热休克及菌胁迫对厚壳贻贝HSP70基因表达的影响

70 ku的热激蛋白(HSP70)具有细胞内分子伴侣和细胞外免疫调节功能,是受到热激、重金属、干旱、疾病、寄生虫感染等条件胁迫后产生的诱导蛋白。本实验利用同源克隆法,获得厚壳贻贝HSP70基因1 700 bp的核心序列,BLAST比对和系统进化分析显示,该序列与已知的双壳贝类相应分子高度同源,其中与紫贻贝HSP70基因相似性高达96%,在进化树中两者位于同一分支。37 ℃热激后,厚壳贻贝肝胰腺和鳃中HSP70基因的表达快速上调,2 h即达到对照组30倍,随后持续上升,肝胰腺中最高表达量达到对照组100倍,说明厚壳贻贝HSP70分子为典型的热诱导蛋白。鉴于大肠杆菌及枯草芽孢杆菌是水体中常见微生物,进一步利用实时荧光定量PCR技术检测上述两种细菌感染后,HSP70基因在厚壳贻贝鳃和肝胰腺中的表达水平,结果显示,两种细菌均可引起厚壳贻贝鳃和肝胰腺的HSP70基因表达上升,但大肠杆菌诱导效应显著,8 h时表达量达到最高,鳃为对照组的4倍,肝胰腺为4.5倍,随后逐渐下降,到48 h已恢复到原始水平;枯草芽孢杆菌刺激后,肝胰腺组织中最高表达量在刺激后6 h约为对照组2.5倍,与注射PBS的效果相差不大,鳃组织的响应较肝胰腺组织明显,表达量缓慢升高,8 h达到最高,为对照组4倍,随后迅速下降,直至生理水平。研究表明,厚壳贻贝HSP70分子在调节环境胁迫及宿主免疫防御中具有重要功能。     

KK-42对凡纳滨对虾热激蛋白基因表达的诱导作用

为探讨咪唑类物质KK-42可能的作用机制,首次克隆了热激蛋白90(HSP90)部分mRNA序列,研究了HSP90以及HSP70基因的时空表达以及KK-42对其表达的影响。将体长3.5～5.0 cm凡纳滨对虾幼虾随机分为2组,分别用1.95×10^-4 mol/L的KK-42溶液或不含KK-42的溶液浸泡处理1 min,之后在相同条件下常规饲养。结果显示, HSP90和HSP70在凡纳滨对虾大颚器官、眼柄、肌肉和肝胰腺都有表达,其中,肝胰腺中的表达水平较高。实验期间,肝胰腺中两种HSP mRNA水平虽有一定波动,但含量相对较低;KK-42处理可显著诱导肝胰腺HSP的表达,在处理后第1、2、3天,HSP70和HSP90 mRNA含量分别比相应的对照组升高了243.4%、141.3%、410.5%和121.6%、505.5%、481.7%。结果表明,KK-42处理可显著提高凡纳滨对虾肝胰腺HSP90和HSP70基因的转录水平。     

温度对拟穴青蟹循环水养殖系统微生物群落结构的影响

为探究温度对拟穴青蟹(Scylla paramamosain)循环养殖水体中微生物群落的影响,深入理解环境因子与养殖水体微生物群落之间的相关性。在水温分别为18℃、22℃、26℃、30℃和32℃的循环养殖系统中进行拟穴青蟹养殖,采用16S rRNA高通量测序技术对不同温度下的养殖水体微生物群落组成进行分析。结果表明:(1) Alpha多样性指数显示5组水样中细菌群落多样性指数大小为t26t22t30t32t18,物种丰度大小为t22t26t32t30t18。温度可以导致养殖水体中微生物群落多样性发生变化,但并未发现二者之间直接的相关性。(2) 5种温度下的养殖系统中细菌群落共有31门、43纲、94目、185科和351属,变形菌门(Proteobacteria)为循环养殖水体中绝对优势菌门。在养殖系统水温为26℃时,养殖水体中氮磷污染物含量相对较高,其水中脱氮除磷菌属的相对丰度也显著高于其他4组。(3)由花瓣图和UPGMA聚类分析图进行相似性分析可知,水体微生物群落对温度的变化有着良好的响应, 5组水样的细菌群落结构总体相似性较低,其中最高温度32℃和最低温度18℃的水体中细菌群落结构最为相似。(4)水体中微生物群落结构和与环境因子的相关性分析显示,氮磷营养盐中的总磷(TP)和硝酸盐(NO3–-N)对水中微生物群落特征具有显著性影响,而温度是导致养殖水体中氮磷营养盐差异化的主要原因。上述研究结果初步解释了温度对拟穴青蟹循环养殖水微生物群落的作用机制,为下一步深入研究拟穴青蟹循环水养殖系统中环境变化与微生物的相互关系提供参考。     

