注射磷脂酰丝氨酸对凡纳滨对虾血蓝蛋白合成、酚氧化酶活性的影响

为研究磷脂酰丝氨酸(PS)对凡纳滨对虾血蓝蛋白合成、酚氧化酶活性的影响,并探讨PS激活凡纳滨对虾血蓝蛋白发挥酚氧化酶活性的机制,实验采用5、10和20μg/mL 3个PS浓度对凡纳滨对虾尾节肌肉进行注射,注射量为50μL,对照组注射生理盐水,各处理组设3个平行组,取样时间为0、6、12、24、36、48和60 h,分别测定了血浆血蓝蛋白含量,肝胰脏血蓝蛋白p75、p77亚基和mRNA的表达以及血浆血蓝蛋白酚氧化酶活性。结果显示:与对照组相比,血蓝蛋白含量在6 h内略有下降,6～36 h内呈峰值变化,24 h时达到最大值(P<0.05);血蓝蛋白亚基p75、p77和mRNA表达在0～36 h内呈峰值变化,12 h时达到最大值(P<0.05);血蓝蛋白酚氧化酶活性在36 h内呈峰值变化,12 h时达到最大值(P<0.05)。研究表明,PS能够激活凡纳滨对虾血蓝蛋白的酚氧化酶活性,表现出明显的时间剂量效应,同时在短时间内引起血蓝蛋白含量下降,随后机体启动血蓝蛋白的合成机制,证实PS是凡纳滨对虾血蓝蛋白发挥酚氧化酶活性的激活因子,为对虾血蓝蛋白免疫活性的研究奠定了理论基础。     

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。     

对虾白斑综合征病毒（WSSV）囊膜蛋白VP110部分重组表达及其与对虾鳃细胞膜蛋白结合分析

将VP110基因的部分序列克隆到pET-28a载体中构建pET28a-vp110b重组质粒并进行原核表达,获得重组表达的蛋白rVP110-B;用rVP110-B注射凡纳滨对虾Litopenaeus vannamei后,经WSSV感染,实验表明,该蛋白注射使凡纳滨对虾感染WSSV的半数死亡时间比对照组延长了20%。用表达纯化的该重组蛋白制备了兔抗rVP110-B多克隆抗体,该抗体用于凡纳滨对虾鳃细胞膜蛋白与rVP110-B的Far-western分析显示,凡纳滨对虾鳃细胞膜蛋白中除90 kDa左右的血蓝蛋白外,在41.7 kDa存在结合条带,经质谱分析表明这条鳃细胞膜蛋白是肌动蛋白。     

凡纳滨对虾血清中直接与病原菌相结合的主要蛋白的鉴定

以凡纳滨对虾为研究对象,将其血清分别与溶藻酸弧菌,哈维氏弧菌,嗜水气单胞菌和金黄色葡萄球菌等4种病原菌孵育,采用亲和蛋白质组学和Western-blotting等分子生物学方法纯化、鉴定和确证其中可与病原菌直接结合的蛋白.结果发现,与对照组相比4种病原菌与虾血清孵育5 h后均可与对虾血清中分子质量约为75 000 (p75) 的蛋白相结合,而孵育15 h后既可与p75相结合,还可与分子质量约为77 000 (p77) 的蛋白以及0～4种不同分子质量的蛋白相结合.经MALDI-TOF/MS分析和Mascot搜索引擎检索,p75和p77蛋白分别与凡纳滨对虾中分子质量约为75 000、77 000的2个血蓝蛋白亚基具有显著一致性.尤其是p75蛋白还可与兔抗血蓝蛋白75 000亚基抗体发生特异性结合.由此推断,凡纳滨对虾血清中与病原菌直接结合的主要蛋白为血蓝蛋白,提示血蓝蛋白全蛋白可能具有抗菌活性.     

