坛紫菜自由丝状体响应琼胶寡糖的激发

为了研究琼胶寡糖对坛紫菜自由丝状体藻丝生长和壳孢子囊枝形成的激发的影响,实验采用琼胶寡糖激发坛紫菜自由丝状体,以液相氧电极检测坛紫菜丝状体净光合放氧速率的变化;以对羟基苯乙酸(POHPAA)化学发光法检测坛紫菜丝状体的H_2O_2释放量;利用LC-MS检测红藻糖苷含量变化;利用实时定量PCR技术检测坛紫菜丝状体H_2O_2产生相关基因(Phrboh、Ph SOD)和红藻糖苷合成相关基因(Phnho1、Phgpdh、Phtps)的表达情况;并利用显微镜观察法检测壳孢子囊枝数量的变化。结果发现,琼胶寡糖能够激发坛紫菜自由丝状体的系列响应,表现在净光合放氧速率以及光合同化产物红藻糖苷的含量和生物合成出现增加;H_2O_2释放量持续增加,与产生H_2O_2相关的2个酶基因Phrboh和Ph SOD的表达增强。此外,琼胶寡糖也能够促进在坛紫菜自由丝状体的发育,在培养第30天时,琼胶寡糖处理组的壳孢子囊枝形成率达到59%,显著高于对照组46%。综上所述,琼胶寡糖能够增加坛紫菜自由丝状体的光合速率和光合同化产物,并通过形成H_2O_2的酶的表达来诱导活性氧的释放;琼胶寡糖还能促进坛紫菜自由丝状体的繁殖发育。     

几丁质脱乙酰酶的特点及应用

几丁质是渔业生产过程中废弃虾蟹壳的主要组成成分。传统生产几丁质、壳聚糖和壳寡糖的方法是化学消化法,此方法的主要问题是环境污染严重,随着人们环保意识的提高,该方法已难以持续。此外由于化学法生产的壳聚糖和壳寡糖难以控制其聚合度、脱乙酰度和脱乙酰模式,因此很难研究壳寡糖的结构与功能之间的关系。几丁质脱乙酰酶(Chitin deacetylase EC 3.5.1.41)是一种可在温和条件下将几丁寡糖转化为壳寡糖的酶,利用此酶规模化生产具有特定乙酰化模式的壳寡糖已成为一个很重要的研究方向。鉴于该酶对于产业发展的重要性,本文对目前已经研究的几丁质脱乙酰酶的来源、结构特征、催化机制、脱乙酰化模式及几丁质脱乙酰化酶的应用等方面进行了综述。     

壳寡糖改善珍珠龙胆石斑鱼非特异性免疫能力的机制

为研究壳寡糖(OCS)对珍珠龙胆石斑鱼非特异性免疫能力的影响及作用机制,实验通过在基础实验饲料中分别添加0(对照组)、200、400和800 mg/kg的壳寡糖投喂珍珠龙胆石斑鱼4周,综合分析壳寡糖对石斑鱼免疫调控作用的影响.实验首先检测了壳寡糖对鱼体内免疫及抗菌相关酶活性的影响.结果 显示,投喂壳寡糖后显著提高珍珠龙...     

两种海洋寡糖对仿刺参免疫活性的影响

研究了褐藻酸钠寡糖(AOS)和壳寡糖(COS)对仿刺参(Apostichopus japonicus)生长及免疫功能的影响。实验分为对照组、COS组和AOS组,对照组投喂企业常规饲料,COS组在对照组饲料基础上添加壳寡糖(1 g·kg~(-1)),AOS组在对照组饲料基础上添加褐藻酸钠寡糖(1 g·kg~(-1))。在第30、60、90天,测定各组仿刺参的质量,并在第90天随机采集50 ind仿刺参的体腔液,测定酸性磷酸酶、碱性磷酸酶、过氧化氢酶和一氧化氮合酶等免疫酶的活力。结果表明,AOS组和COS组仿刺参体质量分别比对照组增长16.4%和18.2%;AOS组仿刺参的酸性、碱性磷酸酶活性分别提高了75.1%和67.3%;而COS组仿刺参的过氧化氢酶和一氧化氮合酶的活性分别提高了190.3%和55.6%。海洋寡糖COS和AOS作为仿刺参的免疫增强剂,可有效提高幼参的生长性能和免疫力,其中COS的作用效果优于AOS。     

