中国对虾Penaeus Chinensis Osbeck卵母细胞发育的初步研究

本文描述了中国对虾Panaeus chinensis (Osbeck)卵母细胞的发育及其内部结构变化和卵巢的周年变化。根据卵母细胞的大小，核及核仁的形态，以及卵黄粒的有无，皮质棒的出现及卵母细胞与滤泡细胞的关系，将中国对虾卵母细胞的发育分为七个主要时期：（1）中央核仁期，（2）核仁染色质期，（3）周边核仁期，（4）滤泡期，（5）卵黄粒期，（6）皮质特化期和（7）长成期。卵原细胞含有酸性粘多糖。从滤泡期始，卵母细胞核外周出现中性粘多糖，并逐渐充满细胞质。根据卵母细胞的发育，内部结构及卵巢的周年变化特征，将卵巢发育分为五期：卵黄未发生期，卵黄发生早期，卵黄发生后期，皮质棒期和产后期。本文还讨论了卵黄的形成，卵巢发育的分期及亲虾产卵少，即孵化率低等问题。

中国对虾 Penaeus chinensis 血细胞及细胞免疫的研究

无脊椎动物免疫是一个新兴的研究领域。本文研究了中国对虾的血细胞形态，亚显微结构以及定量测定细胞吞噬活性的方法。中国对虾血细胞分三种：透明细胞，呈现球形或梭形，内部不含颗粒，半颗粒细胞及颗粒细胞卵球形椭球形，内部含有电子致密颗粒，颗粒又分均匀和非均匀颗粒。半颗粒细胞富含线粒体，可能与它对异物敏感易发生胞吐作用有关，半颗粒细胞是识别外源异物的关键细胞。可以观察到高尔基器，它可能与酶原颗粒的形成有关。姬姆萨染色细胞为紫红色，细胞核与颗粒染色较深，呈现蓝紫色与人白细胞中的粒细胞染色结果相近，这或许与两者功能相似有关。应用医学上常用的化学发光法（CL）定量测定中国对虾血细胞的吞噬活性，加入脂多糖及弧菌悬液的血细胞的发光值较只另菌液未加脂多糖的血细胞和对照组的发光值高。实验表明，脂多糖（<10μg/ml）对中国对虾血细胞的吞噬活性具有激活加强作用，而酵母聚糖不能引发血细胞的吞噬作用。上述测定方法可为监测对虾免疫机能的强弱提供定量指标，在对虾养殖生产实战中具有潜在的应用前景。

