卵黄抗体对凡纳滨对虾生长、免疫及抗VpAHPND感染的影响

为探讨特异性卵黄抗体抗致急性肝胰腺坏死病副溶血弧菌 (Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease, VpAHPND) 感染的效果及其机制,防控对虾急性肝胰腺坏死病 (Acute hepatopancreatic necrosis disease, AHPND),本研究以添加不同剂量VpAHPND卵黄抗体制剂 (0、0.2%和0.5%) 的饲料投喂凡纳滨对虾幼虾,测定对虾的生长率和存活率、对虾肝胰腺免疫酶活力和免疫基因相对表达水平,通过浸浴感染实验测定免疫对虾抗VpAHPND感染的能力。生长实验结果显示,免疫28 d后,免疫组与未添加卵黄抗体制剂的对照组对虾在平均生长率、特定生长率和存活率方面均无显著性差异。免疫功能实验结果显示,免疫14 d后,与对照组相比,0.2%免疫组对虾肝胰腺的酚氧化酶 (PO)、超氧化物歧化酶 (SOD)、溶菌酶 (LZM) 活力显著升高,抗菌肽 (Crustin) 基因的相对表达水平也显著升高,而β-1,3-葡聚糖结合蛋白-脂蛋白 (β-GBP-HDL) 基因的相对表达水平则显著降低。浸浴感染实验结果显示,0.2%免疫组对虾的存活率显著高于对照组;0.2% VpAHPND卵黄抗体制剂对凡纳滨对虾的相对免疫保护率为63.77%。研究表明,口服卵黄抗体不会对凡纳滨对虾的生长和存活产生不良影响,0.2%的VpAHPND卵黄抗体制剂通过提升凡纳滨对虾的PO、SOD、LZM活力和Crustin基因表达水平,增强凡纳滨对虾的免疫功能,从而提高对虾抗VpAHPND感染的能力,具有很强的应用潜力。本研究为使用特异性卵黄抗体防控AHPND提供了依据,也为其作用机制研究提供了参考。     

恩诺沙星在凡纳滨对虾体内和体外肝微粒体中的代谢产物比较分析

采用超高效液相色谱-四级杆-飞行时间质谱(UPLC/Q-TOF MS)对恩诺沙星在凡纳滨对虾(Litopenaeus vannamei)体内和体外肝胰腺微粒体孵育下的代谢产物进行定性分析。体内试验为凡纳滨对虾口灌30 mg.kg-1恩诺沙星,24 h后采集血浆用于代谢产物分析。体外试验为在含有肝微粒体的NADPH体系中加入恩诺沙星(100μM)进行孵育,检测孵育液中的代谢产物;肝胰腺采集自未给药的凡纳滨对虾,超速离心法制备而得。凡纳滨对虾血淋巴中除以恩诺沙星原形药物为主外,还可检测到少量的N-去乙基代谢产物环丙沙星、环丙沙星哌嗪环开环代谢物及其同分异构体;而在肝胰腺微粒体体外孵育体系中除检测到原形药物外,只有少量的N-去乙基代谢物环丙沙星。无论是体内还是体外,代谢产物生成量都很少,但环丙沙星是主要代谢产物。推测恩诺沙星在凡纳滨对虾体内的代谢反应主要是脱乙基反应和加氢还原反应,而在体外肝胰腺微粒体恩诺沙星代谢的反应主要是脱乙基反应。     

碳酸盐类盐碱水驯化南美白对虾的试验

以淡化虾塘水添加天然盐碱水为试验用水驯化南美白对虾幼虾,探讨东北地区碳酸盐类盐碱水养殖对虾的技术。结果表明,每隔36、24、12 h各提高1次碱度2 mmol/L,经过12 d,幼虾从碱度3.17 mmol/L分别驯化至17.25、25.33、38.78 mmol/L的存活率为37%、44%及24%。通过驯化提高了幼虾对盐碱水环境一系列生态因子的综合适应能力,其生存环境由海水类型逐渐适应于内陆碳酸盐类盐碱水,是东北地区碳酸盐类盐碱水养殖对虾的有效措施。     

Changes in lipid class and fatty acid composition of adult male Litopenaeus vannamei (Boone) in response to culture temperature and food deprivation

