岩牡蛎正常和低盐胁迫下定量PCR内参基因的筛选与验证

实时荧光定量 PCR 已广泛用于基因表达的分析,合适的内参基因选择是获得准确分析结果的关键。本研究选用正常生理状态下岩牡蛎(Crassostrea nippona)的鳃、外套膜、闭壳肌和内脏团 4 种组织以及盐度 10、20 和 30暂养 1 周后的鳃组织为材料,对已报道的常见内参基因(EF1A、GAPDH、RO21、TUB 和 TUA)的稳定性通过 3 种方法(geNorm、NormFinder 和 BestKeeper)进行分析和筛选,发现针对鳃单一组织在不同盐度胁迫下, GAPDH 和RO21 可以作为合适内参基因,而在不同组织中,需要更多的内参基因联合分析才能获得准确的定量表达结果。本研究是首次利用 q-PCR 对岩牡蛎进行内参基因的筛选和验证,为今后该物种低盐胁迫相关的基因表达和功能基因的研究提供重要依据。     

牡蛎的品质及其开发利用

牡蛎是沿海习见且大量生长的一种贝类，属于软体动物门、瓣鳃纲、牡蛎科。我国沿海盛产的牡蛎，主要有近江牡蛎、长牡蛎、褶牡蛎、花缘牡蛎、大连湾牡蛎、密鳞牡蛎等。牡蛎是广东沿海养殖的主要贝类之一，碍石湾、红海湾、深圳湾、唐家湾、镇海湾和海陵湾等地有大量养殖。     

长牡蛎诱导三倍体与二倍体的养殖生物学比较研究

许多水产动物的三倍体在养殖生产中显示出很大的增产潜力 ,因此 ,采用染色体操作技术诱导三倍体的研究越来越受到人们的重视。长牡蛎( Crassostrea gigas)是我国沿海重要的经济贝类 ,长期以来 ,由于牡蛎繁殖季节大量排放精、卵 ,一是引起蛎肉瘪小充水 (成为透明状水蛎 ) ,致使肉质变劣而无法上市。二是雌性牡蛎大量排卵导致生理活性降低 ,出现大批死亡。近年来 ,笔者对长牡蛎三倍体的诱导和培育技术进行了深入、系统的研究 ,在三倍体生长发育、生殖腺组织学和超微结构变化、三倍体软体组织生化成分含量周年季节变化以及二倍体和三倍体的鉴…     

悬沙胁迫下熊本牡蛎的损伤及恢复

首先将熊本牡蛎(Crassostrea sikamea)暴露于不同质量浓度(0、50、100、500、1000和5000 mg/L)悬沙中15d后,再将受试牡蛎转移到干净海水中恢复培养15 d,观察牡蛎的存活率,检测鳃丝中Na+-K+-ATP酶、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性变化、鳃细胞DNA损伤情况和肌肉中RNA/DNA比率变化,探讨悬沙对牡蛎的影响。结果表明,悬沙暴露试验后,本研究所设悬沙浓度对牡蛎存活率几乎无影响,但当悬沙质量浓度≥500mg/L时,鳃丝中Na+-K+-ATP酶、SOD和CAT活性开始显著降低,鳃细胞中开始检测到明显的DNA损伤,反映牡蛎生长状况的RNA/DNA比率也较对照组明显减小(P<0.05)。这些受损牡蛎经干净海水培养15 d后,检测的生理生化指标虽有一定程度的恢复,但仍未恢复到对照组和低浓度悬沙组的水平。可见,较高水平的悬沙胁迫对牡蛎并没有产生致命的影响,依据常规的生态毒理学指标如存活率并不能得出有效的评价结果,但分子细胞水平上的各指标就能比较灵敏地反映悬沙胁迫的影响。     

僧帽牡蛎和华贵栉孔扇贝染色体的制作

本实验以僧帽牡蛎和华贵栉孔扇贝的胚胎、担轮幼虫和鳃组织等为材料，经秋水仙素处理，海水蒸馏水低渗，甲醇、冰醋酸固定液固定后得到的细胞悬液、压片或滴片，醋酸洋红或吉姆莎染料染色，最后获得较理想的染色体玻片标本。     

