The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Diet-induced changes in lipid and fatty acid composition of Artemia salina

The influence of diet on lipid and fatty acid composition of the brine shrimp Artemia salina nauplii was investigated. Various diets with different lipid composition and fatty acid profiles were fed to nauplii for 2 weeks. The lipid composition of microalgal diets, Isochrysis galbana, Phaeodactylum tricornutum and Nannochloropsis oculata and baker's yeast was analyzed. Newly hatched nauplii were examined before the feeding experiment. It was shown that Artemia was able to incorporate and selectively concentrate some dietary lipids. Depot lipids were more sensitive to changes in the dietary lipid composition than the main structural lipids, polar lipids and sterols. Variations in the content of the lipid classes correlated with stage of development of the animal. The fatty acid composition of the animal varied with that of diet. The concentrations of saturated fatty acids were apparently supported in the nauplii by biosynthesis de novo. The acid 16:1(n-7) originated from the food. The concentration range of n-6 polyunsaturated fatty acids (PUFAs) remained constant through the accumulation from the diet. The proportion of n-3 PUFAs varied with their level in the diet. The dynamics of alteration of 20:5(n-3) content in Artemia fed on Isochrysis, which is poor in this acid, suggested a limited capacity for elongation and desaturation of 18:3(n-3) to 20:5(n-3). None of the diets provided dietary input of 22:6(n-3). (C) 1998 Elsevier Science Inc. All rights reserved.

