企鹅珍珠贝微卫星分子标记的筛选

运用生物素与链霉亲和素的强亲和性原理,用生物素-磁珠吸附微卫星富集法,筛选企鹅珍珠贝(Pteria penguin)的微卫星分子标记序列,进而对南海区企鹅珍珠贝的遗传多样性进行分析。对136个菌落中的85个阳性克隆进行微卫星测序,获得64个序列,达到85.93%。所得到的55个重复6次以上的微卫星序列中,除生物素探针中使用的CA重复外,还得到TG、TC、GA的重复序列。重复序列中完美型36个,占65.5%;不完美型14个,占25.5%;混合型5个,占9.1%。利用Primer Premier5.0设计引物40对,合成其中的20对并进行PCR筛选,15对可扩增出特异性条带。研究表明,筛选出的企鹅珍珠贝微卫星分子标记可用于进一步的遗传多样性分析。     

磁珠富集法筛选西施舌微卫星序列

采用生物素-磁珠吸附微卫星富集法（FIASCO）筛选西施舌（Coelomactra antiquata）微卫星分子标记。结果获得含有重复次数不少于5次的微卫星序列38个,其中完美型占68.4%,非完美型26.3%,混合型5.3%。除探针中使用的CA和GA 2种重复外,还得到ATTG、CGTG和TGTTG的重复序列。微卫星重复次数主要集中在5～46次,最高为70次。利用这些微卫星序列设计了14对引物。该研究筛选出的微卫星标记为进一步研究西施舌的遗传多样性提供帮助。     

云斑尖塘鳢微卫星分子标记的筛选与检测

用磁珠富集法分离云斑尖塘鳢的微卫星序列,由所获得的1032个克隆中筛选出146个阳性克隆,经测序81个含有微卫星序列,52个为完美型,27个为非完美型,2个为复合型,其中43个微卫星序列重复次数在10以上。所获得的云斑尖塘鳢微卫星序列中除探针中使用的CA重复单元和GA重复单元外,还有TAC等其他类型的重复单元。设计合成38对微卫星引物,其中29对引物可稳定扩增出条带,使用这些引物对云斑尖塘鳢48个个体进行检测显示:观测杂合度平均值为0.63,期望杂合度平均值为0.43。29对引物中1对引物表现为单态,7对表现为高度多态,14对表现为中度多态,7对表现为低度多态,多态性较为丰富,说明本研究开发的绝大部分微卫星分子标记较为理想。     

牙鲆基因组 (CAG)n微卫星 DNA 特征分析

通过磁珠富集法筛选牙鲆(Paralichthys olivaceus)的微卫星分子标记,采用限制性内切酶Sau 3A Ⅰ对牙鲆完整基因组DNA进行酶切;通过蔗糖溶液梯度离心,收集400～900 bp大小的片段,连接Brown接头,构建牙鲆基因组文库.用生物素标记的微卫星探针(CAG)15,对基因组文库进行杂交,利用磁珠富集含有微卫星的DNA单链序列,并对其进行PCR扩增;将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星序列文库.利用大量质粒检测法进行二次筛选,成功地从牙鲆基因组中分离出含有CAG重复的微卫星序列,测序其中的3000个单菌落,获得2805个(占93.5%)含有微卫星序列的克隆,其中含有微卫星座位3120个,完美型1808个,占57.97%;非完美型226个,占7.25%;混合型1085个,占34.78%.从中选出186个微卫星序列设计120对引物并合成,经过筛选,74对引物可扩增清晰条带,其中68对呈多态性.     

兰州鲇（CAG）n、（GATA）n微卫星文库构建与鉴定

应用磁珠富集法构建兰州鲇（ Silurus lanzhouensis） CAG重复和GATA重复的微卫星文库,并分析其序列特征。兰州鲇基因组DNA经MseI酶切,选取200～800 bp的片段与生物素标记的探针（CAG）8和（GATA）6杂交,捕获到含有微卫星序列的目的DNA片段连接到pMD19-T载体,转化到大肠杆菌DH5α菌株中构建微卫星富集文库,经PCR检测筛选出阳性克隆进行测序。从126个阳性克隆中随机选取96个进行测序,获得59个微卫星序列（ GenBank登录号： KJ545973～KJ545998, KJ598088～KJ598120）。其中完美型31个（52.54％）、非完美型20个（33.9％）、混合型为8个（13.56％）。根据侧翼序列,成功设计48对引物,选取25对微卫星引物在10个个体进行扩增与多态性筛选,共获得10对多态性引物。结果表明,经优化的磁珠富集法能够高效地获得兰州鲇微卫星标记,这些标记将为兰州鲇种质资源保护、微卫星连锁图谱构建、经济性状的QTL定位及分子标记辅助选育奠定基础。     

磁珠富集法与小片段克隆法筛选鲤微卫星的比较研究

用2种方法克隆黑龙江鲤(Cyprinus(Cyprinus)carpio haonatopterus)的微卫星序列。这2种方法分别是：①经典的小片段DNA克隆库,用末端标记的[γ^-32]ATP的CA重复序列为探针筛选;②将酶切获得的小片段DNA用结合有磁珠的并连接有15个CA重复序列的生物素进行富集,获得的含有CA重复的DNA片段并经过两次PCR扩增再克隆的方法获得富集微卫星片段的克隆库。从前一个方法的克隆库中筛选2000个菌落,获得阳性克隆45个,有22个含有微卫星,完美型的占63．6％,非完美型的占22．7％,混合型的占13．7％,重复次数超过10的有9个,占40．9％;从方法②的克隆库中筛选2600个菌落,获得阳性克隆1300个,测序其中的390个克隆,微卫星314个,完美型的占79．0％;非完美型的占14．3％;混合型的占6．7％,重复次数超过10的有293个,占93．3％。结果表明,用生物素结合磁珠富集法克隆微卫星效率高,成本低,所获微卫星质量高,是一种值得推荐的微卫星制备方法。     

