基于RNA-seq技术的江鳕转录组SSR位点信息分析

为开展江鳕(Lota lota)遗传多样性、系统分化分析,开发有效分子标记,采用转录组测序RNA-seq方法,挖掘江鳕微卫星标记。结果显示:通过聚类、从头组装和拼接,获得Unigene共计106 084条。所有的Unigene中,共识别17 619个SSR位点,包含SSR位点的Unigene序列数量为10 893条,占总Unigene的10.27%。江鳕转录组中SSR含量较为丰富,一至六核苷酸重复类型均存在。其中,单核苷酸重复类型的数量最多(7 102个),占总SSR位点数的40.31%。江鳕转录组SSR位点中,以10次重复次数最多,达2 788个位点,占总SSR位点的15.82%。江鳕转录组SSR的片段长度从10～66 bp均有分布,大部分集中在10～24 bp,占SSR总数的87.24%。     

7种珍珠贝RAPD鉴别标记的初步研究

采用RAPD分子标记技术对珠母贝属的大珠母贝、珠母贝、黑珠母贝、白珠母贝、合浦珠母贝、长耳珠母贝和珍珠贝属的企鹅珍珠贝的基因组DNA的特异性遗传标记进行分析。从21个OPM和S系列中筛选出4个引物,共扩增出57个位点,每条引物平均产生14·3个位点。扩增片段大小在250~2000bp间,平均每种贝每条引物产生4·9条带。其中引物S10对7种珍珠贝的RAPD产物呈现出物种的特异性,可同时将7种珍珠贝分开,其余引物可以将2种或2种以上的珍珠贝区别开来。引物S10可以作为种间鉴定的标记。     

太平洋鳕RAG1和IgM基因荧光定量PCR方法的建立及初步应用

为研究太平洋鳕发育早期特异免疫系统形成的机制,通过RAG1和IgM基因的转录水平衡量特异免疫系统的发育特点.根据GenBank中RAG1和IgM的序列信息,分别设计1对特异引物,从太平洋鳕头肾中扩增得到RAG1和IgM的基因片段.将所获基因片段分别插入到克隆载体pMD18-T中,从而构建太平洋鳕RAG1和IgM基因的质粒标准品.建立并优化太平洋鳕RAG1和IgM基因绝对荧光定量PCR方法.为进一步验证该方法的可靠性,分别利用绝对定量和相对定量检验目的基因在太平洋鳕早期发育过程不同组织内的表达差异.以优化后的绝对荧光定量PCR方法检测不同发育时期太平洋鳕RAG1和IgM的表达情况.结果显示,RAG1的回归方程为y=-3.266x+33.77,回归系数R2=0.996;IgM的回归方程为y=-3.119x +27.61,回归系数R2 =0.998.绝对定量和相对定量结果在基因转录趋势上显现出一致性,即RAG1基因在胸腺和头肾中表达,且在胸腺中的表达量显著高于头肾中的表达量,在肝脏和脾脏中无表达;IgM基因在胸腺、头肾、肝脏和脾脏中均有表达,其中脾脏中表达量最高,其次是头肾.RAG1基因在太平洋鳕发育早期的表达水平很低,到61日龄(days posthatching,dph)至95 dph表达量显著提高;IgM基因在早期表达水平同样很低,到33 dph至61 dph才有明显表达,在95 dph时表达量显著提高.研究表明,本实验方法可靠,特异性较强,可成功对目标基因转录水平进行检测.     

三疣梭子蟹多态性微卫星DNA标记的筛选及评价

根据前人FIASCO方法构建的三疣梭子蟹微卫星富集文库,对含微卫星的DNA序列设计了71对引物并对其进行了多态性筛选,筛选出21对多态的微卫星引物,对三疣梭子蟹1个野生群体的30个个体进行遗传多样性检测,同时对引物进行评价。21个位点共获得了188个等位基因,平均每个位点扩增得到8.9个等位基因。不同引物获得的等位基因数差异较大,从3～13个不等,其中Pot8、Pot37、Pot48、Pot53、Pot54、Pot66六个位点分别获得了11、12、12、11、13、11个等位基因,而Pot46仅获得了3个等位基因。等位基因的大小分布在131～312bp,基本符合引物设计时理论产物长度。21个微卫星位点的期望杂合度的范围为0.659～0.889,PIC值均高于0.5,表明它们都有很高的杂合度,均可用于三疣梭子蟹种群遗传结构分析,为三疣梭子蟹品种选育、种系评估提供更多的微卫星DNA信息。     