Serum haemolytic and agglutinating activity as indicators of fish immunocompetence: their suitability in stress and dietary studies

Gilthead sea bream, Sparus aurata, were subjected to different stressful situations and the haemolytic activity mediated by the alternative complement and the agglutinating serum activity were tested as indicators of immunocompetence. The results show that both parameters were significantly compromised after chronic or repeated acute stress as well as in groups fed with vitamin E or 3 highly unsaturated fatty acids (HUFA) deficient diets and in infected fish. These results suggest that both haemolytic and agglutinating activity are suitable indicators for immunocompetence as they are non-specific responses induced by non-specific stressors. In addition, a comparison is performed with other immune indicators subjected to the same stressors.     

脊尾白虾热休克蛋白HSP70基因的克隆及其表达分析

克隆了脊尾白虾热休克蛋白基因全长cDNA,并进行了序列分析。该基因由2 250 bp的碱基组成,开放阅读框长1 959 bp,编码由652个氨基酸组成的蛋白,基因两翼分别存在83 bp(5′端)和208 bp(3′端)的非翻译区,将该基因命名为Ec-HSP70。与其它物种HSP70s氨基酸序列进行同源性比较发现,与甲壳动物的同源性都在90%以上,表明该蛋白属于热休克蛋白HSP70家族。聚类分析表明,脊尾白虾热休克蛋白氨基酸序列与中国明对虾和凡纳滨对虾紧密聚为一支,之后聚类顺序依次为斑节对虾、日本囊对虾、刀额新对虾等。通过荧光定量RT-PCR对该基因在肝胰腺、肌肉的表达分析表明,温度、pH和氨氮胁迫都可以引起该基因的高表达,而且肝胰腺中的高表达时间相对肌肉中出现的较早。试验结果表明,温度、pH和氨氮胁迫对脊尾白虾HSP70基因表达有一定的诱导效果,但胁迫的时间过长则抑制其表达,肝胰腺对胁迫比肌肉较敏感。     

Identification of Stress Sensitive Genes in Hyperthermic Atlantic Salmon (Salmo salar) Embryos by RAP-PCR

Deformities of skeletal structures, the heart, and other organs are a recurrent problem in species used in intensive aquaculture. Elevated egg incubation temperature appears to be a high risk factor in the development of these malformations, but the causal relation has not been established. Our aim was to identify candidate genes involved in the development of heat induced deformities in Atlantic salmon (Salmo salar). Temperature sensitive genes were isolated by RNA Arbitrarily Primed (RAP)-PCR. A total of 33 RAP-PCR products were successfully sequenced, and the expression of eight identified genes was further examined by RT-PCR from pooled samples of heat exposed embryos. Five of these genes were demonstrated to be temperature sensitive, of which four were shown to be up-regulated and one was down-regulated at elevated water temperatures. The atrial natriuretic peptide (ANP) gene, which is a promising candidate for heart deformities, showed the highest level of heat induction. Three additional RAP-PCR products identified as heterogeneous nuclear ribonucleprotein (hnRNP) A0, acyl CoA binding protein (ACBP) and mitochondrial (mt)-HSP70 showed up-regulated mRNA expression in response to elevated water temperature. The single down-regulated gene was identified as an Atlantic salmon (Salmo salar) homolog of the cysteine and tyrosine-rich 1 (CYYR1) gene. This study demonstrated that a temperature elevation of only 4 °C during the early stages of the organogenesis in Atlantic salmon induce altered expression of a number of genes, which are candidates for the development of heat induced deformities.     

