Partial characterization of the hemolytic activity of the nematocyst venom from the jellyfish Cyanea nozakii Kishinouye

Using a recently developed technique to extract jellyfish venom from nematocysts, the present study investigated the hemolytic activity of Cyanea nozakii Kishinouye nematocyst venom on chicken erythrocytes. Venom extract caused a significant concentration-dependent hemolytic effect. The extract could retain its activity at -80 degrees C but was unstable when kept at 4 degrees C and -20 degrees C for 2 days. The hemolytic activity was inhibited by heating within the range of 37-100 degrees C. The extract was active over a pH range of 5.0-8.63 and the pH optima for the extract was 7.8. Incubation of the venom with sphingomyelin specially inhibited hemolytic activity by up to 70%. Cu2+ and Mn2+ greatly reduced the hemolytic activity while Mg2+, Sr2+ and Ba2+ produced a relatively low inhibiting effect on the hemolytic activity. Treatment with Ca2+ induced a concentration-dependent increase in the hemolytic activity. In the presence of 5 mM EDTA, all the hemolytic activity was lost, however, the venom containing 1.5 mM EDTA was stable in the long-term storage. PLA(2) activity was also found in the nematocyst venom of C. nozakii. These characteristics provide us a fundamental knowledge in the C. nozakii nematocyst venom which would benefit future research. (C) 2010 Published by Elsevier Ltd.

两种大型水母毒素的分离纯化及其蛋白酶活性和抗氧化活性的研究

水母在我国分布广，种类多，数量大，是一种丰富的海洋生物资源。已研究的水母毒素主要分布于触角和刺丝囊中，是一类结构新颖独特的肽类毒素。水母种类的不同，其毒素的结构、生物活性往往不同。本文首次报道了海蜇（Rhopilema esculentum Kishinouye）毒素 和白色霞水母（Cyanea nozakii Kishinouye）毒素的提取、纯化、理化性质及蛋白酶和抗氧化方面的生物学活性，主要结果如下： 1.用DEAE Sepharose Fast Flow分离海蜇毒素，用含0.4 mol/L NaCl 的PBS (0.02 M，pH 8.0) 进行阶段洗脱时，得到的组分是组蛋白H4。 2.水母毒素的蛋白酶活性实验结果为：海蜇毒素和白色霞水母毒素都有蛋白酶活性。该两种毒素的蛋白酶活性都受理化因素的影响，其中0.5%甘油和1%邻菲罗啉可以有效的抑制海蜇毒素的蛋白酶活性。 3.对海蜇毒素蛋白的体外抗氧化活性研究表明：蛋白样品均具有抗氧化性，它们对超氧阴离子和羟自由基具有显著的清除作用。对白色霞水母毒素蛋白的体外抗氧化活性研究表明：白色霞水母蛋白具有较高的清除羟自由基的能力，白色霞水母蛋白对超氧阴离子的清除作用较弱。白色霞水母蛋白有较强的还原能力，但没有螯合能力。 通过本文的研究表明水母毒素具有蛋白酶活性和抗氧化活性，能够清除氧自由基。这些研究结果为开发利用水母资源和毒素的合理、综合利用打下了坚实的基础。

Female Reproductive Biology and Spawning Periodicity of Two Species of King Prawns, Penaeus longistylus Kubo and Penaeus latisulcatus Kishinouye, from Queensland's East Coast Fishery

In the coastal region of central Queensland female red-spot king prawns, P. longistylus, and the western or blue-leg king prawns, P. latisulcatus, had high mean ovary weights and high proportions of advanced ovary development during the winter months of July and August of 1985 and 1986. On the basis of insemination, both species began copulating at the size of 26-27 mm CL, but P. longistylus matured and spawned at a smaller size than P. latisulcatus. Abundance of P. longistylus was generally three to four times greater than that of P. latisulcatus but the latter was subject to greater variation in abundance. Low mean ovary weight and low proportions of females with advanced ovaries were associated with the maximum mean bottom sea-water temperature (28.5ºC) for both species. Population fecundity indices indicated that peaks in yolk or egg production (a) displayed a similar pattern for both species, (b) varied in timing from year to year for both species and (c) were strongly influenced by abundance. Generally, sample estimates of abundance and commercial catch rates (CPUE) showed similar trends. Differences between the two may have been due to changes in targeted commercial effort in this multi-species fishery.