利用虾壳清洁化生产不同脱乙酰度壳寡糖及其抗TMV效果研究

壳寡糖的传统生产工艺是采用HCl和NaOH处理甲壳类制备壳聚糖,再酶解获得壳寡糖,生产过程中含高浓度Cl–和Na+的废水对环境造成严重污染。本研究采用H3PO4和KOH为反应溶液建立壳聚糖的绿色生产工艺,并制得不同脱乙酰度的壳寡糖,探究了不同脱乙酰度壳寡糖抗烟草花叶病毒(TMV)的效果。结果显示,通过该生产工艺制得壳寡糖的脱乙酰度分别为63.79%、72.12%、79.34%和88.15%,分子量均为1500 Da左右。脱乙酰度为79.34%和88.15%的壳寡糖诱导植株对TMV产生抗病性,表现出对TMV进行体外钝化、抑制TMV在寄主内的复制和提高植物体内过氧化氢酶、过氧化物酶和多酚氧化酶的活性。     

基于单链DNA浓度法的哈维氏弧菌核酸适配体亲和特异性研究

本研究采用核酸适配体的单链DNA浓度来表征其亲和力,通过测定核酸适配体与靶细菌哈维氏弧菌(Vibrio harveyi)结合后ssDNA的浓度,来研究该核酸适配体的亲和特异性和亲和常数,并通过荧光显微镜法对其亲和特异性进行验证。结果显示,采用单链DNA浓度法测得该核酸适配体对靶细菌哈维氏弧菌的亲和力是非目标菌的15.2倍以上;荧光显微镜直接观察发现只有靶细菌能较好结合有荧光标记的核酸适配体,并呈现明显荧光;荧光阻断法发现靶细菌和非靶细菌都未呈现明显荧光,证明了该适配体有较好的亲和特异性,也进一步验证了单链DNA法的测定结果。在亲和常数的测定方面,利用单链DNA浓度法测得该核酸适配体的亲和常数K_d=(33.70±7.83) nmol/L,相应的拟合系数为R~2=0.960,有较好的准确性和可靠性,说明采用单链DNA浓度法来测定适配体的亲和力和亲和常数是可行的。     

基于核酸适配体的PCR法检测溶藻弧菌及其灭活菌

溶藻弧菌(Vibrio alginolyticus)分布广,数量多,发病率高,是水产养殖中常见的条件致病菌,而对溶藻弧菌进行快速准确的识别鉴定是其病害防治的前提和基础。核酸适配体,因为具有较高的亲和特异性,在微生物的识别鉴定方面展现出了巨大的优势。本文利用核酸适配体和适配体筛选产物,通过结合、洗涤、加热分离、PCR扩增以及电泳检测等步骤,对溶藻弧菌进行了检测鉴定。结果表明,适配体和筛选产物都能对溶藻弧菌及其灭活菌进行较好的识别鉴定,适配体筛选产物对溶藻弧菌的检测下限为10~3cfu/mL,而对其灭活菌的检测下限为10~2cfu/mL,适配体对溶藻弧菌及其灭活菌的检测下限都可达到10 cfu/mL。该方法对溶藻弧菌有较好的亲和特异性,并能较好地区分溶藻弧菌与哈维氏弧菌等水产常见病原菌,在水产病害的检测中显示了较好的应用前景。     

壳寡糖对吉富罗非鱼幼鱼生长性能、前肠组织结构及肠道主要菌群的影响

选用初始体质量(3.02±0.16) g的吉富罗非鱼幼鱼(Oreochromis niloticus)450尾,随机分为5组,每组3个重复,每重复30尾实验鱼,分别饲喂添加壳寡糖质量分数为0.00%(对照组)、0.10%、0.30%、0.50%和0.70%的饲料8周,考查壳寡糖对吉富罗非鱼幼鱼生长性能、前肠组织结构及肠道主要菌群的影响.结果表明,在生长性能方面,添加0.30%、0.50%和0.70%壳寡糖组增重率分别较对照组显著提高12.53%、16.17%和9.47%(P<0.05);添加壳寡糖各组较对照组饲料系数显著降低,饲料干物质和蛋白质的表观消化率均显著升高(P<0.05).在肠道组织结构方面,与对照组相比,添加0.30%和0.50%壳寡糖组的幼鱼前肠绒毛长度显著增加了18.02%和23.21%,宽度显著增加了45.21%和54.06%,密度显著增加了15.18%和19.37%(P<0.05);添加0.30%、0.50%和0.70%壳寡糖组的幼鱼肠壁厚度较对照组分别减少了16.41%、19.96%和15.00%(P<0.05).在肠道主要菌群方面,各壳寡糖添加组大肠杆菌数量均显著降低,乳酸杆菌数量显著增加(P<0.05).上述结果表明,吉富罗非鱼幼鱼饲料中添加壳寡糖可提高其生长性能,并改善肠道内环境,推荐适宜添加量为0.30%~0.50%.     