Disinfection of Sparus aurata eggs with glutaraldehyde

This study was conducted to determine suitable conditions fordisinfecting eggs of gilthead sea bream (Sparus aurata)with glutaraldehyde. Effects of the developmental stage (4–8 cells,morula, blastopore closure or heart beating), of the concentration (C = 200,300or 400 ppm) and of the duration of the glutaraldehyde treatment (T= 2 to 10 min) were investigated. Before the blastopore closurestage, egg manipulation and treatment induced mortality. After this stage, thetoxicity of the glutaraldehyde treatment was negligible if the value of theproduct C × T was less than 1000. Above this value, the percentage ofhatching and of normal larvae decreased and the percentage of imprisoned larvaeincreased. Toxic effects of glutaraldehyde varied according to the egg qualityat the time of the treatment. It was concluded that 200 ppmglutaraldehyde for 4 min, at the blastopore closure stage or attheheart beating stage, were acceptable conditions for disinfecting gilthead seabream eggs at 18 °C.     

Mass production of Rhodopseudomonas palustris as diet for aquaculture

Three different types of anaerobic fermentations were used for the mass production of the photosynthetic bacterium Rhodopseudomonas palustris as diet for aquaculture. The optimum agitation speed and malate concentration were 300 r.p.m. and 0.2% in the modified MYC medium, respectively. In batch fermentations of R. palustris, the maximum number of viable cells was 1.1×10¹⁰ c.f.u. ml⁻¹ with 2.65 g l⁻¹ of DCW, and the maximum specific growth rate and biomass productivity were estimated to be 0.12 h⁻¹ and 55 mg l⁻¹ h⁻¹, respectively. Crude protein content of R. palustris was about 72–74%. The composition of stearic acid and oleic acid of R. palustris was superior to those of Chlorella and yeasts, while that of other fatty acids tested was not. The amino acid profiles of the protein hydrolysate compared favorably with Food Agricultural Organization (FAO) guidelines. The biomass productivities from fed-batch experiments were found to be 50, 47 and 49 mg l⁻¹ h⁻¹ for linear, exponential, and sigmoidal feeding strategy, respectively. The maximum biomass productivity was found to be 112 mg l⁻¹ h⁻¹ in chemostat. Compared to growth in batch cultures, continuous fermentation yielded two times higher biomass productivity.     

Optimal stocking in intensive aquaculture under sinusoidal temperature, price and marketing conditions

When the environmental and/or market conditions of an intensive aquacultural operation vary with time, maximization of profit may require a time dependent fish stocking rate. A simplified stocking problem is formulated where temperature, market price and/or market demand change sinusoidally over the annual cycle. The fish biomass sustaining-capacity is limited by the water treatment equipment, and is expressed in terms of maximum feeding rate per unit volume of culture tanks.It is demonstrated that under such conditions, sinusoidal stocking rates produce good sub-optimal solutions. A critical element of the solution is the time delay (phase-shift) between the constraining (restricting) conditions (temperature and market), and the stocking cycle. Furthermore, if the solutions for two individual conditions have very different time delays, a combination of the two conflicting restrictions is likely to produce poor returns.     

