虾夷扇贝脓胞病病原的分离、鉴定与致病性

开展对大连市长海县附近养殖海域近几年夏季出现的大规模虾夷扇贝死亡现象的研究,能为虾夷扇贝养殖过程中的疾病防治提供基础理论支持。以患病虾夷扇贝为研究对象,通过对病变组织进行细菌分离、提纯培养,并采用人工感染试验确定虾夷扇贝脓胞病的病原性质;通过生理生化特性、16SrRNA基因部分序列和药物敏感性测定,对虾夷扇贝脓胞病进行了鉴定。结果表明,从患病组织中分离出的其中一株细菌经人工感染试验证实为虾夷扇贝脓胞病的病原菌,在水温19℃、注射浓度为1.09×105CFU/mL的条件下,该菌有较强的致病性。通过对该病原菌的生理生化特性测定和16SrRNA基因部分序列分析,确定虾夷扇贝脓胞病的病原为查氏弧菌。     

微酸性电解水对活品虾夷扇贝存活率的影响及杀菌效果

以活品虾夷扇贝(Patinopecten yessoensis)为实验对象,用不同理化性质的微酸性电解水(slightly acidic electrolyzed water,SAEW)处理受试扇贝,并用副溶血性弧菌人工浸染经微酸性电解水处理过的扇贝,检测其存活率、微酸性电解水杀菌效果、副溶血性弧菌的变化规律,以及虾夷扇贝在不同电解水处理阶段的菌相。结果显示,微酸性电解水处理1 min、2 min、4 min,扇贝存活率均为80%,处理8 min存活率为82%,均高于染菌组和对照组;电解水处理时间与细菌总数和大肠菌群呈显著负相关(P0.05),与处理时采用的电解水有效氯浓度没有显著性关系(P0.05)。采用电解水处理8 min后,活品虾夷扇贝体内染上的副溶血性弧菌数量从1 100 MPN/g降至28 MPN/g。扇贝初期主要菌群为假单胞菌和弧菌,在经过微酸性电解水处理后,其体内的菌群趋于复杂化,优势菌群有所改变,细菌总数有所下降。24 h后经电解水处理或者未处理的虾夷扇贝体内优势腐败菌均为假单胞菌。研究表明,SAEW在虾夷扇贝净化中具有一定的应用潜力。     

虾夷扇贝C型凝集素母源传递与抑菌作用的初步研究

为研究虾夷扇贝C型凝集素的母源传递及其抑菌作用,实验运用qRT-PCR技术检测了经鳗弧菌刺激后虾夷扇贝卵巢中C型凝集素的表达模式,比较分析了C型凝集素基因在正常虾夷扇贝和鳗弧菌刺激的虾夷扇贝所产的卵及其胚胎发育前期的存在与变化;通过抑菌实验研究了卵胞浆中C型凝集素抑制细菌存活的作用.结果表明,鳗弧菌刺激能够诱导虾夷扇贝卵巢中的C型凝集素mRNA表达量显著变化,最高表达量出现在刺激8h后,为对照组的6.2倍;母体中C型凝集素可以传递给卵和胚胎,刺激组表达量极显著高于正常组,且表达量都随胚胎发育逐渐降低,至受精36 h时分别为正常对照卵的0.3和0.2倍;蛋白终浓度为200和400 μg/mL的卵无细胞体系都具有一定的抑菌作用,与C型凝集素家族抗体反应后,细菌存活率显著上升,说明母源C型凝集素在抑制细菌存活方面发挥了重要的作用.     

3种扇贝遗传结构的RAPD分析

用RAPD方法对扇贝科中的虾夷扇贝、海湾扇贝和栉孔扇贝的基因组DNA的特异性遗传标记进行分析,筛选了43个随机引物,有13个引物获得了片段长度在250～2000 bp之间且重复性良好的谱带,每个引物分别获得了4～17个大小不等的片段,共获得了170个扩增片段,其中12个引物得到特异性片段58个,可以将两种或者3种扇贝区别开来。通过Popgene 3.2分析软件包统计表明,虾夷扇贝与海湾扇贝的遗传距离最大,为0.2481;栉孔扇贝与海湾扇贝的遗传距离次之,为0.2161;虾夷扇贝和栉孔扇贝遗传距离最小,其值为0.2133。根据遗传距离指数,用MEGA3统计软件中的UPGMA方法进行聚类分析,构建系统树。     