双刺纺锤水蚤（Acartia bifilosa）基础生物学与生态学研究

20世纪90年代以来，为了确保日益增长的人口对蛋白质的需求，海洋鱼类养殖在全球范围内日趋发展。然而，许多养殖鱼类的品质如抗病力、口味等与野生种类相比大为降低。饵料对于鱼类品质的好坏起着至关重要的作用。在海洋的自然环境中，浮游动物，特别是数量庞大、种类繁多的桡足类是野生鱼类的天然活饵料。哪些桡足类适于作养殖饵料，如何获得、从何处获得桡足类，人工培养是否可行，能否通过加入桡足类来改善养殖鱼类的品质是长期以来业内人士一直关注的问题，需要大量的基础性探索研究工作。 开展有潜在开发价值种的生物学特性及室内培养的基础研究，进而开展大量生产技术的研究与应用，不仅是开发利用桡足类的一个重要途径，而且可以获得一些重要的生态学参数。 本论文自2003年10月－2004年9月之间，在胶州湾采集不同的桡足类种类，通过室内比较培养实验，选定双刺纺锤水蚤作为具有开发潜力的研究培养对象，对其展开一系列培养条件及生物学特性研究，在此基础上进行了小水体增殖培养，结合现场调查资料对与其生活策略相关的生态学问题进行了初步研究探索。结果如下： 筛选：2003年10月－2004年9月全年不同季节共采集11 种桡足类，在室内自然温度、自然海水（盐度30－32）下长时间培养，粗略筛选出能够经受实验室人工养殖水体生活的种类有：强额拟哲水蚤（Paracalanus. crassirostris）、汤氏长足水蚤（Calanopia thompson）、太平洋真宽水蚤（Eurytemora pacifica）、双刺纺锤水蚤（Acartia bifilosa）。对上述种类的成体和子代幼体分别测定其对不同盐度、温度的耐受能力。培养结果表明：18℃室温下，强额拟哲水蚤幼体在盐度20以上的环境中，存活时间不超过11天；汤氏长足水蚤雌体在培养温度低于20℃时，只能存活10天；25℃室温下，当盐度低于20时，汤氏长足水蚤雌体存活时间不超过9天，子代的存活时间不超过7天；太平洋真宽水蚤不适宜在温度较高的夏秋季培养，幼体在不同盐度的实验条件下存活时间不超过5天，不适宜长期培养；双刺纺锤水蚤在全年8－24℃的室内培养温度范围内保持了24－85％的存活率，雌体和子代在盐度10－35的范围内都能存活，最终结果表明双刺纺锤水蚤是其中最适宜进行人工培养的种类。 繁殖：对双刺纺锤水蚤雌体的培养条件和繁殖生物学的研究结果表明：在本实验所使用的6种微藻饵料：微绿球藻（Nannochloropsis oculata）、小球藻（Chlorella.sp）、等鞭金藻（Isochrysis galbana）、三角褐指藻(Phaeodactylum tricornutum)、亚心型扁藻(Platymonas subordiformis)、中肋骨条藻(Skeletonema costatum)中，亚心型扁藻和中肋骨条藻适宜成体培养，亚心型扁藻对雌体存活有利，排粪率也要比中肋骨条藻低得多，亚心型扁藻在高温条件下的饵料利用效率要高于中肋骨条藻，中肋骨条藻对产卵有利，二者混合优势互补；预饥饿处理的双刺纺锤水蚤恢复到最高产卵率需要较长的时间，并且一直保持较低的产卵率；该种繁殖的最适温度范围15－20℃；在10－25℃温度范围内的平均产卵率差异并不显著。 生长及发育：对双刺纺锤水蚤幼体培养条件及发育生物学研究结果表明：在本实验所使用的6种微藻饵料中，微绿球藻是比较理想的开口饵料；粒径小（ 4 m）的微藻——微绿球藻和小球藻不能保证双刺纺锤水蚤后期无节幼体发育至桡足阶段，22℃以下采用微绿球藻 + 亚心型扁藻 + 中肋骨条藻的食物搭配，22℃以上需加入粒径在4m左右的等鞭金藻。 世代时间：通过一系列的温度梯度实验，证明在相同饵料的情况下，温度对双刺纺锤水蚤的发育具有显著的影响，在15－25℃的范围内，随着温度的升高，生长速度加快、世代周期缩短；在温度条件为15、18、20、22、25℃下的世代时间分别为25.5, 18.5, 13, 11.5, 9.5天。 群体培养：研究了适宜的微藻饵料种类搭配比例以及总饵料浓度对种群日均增值率的影响。结果表明：20℃下培养宜采用亚心型扁藻：中肋骨条藻：微绿球藻按含碳量2:1:1的比例组成混合饵料，达到最高增殖率的混合饵料浓度范围在1.0－4.0 μg C ml-1之间；25℃下培养宜采用亚心型扁藻：中肋骨条藻：微绿球藻：等鞭金藻按含碳量2:1:1:2的比例组成混合饵料，日均增殖率在混合饵料总浓度为1.0 μg C ml-1 时最高，低于和高于此浓度都会降低日增值率。 度夏机制：针对野外大面调查发现双刺纺锤水蚤在高温季节的胶州湾内仍然存在的事实（传统观点认为该种在夏季从水体中消失，通过休眠卵度夏），本论文从基础生态学研究出发，根据胶州湾夏季的温度和叶绿素浓度资料，设计实验研究了高温和饵料浓度对成体繁殖和幼体生长发育的影响。实验发现，饵料浓度对高温下雌体的繁殖有着明显的影响，15g Chla l-1浓度下的雌体在28℃都可以保持产卵状态，而且卵的孵化率也在50％以上；各处理中的卵都很快孵化，并保持了60％以上的孵化率；高浓度组15 g Chla l-1和10 g Chla l-1，无节幼体都能发育至成体，低浓度5 g Chla l-1处理组中，28℃下，不能发育至桡足阶段，而25℃下也只能发育至C4期。在本实验中没有发现双刺纺锤水蚤产生休眠卵。在胶州湾自然环境中发现该种全年存在。胶州湾中的双刺纺锤水蚤在夏季能够在不产生休眠卵的情况下安全度夏。

Sorption and desorption of Cu and Cd by macroalgae and microalgae

The sorption and desorption of Cu and Cd by two species of brown macroalgae and five species of microalgae were studied. The two brown macroalgae, Laminaria japonica and Sargassum kjellmanianum, were found to have high capacities at pHs between 4.0 and 5.0 while for microalgae, optimum pH lay at 6.7. The presence of other cations in solution was found to reduce the sorption of the target cation, suggesting a competition for sorption sites on organisms. Sorption isotherms obeyed the Freundlich equation, suggesting involvement of a multiplicity of mechanisms and sorption sites. For the microalgae tested, Spirulina platensis had the highest capacity for Cd, followed by Nannochloropsis oculata, Phaeodactylum tricornutum, Platymonas cordifolia and Chaetoceros minutissimus. The reversibility of metal sorption by macroalgae was examined and the results show that both HCl and EDTA solutions were very effective in desorbing sorbed metal ions from macroalgae, with up to 99.5% of metals being recovered. The regenerated biomass showed undiminished sorption performance for the two metals studied, suggesting the potential of such material for use in water and wastewater treatment. (C) 1998 Elsevier Science Ltd. All rights reserved.