5种壳色长牡蛎不同组织中金属元素的分析与评价

为评估不同壳色长牡蛎金属元素价值,实采用电感耦合等离子体原子发射光谱仪(ICP-OES)和近红外(NIR)分析模型对5种壳色(壳黑、壳紫、壳橙、壳金和壳白)长牡蛎4个组织(外套膜、鳃、闭壳肌和性腺—内脏团)中的Mg、Fe、Zn、Cu、Mn和Se元素含量进行检测分析,并比较与对照群体金属元素含量的差异以及5种壳色长牡蛎各组织间含量的差异。结果发现,在外套膜中,壳紫、壳橙和壳黑长牡蛎中Zn含量显著高于对照群体,壳紫长牡蛎Cu含量显著高于壳金和对照群体,壳金长牡蛎Mn含量显著高于壳紫和壳黑长牡蛎,其他元素在各群体中含量无显著差异;在鳃中,壳金长牡蛎Mn含量显著高于壳黑和对照群体,其他组份在6个群体无显著性差异;在闭壳肌中,壳橙和壳紫长牡蛎Zn的含量显著高于对照群体,其他组份在各群体中差异不显著;在性腺—内脏团中,壳橙长牡蛎Zn和Mn含量均显著高于对照群体,且Cu的含量显著高于壳金长牡蛎,其他元素在6个群体中含量无显著性差异。4个组织中各金属元素含量差异显著,其中闭壳肌内各金属元素含量显著低于鳃、外套膜和性腺—内脏团。研究表明,不同壳色长牡蛎之间以及不同壳色与对照群体之间在部分金属元素含量上已表现出分化,这为长牡蛎壳色新品系选育提供了重要的基础资料。     

三疣梭子蟹鳃病的病因与防治

鳃是三疣梭子蟹重要的呼吸器官。三疣梭子蟹鳃器官由左右 5对主鳃叶和 1对小鳃叶组成 ,每一鳃叶由许多鳃片构成 ,鳃片表面密布许多刺状或疣状突起 ,从而大大增加了鳃与水的接触面积 ,使机体与水环境的气体交换能够高效率进行。当鳃发生病变或堵塞时 ,则会影响三疣梭子蟹的正常呼吸。病蟹会通过自身调节 ,提高呼吸运动节律来弥补鳃病引起的气体交换的不足。当鳃的气体交换量降低到不能维持最低生命活动需要时 ,三疣梭子蟹就会发生窒息死亡。海洋环境污染和养殖生产自身带来的污染使鳃病成为现代水产养殖业的通病 ,鳃病也成了三疣梭子蟹人工养…     

0号柴油水溶性成分对僧帽牡蛎脂质过氧化的影响

在实验条件下,将僧帽牡蛎分别置于含有0.5、2、5 mg·L-10号柴油水溶性成分的海水中,在污染后1、4、7、11、15 d采样,15 d后转入清洁海水中进行6 d的恢复实验,采样,观察消化腺和鳃脂质过氧化的程度。结果表明:(1)正常生理条件下消化腺和鳃也有一定程度的脂质过氧化,消化腺显著高于鳃;(2)脂质过氧化的程度随污染时间的延长而增加,并存在一定的剂量-效应关系,可作为监测海洋石油污染的生物标志物;(3)解除污染后,脂质过氧化恢复到对照水平。     

鱼类几种烂鳃病的应对措施

鳃是鱼类重要的呼吸器官,由鳃弓、鳃耙和鳃丝组成.鳃丝组织分布许多毛细血管,水中溶氧通过鳃丝表皮细胞渗透进入血液达到气体交换.鲢鳙等滤食性鱼类通过鳃耙将水体浮游生物过滤进入食道而摄食.同时鳃具有调节体内渗透压作用,可以将过量氨氮等机体代谢产物经鳃器官排泄到体外,如果鳃组织发生病变,将直接破坏呼吸功能,威胁鱼类的生存.所以,在水产养殖过程中及时预防和治疗鳃病显得尤为重要.根据笔者多年生产实践总结,将鱼类烂鳃病的分类及应对措施介绍如下,供水产同仁参考. 1 寄生性烂鳃 1.1 流行季节 流行时间为每年4-9月. 1.2 主要症状 病鱼鱼体发黑,尾柄灰白,鳃盖张开难以闭合,鳃丝发白并参差不齐,鳃片表面黏液增多,鳃片腐烂,镜检可见大量寄生虫如:中华鳋、锚头鳋、指环虫、三代虫、车轮虫、孢子虫等. 1.3 防治方法     

原子吸收法测定大嶝海域牡蛎中微量元素含量

采用火焰原子吸收光谱法对大嶝海域牡蛎鳃和肚腹中微量元素的含量进行测定,同时用加标回收法检验方法的可靠性。研究结果表明,牡蛎含有丰富的微量元素,鳃中Cu、Zn、Mn、Fe的含量分别为15.35±0.36μg/g、72.86±1.12μg/g、11.39±0.76μg/g、40.07±0.40μg/g;肚腹中Cu、Zn、Mn、Fe的含量分别为5.37±0.40μg/g、41.93±0.81μg/g、6.74±0.34μg/mg、24.89±0.53μg/mg。牡蛎鳃中Cu、Zn、Mn、Fe的含量均高于肚腹。用原子吸收方法测定牡蛎中微量元素的效果较好,回收率为89.0%～102.5之间,相对标准偏差在1.4%以下。     