急性温度胁迫对太平洋鳕仔稚鱼成活率、生理生化指标的影响

为探究温度胁迫对太平洋鳕的影响,以太平洋鳕仔稚鱼为研究对象,研究了温度胁迫对太平洋鳕仔稚鱼的成活率、总超氧化物歧化酶、过氧化氢酶、丙二醛的活性的影响。试验结果表明,培育温度8℃组(对照组)成活率最高(99%),15℃组的成活率最低(43.3±10.3)%,差异显著(P0.05)。2、5、11、15℃胁迫组的总超氧化物歧化酶活性在24h后急剧升高,与8℃组差异显著(P0.05)。72h后各组总超氧化物歧化酶活性开始下降,与8℃组差异不显著(P0.05)。各胁迫组的过氧化氢酶活性在48h时开始显著升高(P0.05),表明过氧化氢酶活性的变化较总超氧化物歧化酶相比具有滞后性。丙二醛活性在胁迫24h时升高(P0.05),但在48h时略有回落,72h时表现出最大的活性。     

太平洋鳕IRF3 基因的克隆及绝对定量表达分析

本研究通过克隆首次得到太平洋鳕(Gadus macrocephalus)干扰素调节因子3(interferon regulatory factor 3,IRF3)的部分序列。该序列长1878 bp,包含长1377 bp的完整开放阅读框(open reading frame,ORF),编码459个氨基酸,其编码的蛋白质分子量约为51 k D。经氨基酸序列比对发现,太平洋鳕IRF3的氨基酸序列包括1个含有5个保守色氨酸残基的DNA结合域(DNA binding domain,DBD)和1个保守的C端IRF关联域(IRF association domain,IAD);系统进化树分析表明,太平洋鳕IRF3与其他物种的IRF3亲缘关系较近。应用绝对荧光定量PCR方法对IRF3在不同组织和不同日龄(days post-hatching,dph)仔鱼的表达水平进行检测,并对干扰素在仔鱼期的免疫作用进行分析。组织表达绝对定量结果显示,性腺,肝和胸腺表达量高于其他组织。不同日龄仔鱼IRF3表达量结果显示,IRF3在受精卵就已经表达,在25 dph表达量大幅提高。本研究的结果为进一步研究太平洋鳕干扰素作用机制以及其在早期发育中的作用奠定了基础。     

泥蚶34个EST-SSR标记的开发及在格粗饰蚶中的通用性检测

利用泥蚶转录组高通量测序、拼接获得的大量EST序列开发SSR标记,在1123条EST序列里筛查到73条含有SSR位点的EST序列,其中54个位点适合设计引物,在位点两侧设计引物并进行PCR扩增.结果显示,46对引物获得稳定扩增的位点,引物在泥蚶奉化群体的多态性检测中发现,有34对引物表现出多态性,共扩增出122个等位基因,各位点的等位基因数Na为2～7个,平均每个位点产生3.59个等位基因,观测杂合度Ho、期望杂合度He、多态信息含量PIC范围分别为0.000 ～ 0.600、0.078 ～0.771、0.106～0.718;Hardy-Weinberg平衡检测显示,13个位点偏离了平衡状态;用Nr和Swiss-Prot蛋白质数据库对含有多态性SSR的EST进行了基因注释,25个SSR位点来自注释基因序列.将34对泥蚶多态性SSR引物在格粗饰蚶中进行了通用性检测,结果有1 1对成功扩增,8对表现为多态,通用率为23.53％,这些通用引物可用于两种蚶的遗传多样性评价、系统进化分析、比较作图和基因发掘等研究.     

AFLP分析黄颡鱼雌雄个体的遗传差异

用20个AFLP引物组合分析了黄颡鱼(Pelteobagru fulvidraco)雌、雄个体的遗传差异,共获得397个位点,雌性个体获得371个位点,多态位点比例为41.24%,雄性个体检测到391个位点,多态位点比例较高,达到93.35%,雌性和雄性的Nei's基因多样度分别为0.1418、0.3691,Shannon's信息指数为0.2094、0.5350,雌雄个体间的相似系数为0.8225～0.9889,遗传距离为0.0112～0.1775,用PHYLIP V3.6软件绘制了个体间的聚类图。另外,在4个AFLP引物组合中,共发现了6个位点在雌雄个体间表现出明显的扩增差异,其中4个位点显性条带个体为(?)性的比例为66.7%～80%,2个位点显性条带个体为(?)性的比例为100%。     

