中华绒螯蟹(Eriocheir sinensis)精子顶体反应的研究

分别用卵水、海水、caCl_2或NaCl水溶液对中华绒螯蟹成熟精子进行人工诱导顶体反应,结果表明:精子的生理性成熟、同种卵或Ca~(++)的存在、碱性环境以及与一定的固体接触均为精子顶体反应触发的重要条件。3月份精子诱导率最高。 电镜观察证明,中华绒螯蟹精子的顶体反应可分四个阶段:(1)辐射臂收缩;(2)顶体囊外翻;(3)顶体管前伸;(4)片层结构脱落。     

中华绒螯蟹(Eriocheir sinensis)雄性生殖系统的组织学研究

中华绒螯蟹雄性生殖系统的组织学研究表明:生精小管一侧为管壁上皮,另一侧为生发区,生殖细胞由生发区基底部同管腔增殖。输精管分为输精细管和贮精囊,管壁上皮具分泌功能,贮精囊有肌肉层。射精管壁肌肉层较厚,粘膜形成纵行皱襞。副性腺内壁为单层立方上皮。生殖系统发育有明显的季节性,8月开始发育加速,10月进入高峰,4月开始发育停滞。     

饥饿对中华绒螯蟹(Eriocheir sinensis)幼体发育的影响

对刚孵化的中华绒螯蟹第一期的蚤状幼体经不同时间的饥饿后再投喂,发现饥饿可以明显降低幼体的存活率和延长幼体的发育期。实验表明:对中华绒螯蟹第一期的蚤状幼体的饥饿时间(t)和发育期长(D)呈线性关系(D=4.6303+1.3226t r=0.970p<0.01)。对于中华绒螯蟹第一期的蚤状幼体,当起始饥饿时间超过了4d,再予以投饵,幼体均不能恢复正常的发育和蜕皮功能,得出中华绒螯蟹的不可恢复点(the point of no-return,PNR)大约为4d。通常以产生50%的幼体死亡的饥饿期即PNR₅₀,来表明幼体对饥饿的抵抗能力,实验得出中华绒螯蟹第一期的蚤状幼体的PNR₅₀大约为48h。     

中华绒螯蟹Eriocheir sinensis H.Milne-Edwards的幼体发育

1.本文所报道毛蟹的幼体发育试验,全部是在实验室内进行的。 2.毛蟹的幼体发育共经五个(氵蚤)状幼体期和一个大眼幼体期。卵孵化出膜的幼体即为第一(氵蚤)状幼体,而不是早期(氵蚤)伏幼体或原(氵蚤)伏幼体。这两种幼体应该是在卵膜内度过的。 3.在水温11—22℃,盐度为9‰的实验室条件下,从幼体出膜到第一期幼蟹的出现,共经39—40天。每蜕一次皮,即进入另一发育时期,其所需的时间,随着温度的升高而缩短。其发育速度见下表: 幼体名称 水温(℃) 日数 第一(氵蚤)状幼体 11—17 7—9 第二(氵蚤)状幼体 17—18 5—6 第三(氵蚤)状幼体 18—20 6—7 第四(氵蚤)状幼体 16—19.5 5—6 第五(氵蚤)状幼体 16.5—19 7—8 大眼幼体 19—22 9—10 4.饵料、盐度、水温和水质等因子对幼体有着不同程度的影响。 5.(氵蚤)状幼体以两对颚足外肢末端的羽伏刚毛的数目,胸、腹肢的大小与形状和尾叉内面中部刚毛的数目为分期的主要依据。     

基于ND4和ND5基因序列分析的鳅超科鱼类系统发育关系

ND4和ND5是线粒体基因组中编码NADH脱氢酶亚基4和亚基5的两个蛋白质编码基因.该研究以鳅超科鱼类为研究对象,新测定了10个物种的ND4和ND5基因全序列以及中间的3个tRNA基因共212 bp的序列,结合从GenBank 下载的15个物种的15条序列进行序列比较和系统发育关系分析.结果显示:鳅超科鱼类ND4基因全长1380～1387 bp,以ATG为起始密码子,终止密码子为不完全终止信号;ND5基因全长1821～1839bp,同样起始密码子为ATG,终止密码子为TAA或TAG;ND4和ND5基因之间插入了3个tRNA基因,分别编码携带组氨酸、丝氨酸、亮氨酸的tRNA.ND4和ND5基因(包含3个tRNA基因)中A、T、G、C的平均含量分别为30.4%、27.3%、14.2%、28.1%,A+T(57.7%)的含量高于G+C(42.3%)的含量.转换与颠换比(Ti/Tv)平均值为1.586.选取斑马鱼和鲤鱼作为外类群,采用最大简约法(MP)、最大似然法(ML)和贝叶斯推断法(BI)进行系统发育树的重建.三种方法的系统发育分析结果都显示:花鳅亚科、条鳅亚科、沙鳅亚科、平鳍鳅科及Vaillantellidae分别构成单系;它们的系统发育关系为:(Vaillantellidae+(沙鳅亚科+(花鳅亚科+(条鳅亚科+平鳍鳅科).这与线粒体全基因组和某些核基因(如RAG1基因)的研究结果类似,且支持率较高,表明ND4和ND5基因用于鳅超科鱼类的系统发育分析是可行的;但是该研究的结果有别于其他线粒体基因的分析结果,如基于cytb和D-loop基因进行的系统发育分析表明,条鳅亚科和花鳅亚科聚为姐妹群,再和平鳍鳅科聚在一起.这种差异可能是由于使用的基因长度差异造成的,长度越长,信息量越大,所反映的系统发育结果可能更加接近真实情况.     