Gastric evacuation and feeding in the gilthead sea bream reared under semi-intensive conditions

Gastric evacuation rates of the gilthead sea bream, Sparus aurata, fed with commercial pelleted food and polychaetes (Nereis diversicolor) were determined under experimental conditions. The estimated gastric evacuation rate for pelleted food was 7.97% h^–1, with a total time of digestion of approximately 9 h. The respective values for the natural food were 6.24% h^–1, with a total digestion time of approximately 12 h. The daily consumption of fish reared in earth ponds in a semi-intensive aquaculture facility was estimated through 24 h cycles performed between April and August. The daily consumption varied from 18.58 to 31.98 mg g^–1. There was a constant increase in the average daily consumption per individual of 1.8–4.6 g (dry weight). During these cycles, samples of stomachs were taken and the contents preserved for further observation. The feeding behaviour of the reared fish was compared with a fish sample caught in the Ria Formosa lagoon. No common species were found between samples. A total of 38 prey were identified, which suggests that the gilthead sea bream is a non-specific predator. Despite the high abundance of natural prey in the ponds, the dependence of sea bream on pelleted food was high.     

Laboratory trials on the effects of different diets on growth and survival of the common whelk, Buccinum undatum L. 1758, as a candidate species for aquaculture

Newly hatched juvenile Buccinum undatum can be reared under laboratory conditions. Good was growth is achieved when juveniles were fed on combined diets (blue mussel, cod, and fish pellets). Juveniles reached shell heights of 33.0 ± 4.2 mm, 26.9 ± 3.8 ± mm, 23.2 ± 2.2 mm, and 20.1 ± 1.6 mm, after 14 months of fedding on a combined diet, blue mussel, cod, and fish pellets, respectively under ambient sea temperature and salinity. After 14 months juveniles fed blue mussel had the highest survival rates (67%) followed by those fed a combination of all other experimental diets (61%), cod waste (53%) and fish-feed pellets (46%). High mortalities were recorded in most treatments during the summer months between June and September. This species appears to have an aquaculture potential, as juveniles readily feed on artificial diets at an early age, show high survival rates and could potentially reach market size in 2 years or less. The major constraint in realising this potential at present, is the relatively low value of the species; if market values increased as a result of serious depletion of natural populations, hatchery production of juveniles for intensive aquaculture or restocking could become economically viable.     

Effect of feed and feeding in the culture of salmonids on the marine aquatic environment: a synthesis for European aquaculture

While marine aquaculture has grown rapidly, so have concerns regarding the environmental impacts caused by the industry. In particular, increasing discharges of solid and dissolved fish excretions, nutrients and therapeutic chemicals have coincided with greater public awareness of the possibility of environmental damage. This has stimulated a number of criticisms, drawn from a wide spectrum of interests, ranging from the use of natural fish stocks to produce fish meal for aqua feeds to the effects of enhanced nutrient input on the coastal marine environment. The present study reviews available information on the environmental effects of feeding practices in salmonid aquaculture in Europe. Accumulation of waste food and fish faecal material results in changes in the sediment under fish cages, characterized by a low redox potential, high content of organic material and accumulation of nitrogenous and phosphorous compounds. Although significant environmental impacts have been reported in the literature at distances of up to 100 m from the cages, in general such impacts are reported to be localized to within 20–50 m around the cages. For farmed salmon and trout, mass balance models have been developed for nitrogen and phosphorus, indicating that 50% of the nitrogen and 28% of the phosphorus supplied with the food is wasted in dissolved form. The maximum nutrient release can be estimated from the hydrographic conditions in the immediate vicinity of the farm, such as water volume, tidal water exchange and currents. At present production levels, improvements in the feeding efficiency and feed quality of aquafeeds could reduce waste and consequent environmental impacts.     

Expression of HSP70 in response to heat-shock and its cDNA cloning from Mediterranean blue mussel

Expression of HSP70 in response to heat-shock was investigated at the protein and mRNA levels in Mediterranean blue mussel. Western and Northern blot analyses revealed that HSP70 was expressed following heat-shock in the mantle at both protein and mRNA levels, suggesting that gene expression of HSP70 is implicated in the cellular response to heat-shock stress in mussel. It was then attempted to clone HSP70 cDNA in order to determine the primary structure of mussel HSP70. As a result, two full-length cDNA encoding HSP70 were isolated from a cDNA library prepared from the heat-shocked mantle. The isolated cDNA consist of single open reading frames of 2067 bp and 1911 bp which encode proteins of 689 amino acids and 637 amino acids, respectively. Both HSP70 cDNA encode an ATPase do main, and a substrate-binding do main in addition to a Glu-Glu-Val-Asp (EEVD) peptide motif that is specific for cytosolic HSP70. These findings suggest that the cDNA clones obtained in the present study encode cytosolic HSP70.     