Comparison of physicochemical properties of muscle proteins in cultured and wild prawns (Penaeus (Fenneropenaeus) orientalis Kishinouye, 1918)

Protein physicochemical properties in cultured and wild prawns (Penaeus (F.) orientalis Kishinouye, 1918) were studied and compared. Protein fractions were separated into water-soluble, salt-soluble, alkali-soluble, and stroma. The results showed that salt- and alkali-soluble proteins were slightly higher in wild prawns and water-soluble proteins were higher in cultured prawns. There were only slight differences in Ca2+-ATPase, MG2+-ATPase, and ATP sensitivities. The textural values of wild prawns were significantly higher than the cultured ones.

Studies on freezing and storage of Psenopsis cyanea

The freezing and storage characteristics of Psenopsis cyanea caught on board FORV Sagar Sampada from a depth of 350 m off Cochin are reported. The fat content of the fish was high (15.58% on the weight of whole fish) and the meat was white in colour. Peroxide value, free fatty acids and thiobarbituric acid values increased during frozen storage and organoleptically the fish was acceptable up to 32 weeks at -22 ± 1°C.

Studies on canning Psenopsis cyanea

Suitability of Psenopsis cyanea, a deep sea fish caught on board FORV Sagar Sampada from a depth of 350m off Cochin for processing into canned product was studied. The fish having high fat content around 52% (DWB) and white attractive meat renders itself well for canning yielding a product good in organoleptic characteristics. However to have a presentable appearance to the product the fish has to be skinned prior to canning. Gentle agitation of P. cyanea in 0.25% aqueous solution of lactic acid at ambient temperature for 15 min followed by thorough washing yields skinless fish. Fish suffers around 8% (DWB) fat loss during skinning. Fish canned in natural style and in brine have better organoleptic characteristics initially. On storage they develop slight rancidity. Fish canned in oil maintains the characteristics over a longer period.

Comparison of physicochemical properties of muscle proteins in cultured and wild prawns (Penaeus (Fenneropenaeus) orientalis Kishinouye, 1918)

Protein physicochemical properties in cultured and wild prawns (Penaeus (F.) orientalis Kishinouye, 1918) were studied and compared. Protein fractions were separated into water-soluble, salt-soluble, alkali-soluble, and stroma. The results showed that salt- and alkali-soluble proteins were slightly higher in wild prawns and water-soluble proteins were higher in cultured prawns. There were only slight differences in Ca super(2+)-ATPase, MG super(2+)-ATPase, and ATP sensitivities. The textural values of wild prawns were significantly higher than the cultured ones.