中国对虾(Penaeus chinensis) 与鹰爪虾(Trachypenaeus curvirostris)生殖生物学及早期发育的比较研究

本文主要对中国对虾及鹰的生殖生物学及早期胚胎的发育进行了较为系统的比较研究。在前人工作的基础上对中国对虾及鹰爪虾的精子及精荚形成，精子的超微结构，纳精囊的形态及结构，卵子的激活，受精卵的卵裂等进行了比较研究。使用激光扫描共聚焦显微镜对中国对虾精管的结构及精子的形成过程进行研究。中国对虾的输精管可分为近端、中段、远端输精管及壶腹四部分。输精管壁的本结构相似，分泌细胞的结构及分泌活动有较大的差别。精管自中段膨大部开始管腔逐渐被一隔膜分为大小不等的两部分，较大的腔内充满了很多精子，输精管较小的腔内充满胶体状物质。中国对精子细胞在精巢内产生，在两条输精管内渐发育成熟，经过细胞质的变化并与精子外部进行物质交换，形成顶体、亚顶体、棘突。核的变化则经历了染色质的解凝和核膜的消失，最后形成成熟的精子。对鹰爪虾精子的形成过程及精子的形态结构特征进行，结果表明鹰爪虾精子细胞产生于精巢的生精小管，在输精管内进一步发育，逐渐形成成熟的精子，许多精子在输精管内聚集成团外包胶质膜，形成许多大小不一的精子囊。精子由体 及棘突两部分组成，入海水中不运动。电镜下可见体部包括核区及细胞质区两部分。核区位于精子中部，核内由均质解凝的细胞核，外包一薄层细胞质带。细胞质区比核区略小，末端变尖，细胞质区内含有大量囊泡及膜层体。棘突从体部前端斜向伸出，由许多纤丝平行紧密排列组成，外包质膜。雄虾精子囊中的精子与已交配雌虾纳精囊内的精子在形态结构上没有明显差别。对中国对虾及鹰爪虾的纳精囊及精子的贮存特征进行研究。中国对虾及鹰爪虾的纳精囊都是封闭型纳精囊，中国对虾的纳精囊腔，表面为两个对称的半圆形甲壳版所覆盖，囊内由前向后伸出三个锥状突起。鹰爪纳精表面为前后两个甲壳版所覆盖。纳精囊中部为单一的腔，向后部两侧呈管状延伸，末端膨大形成两个袋状囊，内部贮存精子囊。纳精囊从功能上分为三个部分，即纳精囊中部的开口，交配时为胶体物质所充塞；内部的囊腔为贮存精子的部位；前端的开口为产卵时精释放口。中国对虾及鹰卵子具有相似的激活和卵裂过程。卵子可以不受精子的激活，只受海水刺激即可发生皮层反应；先排出皮质棒或皮质囊泡，形成胶质层，进而释放出皮层颗粒，形成孵化膜。中国对虾的孵化膜在卵子表面形成以后再举起；鹰爪虾的孵化膜远离卵子表面形成，然后进一步举起，鹰爪虾卵周隙较大。发生皮层反应的过程中，减数分裂重新启动，排出第一极体和第二极体。中国对虾与鹰爪虾卵子的皮层反应和减数分裂都可由海水刺激而进行，与受精与否无关，但未受精卵不能分裂或公分裂为大小不等的子细胞。中国对虾与鹰爪虾受精卵都具有原始的螺旋卵裂的特性。对海水中的Mg~(2+)、 Ca~(2+)、 K~+及胰蛋白酶和胰蛋白酶抑制剂对中国对虾卵子激活的影响进行研究。结果表明海水中的Mg~(2+)对中国虾卵子的激活是必须的，Ca~(2+)、 K~+是非必须的，而且它们对受精卵和非受精卵都具有相同的作用。胰蛋白酶（0．1％）在卵子产出后可以破坏胶质层及孵化膜的形成并影响受精卵的正常卵裂。胰蛋白酶抑制剂（0．01％）可以完全抑制卵子的激活及受精，也可以使发生皮层反应的卵子处于停滞状态。卵子产出后30min胰蛋白酶及胰蛋白酶抑制剂对卵子失去作用，表明胶膜及孵化膜完全形成后具有抵抗胰蛋白酶及胰蛋白酶抑制剂的作用。

Responses of Artemisia frigida Willd. (Compositae) and Leymus chinensis (Trin.) Tzvel. (Poaceae) to sheep saliva

Although studies show that grazing and browsing by herbivores have marked effects on host plants, the mechanisms remain unclear. The objective of this study is to determine the effects of sheep saliva on host plant growth. Sheep saliva was manually applied to clipped plants of two different life forms, a semi-shrub, Artemisia frigida Willd., and a herbaceous species, Leymus chinensis (Trin.) Tzevel. The results showed that sheep saliva significantly enhanced aboveground net primary productivity (ANPP) and the ratio of ANPP to belowground net primary productivity (BNPP) for both species. This indicated that sheep saliva promotes aboveground compensatory growth and allocation of photosynthate to aboveground for both plant species. Sheep saliva stimulated only tillering of L. chinensis. Regardless of saliva application, clipping significantly decreased BNPP and plant height, but significantly increased the number of branches or tillers for both plant species. The relative growth rates (RGRs) on both species were significantly greater after clipping with saliva compared with control and clipping without saliva treatments. In addition, RGR of the herbaceous species L. chinensis was faster than that of the semi-shrub A. frigida after application of saliva. (c) 2006 Elsevier Ltd. All rights reserved.