低温胁迫对暗纹东方鲀肠道氧化应激、细胞凋亡及肠道微生物组成的影响

为了探究低温胁迫对暗纹东方鲀肠道氧化应激、细胞凋亡及肠道微生物组成的影响,实验测定了3个处理温度 (25、19和13 °C),4个取样时间点 (0、6、24和96 h)下氧化应激、细胞凋亡与肠道微生物相关指标。结果显示,与对照组相比,随着温度降低肠道绒毛发生坏死、增生和杯细胞肿大。总超氧化物歧化酶 (T-SOD)、过氧化氢酶 (CAT)、谷胱甘肽过氧化物酶 (GSH-Px)的活性以及丙二醛 (MDA)含量显著增加。肠道微生物组成多样性降低。肠道微生物丰度发生改变,如黄色杆菌属、副球菌属、乳球菌属和双歧杆菌属的丰度随温度降低而上升,梭菌属、气单胞菌属和嗜热芽孢杆菌属的丰度随温度上升而下降。细胞凋亡相关基因 (caspase-3、caspase-7、caspase-9、p53、Bax和Bcl-2)的表达随温度降低升高。此外,实验发现副球菌属和嗜热芽孢杆菌属丰度的改变与肠道氧化应激、细胞凋亡均具有Pearson相关性,其可作为潜在微生物用来反映暗纹东方鲀是否处于氧化应激和细胞凋亡状态。本研究结果可为暗纹东方鲀健康养殖提供基础资料。     

急性氨氮胁迫对黄颡鱼组织抗氧化酶活性及HSP70和HSP90基因mRNA表达水平的影响

为了探讨急性氨氮胁迫对黄颡鱼组织中抗氧化酶活性及HSP70和HSP90基因mRNA表达水平的影响,实验随机挑选了360尾黄颡鱼[初体质量(17.25±0.05) g],分别暴露于含有0(对照)、5.70(低浓度组)、28.50(中浓度组)和57.00 (高浓度组) mg/L总氨氮浓度的水体中,进行96 h的急性胁迫实验。实验开始后,分别于0、12、24、48和96 h取样。结果显示,氨氮胁迫发生后,低、中浓度组实验鱼肝脏中超氧化物歧化酶(SOD)活性呈先升高后降低趋势,而高浓度组则持续降低;低、中、高浓度组实验鱼肝脏中丙二醛(MDA)含量在胁迫开始后显著升高;3 h时,高浓度组实验鱼肝脏中SOD活性达到最低,而MDA含量最高;24 h后,高浓度组实验鱼肝脏中过氧化氢酶(CAT)活性显著升高;低、中、高浓度组实验鱼肝脏中HSP70基因的mRNA表达量呈先降低后升高趋势,而鳃中HSP70基因表达量持续升高,但脑中HSP70基因在0 h后显著降低;氨氮胁迫3 h时,低、中、高浓度组实验鱼肝脏和脑中HSP70基因表达量显著低于对照组,而在鳃中正好相反;相比HSP70基因,高氨氮浓度组实验鱼肝脏...     

Physiological stress responses in spotted wolffish (Anarhichas minor) subjected to acute disturbance and progressive hypoxia