凡纳滨对虾28.5D血蓝蛋白的降解新片段

血蓝蛋白是一种具有多种非特异性免疫学活性且可以产生免疫学活性降解片段的多功能蛋白。为了探索血蓝蛋白新的降解片段，本研究以凡纳滨对虾旺itopenaeus vannamei）为研究对象，采用比较蛋白质学技术分析对虾感染病原菌后血清蛋白质组的变化。结果发现，对虾感染副溶血弧菌（Hbrio parahaemolyticus）12h后，血清中新增一种28．5kD的蛋白（命名为p28．5）。经MALDI-TOF／MS分析，其与凡纳滨对虾血蓝蛋白75kD亚基具有高度同源性。进一步研究显示，p28．5蛋白不仅可与抗血蓝蛋白抗体发生特异性结合，而且还与对虾抗感染能力呈正相关。由此推测，p28．5蛋白应该是对虾感染病原菌后产生的一种28．5kD血蓝蛋白降解新片段，其可能是血蓝蛋白发挥免疫学功能的一种新方式的产物。[中国水产科学，2008，15（3）：425-430]     

凡纳滨对虾28.5kD血蓝蛋白的降解新片段

血蓝蛋白是一种具有多种非特异性免疫学活性且可以产生免疫学活性降解片段的多功能蛋白。为了探索血蓝蛋白新的降解片段，本研究以凡纳滨对虾(Litopenaeus vannamei) 为研究对象，采用比较蛋白质学技术分析对虾感染病原菌后血清蛋白质组的变化。结果发现，对虾感染副溶血弧菌(V     

壳聚糖硫酸酯提高凡纳滨对虾抗白斑综合征病毒感染力的研究

论述如下一项研究,采用壳聚糖硫酸酯添加到饲料中投喂凡纳滨对虾,4周后检测对虾血清酚氧化酶、超氧化物歧化酶和溶菌酶活性;同时进行白斑综合征病毒(WSSV)的肌肉注射感染实验,以检测壳聚糖硫酸酯对病毒的抗感染能力。实验结果显示,饲料中壳聚糖硫酸酯添加量为0.15‰和0.50‰,能显著提高凡纳滨对虾血清酚氧化酶活性;添加量为0.15‰时,能显著提高对虾血清超氧化物歧化酶活性,但添加量继续增大时反而下降;在低添加量时,对虾血清溶菌酶活性与对照组相近,添加量为0.15‰和0.50‰时,对虾血清溶菌酶活性随添加量增大而升高。凡纳滨对虾摄食添加壳聚糖硫酸酯饲料4周后,经注射WSSV攻毒感染,壳聚糖硫酸酯添加量为0.04‰、0.15‰和0.50‰试验组,对虾比成活率分别为39.3%、42.9%和53.6%,而未摄食壳聚糖硫酸酯的对照组成活率仅为17.9%。结果表明,摄食壳聚糖硫酸酯可以明显提高对虾抵御WSSV感染的能力。     

酵母提取物替代鱼粉对凡纳滨对虾消化机能的影响

为探明使用经氨基酸平衡后的酵母提取物替代鱼粉对凡纳滨对虾消化机能的影响,分别用酵母提取物替代饲料中15 %(B)、30 %(C)、45 %(D)、60 %(E)和100 %(F)的鱼粉,配制6种等氮、等能的试验饲料投喂凡纳滨对虾(6.328±0.630 g),以鱼粉含量为25 %的饲料为对照组(A)。饲养7 d后开始收集粪便,测定凡纳滨对虾对饲料的表观消化率。养殖试验结束后,研究酵母提取物替代鱼粉对凡纳滨对虾消化酶的影响。试验结果:凡纳滨对虾对各组饲料均具有较高的干物质表观消化率(77.96 %~81.21 %)和粗蛋白表观消化率(87.19 %~92.82 %)；各替代组饲料的表观消化率均高于对照组,其中C、E和F组的干物质表观消化率显著高于对照组(P<0.05),粗蛋白表观消化率随酵母提取物用量的增加而显著增加(P<0.05)；凡纳滨对虾肝胰腺中类胰蛋白酶的活性随饲料中酵母提取物用量的增加而显著升高(P<0.05),脂肪酶的活性随饲料中酵母提取物用量的增加而显著降低(P<0.05)；肝胰腺中淀粉酶活性最高的为C组；各替代组凡纳滨对虾的肠胰蛋白酶活性均高于对照组。结果表明,以氨基酸平衡酵母提取物替代饲料中的鱼粉可以提高凡纳滨对虾对饲料的消化机能。     

与凡纳滨对虾抗副溶血弧菌有关的6.0 kD 血蓝蛋白降解肽段

以凡纳滨对虾(Litopenaeus vannamei)为研究对象,采用人工感染、Tricine-SDS-PAGE、Western-blotting、MALDI-TOF/TOF、抑菌实验等方法对血蓝蛋白小分子降解肽段进行研究。结果发现,副溶血弧菌(Vibrio parahaemolyticus)刺激对虾24 h后,与对照组相比,其血淋巴中新出现5个分子量为6.0~31.0 k D的与兔抗血蓝蛋白抗体呈阳性的小分子条带。其中,分子量为6.0 k D的a条带(命名为HMCp6肽段)与凡纳滨对虾血蓝蛋白具有高度同源性,对副溶血弧菌具有明显的抑菌活性,与对照组相比,存在极显著性差异(P0.01)。由此说明,HMCp6应该是对虾感染副溶血弧菌后所产生的一种新的小分子降解肽段,推测其在对虾抗感染防御中发挥重要作用。     