壳寡糖添加量对青鱼幼鱼血清特定生化指标、肝和肠白介素IL-2、IL-8和IL-10 mRNA表达量的影响

将30日龄、体质量约4.7g的青鱼放养在12个网箱中,每箱40尾,分别投喂添加0(对照组)、0.3%、0.6%和1.2%壳寡糖的饲料50d,每种饲料3个重复,研究壳寡糖添加量对青鱼血清特定生化指标、肝和肠白介素-2(IL-2)、白介素-8(IL-8)和白介素-10(IL-10)mRNA表达量的影响。试验结果显示,与对照组比较,0.3%壳寡糖组青鱼血清乳酸脱氢酶的活性显著降低(P0.05),0.6%壳寡糖组青鱼血清甘油三酯、葡萄糖的含量和乳酸脱氢酶的活性显著降低(P0.05),0.3%组的肝IL-2和IL-10的表达量显著提高(P0.05),0.6%壳寡糖组的肝IL-2和IL-8的表达量显著提高(P0.05)。试验结果表明,在饲料中添加壳寡糖能促进青鱼幼鱼肝和肠白介素的表达,增强免疫力,降低血糖血脂含量,其中以添加0.6%壳寡糖的效果最佳。     

壳寡糖与低聚木糖对大菱鲆(Scophthalmus maximus)幼鱼生长、体组成和血液生化指标的影响

本研究以初始体重为(15.46±0.06) g的大菱鲆(Scophthalmus maximus)幼鱼为实验对象,采用2×3双因素实验设计,研究饲料中壳寡糖(Chitosan oligosaccharide,COS)和低聚木糖(Xylo-oligosaccharide,XOS)对大菱鲆幼鱼生长、体组成和血液生化指标的影响。养殖实验在全封闭循环水养殖系统中进行,养殖周期为60 d。9组实验饲料粗蛋白和粗脂肪含量分别为53%和11%;每组饲料随机投喂3桶,每桶30尾鱼。结果显示,饲料中同时添加0.5%的壳寡糖、1.0%的低聚木糖对大菱鲆幼鱼的促生长作用最明显,相对增重率显著提高。饲料中壳寡糖和低聚木糖对大菱鲆幼鱼增重率、特定生长率、饵料系数、蛋白质效率均有显著影响(P<0.05),但对大菱鲆体成分影响不显著(P>0.05);低聚木糖和壳寡糖对大菱鲆幼鱼特定生长率、增重率、全鱼粗脂肪和灰分、血清甘油三酯、溶菌酶以及碱性磷酸酶均存在显著交互作用(P<0.05)。研究表明,低聚木糖和壳寡糖配合使用可以显著提高大菱鲆幼鱼的生长效果,并且可在一定程度上增强其非特异性免疫能力,降低血脂含量。     

Low-pH increases the binding of haemorrhagic septicaemia rhabdovirus to membrane phospholipids

Abstract. The rhabdovirus causing viral haemorrhagic septicaemia (VHS), a disease of coldwater fish, especially salmonids, binds to sonicated radioactively labelled membrane phospholipids [phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC)]. The extent of binding of VHS V to membrane phospholipids is dependent on pH, occurring at physiological pH, but being maximal at about pH 5.5 and suggesting that a pH-induced conformation] change is needed for the VHSV to maximally bind phospholipids. VHSV bound PS more effectively than either PE or PC, as demonstrated by ultracentrifugation and competition experiments.     

Larval sea lamprey release two unique bile acids** to the water at a rate sufficient to produce detectable riverine pheromone plumes

The discovery that the olfactory system of the anadromous sea lamprey is extremely sensitive to two unique bile acids (petromyzonol sulfate [PS] and allocholic acid [ACA]) produced by stream-resident larval conspecifics has lead us to hypothesize that these compounds function as a migratory pheromone. Here, we test whether lamprey release these bile acids to the water in quantities sufficient for them to function as a long distance attractant. Five experiments were conducted. First, high performance liquid chromatography (HPLC) of liver extracts from all three life history stages of this species established that only larvae produce PS and ACA; parasites and maturing adults produced no identifiable bile acids. Large quantities of PS and ACA were found in larval gall bladders. Second, HPLC analyses of larval lamprey holding waters established that recently-fed larvae held in the laboratory release these bile acids to the water, with PS being released at a rate of approximately 16 ng h^–1 animal^–1 and ACA at 5 ng h^–1 animal^–1. Fasted animals released little bile acid. Third, an investigation of bile acid release routes demonstrated that larvae release bile acids primarily via their feces. Fourth, a study of the stability of ACA and PS in river water found both to have a half-life of a day. Finally, theoretical extrapolations using these data suggest that PS and ACA are present in picomolar concentrations in lamprey streams, a concentration within the detection range of adults. In conclusion, these data demonstrate for the first time in a fish that bile acid release rates and modes are adequate for these compounds to have pheromonal function.     

