定影响,且养殖水体的影响高于颗粒饵料。本研究首次报道了斑石鲷肠道菌群结构,其研究结果为今后斑石鲷的健康养殖、疾病防控及其微生态学研究提供了参考依据和技术支撑。     

一种中草药复方和11种单方的体外抑菌效果及对大菱鲆肠道菌群的影响

用平板打孔法测定了一种中草药复方制剂YCJ及11种中草药单方水提液对11种鱼类致病菌和肠道菌的体外抑菌效果。试验结果显示,石榴皮、五倍子抑菌效果明显,其次为半枝莲,但败酱草、金银花等效果不明显;中草药复方制剂水提液对不同菌株表现出差异抑菌效果,其中对副溶血弧菌、哈维氏弧菌、金黄色葡萄球菌的抑制效果较好,其次为嗜水气单胞菌与爱德华氏菌,而对产气肠杆菌、奇异变形杆菌、鲁菲不动杆菌3种肠道细菌的抑菌效果不明显。利用中草药复方粉剂拌饲投喂患肠炎的大菱鲆,连续投喂30d,与普通饲料投喂组相比中草药投喂组鱼体的死亡率显著降低,计算其相对保护率为47%。同时,运用高通量测序方法研究了中草药投喂组大菱鲆在投喂前后肠道与肠道内容物中的微生物菌群结构变化情况,结果显示,中草药投喂后肠道及肠道内容物中的菌落丰富度略高于投喂前,且其菌落组成的均匀度也高于投喂前,不同样品中的细菌门类及相对丰度的统计结果显示,变形菌门与厚壁菌门是最主要的优势门类,占总菌群的90%以上。通过对各样品中目的优势细菌统计结果对比分析显示,在肠道内容物样品中乳杆菌目与芽孢杆菌目细菌比例在中草药投喂后的比例较投喂前有明显地提升,而弧菌目、假单胞菌目及支原体目细菌的比例有明显地下降,而在肠道样品中除了支原体目与假单胞菌目细菌比例有明显降低外,其他目的细菌比例无明显差异。研究结果表明,中草药复方制剂对大菱鲆肠炎的治疗效果良好,且可对大菱鲆肠道微生态菌群结构造成一定影响。     

Microbially matured water: a technique for selection of a non-opportunistic bacterial flora in water that may improve performance of marine larvae

Before transfer to larval incubators, water was membrane filtered to remove >95% of the bacteria and then transiently maintained in a biofilter that promoted recolonization of the water by non-opportunistic bacteria. The process is termed microbial maturation of the water. Hypothetically the bacterial flora in the matured water should protect the marine larvae from colonization and proliferation by opportunistic bacteria. Testing of the hypothesis demonstrated 76% higher survival of yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) in matured than in membrane filtered water. Proliferation of opportunistic bacteria was observed in the rearing water after hatching of turbot eggs (Scophthalmus maximus), but to a less extent in the microbially matured water. In the early phase of first feeding of turbot larvae, the matured water induced qualitative differences in the gut microflora. Significantly higher initial growth rate of the turbot larvae in the matured water affected 51% higher average weight of 13 days old larvae than in membrane filtered water. Algal addition to the matured water enhanced the larval growth further. The experiments conducted supported the proposed hypothesis that microbial maturation selects for non-opportunistic bacteria, which protects the marine larvae from proliferation of detrimental opportunistic bacteria.     

An aerolysin-like enterotoxin from Vibrio splendidus may be involved in intestinal tract damage and mortalities in turbot, Scophthalmus maximus (L.), and cod, Gadus morhua L., larvae