江鳕人工繁殖试验

江鳕(Lota lota)属鳕形目,鳕科,江鳕属。肉食性。环境条件好、食物充足时,当年鱼可长到300克、两年鱼可长到800克以上,笔者所见最大个体达8千克。在0～26℃的水域中生存,体被暗纹,有细小圆鳞,体表有金钱豹类似的花纹,所以在新疆当地俗称"金钱豹",在黑龙江有地方名:山鲶鱼、山怀子。江鳕的肉质细腻鲜美,无肌间刺,特点是其肝脏大而味美,是鳕科鱼类中制造鱼肝油的唯一淡水品种。在我国仅分布于新疆的额尔齐斯河及东北的黑龙江水系及鸭绿江上游。     

三角帆蚌微卫星位点筛选及多态性分析

采用磁珠富集法,以生物素标记的(CA)10寡核苷酸为探针,构建了三角帆蚌(Hyriopsis cumingii)基因组微卫星富集文库。根据微卫星位点的侧翼序列设计引物,随机挑选扩增出与预期大小相符的32对引物,引物荧光标记后对24个三角帆蚌个体分别进行PCR扩增,共筛选出20对多态性较好的引物。结果表明,20个微卫星位点的等位基因数为5～20,有效等位基因数2.321 3～13.260 3。观察杂合度和期望杂合度分别为0.318 2～1.000 0和0.582 5～0.946 1;PIC值0.547 2～0.919 9;其中14个位点符合Hardy-Weinberg平衡(P>0.05)。本研究筛选的20个微卫星标记可作为三角帆蚌遗传多样性、种群遗传结构等研究的理想分子标记。     

Characterization of Acid-Soluble Collagen From Bone of Pacific Cod (Gadus macrocephalus)

Acid-soluble collagen (ASC) was isolated from Pacific cod (Gadus macrocephalus) bone. The ASC was rich in glycine. The amount of imino acid was lower than that of calf skin collagen, as was the transition temperature (48.6°C). Electrophoresis revealed two different α chains (α₁ and α₂), β-component, and γ-component. Fourier transform infrared spectroscopy measurement showed that ASC was in triple-helix structure. ASC had a solubility greater than 90% in a very acidic pH range (pH 1–4), and the solubility decreased with increasing NaCl concentrations up to 3%. Lyophilized ASC had a network ultrastructure with lace-like fibers, similar to calf skin collagen sponge.     

Evaluation of Electrical Stunning of Atlantic Cod (Gadus morhua) and Turbot (Psetta maxima) in Seawater

The aim of this study was to assess electrical stunning of Atlantic cod and turbot in seawater to develop a protocol for the process of stunning and killing. An induced general epileptiform insult (unconscious) had a duration of 40 ± 27 s (n =14) in cod (2.6 ± 0.5 kg) and 34 ± 18 s (n = 19) in turbot (520 ± 65 g). Seven cod and 3 turbot displayed a physical reaction, and 11 turbot registered an electroencephalogram (EEG) response to pain stimuli administered 30 s post-stun. The heart rate was 32 ± 6 beats/min in cod and 25 ± 7 beats/min in turbot prior to stunning. Post-stunning, the electrocardiogram (ECG) revealed fibrillation and reduced activity post-stun. EEG, ECG recordings, and behavioral observations indicate that when a bipolar square wave current was applied with a frequency of 133 Hz and 43% duty cycle side to side (turbot) and at 170 Hz and 33% duty cycle (cod) head to tail, both species were stunned in seawater at current densities of 3.2 A/dm² and 2.5 A/dm², respectively. For turbot, a 5 s exposure to electricity followed by chilling in ice water for 15 min is sufficient to prevent recovery. For cod, a killing method needs to be established.     

太平洋鳕(Gadus macrocephalus)亲鱼驯化培育与早期发育特征

采捕山东威海外海黄海海域的太平洋鳕(Gadus macrocephalus)亲鱼进行驯化和培育,在人工条件下成功驯化存活野生亲鱼49尾,经短期促熟培育后,通过人工授精方式获得了多批次受精卵.对胚胎和早期仔鱼发育过程进行了观察,详细描述了从受精卵到早期仔鱼各发育时期的形态特征.结果显示,太平洋鳕成熟卵子为沉性卵,圆球形,卵径为0.9-1.1 mm,无油球.胚胎发育分为5个时期,分别为卵裂期、囊胚期、原肠胚期、神经胚期和器官形成期.在水温为9-10℃、盐度为27-29的海水中孵化,受精卵历时312 h 30 min孵化出膜.初孵仔鱼全长为(3.85±0.12) mm,6日龄仔鱼开口,肛门与外界相通,进入混合营养期.8日龄仔鱼卵黄囊消耗殆尽,开始进入外源性营养阶段.仔鱼开口饵料为轮虫(Rotifer),12日龄开始摄食卤虫(Artemia saline)无节幼体.6日龄仔鱼鳔原基形成,16日龄鳔充气成为亮泡状.12日龄仔鱼形成肠道第1个生理弯曲,22日龄仔鱼第2个肠道生理弯曲形成.研究结果可为太平洋鳕亲鱼驯化培育和苗种培育提供基础资料.     