Depression of net ecosystem CO2 exchange in semi-arid Leymus chinensis steppe and alpine shrub

Uptake and release of carbon in grassland ecosystems is very critical to the global carbon balance and carbon storage. In this study, the dynamics of net ecosystem CO2 exchange (FNEE) of two grassland ecosystems were observed continuously using the eddy covariance technique during the growing season of 2003. One is the alpine shrub on the Tibet Plateau, and the other is the sem-arid Leymus chinensis steppe in Inner Mongolia of China. It was found that the FNEE of both ecosystems was significantly depressed under high solar radiation. Comprehensive analysis indicates that the depression of FNEE in the L. chinensis steppe was the results of decreased plant photosynthesis and increased ecosystem respiration (R-eco) under high temperature. Soil water stress in addition to the high atmospheric demand under the strong radiation was the primary factor limiting the stomatal conductance. In contrast, the depression of FNEE in the alpine shrub was closely related to the effects of temperature on both photosynthesis and ecosystem respiration, coupled with the reduction of plant photosynthesis due to partial stomatal closure under high temperature at mid-day. The R,c of the alpine shrub was sensitive to soil temperature during high turbulence (u* > 0.2 m s(-1)) but its FNEE decreased markedly when the temperature was higher than the optimal value of about 12 degrees C. Such low optimal temperature contrasted the optimal value (about 20 degrees C) for the steppe, and was likely due to the acclimation of most alpine plants to the long-term low temperature on the Tibet Plateau. We inferred that water stress was the primary factor causing depression of the FNEE in the semi-arid steppe ecosystem, while relative high temperature under strong solar radiation was the main reason for the decrease of FNEE in the alpine shrub. This study implies that different grassland ecosystems may respond differently to climate change in the future. (c) 2006 Elsevier B.V All rights reserved.

Effect of Bacillus thuringiensis naturally colonising Brassica campestris var. chinensis leaves on neonate larvae of Pieris brassicae

The feeding of neonate larvae of Pieris brassicae (Order Lepidoptera) on leaves of brassica plants that had been colonised by Bacillus thuringiensis resulted in the death of 35% of the population within 72 h. The bacteria Multiplied in the cadavers, resulting in an increase of about 50-fold compared to the living insects. Surviving insects showed no ill effects during the time of the study. There was negligible multiplication of B. thuringiensis in the frass. (C) 2009 Elsevier Inc. All rights reserved.

Triterpenoids from Pulsatilla chinensis

A new lupane type triterpenic acid, pulsatillic acid, and two new lupane type triterpenoid glycosides, pulsatilloside A and B, along with the known 23-hydroxybetulinic acid were isolated from the roots of Pulsatilla chinensis. Their structures were characterized as 3-oxo-23-hydroxy-lup-20(29)-en-28-oic acid, 3 beta, 23-dihydroxy-lup-20(29)-en-28-oic acid 3-O-alpha-L-arabinopyranoside and 3 beta, 23-dihydroxy-lup-20(29)-en-28-oic acid 28-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside on the basis of hydrolysis and spectral evidence including two-dimensional relay HOHAHA, one-dimensional multiple relay COSY and ROESY NMR techniques. Pulsatillic acid exhibited cytotoxic activities against P-388, Lewis lung carcinoma and human large-cell lung carcinoma.