氨氮胁迫下白斑综合征病毒对凡纳滨对虾的致病性

为了评价养殖水环境中氨氮(NH_4-N)对凡纳滨对虾(Litopenaeus vannamei)的危害性,开展了NH_4-N胁迫对凡纳滨对虾感染白斑综合征病毒(WSSV)后的死亡率、WSSV增殖速率和对虾主要免疫相关酶活性影响的实验。在NH_4-N胁迫质量浓度为15.6 mg·L-1,分别注射2×105和2×106个WSSV粒子,结果显示,NH_4-N胁迫下注射2×105个WSSV粒子的凡纳滨对虾第144小时死亡率达到53.3%,显著高于无胁迫组(40.0%)。对虾鳃组织WSSV荧光定量PCR检测结果显示,NH_4-N胁迫下凡纳滨对虾鳃组织内WSSV的增殖加快。此外,免疫相关酶活性结果显示,NH_4-N浓度突变会促使对虾血清中酚氧化酶(PO)、酸性磷酸酶(ACP)和碱性磷酸酶(AKP)活性短暂升高后持续降低。由此可见,NH_4-N胁迫会加快WSSV在患病凡纳滨对虾体内的增殖,导致更高死亡率,这可能是因为胁迫造成了对虾免疫相关酶活性降低和抗病原感染能力下降。     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.     

美人鱼发光杆菌对凡纳滨对虾非特异性 免疫功能及抗病力的影响

在基础饲料中分别添加0.1%和1%美人鱼发光杆菌灭活菌、0.1%美人鱼发光杆菌活菌配制成3种免疫实验饲料,以基础饲料为空白对照组饲料,每组设3个平行样。对个体质量为(4.83±0.36)g的凡纳滨对虾进行为期20 d的饲养实验,分别在0、5、10、15和20d进行取样,以血清中的酚氧化酶(PO)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和溶菌酶(UL)活性为免疫指标,探讨了美人鱼发光杆菌作为免疫制剂对凡纳滨对虾非特异性免疫效应的影响;在投喂免疫饲料后的第22天,按0.004 2 kg/kg体重的剂量,直接投喂对虾白斑综合征病毒(WSSV)病料,并记录累积死亡率。结果表明,美人鱼发光杆菌免疫实验组对凡纳滨对虾血清中PO、ACP、AKP、UL和SOD活性影响明显高于对照组,并且在饲料中添加美人鱼发光杆菌后,明显提高了对虾抵御WSSV感染的能力。其中0.1%美人鱼发光杆菌活菌实验组的抗病毒感染能力最强,WSSV感染14d内累计死亡率为63.3%±5.8%;而对照组为96.7%±3.3%。研究表明,美人鱼发光杆菌添加在对虾饲料中能提高凡纳滨对虾非特异性免疫水平,增强抵抗疾病的能力,将其作为对虾免疫增强剂具有良好的应用前景。     

Effects of Spirulina platensis as a feed additive on growth and coloration of blue dolphin cichlids (Cyrtocara moorii Boulunger, 1902)

This study was carried out to investigate the effects of replacing fish meal with dietary Spirulina as a feed supplement on the growth performance and coloration of blue dolphin cichlids (Cyrtocara moorii). Five isonitrogenous (47% crude protein) and isocaloric (17.36 kJ/g digestible energy) diets were for formulated to replace FM with 0, 5, 10, 15 and 20% Spirulina (designated as Control, SP5, SP10, SP15 and SP20 respectively) and fed to the fish (initial body weight, 3.15 ± 0.01 g). Fish were randomly distributed into fifteen 120 L aquariums (26.5 ± 1.00°C), 15 fish per aquarium. The diets were tested in triplicate for 12 weeks. Experimental groups were fed twice daily (09:00 and 17:00) by hand to satiation. At the end of the feeding trial, significantly (p < 0.05) higher weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER) and lower feed conversion ratio (FCR) were observed in fish fed the SP10 diet when compared to the SP20 diet. There was no significant difference in these parameters between the other groups. The skin coloration of blue dolphin cichlid fed a diet containing Spirulina meal was enhanced. The best coloration was observed in the SP15 group. These impressions were objectively validated by chemical determinations of carotenoids extracted from fish skins and passed statistical tests of significance. The study findings show that Spirulina meal does not diminish growth rates except at very high levels.     