黄东海胶质浮游动物水母类研究

近年来，胶质类浮游动物尤其是水母类在近岸高生产力的海洋生态系统中的作用越来越受到全球科学家们的关注。这种关注很大程度上是由于近年来很多海域或海湾出现水母类大量繁殖的现象而引起的，这种现象与过度捕捞、气候变化、富营养化以及生物入侵等因素有着内在的联系，给人们带来经济，社会和生态等各方面的损失。这种损失包括：当水母大量出现时，由于它们与鱼类存在食物和捕食竞争，使渔业资源和渔业生产锐减；由于它们会堵塞和撕破渔业生产网，干扰了人们的渔业生产活动；另外水母增多增加了海滨游泳爱好者被蛰伤甚至死亡事件发生的频率，对旅游者们的娱乐行为造成不便。到目前为止全球范围内报道了许多水母大量繁殖并对经济社会或海洋生态系统造成负面影响的事件。在北海水母数量爆发的频率与气候相关的事实表明了该海域将要面临一个更多的胶质类浮游动物的未来。所有这些都表明海洋中水母的生态学问题仍旧是全球的一个研究热点。到目前为止，在世界上许多海湾和海区有关水母生态学问题的研究上，均已取得方方面面有意义的进展。但在我国，由于对水母功能群的研究缺乏足够的重视，对于大型水母来讲它们被认为是渔业生产的副产品或者‘垃圾’，在渔业拖网捕获后把它们直接扔入海中并不去研究；对于小型水母来讲，因为它们为非饵料生物，并且有易碎，粘糊糊等难操作的特点，对浮游生物网同步采集到的水母的研究力度远不如对其他浮游动物类群的研究力度，因此我国对水母功能群生态学的研究基础相当薄弱。 本论文基于以上背景对黄东海大型和小型水母类的生物地理分布格局进行了研究，对其生物量或丰度等生态学指标进行了定量研究。（1）利用浮游生物网样品对黄海小型水母类的种类组成及其丰度的时空变化进行了研究，表明黄海测区内小型水母整体丰度很低，且主要分布在50 m等深线以浅海域，各水母类群以及优势种类的季节更替非常明显。比较不同海区的小型水母的丰度水平及其占浮游动物丰度的比例，发现黄海的小型水母丰度水平最低，为0.8（0.04–1.3）ind. m-3，只占浮游动物丰度的 < 0.5 %，表明小型水母在黄海海域并非占优势的功能群。（2）在中国黄东海采集的沙海蜇和在日本采集的越前水母的COI基因序列差异（0.2%）处于种内水平，从分子水平上证明两者为同一个种类，基于形态学和分子生物学的证据，我国大型水母沙海蜇的分类地位可初步订正为：Nemopilema nomurai Kishinouye, 1922（Scyphozoa：Rhizostomeae：Rhizostomatidae）。（3）利用渔业底层拖网的方法对黄东海大型水母的种类组成，总生物量及各优势种类生物量的时空分布进行了半定量研究。结果共鉴定到11个种（类），其中沙海蜇（Nemopilema nomurai），洋须水母（Ulmaridae genus sp.）以及多管水母（Aequorea spp.）为黄海的优势种（类），沙海蜇（只在东海北部）和霞水母（Cyanea spp.）为东海的优势种（类）；这四个种（类）的湿重随着伞径的增大成冪增长的方式。大型水母平均总生物量的季节变化模式为：3月份水母总生物量最低，为4.6 ± 9.4 kg km–2，春夏季随着海水表层温度的升高，大型水母的生物量逐渐增加，9月初水母的总生物量达到最高值，为22891 ± 25888 kg km–2，随后随着海水的温度下降，生物量也逐渐下降。洋须水母的生物量在10月份达到最高值（2780 ind. km–2，1807 kg km–2），主要在黄海中部出现。多管水母在5月份丰度最高，为8262 kg km–2，且主要分布在30N以北海域。 聚焦大量爆发的水母种（类）沙海蜇和霞水母，基于实测的拖网资料，提供了该种可见的浮游阶段1周年的地理发生和生物量数据；结果表明，这两个种（类）表现了不同的季节出现和生物量格局：首先是黄海出现的沙海蜇（5–12月在黄海出现），5月份少量幼水母体在黄东海的交界处出现，6月它们的分布范围在南黄海扩大，到8月末及9月初沙海蜇几乎遍布黄海，其生物量和丰度以压倒其他大型水母类的优势形成“bloom”（生物量占所有大型水母的96.7%，丰度占93%，在南黄海平均生物量20446 kg km-2，平均占渔获物生物量的86.1 %），10月份至12月，沙海蜇的生物量逐渐减少甚至为零，其分布区域也向北回缩。其次为东海出现的霞水母（5月–10月）：霞水母在5月初达到生物量的高值（平均生物量为380 kg km-2），其伞径随纬度的升高而变小，8月至10月其生物量骤然下降。这两个种类的生物量高值分布在温度或潮汐锋区。结合水文条件及沙海蜇和霞水母关键的生活史策略推测了黄东海该种浮游阶段的生活史模型。 最后，通过对黄海8、9月份沙海蜇生物量的最高峰或暴发时期的呼吸率，摄食率进行估算，获得其每天对浮游动物现存量及生产力的潜在摄食压力，结果表明2006年9月在对沙海蜇最大捕获率的情况下，沙海蜇的摄食率为8.37（0.12–37.83）mg C m-2d-1，假设都以浮游动物为食，这时每天对浮游动物现存量及生产力的摄食压力分别为11.2%（0.17–50.6%），134.1%（1.98–605.7%）。因此在沙海蜇暴发期间对浮游动物的潜在的消耗非常大，甚至为毁灭性的。