Parasites, pathological conditions and mortality in QX-resistant and wild-caught Sydney rock oysters, Saccostrea glomerata

Differences in the survival of QX-resistant fifth generation (QXR₅) and wild-caught (Wild-Caught) Sydney rock oysters were assessed over the QX-disease risk period in the Pimpama River, SE Queensland. Cumulative mortality of Wild-Caught oysters (31.7%) was significantly greater than QXR₅ oysters (0.0%) over the 118 days of the experiment. PCR and histological results showed that Wild-Caught oyster did not die from QX disease. Histology revealed oysters were infected with disseminating hemocytic neoplasia, a Steinhausia-like infection, a Rickettsia-like organism infecting epithelial cells of the gill, digenean flukes encysted in the gonadal tissue and a gill response to an unknown toxin. The cause of mortality is attributed to disseminating hemocytic neoplasia.     

Pathology and mass mortality of Pacific oysters, Crassostrea gigas (Thunberg), in 2005 at the East Frisian coast, Germany

In 2005, Pacific oysters, Crassostrea gigas , were collected from May to September along the East Frisian coast and processed for histology. Because of mass mortalities in September, additional samples of moribund oysters and apparently healthy blue mussels, Mytilus edulis , were subjected to virological and ultrastructural investigation. The oysters displayed a variety of pathological conditions including viral gametocytic hypertrophy which is reported here for the first time from the German coast. Haemocyte aggregations in the digestive tract, in the intestinal mucosa and submucosa, in the mid-gut gland and in the ventricle of the heart were commonly observed at some stations. In association with mass mortalities, severe gill necrosis occurred which may have contributed to the high mortality rates. Total mortality rates of up to approximately 60% were seen. All size classes and thus age classes of oysters were affected, with highest mortality rates within the youngest age classes which had just reached sexual maturity (shell lengths <40 mm). The smallest dead oysters had shell lengths of 10 mm. The phenomenon was mainly restricted to C. gigas stocks in harbours, probably because of favourable conditions for infection, i.e. limited water exchange, less food availability, reduced oxygen content and higher pollution levels.     

Biochemical changes in oyster tissues and hemolymph during long-term air exposure

The Pacific oyster Crassostrea gigas is a sessile bivalve that inhabits the intertidal zone and therefore frequently exposed to air during the tidal cycle. It is highly adaptive to hypoxic conditions. We have studied the physiological state of oysters during long-term exposure to air. The oysters became hypoxic when exposed to air or hypoxic seawater. The 50% lethal time of oysters exposed to air at 4, 15 and 20°C was 47.8, 15.9 and 12.2 days, respectively. The hemolymph pH decreased by day 3; however, it showed a slight increase by day 5 at both 4 and 20°C. The adenylate energy charge (AEC) values decreased rapidly on the first day of air exposure in the adductor muscle, mantle, gill and body trunk, and these decreases were accompanied by decreases in ATP concentrations and increases in AMP concentrations. The AEC values in all of the tissues had fallen to below 30% by day 50 of air exposure at 4°C. These data suggest that the energy state of oysters deteriorates rapidly with air exposure. Consequently, AEC values may be useful indices of the physiological state of the oyster during long-term exposure to air.     

长牡蛎壳金选育群体性腺发育与营养成分的周年变化

为了评估长牡蛎(Crassostrea gigas)壳金选育群体的繁殖周期及营养价值,自2013年11月至2014年10月,对山东省刘公岛海区长牡蛎第三代壳金选育群体的性腺发育、营养成分(总蛋白、总脂肪、糖原)季节变化及其与环境因子的关系进行了研究,并比较了与普通养殖群体营养成分的差异。结果表明,长牡蛎壳金选育群体雌雄比例接近1︰1,性腺同步发育;配子发生开始于2月,全年只有1个产卵季节(6―8月),7月为两性配子排放高峰期。配子成熟及排放期间,水温处于较高水平,食物充足,可为性腺发育提供能量,也有利于幼体的存活和生长。卵母细胞直径从配子发生开始逐渐增大,并在产卵前达到最大值(36.88μm),产卵后则降低。营养成分分析表明,脂肪含量在性腺–内脏团中随配子发育积累储存,产卵后显著降低;糖原在性腺发育到4月时开始下降,为配子的发育提供能量;蛋白质在配子发育过程中大量合成,与繁殖活动存在密切联系。长牡蛎壳金选育群体外套膜的总蛋白含量显著高于普通养殖群体(P0.05),糖原和总脂肪含量显著低于普通养殖群体(P0.05),其他组织的一般营养成分未出现显著差异。研究表明,长牡蛎在壳金群体选育过程中营养成分已表现出分化,这为长牡蛎壳色选育提供了重要参考依据。     