Dietary thiamin and pyridoxine requirements of fingerling Indian major carp,Cirrhinus mrigala (Hamilton)

Two experiments were conducted to quantify the dietary thiamin (experiment I) and pyridoxine (experiment II) requirements of fingerling Cirrhinus mrigala for 16 weeks. In experiment I, dietary thiamin requirement was determined by feeding seven casein–gelatin‐based diets (400 g kg^?1 CP; 18.69 kJ g^?1 GE) with graded levels of thiamin (0, 0.5, 1, 2, 4, 8 and 16 mg kg^?1 diet) to triplicate groups of fish (6.15 ± 0.37 cm; 1.89 ± 0.12 g). Fish fed diet with 2 mg kg^?1 thiamin had highest specific growth rate (SGR), protein retention (PR), RNA/DNA ratio, haemoglobin (Hb), haematocrit (Hct), RBCs and best feed conversion ratio (FCR). However, highest liver thiamin concentration was recorded in fish fed 4 mg thiamin kg^?1 diet. Broken‐line analysis of SGR, PR and liver thiamin concentrations exhibited the thiamin requirement in the range of 1.79–3.34 mg kg^?1 diet (0.096–0.179 μg thiamin kJ^?1 gross energy). In experiment II, six casein–gelatin‐based diets (400 g kg^?1 CP; 18.69 kJ g^?1 GE) containing graded levels of pyridoxine (0, 2, 4, 6, 8 and 10 mg kg^?1 diet) were fed to triplicate groups of fish (6.35 ± 0.37 cm; 1.97 ± 0.12 g). Fish fed diet containing 6 mg kg^?1 pyridoxine showed best SGR, FCR, PR, RNA/DNA ratio, Hb, Hct and RBCs, whereas maximum liver pyridoxine concentration was recorded in fish fed 8 mg kg^?1 dietary pyridoxine. Broken‐line analysis of SGR, PR and liver pyridoxine concentrations reflected the pyridoxine requirement from 5.63 to 8.61 mg kg^?1 diet. Data generated during this study would be useful in formulating thiamin‐ and pyridoxine‐balanced feeds for the intensive culture of this fish.     

Sea bream culture in Egypt; status, constraints and potential

Marine fish farming in Egypt began in 1976 with the culture of gilthead sea bream, (Sparus aurata) as this fish was notably adaptable to brackish and marine pond conditions. Today, marine fish and shrimp farms amount to about 19,000 ha, out of which 42% is already in production while the rest, i.e., 58% is still under construction. In 1997, cultured gilthead sea bream production of 2,250 tons made up 3% of the 75,000 tons total aquaculture catch. In polyculture, usually with the grey mullet and sea bass, gilthead sea bream contributed 440 kg ha^–1 to the total yield of 1,700 kg ha^–1 (26%) over a period of 16 months. For the same period, the yield of monoculture ponds averaged 100 kg ha^–1, while in marine cages, yields ranged from 4–10 kg m³. In 1996–1997, fry of 0.25–1 g and fingerlings 1–10 g with a total of 3 million, were collected from the wild and 1 million fry were produced in the three marine hatcheries out of the four existing ones. The development of sea bream culture in Egypt is now severely inhibited by the shortage of seeds and adequate feeds. Exports of both sea bream and sea bass, during 1994–1996 averaged 1,300 tons per year.     

A Minchinia mercenariae‐like parasite infects cockles Cerastoderma edule in Galicia (NW Spain)

The cockle Cerastoderma edule fishery has traditionally been the most important shellfish species in terms of biomass in Galicia (NW Spain). In the course of a survey of the histopathological conditions affecting this species in the Ria of Arousa, a haplosporidan parasite that had not been observed in Galicia was detected in one of the most productive cockle beds of Galicia. Uni‐ and binucleate cells and multinucleate plasmodia were observed in the connective tissue mainly in the digestive area, gills and gonad. The parasite showed low prevalence, and it was not associated with abnormal cockle mortality. Molecular identification showed that this parasite was closely related to the haplosporidan Minchinia mercenariae that had been reported infecting hard clams Mercenaria mercenaria from the Atlantic coast of the United States. The molecular characterization of its SSU rDNA region allowed obtaining a fragment of 1,796 bp showing 98% homology with M. mercenariae parasite. Phylogenetic analysis supported this identification as this parasite was clustered in the same clade as M. mercenariae from the United States and other M. mercenariae‐like sequences from the UK, with bootstrap value of 99%. The occurrence of M. mercenariae‐like parasites infecting molluscs outside the United States is confirmed.     