Effects of different diets on the reproduction and naupliar development of the copepod Acartia bifilosa

The influence of diatoms on the reproduction and naupliar development of Acartia bifilosa was investigated under laboratory conditions, comparing initial in situ values and laboratory-food treatments. Egg production by A. bifilosa was significantly reduced by one diatom diet (Phaeodactylum tricornutum: Pt) and by two non-diatom diets (Platymonas subordiformis: Ps and Nannochloropsis oculata: No). It was less affected by the other diatom diet (Skeletonema costatum: Sc) or by two mixed-food treatments (D-mix and DG-mix), composed of two diatoms (Pt, Sc) and four species (Pt, Sc, Ps and No), respectively. The negative effect of Pt was eliminated when adult copepods were offered mixed-food diets. There were no significant differences between the hatching success values observed in filtered seawater and in algal exudates, indicating that diatoms did not produce active dissolved toxic substances under the different food concentrations tested. The mortality rate of nauplii was higher with Pt than the other diets, suggesting that this diatom species had a negative effect on egg production, hatching success and naupliar survival simultaneously. Compared to other diets, No and Pt were not beneficial food sources for reproduction and for female and larval survival. (c) 2007 Elsevier B.V. All rights reserved.

CO_2对微拟球藻(Nannochloropsis sp.)利用有机碳和光合作用的影响

CO2浓度提高时,微拟球藻吸收醋酸钠的速率增加2倍。混养生长的藻细胞最大光合作用速率、光合作用效率、无机碳半饱和常数和无机碳饱和的光合作用速率均显著低于光自养条件下生长的。

Response of growth and fatty acid compositions of Nannochloropsis sp to environmental factors under elevated CO2 concentration

Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO2 at 2,800 mu l l(-1). Increased levels of NaNO3 and KH2PO4 raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l(-1), and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5 omega 3) content based on the dry mass was above 3% under low N (150 mu M NaNO3) or high N (3000 mu M NaNO3) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.

Optimization of growth and fatty acid composition of a unicellular marine picoplankton, Nannochloropsis sp., with enriched carbon sources

A unicellular marine picoplankton, Nannochloropsis sp., was grown under CO2-enriched photoautotrophic or/and acetate-added mixotrophic conditions. Photoautotrophic conditions with enriched CO2 of 2800 mul CO2 l(-1) and aeration gave the highest biomass yield (634 mg dry wt l(-1)), the highest total lipid content (9% of dry wt), total fatty acids (64 mg g(-1) dry wt), polyunsaturated fatty acids (35% total fatty acids) and eicosapentaenoic acid (EPA, 20:5omega3) (16 mg g(-1) dry wt or 25% of total fatty acids). Mixotrophic cultures gave a greater protein content but less carbohydrates. Adding sodium acetate (2 mM) decreased the amounts of the total fatty acids and EPA. Elevation of CO2 in photoautotrophic culture thus enhances growth and raises the production of EPA in Nannochloropsis sp.