合浦珠母贝热休克蛋白hsp70基因的克隆与表达分析

采用同源克隆和RT-PCR技术对合浦珠母贝(Pinctada fucata)热休克蛋白hsp70基因进行了克隆和表达分析。获得cDNA全长序列2 365 bp,其中3’非编码区域(UTR)为318 bp,5’UTR为88 bp,开放阅读框(ORF)为1 959 bp,编码652个氨基酸,分子量约为71.39 kD,理论等电点为5.22,并含有3个HSP70家族的签名序列IDLGTTYS、DLGGGTFD和EEVD。同源性分析表明,合浦珠母贝HSP70的氨基酸序列与太平洋牡蛎(Crassostrea gigas)等双壳贝类的相似性高达86%以上,基于氨基酸序列的聚类分析表明,合浦珠母贝与牡蛎属种类亲缘关系最近。高温、高盐刺激后,半定量RT-PCR检测发现hsp70基因的表达明显增加,高温刺激的表达量高于高盐刺激,高温刺激组不同组织的表达量由大到小依次为鳃、消化腺、外套膜、肌肉、性腺,高盐刺激组不同组织的表达量由大到小依次为鳃、外套膜、肌肉、消化腺、性腺,表明HSP70参与了机体对刺激的应答过程。该基因的克隆为进一步深入研究合浦珠母贝的抗逆机理及其遗传改良奠定了重要基础。     

Monthly variation of condition index,energy reserves and antibacterial activity in Pacific oysters,Crassostrea gigas,in Stansbury (South Australia)

This paper investigates the temporal responses of 2-year old Crassostrea gigas to environmental changes in Stansbury, South Australia from September 2005 to October 2006. A total of 360 oysters were grown in one-line baskets on the farm using six replicates that were sampled monthly. A range of environmental parameters were assessed and correlated against biological indicators for oyster condition, metabolism and antimicrobial activity. Food availability by chlorophyll a, was low throughout the study period (0.5–1.5 µg L^? 1) and was significantly correlated to phosphorus concentrations. The condition index and shell weight of oysters significantly increased over the year, with the condition index dropping after spawning but then recovering within one month. Significant temporal variation in energy storage and utilization were observed in different tissues over the year. Glycogen in the mantle tissue was influenced by reproduction and correlated to chlorophyll a levels, but not in the gill or adductor muscle. The mantle glycogen and gill protein reached the lowest level in February when spawning occurred and presented evidence for seasonal variation in oyster metabolic activity. However, mantle and adductor muscle proteins did not drop after spawning indicating that these proteins contribute little to gametogenesis. Hemolymph protein was negatively correlated to water temperature and chlorophyll a, reaching the lowest level during summer. Hemolymph antibacterial activity significantly decreased after spawning, implying that the period of post-spawning is critical for oyster health. This study revealed trade-offs in the energy budget between immune resistance, growth, and reproduction. The results indicate that in a lean water environment, spawning events significantly regulate metabolic and immune capacities of oysters and a second year of rearing increased meat and shell weight but not the shell length. These findings are applicable to the management and development of oyster aquaculture within temperate southern hemisphere.     

育珠对合浦珠母贝N19和Prismalin-14基因表达水平的影响

为探讨育珠对壳基质蛋白基因表达的影响,开展了合浦珠母贝（Pinctada fucata）N19和Prismalin-14基因在育珠贝和非育珠贝中的荧光定量表达研究。结果显示,N19和Prismalin-14在育珠贝与非育珠贝的闭壳肌、肝胰脏、性腺、肠、鳃等组织中均无表达,在外套膜均有表达;N19在珍珠囊表达,而Prismalin-14则不表达。N19勺Prismalin．14在育珠贝外套膜的表达量均大于非育珠贝,表明育珠可能对某些壳基质蛋白的表达具有一定的促进作用。其中Prismalin-14在育珠贝外套膜的表达量最高（P〈0．01）,是非育珠贝外套膜Prismalin-14的1．23倍,是育珠贝外套膜N19的25．04倍,是非育珠贝外套膜N19的41．04倍,推测在珍珠贝生长过程中,对Prismalil7-14的需求量可能比N19大。N19在育珠贝珍珠囊中表达量最高（P〈0．01）,是育珠贝N19外套膜的11．58倍,是非育珠贝外套膜的18．97倍,推测在珍珠形成的过程中,对N19的需求量可能比贝壳自身生长对N19的需求量大。     