Selection and characterization of ssDNA aptamers specifically recognizing pathogenic Vibrio alginolyticus

Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single‐stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem‐loop structures, which could form the basis of aptamers’ specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.     

Generation and characterization of aptamers against grass carp reovirus infection for the development of rapid detection assay

Grass carp reovirus (GCRV) causes devastating viral haemorrhagic disease in farmed grass carp (Ctenopharyngon idellus). As novel molecular probes, aptamers have been widely applied in rapid diagnosis and efficient therapies against virus or diseases. In this study, three single‐stranded DNA (ssDNA) aptamers were selected against GCRV‐infected CIK cells via SELEX (systematic evolution of ligands by exponential enrichment technology). Secondary structures predicted by MFOLD indicated that aptamers formed stem‐loop structures, and GVI‐11 had the lowest ΔG value of ?30.84 KJ/mol. Three aptamers could specifically recognize GCRV‐infected CIK cells, with calculated dissociation constants (Kd) of 220.86, 176.63 and 278.66 nM for aptamers GVI‐1, GVI‐7 and GVI‐11, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. The targets of aptamers GVI‐1, GVI‐7 and GVI‐11 on the surface of GCRV‐infected cells could be membrane proteins, which were trypsin‐sensitive. Furthermore, FAM‐labelled aptamer GVI‐7 could be applied to detect GCRV infection in vivo. It is the first time to generate and characterize aptamers against GCRV‐infected cells. These aptamers have great potentials in development of rapid diagnosis technology and antiviral agents against GCRV infection in aquaculture.     

Amino acid composition of edible parts of three-year-old experimental scaly crossbreds of common carp (Cyprinus carpio, Linnaeus 1758)

The aim of this study was to compare the amino acid (AA) composition of edible parts of three experimental groups of carp, i.e. a pure line of Přerov scaly carp (PS), a hybrid line of Přerov scaly carp and Northern mirror carp (PS × M72), and a hybrid line of Přerov scaly carp and Ropsha scaly carp (PS × ROP), with the quality of the edible parts of control hybrids of Hungarian and Northern mirror carp (M2 × M72) in harvest size (K₃). A comparison between the controls (M2 × M72) and experimental carp (PS, PS × M72, PS × ROP) showed that their muscle tissues contained the same amounts of 10 AA [essential amino acids (EAA): Thr, Val, Leu, Phe, Lys, His; non‐essential amino acids (NEAA): Asp, Gly, Ala, Tyr] of the 16 AA determined. Glu, Asp, Lys and Leu were the AA with the highest muscle concentrations. The total EAA_sum and NEAA_sum contents in the fastest‐growing PS × ROP hybrid, in spite of specific differences found (P<0.05: Arg, Met; P<0.01: Pro), were practically identical to those found in the control group of M2 × M72 mirror carp. PS × ROP hybrid female and male muscle tissues differed (P<0.05) only in Met and Ala levels. Hard roes of experimental female carp (PS, PS × M72, PS × ROP) contained the largest quantities of Glu and Val, and that of control female carp (M2 × M72) the largest quantities of Glu and Gly. Hard roes of PS × ROP hybrids contained the largest quantities (P<0.01) of EAA_sum (52.44±0.19%). Compared with hard roes, soft roes from all groups of carp contained more EAA_sum (PS × ROP: 55.03±0.26%). The two most abundant AA in soft roes were Lys and Arg. The most abundant AA in the hepatopancreas in all carp groups were Glu, Asp, Leu and Arg. Hepatopancreas EAA_sum levels in experimental carp (PS, PS × M72, PS × ROP) were significantly (P<0.05) lower than those in controls (M2 × M72).     

Development of novel aptamer‐based enzyme‐linked apta‐sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 10⁴/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.     