海洋小球藻Chlorella sp.甘油脂及脂肪酸、光合色素特征的研究

海洋微藻是海洋生态系统中最主要的初级生产者，也是海洋生物资源的重要来源。许多海洋微藻富含对人体具有重要的生理作用与保健功能的长链多不饱和脂肪酸，因此，筛选富含EPA、DHA等长链多不饱和脂肪酸的微藻和利用人工培养方法提高这些脂肪酸的产量是当前海洋生物学研究领域的热点之一。在本研究中，我们对被中科院海洋所定名为“Chlorella sp”（编号为1061）的一种海洋微藻的化学分类、甘油脂及其脂肪酸组成和外源葡萄糖和抗氧化剂（硫代硫酸钠）对这种微藻的脂肪酸含量的影响进行了研究，取得了以下主要结果。 海洋微藻是我固海水养殖中广泛使用的优良饵料藻。脂类物质是微藻最重要的营养指标之一，在本研究中，我们首先分析了被中科院海洋所定名为“Chlorellasp”的海洋微藻中的甘油脂及其脂肪酸种类和组成特点。结果表明，Chlorella sp.中的非极性脂主要为三脂酰甘油，极性甘油脂有10种。其中，一般培养条件下（温度23℃：光照，周期L/D14:10，强度60umolm-2-S-l）三脂酰甘油约占总脂的31 mol%，极性甘油脂约占总脂的69 rriol%。10种极性甘油脂是单半乳糖甘油二脂( monogalactosyldiacylglycerol． MGDG)、 双半乳糖甘油二脂( diagalactosyldiacylglycerol ， DGDG)、 硫代异鼠李糖甘油二脂( sulfoquinovosyldiacylglycerol，SQDG)、磷脂酰甘油(phosphatatidylglycerol，PG)、磷脂酰乙醇胺(phosphatidylethanolamine，PE)、磷脂酰胆碱( phosphatidylcholine，PC)、磷脂酰肌醇(phosphatidylinositol，PI)、磷脂酰丝氨醴(phosphatidylserine，Ps)、l，2-二酰基甘油-0-4，．（ⅣMⅣ-三甲基）高丝氨酸（diacylglyceryltrimethylhomoserine，DGTS）以及一种未能完全肯定，但可能是一中氯硫脂( chlorosulfolipid，CSL)。其中MGDG、DGDG、SQDG和PG是构成光合膜的主要成分，也是Chlorella sp中的主要极性脂。甜菜碱脂DGTS和磷脂PC是构成非光合膜的主要组分。Chlorella sp.中的主要脂肪酸为C16:0、C16:1耜C20：5（EPA)，后者主要存在于MGDG、DGDG和DGTS中，而三脂酰甘油也含有接近7%的EPA。 海洋微藻Chlorella sp.1061虽然被归属到绿藻纲绿藻目小球藻属，但是我们的研究表明，其色素、极性脂皮其脂肪酸组成与其它小球藻属藻类存在这很大差异Chl b是绿藻纲藻类中最主要的光合色素之一，1 6：4(n-3)和l 8：3(n-3)是绿藻微藻的主要脂肪酸，然而所有这些绿藻的特征化合物均未在Chlorella sp. 1061中检测到。DGTS和20:5(n-3)存在于很多的海洋微藻中，我们从Chlorella sp. 1061 中分离到占总极性甘油脂8 mo1%的DGTS，并从MGDG、DGDG和DGTS等极性甘油脂中检测到大量的20:5(n-3)。但是一般认为，小球藻属藻类中不舍这两种化合物。根据Chlorella sp. 1061的以上特点，这种藻不应该被归到小球藻属中。另外，由于Chlorella sp. 1061在色素、膜脂和脂肪酸组成特征方面大眼藻纲( Eustigmatophyceae)中的微绿球藻（Nannochloropsis）非常相似，因此，我们认为ChloreHa sp. 1061可能是Nannochloropsis中的一个种。但是未得到更进一步的证明和权威的认可之前，本文中我们仍然沿用ChloreHa sp,这一名称。 许多藻类中DGTS和PC -般不会同时存在，或者说一个存在时另外的一个的含量非常低。由此有人认为DGTS和PC之间存在着相互替代的关系。然而本研究中发现正常培养条件下Chlorella sp.中的DGTS和PC含量均较高（约10%）。磷处理实验结果表明，磷缺乏时Chlore Ha sp,中DGTS舍量大幅升高，而同时PC含量相应下降许多：但高浓度的磷并不能提高PC含量和降低DGTS含量，说明Chlorella sp,中DGTS仍可起替代PC的作用，然而PC可能并不能替代DGTS。Chlorella sp.中MGDG和DGTS脂肪酸组成及其位置分布结果显示，它们的组成和分布相似;在老化培养过程中MGDG和DGTS表现出周期性的相反的含量升高、降低的趋势，这进一步说明MGDG和DGTS之间存在着特殊的关系，MGDG可能合成自DGTS。 海洋微藻富含有利于人体健康的长链不饱和脂肪酸，如何提高微藻脂肪酸特别是多不饱和脂肪酸产量是目前研究的热点之一。本文首次报道了同时加入葡萄糖和硫代硫酸钠对Chlorelta sp,的生长、脂类组成和脂肪酸总产量的影响，结果显示葡萄糖和硫代硫酸钠存在明显而且强烈的互作，二者在培养液中的同时存在显著刺激了脂肪酸总产量的积累，在培养液中分别加入2.5 mM的葡萄糖和5mM的硫代硫酸钠，脂肪酸的产量可以比对照提高78%。而低浓度的葡萄糖和硫代硫酸钠对Chlorella sp.脂肪酸组成影响变化不明显，甚至在硫代硫酸钠存在下令人感兴趣的EPA含量还略有升高。显然，在Chlorella sp.培养中同时加入低浓度的葡萄糖和硫代硫酸钠是极具潜力的提高脂产量的方法，也可作为提高培养微藻其它活性物质产量借鉴的方法。在不久的将来，这种培养方法很可能发展成为生产实践中提高Chlore sp．乃至其它微藻脂肪酸、EPA和其它活性物质产量的经济有效的新途径。