虾夷扇贝Dmrt1基因的分子鉴定及表达模式分析

Dmrt1 是 Dmrt 双性和 mab-3 相关转录因子基因家族的重要成员之一, 在生殖细胞分化和性别调控中发挥着重要作用。为探究其在虾夷扇贝(Patinopecten yessoensis)性别分化中的表达调控模式, 本研究利用生物信息学的方法分析了虾夷扇贝 Dmrt1 的序列特征; 采用半定量 PCR (RT-PCR)检测了 Dmrt1 在不同组织中的表达量差异; 应用实时荧光定量 PCR (qRT-PCR)和原位杂交技术 ISH (in situ hybridization)揭示了 Dmrt1 在性腺发育 4 个时期(增殖期、 生长期、成熟期、排放期)中的时空表达变化。结果显示, 虾夷扇贝 Dmrt1 序列包含 Dmrt 基因家族共有的 DM 结构域; 与已知物种同源序列比对后发现, 虾夷扇贝 Dmrt1 序列与欧洲大扇贝(Pecten maximus)同源性最高; 原位杂交检测到的阳性信号主要定位在生殖细胞的细胞质中; qRT-PCR 定量结果发现, Dmrt1 在精巢生长期和成熟期的表达水平显著高于卵巢, 在卵巢各发育时期中的表达量无明显变化且维持低水平状态。此外, 在外套膜、鳃、肾、和闭壳肌中检测到少量表达的 Dmrt1 转录本, 而在肝胰腺中未检测到。研究结果表明, Dmrt1 在虾夷扇贝生殖细胞分化过程中的表达特征与在其他动物性腺发育中的表达特征基本一致, 推测其是虾夷扇贝性别分化调控中的关键基因。     

虾夷扇贝EST-SSR标记在栉孔扇贝中的通用性研究

为研究虾夷扇贝(Patinopecten yessoensis)和栉孔扇贝(Chlamys farreri)的群体遗传多样性并分析其亲缘关系,对在虾夷扇贝中开发的60个EST-SSR位点在栉孔扇贝中的通用性进行了研究。结果显示,21个位点可在栉孔扇贝中扩增出特异性条带,通用性比例达35.00%,其中,17个位点具有多态性,多态性比例达28.33%。在虾夷扇贝和栉孔扇贝群体中,平均等位基因数(N_a)分别为2.7647和2.3529;平均有效等位基因数(N_e)分别为1.9487和1.6350;平均观测杂合度(H_o)为0.6314和0.3333;平均期望杂合度(H_e)为0.4569和0.3139;平均多态信息含量(PIC)为0.3726和0.2597;Nei's(1973)基因多样性指数(I)分别为0.4493和0.3087;Shannon信息指数分别为0.7176和0.5041;固定指数(F_(is))检测发现,7个位点偏离了Hardy-Weinberg平衡;遗传分化系数(F_(st))为0.2398。本研究开发的21对通用性EST-SSR标记,为进一步开展虾夷扇贝和栉孔扇贝遗传多样性分析、分子辅助育种、基因发掘和种质资源评价奠定了基础。     

急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组DNA甲基化的影响

为探讨急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组 DNA 甲基化水平的影响,本研究从表观遗传学角度,运用甲基化敏感扩增多态性(Methylation-sensitive amplification polymorphism,MSAP)技术比较分析了升温海水(17℃和24℃)急性胁迫虾夷扇贝(原暂养于9℃的海水中)9 h 和24 h 后基因组 DNA 甲基化的变化情况。结果显示,9对引物在各组的总扩增位点数为314–337,总甲基化位点为79–94,所占比例为23.45%–28.51%;所有处理组基因组 DNA 总甲基化率低于对照组,急性升温胁迫使虾夷扇贝基因组 DNA 发生去甲基化,而且去甲基化程度随胁迫温差增大和胁迫时间增长而增强,说明急性升温胁迫能够改变 DNA 甲基化水平和模式。本研究为进行虾夷扇贝抗逆基因的筛查提供了新思路和研究基础,丰富了表观遗传学在扇贝中的研究资料。     

急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组DNA甲基化的影响

为探讨急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组DNA甲基化水平的影响,本研究从表观遗传学角度,运用甲基化敏感扩增多态性(Methylation-sensitive amplification polymorphism,MSAP)技术比较分析了升温海水(17℃和24℃)急性胁迫虾夷扇贝(原暂养于9℃的海水中)9 h和24 h后基因组DNA甲基化的变化情况。结果显示,9对引物在各组的总扩增位点数为314–337,总甲基化位点为79–94,所占比例为23.45%–28.51%;所有处理组基因组DNA总甲基化率低于对照组,急性升温胁迫使虾夷扇贝基因组DNA发生去甲基化,而且去甲基化程度随胁迫温差增大和胁迫时间增长而增强,说明急性升温胁迫能够改变DNA甲基化水平和模式。本研究为进行虾夷扇贝抗逆基因的筛查提供了新思路和研究基础,丰富了表观遗传学在扇贝中的研究资料。     

科技信息

栉孔扇贝与虾夷扇贝杂交育种技术研究及应用日前,中国水产科学研究院黄海水产研究所杨爱国研究员主持完成的"栉孔扇贝与虾夷扇贝杂交育种技术研究及应用"荣获2008年度山东省科技进步一等奖。该成果对栉孔扇贝和虾夷扇贝杂交的受精生物学过程和受精机制、同步生殖发育调控技术、杂交子代的遗传性     

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.     

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.     

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.     

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).