Movements and spatiotemporal distribution of escaped farmed and local wild Atlantic cod (Gadus morhua L.)

Commercial farming of Atlantic cod (Gadus morhua L.) is now being developed in several countries. The ecological consequences of cod culture are poorly understood, but recent research suggests that Atlantic cod are more prone to escape from net pens than Atlantic salmon. Here, we describe the movements and the spatiotemporal distribution of farmed cod after escape relative to wild cod, both during and outside the natural spawning season. The experimental design included simulating escape incidents of farmed cod tagged with acoustic transmitters and using an array of automatic listening stations to monitor their dispersal and distribution. For comparison, local wild cod were monitored using the same array of receivers. The farmed cod dispersed rapidly after a simulated escape, they randomly distributed over large areas and their distribution overlapped with local wild cod. Moreover, escaped farmed fish were found at local cod spawning areas during the spawning season. The study also indicated that the recapture rate of escaped farmed cod was high compared with that of escaped farmed salmon. Thus, while our results showed that there is a considerable potential for ecosystem effects caused by escaped farmed cod, mitigating actions such as an efficient recapture fishery for escapees may be possible.     

Effects of ontogeny, temperature, and light on vertical movements of larval Pacific cod (Gadus macrocephalus)

The role of behavior, especially vertical migration, is recognized as a critical component of realistic models of larval fish dispersion. Unfortunately, our understanding of these behaviors lags well behind our ability to construct three-dimensional flow-field models. Previous field studies of vertical behavior of larval Pacific cod ( Gadus macrocephalus ) were limited to small, preflexion stages (≤11 mm SL) in a narrow range of thermal conditions. To develop a more complete picture of larval behavior, we examined the effects of ontogeny, temperature, and light on vertical responses of larval Pacific cod in experimental columns. While eggs were strictly demersal, yolk-sac larvae displayed a strong surface orientation as early as 1 day post hatch (∼ 5 mm SL). Consistent with field observations, small preflexion larvae (<10 mm SL) showed no response to varying light levels. However, there was a direct effect of temperature on larval behavior: Pacific cod larvae exhibited a stronger surface orientation at 4°C than at 8°C. The behavior of larger, postflexion larvae (>15 mm SL) in experimental columns was consistent with a diel vertical migration and independent of water temperature: fish were more widely distributed in the column, and median positions were consistently deeper at higher light levels. These laboratory observations are combined with observations from discrete-depth (MOCNESS) sampling in the Gulf of Alaska to characterize the vertical distribution of larval Pacific cod and contrast ontogenetic patterns with walleye pollock ( Theragra chalcogramma ). The vertical movements of larval Pacific cod described here will be applied in the development of dispersal projections from Gulf of Alaska spawning grounds.     

Physical and biochemical properties of effluent leaving an onshore Atlantic cod (Gadus morhua,Linnaeus 1758; Gadiformes: Gadidae) aquaculture facility and potential use in integrated multi‐trophic aquaculture

The physical and biochemical properties of Atlantic cod (Gadus morhua) wastes were analysed, and the waste remediation potential of blue mussels (Mytilus edulis) was assessed. Waste generated daily by Atlantic cod represented 24.9% of the cod feed added to the system. Particle distributions determined using a Coulter Multisizer and image analysis revealed that the majority of the particles in terms of numbers occupy the smaller size ranges; however, larger particles occupy a larger proportion of the volume. Effluent was composed of particles <70 μm (36%), 70–500 μm (31%) and particles >500 μm (33%) by weight. The amount of dissolved carbon and nitrogen associated with the effluent represented 3.1% and 3.7%, respectively, of the total feed added to the system daily. Particles <70 μm had significantly less organic matter, lipids and fatty acids and were expected to be ingested more by mussels than larger particles. The major lipid classes present in effluent were free fatty acids, triacylglycerols, phospholipids, acetone mobile polar lipids and sterol. Cod effluent contained two essential fatty acids DHA and EPA, a diatom marker (16:1ω7), as well as two zooplankton markers 22:1ω11 and 20:1ω9, which accumulated in mussels and may serve as markers for aquaculture wastes. Although only 36% of the effluent was of a size suitable for mussel ingestion, this size fraction has the greatest potential to spread to surrounding areas. These particulates may be useful as an alternate food source when natural seston is low.     