Isolation and characterization of venom from nematocysts of jellyfish Rhopilema esculentum Kishinouye

The present work is the first report of the biochemical characterization of the venom from nematocysts of the jellyfish Rhopilema esculentum Kishinouye. The nematocysts were isolated by autolysis and centrifugation and separated by flow cytometry. Four types of nematocysts were identified: mastigophores, euryteles, and atrichous and holotrichous isorhiza. SDS-PAGE and amino acid analyses demonstrated that most of the proteins in the nematocyst extract were between 10 kDa and 40 kDa, and that glutamic acid was the main amino acid. A hemolytic activity assay showed that the activity of the nematocyst venom (RNV) was strongest in Tris-HCl buffer (50 mmol/L, pH 7.8, 5% glycerol, 0.5 mmol/L EDTA, 0.1 mol/L NaCl). The hemolytic activity was related to protein concentration and the HU50 against chicken erythrocytes was 0.91 A mu g/mL.

Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well.

Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye

In this paper, the effects of some chemical and physical factors such as temperature, pH values, glycerol, and divalent metal cations on the protease activity of venom from jellyfish, Rhopilema esculentum Kishinouye, were assayed. Protease activity was dependent on temperature and pH values. Zn2+, Mg2+, and Mn2+ in sodium phosphate buffer (0.02 M, pH 8.0) could increase protease activity. Mn2+ had the best effects among the three metal cations and the effect was about 20 times of that of Zn2+ or Mg2+ and its maximal protease activity was 2.3 x 10(5) U/mL. EDTA could increase protease activity. PMSF had hardly affected protease activity. O-Phenanthroline and glycerol played an important part in inhibiting protease activity and their maximal inhibiting rates were 87.5% and 82.1%, respectively. (c) 2005 Elsevier Ltd. All rights reserved.

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye was determined against three pest species, Stephanitis pyri Fabriciusa, Aphis medicaginis Koch, and Myzus persicae Sulzer. R. esculentum full proteinous venom had different insecticidal activity against S. pyri Fabriciusa, A. niedicaginis Koch, and M. persicae Sulzer. The 48 It LC50 values were 123.1, 581.6, and 716.3 mu g/mL, respectively. Of the three pests, R. esculentuin full proteinous venom had the most potent toxicity against S. pyri Fabriciusa, and the corrected mortality recorded at 48 It was 97.86%. So, S. pyri Fabriciusa could be a potential target pest of R. esculentum full proteinous venom. (c) 2005 Elsevier Ltd. All rights reserved.

Factors influencing hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye

In this study, hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye and some factors affecting it were assayed. The HU50 of R. esculentum full venom (RFV) against chicken erythrocytes was 3.40 mu g/ml and a Hill coefficient value was 1.73 suggesting at least two molecules participated in hemolytic activity. The hemolytic activity of RFV was affected by some chemical and physical factors such as divalent cations, EDTA, (NH4)(2)SO4, pH and temperature. In the presence of Mg2+, Cu2+, Zn2+, Fe2+, Ca2+ ( >= 2 mM), Mn2+ (>= 1 mM), EDTA (>= 2 mM) and (NH4)(2)SO4, the hemolytic activity of RFV was reduced. RFV had strong hemolytic activity at the pH 6-10 and the hemolytic ratios were 0.95-1.19. Hemolytic activity was temperature-sensitive and when RFV was pre-incubated at temperatures over 40 degrees C, it was sharply reduced. (c) 2007 Elsevier Ltd. All rights reserved.

Studies on the hemolytic activity of tentacle extracts of jellyfish Rhopilema esculentum Kishinouye: Application of orthogonal test

The present work is first reporting the hemolytic activity of venom from jellyfish Rhopilema esculentum Kishinouye extracted by different phosphate buffer solutions and incubated at different temperature according to the orthogonal test L6(1) x 3(6). Of the seven controllable independent variables, incubated temperature and phenylmethylsulfonyl fluoride (PMSF) had strongest effect on the hemolytic activity. (c) 2006 Elsevier B.V. All rights reserved.