Nutritional responses in Indian white shrimp Fenneropenaeus indicus to varying protein: energy combinations in compounded artificial feeds

Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology

Cellular and humoral factors involved in defense mechanisms of Fenneropenaeus indicus (H. Milne Edwards, 1837)

The present investigation revealed three types of circulating haemocytes in the haemolymph of F. indicus: hyalinocytes, small-granule haemocytes, and large-granule haemocytes. Intermediate stages indicate the maturing process of a single cell. The presence of enzymes such as peroxidase, phenoloxidase and acid phosphatase in the haemocytes, and the substantial production of oxygen radicals during phagocytosis show that the haemocytes are capable of mounting a fme cellular defense mechanism. The enzyme activities of the serum and the presence of agglutinins in the serum, which may act as opsonins, agglutinate foreign particles and augment phagocytosis, confirm the presence of a superior humoral immune system in F. indicus.Bacterial infection caused considerable variations in the cellular and humoral factors, such as the number of circulating cells and haemagglutinating activity, especially in the initial hours of infection. The total haemocyte count, haemagglutination titer and phenoloxidase enzyme showed significant reductions on bacterial presence and could be used as indicators of bacterial infection.The number of circulating cells showed drastic fluctuation on exposure to pollutants. Nuvan at low concentrations was able to produce changes in the haemolymph factors and in the tissue organization, which implies that the animal is under stress and is easily prone to infections. Exposure to nuvan resulted in significant variation in all of the cellular and humoral factors, especially, the total haemocyte count, percentage of small granule haemocytes, phagocytic activity and the haemagglutinating activity, which might be good indicators of pesticide pollution. Heavy metal exposure caused significant increase in total haemocyte count and reduction in phenoloxidase enzyme activity Even changes in the physio-chemical parameters, such as salinity caused fluctuations in the defense factors, indicating stress in this euryhaline species. The dietary incorporation of a commercial immunostimulant containing P-l,3 glucan resulted in stimulation of some of the humoral defense factors of F indicus, but was time dependent. The modulations, on exposure to various external factors, in the cellular and humoral factors, especially, total haemocyte count, phagocytic activity, haemagglutinating activity and the phenoloxidase and acid phosphatase enzymes suggest that these parameters could be used as indicators of the health status of F indicus, which assist in better monitoring and effective health management of this important cultured species.

Haematology of Fenneropenaeus indicus in response to environmental alterations and microbial infections

Aquaculture has developed to become one of the fastest growing food producing sectors in the world.Today India is one among the major shrimp producing countries in the world.There are extensive and intensive shrimp culture practices. In extensive shrimp culture, shrimps are stocked at low densities (< 25 PLs m'2)in large ponds or tidal enclosures in which little or no management is exercised or possible. Farmers depend almost entirely on natural conditions in extensive cultures. Intensive shrimp culture is carried out in high densities (>200 PLs m'2). Much of the world shrimp production still comes from extensive culture.There is a growing demand for fish and marine products for human and animal consumption. This demand has led to rapid growth of aquaculture, which some times has been accompanied by ecological impacts and economic loss due to diseases. The expansion of shrimp culture always accompanies local environmental degradation and occurrence of diseases.Disease out breaks is recognised as a significant constraint to aquaculture production. Environmental factors, water quality, pollution due to effluent discharge and pathogenic invasion due to vertical and horizontal transmission are the main causes of shrimp disease out breaks. Nutritional imbalance, toxicant and other pollutants also account for the onset of diseases. pathogens include viruses, bacteria, fungi and parasites.Viruses are the most economically significant pathogens of the cultured shrimps world wide. Disease control in shrimp aquaculture should focus first on preventive measures for eliminating disease promoting factors.ln order to design prophylactic and proactive measures against shrimp diseases, it is mandatory to understand the immune make up of the cultivable species, its optimum culture conditions and the physico chemical parameters of the rearing environment. It has been proven beyond doubt that disease is an end result of complex interaction of environment, pathogen and the host animal. The aquatic environment is abounded with infectious microbes.The transmission of disease in this environment is extremely easy, especially under dense, culture conditions. Therefore, a better understanding of the immune responses of the cultured animal in relation to its environmental alterations and microbial invasions is essential indevising strategic measures against aquaculture loss due to diseases. This study accentuate the importance of proper and regular health monitoring in shrimps employing the most appropriate haematological biomarkers for application of suitable prophylactic measures in order to avoid serious health hazards in shrimp culture systems.