A novel approach to denitrification processes in a zero-discharge recirculating system for small-scale urban aquaculture

This paper presents an innovative process to solve the nitrate build-up problem in recirculating aquaculture systems (RAS). The novel aspects of the process lie in a denitrification bioreactor system that uses solid cotton wool as the primary carbon source and a unique degassing chamber. In the latter, the water is physically stripped of dissolved gaseous O₂ (by means of a Venturi vacuum tube), and the subsequent denitrification becomes more efficient due to elimination of the problems of oxygen inhibition of denitrification and aerobic consumption of cotton wool. The cotton wool medium also serves as a physical barrier that traps organic particles, which, in turn, act as an additional carbon source for denitrification. Operation in the proposed system gives an extremely low C/N ratio of 0.82 g of cotton wool/g of nitrate N, which contributes to a significant reduction of biofilter volume. The additional advantage of using solid cotton wool as the carbon source is that it does not release organic residuals into the liquid to be recycled. Operation of the system over a long period consistently produced effluents with low nitrate levels (below 10 mg N/l), and there was only a very small need to replace system water. The overall treatment scheme, also incorporating an aerobic nitrification biofilter and a granular filtration device, produced water of excellent quality, i.e., with near-zero levels of nitrite and ammonia, a sufficiently high pH for aquaculture, and low turbidity. The proposed system thus provides a solution for sustainable small-scale, urban aquaculture operation with a very high recovery of water (over 99%) and minimal waste disposal.     

Fertilization by refrigerator stored sperm of the Indian major carp, Labeo calbasu (Hamilton, 1822)

This study reports on the spermatological properties, and on the development of a protocol for refrigerator storage (4°C) of Labeo calbasu (Hamilton, 1822) sperm for artificial breeding. Volume, motility, concentration and pH of the freshly collected sperm were 2.21 ± 0.53 (μL g^?1 of fish weight) (mean ± SD), 95 ± 1 (%), 1.93 ± 0.44 × 10⁹ (cells mL^?1) and 7.56 ± 0.17 respectively. Sperm activation was evaluated at different osmolalities of NaCl solution, and motility ceased completely when osmolality of the extender was ≥287 mOsmol kg^?1. Sperm retained motility for 24, 72 and 108 h, after refrigerator storage when sperm were undiluted, suspended in Alsever's solution and suspended in Alsever's solution containing 5% methanol respectively. Fertilization rate of fresh eggs with sperm stored at 4°C in Alsever's solution and Alsever's solution containing 5% methanol was 77% and 60% with a hatching rate of 60% and 43% respectively. The fertilization and hatching success of the stored sperm suggests potential to use refrigeration for transporting genetic material to hatcheries for artificial breeding of L. calbasu in Bangladesh.     

Effects of metomidate anaesthesia or transfer to pur sea water on plasma parameters in ammonia-exposed Atlantic salmon (Salmo salar L) in sea water

Atlantic salmon (Salmo salar L) postsmolts weighing 150 ± 53 g were exposed to 14–15 mg l^–1 TA-N (total ammonia-N) in sea water in 1 m³ tanks for 24h. Blood samples were then taken A) immediately after the fish were netted from the exposure tanks and stunned by a blow to the head; B) 2–20 min after the fish were transferred to 15 l of an anaesthetic solution of metomidate in ammonia-free sea water; or C) 2–20 min after the fish were transferred to 15 l of ammonia-free sea water. Plasma TA-N level was 18% lower in the anaesthetised fish compared to in the fish sampled directly from the exposure tanks (p 0.05), and accordingly 16% lower in the fish transferred to pure sea water although this difference was not significant (p = 0.07). Plasma glucose level was higher in the fish transferred to pure sea water than in the fish receiving the two other treatments (p 0.05), but plasma urea, osmolality, Na^{+, Cl–, Ca2+ or Mg2+} levels did not vary significantly between the different treatments. Plasma TA-N level increased with time in the fish in the metomidate solution (p 0.02).