The effects of culture temperature and food deprivation on lipid class and fatty acid composition of adult male Litopenaeus vannamei (Boone) were investigated. Shrimp were maintained in recirculating seawater systems at temperatures of 26 and 32°C and fed 75% dry commercial feed and 25% fresh‐frozen squid for 42 days. Additionally, groups of fed and non‐fed shrimp were maintained at 26°C for 17 days. In shrimp fed at either 26 or 32°C, polar lipids were the main constituents of total identified lipid classes in muscle tissue (66–71%), while neutral lipids were more abundant in hepatopancreas (82–88%). Higher levels of triglycerides were observed in lipids of shrimp hepatopancreas kept at 32°C, but no other lipid class was affected by temperature. A significantly higher proportion of 22:6n‐3 was consistent in muscle and hepatopancreas polar and neutral lipids of shrimp maintained at 26°C. In response to food deprivation, the amount of polar lipids, but not neutral lipids, was reduced by approximately 28% in muscle tissue, whereas all lipid reserves were almost depleted in the hepatopancreas. The variable consumption of some individual fatty acids was observed in polar and neutral lipids of both tissues.     

凡纳滨对虾血蓝蛋白的细菌凝集活性

以凡纳滨对虾（Litopenaeaus vannarnei）为研究对象，运用亲和层析、PAGE、SDS-PAGE、Western-blotting和细菌凝集实验等方法探索凡纳滨对虾血蓝蛋白的细菌凝集活性。结果发现，血蓝蛋白对副溶血弧菌（Vibrio parahaemolyticus）、溶藻酸弧菌（Vibrio alginolyticus）、河弧菌（Vibrio fluvialis）、哈维氏弧菌（Vibrio harveyi）、嗜水气单胞菌（Agromona hydrophila）和金黄色葡萄球菌（Stphylococcus aureus）等6种虾类致病菌具有凝集活性，其凝集活性大小为0．27-1．08mg／L，其中对副溶血弧菌和溶藻酸弧菌凝集活性最大，是哈维氏弧菌的4倍。在此基础之上，选取α-D-葡萄糖、D-半乳糖、D-木糖、D-山梨糖、蔗糖、麦芽糖、α-D-乳糖、甘露醇和N-乙酰神经氨酸等9种糖考察其对血蓝蛋白细菌凝集活性的影响。结果显示，血蓝蛋白的细菌凝集活性可被α-D-葡萄糖和N-乙酰神经氨酸所部分抑制和全部抑制，其最低抑制浓度范围为50～100mmol／L。由此推测，血蓝蛋白确实具有细菌凝集活性，这对进一步研究血蓝蛋白的抗菌机理具有重要意义。     

纳豆菌抗菌肽APNT-6对凡纳滨对虾的低温保鲜效果

为了解决因凡纳滨对虾货架期短而滥用化学保鲜剂等问题,实验通过微生物发酵,柱状色谱纯化得到一种新型生物保鲜剂——纳豆菌抗菌肽APNT-6,采用牛津杯法测定了其对凡纳滨对虾腐败菌的抑菌效果,并用浸保鲜剂方式将抗菌肽应用于凡纳滨对虾保鲜,通过测定对照组和保鲜剂组在(4±1)℃贮藏过程中的pH、挥发性盐基氮(TVB-N)、细菌总数、感官评分等鲜度指标的变化规律,评价了抗菌肽对凡纳滨对虾在(4±1)℃贮藏条件下保鲜效果。结果表明,纳豆菌抗菌肽APNT-6对凡纳滨对虾中Pseudomonas、Shewanella等8株腐败菌有良好的抑菌效果,经0.5 mg/mL抗菌肽保鲜剂处理后,保鲜剂组在(4±1)℃贮藏过程中pH、挥发性盐基氮(TVB-N)和细菌总数增加缓慢,货架期延长了2～3 d。     

Lipid nutrition of juvenile Litopenaeus vannamei: I. Dietary cholesterol and de-oiled soy lecithin requirements and their interaction

Two 6-week experiments were conducted in recirculating systems to determine dietary requirements of Litopenaeus vannamei for phospholipids (PL) and cholesterol, and their potential interaction. A 3×4 factorial design consisting of three cholesterol levels (0%, 0.2%, and 0.5% of diet) and four PL levels (0%, 1.5%, 3.0%, and 5.0% of diet) provided by de-oiled soybean lecithin (SL) was applied. Overall survival was 97.7% with no significant difference among dietary treatments. There was a highly significant interaction between PL and cholesterol on shrimp growth (P=0.0001). Dietary cholesterol at 0.2% or 0.5% gave similar shrimp growth at any given level of PL.