A marine candida sake as source of immunostimulants to fenneropenaeus indicus

Aquaculture has developed rapidly over the last three decades to become an important activity worldwide.The Food and Agricultural Organization (FAO) of the UN acknowledge that global fishery output must be increased by at least 50% to offset projected shortfalls in dietary protein by 2030.LAquaculture has developed rapidly over the last three decades and has become an importat industry as today’s demand for fish exceeds the natural supply.lmmunostimulants are chemical compounds that activate the immune system of animals and render them more resistant to infections by viruses, bacteria, fungi, and parasites. lmmunostimulants have been obtained from diverse natural sources where, microbial cell wall acts as the main source.The salient findings of the study are summariseSeven marine yeasts were screened for growth promoting and immunostimulant property in F. indicus. Candida sake S165 was found to be best in terms of its support for growth and protection against white spot virus infection.The study revealed that marine yeast Candida sake can be effectively used as potential source of immunostimulants for application in penaeid prawns culture systems. The study emphasise the fact that the dose and frequency of application of immunostimulants are to be standardised and validated before commercialisation to achieve optimum stimulation of the immune system and to avoid immune fatigue die to verdose.Marine yeast (whole cell) was found to support better immunostimulation compared to its cell wall component B-1,3-glucan. This study shows that administration of marine yeast (whole cell) or B-1,3-glucan as immunostimulants in aquaculture would definitely help in protection of the stock to a few more days even though total protection is not being imparted. This partial protection itself would be highly helpful to the farming industry so that they can get sufficient time to plan for a safe harvest and save the crop from cent percent mortality.

Immunostimulatory effect of a marine yeast Candida sake S165 in Fenneropenaeus indicus

The efficacy of a marine yeast Candida sake as source of immunostimulant to Indian white shrimp Fenneropenaeus indicus was estimated. Biomass of C. sake was prepared using malt extract agar and incorporated at graded levels into a standard diet to prepare yeast diets of varying biomass concentrations (1%, 10% and 20%). F. indicus were fed on these diets for a period of 28 days and challenged orally with white spot syndrome virus (WSSV) and immune parameters such as total haemocyte count, phenoloxidase and nitroblue tetrazolium reduction (NBT) were determined. Ten per cent C. sake in the diet was found to support an optimum immune response in the animals in general and their enhancement could be observed on the second and third day following challenge with the virus. The study has demonstrated that marine yeast C. sake at 10% in diet (w/w) may be used as an effective source of immunostimulants in F. indicus

Probiotic Effects of Lactic Acid Bacteria Against Vibrio Alginolyticus in Penaeus (Fenneropenaeus) Indicus (H. Milne Edwards)

Cell free extracts of four strains of Lactic acid bacteria (LAB) viz. Lactobacillus. acidophilus, Streptococcus.cremoris, Lactobacillus bulgaricus –56 and Lactobacillus bulgaricus –57 inhibited growth of Vibrio alginolyticus in nutrient broth. The antagonism of LAB to Vibrio alginolyticus was further confirmed by streak plating wherein suppression of growth of Vibrio was obtained. Juveniles of Penaeus indicus (average weight 0.985 ± 0.1 g) on administering orally a moist feed base containing 5 × 106 cells·g of the four LAB probionts for a period of four weeks showed better survival (56 to 72%) when challenged with V. alginolyticus by intra-muscular injection of 0.1 ml containing 3 × 109 cells·ml. Animals maintained on a diet devoid of bacterial biomass exhibited 80% mortality. No external or internal pathological changes were observed in shrimp fed with the LAB incorporated diets. Results showed inhibition of V. alginolyticus by LAB and stimulation of the non-specific immune response resulting in resistance to disease in the shrimp fed on LAB incorporated diets.