太湖新银鱼微卫星位点的分离与序列特征分析

为了开发太湖新银鱼（Neosalanx taihuensis）的微卫星分子标记,采用生物素标记探针（AC）12、（AAC）10、（AAG）10和（GATA）8对其微卫星位点进行了筛选,并对其序列特征进行了分析。共获得490个微卫星序列,筛选的总效率为78．09％。筛选出725个微卫星位点,其中完美型592个,占81．66％;混合型82个,占11．31％;非完美型51个,占7．03％。探针（AC）12富集到的微卫星重复次数大多集中在14～26次,最高44次;探针（AAC）10和探针（AAG）10富集得到的微卫星重复次数主要集中在8～20次,最高42次;探针（GATA）8富集得到的微卫星重复次数一般在6～15次,最高32次。探针（AC）12、（AAC）10、（AAG）10和（GATA）8的杂交效率分别为85．19％、60．19％、5．16％和10．64％。筛选得到的725个微卫星位点中,二碱基重复位点390个（53．79％）,三碱基重复位点284个（39．17％）,四碱基重复位点47个（6．48％）,五碱基重复位点 1个（0．14％）和六碱基重复位点3 个（0.42％）。根据二碱基重复核心序列进行引物设计,对抚仙湖24尾太湖新银鱼样本进行 PCR 扩增,电泳结果显示部分微卫星位点有较高的多态性,同时本研究获得的三碱基、四碱基、五碱基和六碱基重复位点也可为长重复单元微卫星引物的开发提供基础。     

大黄鱼EST微卫星标记初步筛选

通过构建大黄鱼性腺线性化cDNA文库，并经测序后获得3535条EST，对其二碱基至六碱基重复序列进行筛选，共发现微卫星位点150个，占EST序列的4．24％；其中包括二碱基重复序列64条，三碱基重复序列80条，四碱基重复序列5条，五碱基重复序列1条；三碱基重复序列是最丰富的重复单元，占53．3％。在这些微卫星序列中，（TG／GT／AC／CA）n形式在二碱基重复中最为常见，（GAG／AGG／GGA／CCT）n形式在三碱基重复序列中最为常见，分别占微卫星序列总数的26％和14％。选取其中的62条微卫星序列进行引物设计、合成与多态性检测，经过PCR扩增，2．0％的琼脂糖凝胶电泳检测，获得呈多态性微卫星引物8对，多态率为12．9％。本研究为开发大黄鱼EST微卫星分子标记和大黄鱼基因编码区微卫星的功能研究提供有价值的信息。     

青石斑鱼微卫星DNA 标记的筛选及群体遗传多样性分析

以中国南海海域青石斑鱼（Epinephelus awoara）为材料，构建青石斑鱼小片段部分基因组DNA文库。以M13通用引物和设计合成的微卫星核心序列引物（CA）15,用PCR法对文库进行筛选，共获得96个微卫星序列，分别分布于28个阳性重组克隆中，其中perfect（完美型）共39个（占40．6％），imperfect（非完美型）30个（占31．3％），compound perfect（混合完美型）7个（占7．3％），compound imperfect（混合非完美型）20个（占20．8％）。同时发现（CA／GT）。序列在青石斑鱼的基因组DNA中含量非常丰富。根据微卫星侧翼序列设计28对引物扩增青石斑鱼基因组DNA，有26对引物能扩增出目的片段，选用其中13对多态性稳定的引物对19尾青石斑鱼进行遗传多样性分析。结果显示，13个位点共检测到48个等位基因，平均观测杂合度（Ho）为0．5982，平均期望杂合度（He）为0．5080，平均多态信息含量（PIC）为0．4722，平均Hardy-Weinberg遗传偏离指数（D）为0．1503。实验初步表明中国南海海域青石斑鱼的遗传多样性较为丰富，但在某种程度上受到了人为的干扰。     

日本蟳微卫星富集文库的建立与多态性标记的筛选

采用磁珠富集法筛选日本蟳微卫星分子标记。日本蟳基因组DNA经Sau3 AⅠ酶切后,收集400~1 200 bp大小的片段并纯化,利用生物素标记的寡核苷酸探针(AC)¹⁵从中筛选出含有微卫星序列的DNA片段,连接到pMD18-T载体中,构建富集微卫星序列的基因组文库,经PCR检测筛选出阳性克隆进行测序。从随机挑选的970个菌落中筛选出369个阳性克隆进行测序,结果86.99%(321个)含有微卫星序列,其中完美型占80.54%,非完美型占15.95%,混合型占4.28%。除使用的探针AC重复外,还得到GA、CT等重复序列。共设计出102对微卫星引物,其中65对能扩增出清晰条带,27对具有多态性。同时筛选出的微卫星标记可为今后研究日本蟳的分子遗传育种提供有效的遗传标记。     

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).