Vibrio splendidus is a pathogen that can cause major losses during the early stages of larval turbot rearing when live feed (rotifers or Artemia) is used. As haemolytic bacteria have often been associated with larval rearing losses, we studied the role of the V. splendidus haemolysin in infection of larvae. From a bank of over 10,000 transposon mutants of V. splendidus, two different types of haemolysin-negative mutants were obtained. Both had lost virulence for larval fish, and immunohistochemistry showed that the transposon mutant studied colonized the turbot larval intestinal tract at a similar level to the wild-type organism but did not cause damage or signs of enteritis found with the wild-type organism. One transposon insertion site was located within a gene with high homology to aerolysin, the cytolytic toxin produced by several Aeromonas spp. The haemolysin, which we have termed vibrioaerolysin, had properties similar to aerolysin and osmotic protection studies showed that it formed pores in the membranes of erythrocytes of similar diameter to those of aerolysin. The Tn10 insertion site of the second transposon mutant was in an adjacent ToxR-like gene, suggesting that this might control expression of the vibrioaerolysin. The gastroenteritis caused by Aeromonas spp. in humans is considered to be due to production of aerolysin causing cyclic AMP-dependent chloride secretion in cells of the gastrointestinal tract. Damage to the intestinal tract of marine fish larvae could occur in a similar way, and it is possible that several Vibrio spp. found in the developing bacterial flora of the larval fish gut can secrete aerolysin-like toxins leading to death of larvae in the early rearing stages. Routine bacteriological screening on blood agar plates of live feed is recommended with measures to reduce the concentrations of haemolytic bacteria in rearing systems.     

Microflora of Arctic charr, Salvelinus alpinus (L.): gastrointestinal microflora of free-living fish and effect of diet and salinity on intestinal microflora

Abstract. The adherent aerobic bacterial flora present in the gastrointestinal tract and faeces of free-living Arctic charr, Salvelinus alpinus (L.), from Lake Takvatn, Northern Norway, were identified both qualitatively and quantitatively. Approximately 10⁵ bacteria g⁻¹ were found in both the small and large intestines. The predominant bacterial species were identified as Aeromonas , Enterobacteriaceae, Micrococcus and Lactobacillus. Other microorganisms isolated included Acinetobacter, Cytophaga, Flavobacterium, Moraxella, Pseudomonas, Vibrio , Coryneforms and Streptococcus. The intestinal microflora of free-living fish was dominated by Aeromonas and Lactobacillus , but the intestinal bacterial flora of wild fish transferred to hatchery was affected by feeding them either a capelin roe diet or a commercial feed in fresh and sea water. Approximately 55% of the bacterial flora in intestinal contents from fish fed the capelin roe diet was Enterobacteriaceae when the fish were held in fresh and sea water. However, when the wild-caught charr were fed a commercial diet in fresh water, Aeromonas and Pseudomonas dominated in faeces, while Vibrio and Pseudomonas were predominant in the diet group held in sea water.     

Bacterial Flora in Turbot Scophthalmus maximus Cultured in Deepwell Seawater of Liaodong Peninsula

The microbial flora in turbot (Scophthalmus maximus) cultured in deepwell seawater from Liaodong Peninsula, China was studied for the first time. The total amount of aerobic meso- and psychrophilic microflora from the culturing water, gills, and skin were 3.46–3.82, 3.19–4.28, and 1.70–1.95 log cfu/g, respectively. Out of 204 strains isolated from fresh turbot meat with skin, Gram-positive bacteria accounted for 19.2% of the total aerobic meso- and psychrophilic microflora, and 72.1% were Gram-negative bacteria. Phylogenetic analysis was performed using 16S rDNA sequences of microbial flora isolated from turbot. The predominant bacteria in fresh turbot were Micrococcus, Serratia liquefaciens, and Enterobacteriaceae. Meanwhile, the bacterial flora of refrigerated turbot fish were investigated, and the results indicated that Shewanella putrefaciens was the predominant spoilage bacteria.     

斜带石斑鱼生态优化与病害防控育苗模式的研究

本文报道了斜带石斑鱼生态优化与病害防控育苗模式的研究,研究重点为如何预防神经坏死病毒（NNV）的感染,包括：1、阻断病毒的传播途径,选择不带病毒的受精卵,并在布卵前,对育苗池、用具以及生物饵料进行严格消毒.2、保持育苗池水小生态相对稳定,使水体中益生菌成为优势种群,消耗水体氨氮等有害物质,同时抑制水体里有害细菌的繁殖.3、进一步规范仔、稚、幼鱼期的饵料系列及其配制,保证鱼苗的营养以提高鱼苗的活力和健康水平,提高其抵抗病毒病侵袭的能力,从而提高鱼苗的成活率.     

Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot,Scophthalmus maximus (L.), larvae

A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection. The bacterial strain was recovered in pure culture from the internal organs of infected fish. Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney. Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C. Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli. In both cell lines, the number of adhered bacteria increased with incubation time.