Molecular cloning and expression of the heat shock protein 70 gene in the Kumamoto oyster <Emphasis Type="Italic">Crassostrea sikamea</Emphasis>

Episodes of summer mortality of the Kumamoto oyster Crassostrea sikamea are a major problem for its cultivation. Expression of the heat shock protein 70 (HSP70) is induced by various environmental stresses, including heat. We cloned and sequenced hsp70 complementary DNA from C. sikamea to investigate the relationship between hsp70 expression and heat tolerance in this oyster. Quantitative real-time polymerase chain reaction was performed using gill tissue dissected from oysters before and after heat shock for 1 h. The results showed hsp70 expression was faster and greater in oysters cultured at 20–22 °C than at 10–12 °C, and survival was lower among oysters cultured at 20–22 °C than at 10–12 °C. Moreover, heat tolerance was investigated by a 1-h pre-heat treatment, followed by exposure to heat shock conditions 5 days later. Survival was higher and hsp70 expression was notably lower in oysters that received the pre-heat treatment compared with those that did not. We conclude that a pre-heat treatment of only 1 h may be useful for inducing heat tolerance in C. sikamea, and that a low level of hsp70 expression after heat shock is an important index in selecting for high heat tolerance in these oysters.     

Histopathology of Pleuronectes platessa L. from Aber Wrac'h and Aber Benoit, Brittany, France: long-term effects of the Amoco Cadiz crude oil spill

Abstract. Organ samples were collected from plaice, Pleuronectes platessa L., captured in the highly oiled Aber Wrac'h and Aber Benoit at five intervals during the period 1978-1980, following the Amoco Cadiz crude oil spill. Reference plaice were obtained along the western and southern coasts of Brittany. Tissue samples were fixed, processed and stained by routine histologic procedures for qualitative and quantitative histopathological examination. The predominant lesions and conditions which characterized fish from the oiled estuaries were: fin and tail necrosis, hyperplasia and hypertrophy of gill lamellar mucous cells, gastric gland degeneration, decreased hepatocellular vacuolation (lipid), increased concentration of hepatic macrophage centres and lateral trunk muscle fibre degeneration. Dilation of Bowman's space, glomerular hypertrophy and abdominal muscle fibre degeneration were pronounced at the last sampling interval. Other types of lesions were seen at a lower frequency: hyperplasia and fusion of gill lamellar epithelium, telangiectasis of gill capillaries, hepatopancreatic degeneration and hepatic necrosis. Fish muscle and liver collected for biochemical analysis to detect the presence or absence of petroleum hydrocarbons were negative for Amoco Cadiz oil fractions. At the same time oysters, Crassostrea gigas Thunberg, collected from the oiled estuaries throughout the study period, showed high levels of Amoco Cadiz oil fractions. These data, along with the type and frequency of lesions observed, suggested that the plaice had been chronically-exposed to petroleum hydrocarbons of Amoco Cadiz origin. Some relief from chronic pollutant exposure was indicated by a decrease in gill mucous cell concentration at the last sampling interval. An increase in abdominal muscle fibre degeneration and an increase in the frequency of dilation of Bowman's space with glomerular hypertrophy at the last sampling interval suggested a latent response to the polluting agent.     

Commercial assessment of growth and mortality of fifth-generation Sydney rock oysters Saccostrea glomerata (Gould, 1850) selectively bred for faster growth

The Sydney rock oyster Saccostrea glomerata (Gould, 1850) industry acquired hatchery-produced spat selected for faster growth for the first time in January 2004. Selectively bred and non-selected (Control) spat produced concurrently were used to compare performance when grown under commercial conditions. Spat were distributed to farmers in seven estuaries in New South Wales. Individual farmers cultivated these oysters using their own techniques and growth and mortality were recorded quarterly. At each site, the two oyster types were cultivated using the same culture method, location and density. Growth was compared when oysters were 27 months of age. At this time, selectively bred oysters were significantly larger and heavier than Control oysters. The same result was obtained when oysters were compared at the point in time when selectively bred oysters had reached 50 g at each site. No significant difference was found for cumulative mortality between the selectively bred oysters and Control oysters across all sites. However, the seven sites had significantly different levels of cumulative mortality. Overall, the performance of selectively bred oysters was superior to the Control oysters and selectively bred oysters reached the 50 g bench mark within 29.3 months when averaged across all sites.