In a second factorial experiment, the same four levels of PL were evaluated in conjunction with smaller intervals between dietary cholesterol levels, 0%, 0.05%, 0.1%, 0.2%, and 0.4%, in an attempt to define dietary requirements and interaction better. Overall survival was 99.7%. A highly significant interaction between dietary PL and cholesterol on growth of shrimp was detected again (P=0.0001). The cholesterol requirement was estimated to be 0.35% of diet in the absence of supplemental PL. At 1.5% and 3% PL, dietary cholesterol requirements were reduced to 0.14% and 0.13% of diet, respectively. When PL were provided at 5% of diet, 0.05% dietary cholesterol was needed for optimal growth. Shrimp growth was significantly enhanced as level of PL increased, but as the level of dietary cholesterol level increased, the growth-promoting effect of PL was diminished. Lipid composition of hepatopancreas (mid-gut gland) and muscle tissue of shrimp fed the different diets was also analyzed. Diets supplemented with PL resulted in higher total lipid in hepatopancreas and lower total lipid in muscle of shrimp than those of shrimp fed diets without supplemental PL. Interaction between dietary PL and cholesterol also affected total lipid and triglycerides (TG) in the hepatopancreas, as well as cholesterol concentration in the muscle.     

海藻糖对南美白对虾免疫活性物的影响

以南美白对虾为实验对象,将海藻糖作为免疫诱导制剂,注射到南美白对虾体内,在连续注射5d和7 d时,分别对南美白对虾血清中的凝血活性、超氧化物歧化酶活性(SOD)、溶血素活性进行测定。结果表明,注射海藻糖5 d和7 d时,南美白对虾血清中凝集活性比对照组分别提高了3倍、2倍,SOD比对照组分别提高了2.5倍、2倍,溶血素活性比对照组分别提高了2倍、1.5倍。说明海藻糖可以明显提高南美白对虾血清中这3种酶的表达水平。     

Investigation of Natural Extracts and Sodium Bisulfite Impact on Thermal Signals and Physicochemical Compositions of Litopenaeus vannamei during Chilled Storage

This research work evaluates the impact of 1.2% pomegranate peel extract (Pp) (Punica granatum), 0.1% grapefruit seed (Gfs) (Citrus paradisi) extract, and synthetic preservative 1.25% sodium bisulfite on shelf life extension of Litopenaeus vannamei stored at 4 and 15°C. The application of isothermal titration calorimetry (ITC) for comparison of the extracts and chemical preservative on shrimp quality retention based on the overall heat production by microbial and enzymatic degradation was addressed. The occurrence of probable bioactive responsible for antimicrobial and antioxidant activity components in the extracts was examined through gas chromatography-mass spectrometry (GCMS). At 4°C, Pp-treated shrimps resulted in 6.73 logCFU/g, 6.93 logCFU/g, 5.31 mgN/100 g, and 0.58 mgMDA/kg for aerobic plate count, psychrotrophic bacterial count, trimethylamine, and thiobarbituric-acid reactive substance, respectively, on the 15th day. At 15°C, Pp-coated shrimps retained quality until day 4. Isothermal calorimetric analysis resulted in a rapid and sharp peak for fresh samples, whereas there was broad and delayed peak for shrimp treated with Pp and sodium bisulfite. These observations prove that Pp counterpart the chemical preservative and outperform Gfs on shrimp quality retention. Results also indicate that isothermal calorimetry can be a useful technique for shelf life predictions and comparative evaluation of extract impact on shrimp quality retention.     

凡纳滨对虾(Litopenaeus vannamei)微卫星多重PCR体系的建立及其在家系亲权鉴定中的应用

为了在遗传分析研究中提高效率并节约成本,本研究从已报道的凡纳滨对虾(Litopenaeus vannamei)微卫星位点中选取多态性较高的位点,基于各位点的扩增片段大小及退火温度等因素进行微卫星位点的组合.通过不断优化位点组合、反应体系及反应程序,成功建立了1个五重、2个四重和1个三重PCR反应体系,并将其应用于11个凡纳滨对虾家系的亲权鉴定.结果显示,四组多重PCR体系中的16个微卫星位点在11个凡纳滨对虾家系中的平均等位基因数(Na)为6,平均多态信息含量(PIC)为0.5813,平均观测杂合度(Ho)为0.513,平均期望杂合度(He)为0.636.利用Cervus3.0软件,对已知系谱关系的11个凡纳滨对虾家系进行亲权分析,其第一亲本累积排除率(CE-1P)、第二亲本累积排除率(CE-2P)和双亲累积排除率(CE-PP)分别为0.99525487、0.99990862和0.99999986.进一步分析表明,当同时使用四组多重PCR体系进行亲权分析时,其模拟配对率和亲权鉴定准确率均为100％,全同胞和半同胞家系鉴别效果良好,表现出准确的鉴别能力.本研究所建立的四组凡纳滨对虾微卫星多重PCR体系均可为后续的凡纳滨对虾遗传多样性分析和亲权鉴定提供高效、准确的检测手段.