中华绒螯蟹(Eriocheir sinensis)肌肉中不饱和脂肪酸含量与越冬死亡、产卵关系

本文报道了中华绒螯蟹越冬时和越冬后不同性别,不同生活状态粗脂肪中不饱和脂肪酸及肌肉中不饱和脂肪酸含量及变化,分析了不饱和脂肪酸含量与越冬死亡,越冬后交配,产卵和孵化关系。越冬前雌,雄河蟹粗脂肪中不饱和脂肪酸碘（I2）值为57和48。越冬（越冬时,越冬后）死亡雌,雄河蟹粗脂肪中不饱和脂肪酸I2值分别低于28和10;而其肌肉中不饱和脂肪酸的I2值分别低于1．25和0．83。越冬后能成功交配的雄河蟹粗脂肪中不饱和脂肪酸I2值高于33。越冬后交配产卵雌河蟹粗脂肪中不饱和脂肪酸I2值高于42,抱卵并使卵正常孵化的雌河蟹粗脂肪中不饱和脂肪酸I2值大于47。脂肪酸在河蟹的细胞内氧化分解,为越冬提供了能量。     

中华绒螯蟹与合浦绒螯蟹两地理亚种的线粒体DNA序列变异

根据线粒体16SrDNA的PCR／RFLP鉴定和对Cyt b基因全序列的分析对中国大陆7水系绒螯蟹地理种群的遗传分化进行探讨。结果表明：中华绒螯蟹与合浦绒螯蟹两亚种在17条Cyt b全序列上以40个固定的碱基变异位点相区别。前者分布在瓯江及其以北的水系;后者主要分布在瓯江及其以南的水系。发现了4种在鸭绿江以南的北方水系中生活的合浦亚种单元型,对这些单元型的形成提出了3种可能的解释[动物学报51（5）：862—866,2005]。     

中华绒螯蟹一龄幼蟹(Eriocheir sinensis)越冬前后脂肪酸和4种类胡萝卜素含量的变化

实验以越冬前后的中华绒螯蟹一龄幼蟹作为研究对象,利用高效气相色谱法分别检测一龄幼蟹越冬前后肝胰腺及头胸甲中脂肪酸含量变化、利用液相二级质谱法分别测定一龄幼蟹越冬前后肝胰腺、头胸甲和血淋巴中4种类胡萝卜素含量变化。结果表明:肝胰腺中的饱和脂肪酸(SFA) C20:0、单不饱和脂肪酸(MUFA) C18:1n9作为主要能量脂肪酸被动用,其含量越冬后显著下降(p0.05),多不饱和脂肪酸(PUFA)中的C18:3n3、C22:6n3越冬后含量显著升高(p0.05)。头胸甲的饱和脂肪酸(SFA) C16:0、C18:0,单不饱和脂肪酸(MUFA)中的C18:1n7作为主要能量脂肪酸被动用,越冬后含量下降显著(p0.05)。多不饱和脂肪酸(PUFA) C18:2n6、C20:3n6越冬后含量显著升高(p0.05)。越冬后头胸甲中4种类胡萝卜素含量均极显著下降(p0.01)。其中,血淋巴中虾青素、叶黄素含量极显著升高(p0.05),β-胡萝卜素含量极显著下降(p0.05)。肝胰腺中虾青素与叶黄素、β-胡萝卜素变化趋势与血淋巴的一致,角黄素含量显著下降(p0.05)。越冬期间气温、水温的短暂升高可能使一龄幼蟹短暂摄食。越冬前经育肥的扣蟹越冬后能量物质无显著变化,越冬后根据肝胰腺颜色来判断一龄幼蟹质量的方法不科学。     