The spatial distribution of cod (Gadus morhua L.) spawning grounds in the Kattegat, eastern North Sea

Similar to many other commercial marine fish species, Atlantic cod (Gadus morhua L.) migrate towards specific sites at spawning. These temporal aggregations are generally the most targeted by the commercial fishery. The Kattegat cod has undergone a substantial reduction over the past 25 years and both stock size and spawning stock biomass have remained at very low levels since the end of the 1990s. There is, therefore, an urgent need to map and document spawning grounds still in use. In the present study, spawning sites of Atlantic cod were identified in the Kattegat using a combination of commercial and fishery independent data from 1996 to 2004. Moreover, putative spawning and non-spawning areas were also sampled before and during the reproductive season between 2002 and 2006, and the proportion of mature females together with the individual physiological status were used to validate and strengthen the spatial analyses.

The spatial analyses identified several spawning areas in the region and two areas in the southeastern part of the Kattegat which appeared to be the most important. The results showed the presence of cod spawning aggregations, although reduced in size, in areas that have been utilized for more than 25 years according to historical information. Some local spawning grounds may have also disappeared. The proportion of mature females was higher in putative spawning than in non-spawning areas (p < 0.001) and females from spawning areas had higher gonadosomatic (p < 0.05) and hepatosomatic (p < 0.001) indices than those from non-spawning areas.

Knowledge of stock spatial and temporal distribution is essential in designing recovery strategies for depleted fish populations. The unambiguous stability of the locations of spawning aggregations over time, as shown in this study, represents a useful aid in order to efficiently implement a recovery plan for the collapsing cod stock in this area.     

Spermatocrit and spermatozoa density in Atlantic cod (Gadus morhua): correlation and variation during the spawning season

The utility of spermatocrit (the proportion of solid packed material in semen after centrifugation) as an indicator of spermatozoa density and male spawning stage was tested in Atlantic cod (Gadus morhua). Semen was collected from captive male cod over three spawning seasons. Spermatocrit was positively and significantly correlated with spermatozoa density measured with a Coulter counter (Multisizer), but not with spermatozoa counts in a haemacytometer. Spermatocrit increased significantly as the spawning season progressed. However, day of season explained only 35% of the variation because spermatocrit varied among individual males. Spermatozoa size remained unchanged throughout the sampling period and was not correlated with spermatocrit, indicating that variation in spermatocrit was due to variation in spermatozoa number and not their size. Spermatocrit is a good estimator of sperm density but is not reliable as indicator of spawning stage because of the variation among individual males.     

Quality and Shelf Life of Liver of Farmed Cod (Gadus morhua)

ABSTRACT

Livers of Atlantic cod (Gadus morhua) are traditionally used in cod liver oil production or consumed cooked or canned. The farming of cod is a relatively new industry in Norway. The aim of this study was to determine quality and shelf life of fresh liver from farmed cod during chilled storage on ice by hydrolysis and oxidation state and sensory quality and the influence on canned liver. In two experiments, livers from farmed cod were stored chilled and sampled from Days 0 to 13, respectively. Quality, measured as hydrolytic and oxidation degradation, was reduced after 7 days of storage, while sensory quality was reduced after 4 days. Free fatty acids increased from Day 7 in both experiments, while peroxide value and anisidine value showed no change when the livers were single wrapped. Rancid odor was the first sign of oxidation and was registered after three to four days of storage. Canning within 2 days of storage prevented leakage of oil from the canned livers. Sensory analyses of oxidation are recommended as a sensitive and rapid method to detect oxidation of chilled cod liver.     

Preliminary Observations on Spoilage Potential of Flora from Desalted Cod (Gadus morhua)

Abstract

Desalted cod products are already marketed, although still presenting some problems. Fresh raw materials and good manufacturing practices during salting, drying and desalting are essential to improve their quality and shelf life. The production of off-odor, hydrogen sulfide, trimethylamine, ornithine, putrescine, cadaverine, histamine, indole, hydrolyzed gelatin and ammonia was investigated in 127 bacterial strains isolated from dried/wet salted cod, later desalted either at 4 or 24°C. Desalting could either introduce or recover off-odor, sulfide hydrogen and/or ornithine producing strains. Cod desalting at 4°C is of great importance in order to improve the shelf life and safety of cod desalted products.