The spotted wolffish (Anarhichas minor) is considered a promising species for coldwater aquaculture. It is a sedentary, bottom-dwelling fish which exhibits a calm and “non-stressed” behaviour in captivity. There are, however, no reports on the physiological responses to stressors in this species. In the present study we investigated primary (cortisol secretion) and secondary (glucose mobilization) responses to common aquaculture stressors like disturbance and hypoxia. Pre-stress plasma cortisol levels were within those generally considered representative for unstressed fish (~ 10 ng ml^− 1), whereas basal glucose levels were unusually low (0.3–0.4 mM). After exposure to a short, but severe disturbance challenge (emptying the tank of water for 10 min), the increase in plasma cortisol level was slow and relatively weak, reaching a peak level of 25 ng ml^− 1 4 to 8 h after disturbance. When the fish were exposed to a gradually decreasing oxygen level in the tank, a significantly elevated plasma cortisol level (35 ng ml^− 1) was seen in the fish that remained in the tank until oxygen saturation had decreased to 20% oxygen saturation after 2.5 h. However, a two-fold higher plasma cortisol level (~ 70 ng ml^− 1) was seen in all fish exposed to reduced oxygen levels (60, 40 and 20% oxygen saturation) after 3.5 h recovery in normoxic water. Plasma glucose levels showed only moderate increases (~ 70%) following disturbance and hypoxia challenges. An in vivo injection of ACTH caused a strong elevation of plasma cortisol (peak level ~ 170 ng ml^− 1), demonstrating a high capacity for interrenal steroidogenesis in the spotted wolffish. The slow and relatively weak cortisol response to stressors, and low plasma glucose levels, may relate to the sedentary lifestyle of the spotted wolffish. The stress-response is characterised by a passive (reactive) coping style, which is considered adaptive for farming of this species.     

青鱼和鲇肠道排泄物对养殖水体铜绿微囊藻休眠体复苏的影响及作用途径

为探讨养殖水体底栖鱼类肠道排泄物对铜绿微囊藻休眠体复苏的影响,将青鱼和鲇肠道排泄物与铜绿微囊藻休眠体用野外养殖水域沉积物(底泥)混匀包埋,在10、15和20°C梯度温度下进行休眠体复苏实验.结果显示,铜绿微囊藻休眠体主要复苏期为第3～15天,在10和15°C条件下,青鱼排泄物组(MP)、鲇排泄物组(SA)和青鱼-鲇排泄...     

水杨酸对龙须菜抗高温生理的影响

以龙须菜为材料,研究了不同浓度水杨酸对高温下龙须菜的生长速率、抗氧化酶、渗透调节物质、藻胆蛋白、叶绿素荧光特性和HSP70基因的影响。结果表明,水杨酸处理组不论是生长、酶活性和膜脂受损情况都不同程度优于对照组,其中5.0、10.0μg/mL处理组效果较好。10.0μg/mL水杨酸处理组效果最为明显,日生长速率与对照组相比增长了340%。在处理第3天,10.0μg/mL处理组各指标达到最大,SOD增长了74%,POD增长了70%,CAT增长了40%。脯氨酸和甘露醇分别增长了70%和26%。藻红蛋白增加了46.2%,藻蓝蛋白增长了40%。与对照组相比,MDA含量第1天下降最为显著,下降了10%。高温胁迫下,龙须菜叶绿素荧光参数Fv/Fm、Fv/Fo、qP和ΦPSⅡ均明显降低,非光化学淬灭(NPQ)呈现先上升后下降的趋势。水杨酸处理能有效减缓5种龙须菜叶绿素荧光参数的降低程度。水杨酸处理后HSP70基因表达量较对照组低。本实验结果表明水杨酸具有增强龙须菜抗高温胁迫的作用。     

Polydorid infestation in the flat oyster, <Emphasis Type="Italic">Tiostrea chilensis</Emphasis>: hyposaline treatment for an aquaculture candidate

Attempts to commercially farm the Chilean oyster, Tiostrea chilensis in New Zealand are hampered by high levels of shell infestation by the polydorid polychaete, Boccardia acus. Wild stock in the warmer, northern waters of New Zealand typically present a mean of 8.6±,0.7 worms per right upper valve from an early stage of development (~17 mm shell height). Both hypersaline and freshwater treatments were effective in reducing worm numbers, but freshwater treatment for 180–300 min was most effective and resulted in >98% worm mortality without producing any obvious harmful effects on the oyster. Hyposaline treatment is therefore recommended as a simple and cost-effective method for the control of B. acus in farmed T. chilensis.