Effects of Sodium Alginate and Chitosan Coating Combined with Three Different Essential Oils on Microbial and Chemical Attributes of Rainbow Trout Fillets

ABSTRACT

Meat products, such as fish meat, are known to be susceptible to undesirable chemical and microbial reactions that characterize spoilage. In this study, the effect of a sodium alginate and chitosan coating incorporated with Mentha piperita, Artemisia dracunculus, and Zataria multiflora essential oils on chemical and microbial attributes of rainbow trout meat was evaluated during storage at 4°C. Chemical and microbial assays were performed on rainbow trout fillets with alginate and chitosan coatings and 0.2% concentration of test essential oils. The results showed that the alginate coating with essential oils significantly decreased production of thiobarbituric acid (TBA) and total volatile basic nitrogen (TVBN) and reduced the growth of foodborne spoilage bacteria during storage at 4ºC. At day 12, the best results were obtained in chitosan coating + Z. multiflora, with 5.96 ± 0.12, 4.93 ± 0.12, and 3.83 ± 0.2 for total viable counts, psychrotrophic bacterial count, and lactic acid bacteria count, respectively. Moreover, the lowest amounts of chemical analysis were observed in chitosan coating + Z. multiflora at the final day (0.54 ± 0.03 and 20.31 ± 0.1 for TBA and TVBN, respectively). Our study revealed that essential oils can be used as effective natural components against undesirable chemical and microbial reactions in fish meat.     

虹鳟肠炎红嘴病病理模型的构建

为探讨鲁氏耶尔森菌侵染虹鳟的致病机制,本实验建立了鲁氏耶尔森菌感染虹鳟引起的肠炎红嘴病的病理模型,制定相应的临床症状及组织病理学评分系统,并对该模型进行研究。将43尾平均体质量约为12 g的健康虹鳟随机分成5组:3个实验组(n=30)、对照组(n=10)和哨兵组(n=3)。3个实验组分别采用2.0×106、2.0×107和2.0×108 CFU/m L的鲁氏耶尔森菌感染浓度,通过腹腔注射方式进行人工感染试验。对感染鲁氏耶尔森菌的虹鳟肠、肝脏、脾脏和肾脏组织进行镜检及临床症状、剖检病变判断,结合细菌学检测,按制定的评分系统评价各组肠炎红嘴病造模效果,确定最佳造模方案。结果显示,各攻毒组虹鳟感染后72 h均出现不同程度死亡,临床症状表现为红嘴、肛门红肿、鳍(胸鳍、腹鳍、臀鳍)等出现不同程度充血,下颌部、腹部出现出血点等。组织病理学可见肝脏、脾脏、肾脏及肠组织均有炎性细胞浸润现象出现,肝细胞、肠上皮细胞和肾小管上皮细胞等实质细胞变性、坏死,脾脏部位淋巴细胞减少、红细胞死亡堆积。综合各组得分发现,2.0×107 CFU/m L组的鲁氏耶尔森菌感染虹鳟造模效果最佳,患病虹鳟的临床症状显著且组内差异较小,病程迁延较长,便于研究。研究表明,对体质量约12 g的虹鳟幼鱼腹腔注射0.1 m L浓度为2.0×107 CFU/m L的鲁氏耶尔森菌可成功构建肠炎红嘴病病理模型。     

The development of oocyte cryopreservation techniques in blue mussels Mytilus galloprovincialis

Reliable techniques for the cryopreservation of both sperm and oocytes of the blue mussel Mytilus galloprovincialis Lamarck would increase the availability of seed supplies out-of-season and enhance efficiency in selective breeding. We have investigated the optimal cryo-technique for blue mussel oocytes. The toxicity of three cryoprotective agents (CPAs) [dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)] at different concentrations (1–5 M) and exposure times (0.25–30 min) were investigated for mussel oocytes at room temperature (20 °C) or on ice. The same CPAs (1, 1.5 and 2 M) as well as three different cryoprotectant mixtures [1.5 M EG + 0.2 M trehalose + 100 % Milli-Q water (EGTM); 1.5 M EG + 0.2 M trehalose + 75 % Milli-Q water + 25 % seawater; 1.5 M EG + 0.2 M sucrose + 100 % Milli-Q water] were tested by comparing the post-thaw oocyte fertilization rate after using the slow-cooling method. Vitrification was also examined; however, this method failed to produce any post-thaw surviving oocytes. Among the tested CPAs, EG was the least toxic to oocytes. There was a tendency for the equilibration of CPAs on ice to achieve a higher oocyte fertilization rate compared with that at room temperature, and this difference was significant at concentrations of 3 and 4 M (P < 0.01). The DMSO, EG and PG treatments all resulted in post-thaw fertilization, with EGTM achieving the highest number of surviving oocytes (32 %). At the optimal seeding temperature (?7 °C), the addition of 0.2 M trehalose to EG resulted in a better fertilization rate of post-thawed oocytes than the addition of 0.2 M sucrose. All of the treatments evaluated produced D-larvae from post-thawed oocytes, although the rates were low.