POPU金钱豹mtDNA ND5基因序列及其遗传变异

测定山西7个不同地域金钱豹9个体mtDNA ND5基因部分序列,结合GenBank下载的37条序列,进行遗传多样性分析,用豹属的狮Panthera leo和虎Panthera tigris作外群,构建其不同单倍型的NJ分子聚类关系。46条序列共产生18个单倍型,单倍型多样度和核苷酸多样度指数表明金钱豹的遗传多样性比较丰富,AMOVA分析显示其种群间出现显著的遗传分化。单倍型聚类分析表明,亚洲和非洲种群尽管有一定的分化,但其置信度很低,不足以形成各自独立的分支。     

Phylogenetic analysis of intestinal bacteria in the Chinese mitten crab (Eriocheir sinensis)

AIMS: To identify the dominant intestinal bacteria in the Chinese mitten crab, and to investigate the differences in the intestinal bacteria between pond-raised and wild crabs. METHODS AND RESULTS: The diversity of intestinal bacteria in the Chinese mitten crabs was investigated by denaturing gradient gel electrophoresis (DGGE) fingerprinting, 16S rRNA gene clone library analysis and real-time quantitative PCR. The principal component analysis of DGGE profiles indicated that substantial intersubject variations existed in intestinal bacteria in pond-raised crab. The sequencing of 16S rRNA genes revealed that 90-95% of the phylotypes in the clone libraries were affiliated with Proteobacteria and Bacteroidetes. Some genera were identified as unique in wild crabs and in pond-raised crabs, whereas Bacteroidetes was found to be common in all sampled crab groups. Real-time quantitative PCR indicated that the abundance of Bacteroides and the total bacterial load were approximately four-to-10 times higher in pond-raised crabs than in wild crabs. A significant portion of the phylotypes shared low similarity with previously sequenced organisms, indicating that the bacteria in the gut of Chinese mitten crabs are yet to be described. CONCLUSIONS: The intestinal bacteria of pond-raised crabs showed higher intersubject variation, total diversity and abundance than that observed in wild crabs. The high proportion of the clones of Proteobacteria and Bacteroidetes in the clone library is an indication that these bacteria may be the dominant population in the gut of the Chinese mitten crab. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated obvious differences in the intestinal bacterial composition of pond-raised crabs and wild crabs. This knowledge will increase our understanding of the effects of aquaculture operations on bacterial community composition in the crab gut and provide necessary data for the development of probiotic products for crab cultivation.     

中华绒螯蟹两种微卫星DNA分型方法的应用比较

为对比荧光标记毛细管电泳和聚丙烯酰胺凝胶电泳在微卫星等位基因分型上的效果,试验选择6对微卫星引物,对60个中华绒螯蟹个体基因组DNA进行PCR扩增,荧光标记法共检测出等位基因129个,平均每个位点21.5个等位变异,聚丙烯酰胺凝胶电泳共检测出等位基因174个,平均每个位点29个等位变异。对位点Esin67的PCR产物分别进行重复检测,发现荧光标记法的重复率为100%,而聚丙烯酰胺凝电泳法两次结果存在较大差异,证实了荧光标记法检测的可靠性显著高于聚丙烯酰胺凝胶电泳。对两种方法的检测效率和经济成本的分析表明,荧光标记法虽然成本较高,但效率高于聚丙烯酰胺凝胶电泳法。建立单重PCR的多重毛细管电泳技术是提高效率、降低成本的有效途径。     

Reproductive function of Selenoprotein M in Chinese mitten crabs (Eriocheir sinesis)

Selenoproteins are present in all major forms of life, including eukaryotes, bacteria and archaea. In eukaryotic animals, selenoproteins often function as antioxidants, but rare or absent in other phyla, such as plants and fungi (except for the green alga Chlamydomonas). Selenoprotein M (SelM) is a selenocysteine containing protein with redox activity, which is involved in the antioxidant response. However, information remains limited about SelM physiology and function in marine invertebrates, particularly in crustaceans. Hence, we investigated the reproductive functionality of SelM in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important yet disease vulnerable aquaculture species. The full-length SelM cDNA (928bp) strand was cloned by using PCR, based on an initial expressed sequence tag (EST) that was isolated from a hepatopancreatic cDNA library. The SelM cDNA contained a 390bp open reading frame (ORF) that encoded a putative 129 amino acid (aa) protein. SelM mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in the hepatopancreas, testis, ovaries and intestines. Based on this information, we then detected the different stages of tissue expression for SelM in the testis, ovary, and male crab hepatopancreas and hemolymph, and the enzyme activity of SelM in the testis. Overall, SelM was isolated successfully from the Chinese mitten crab, and its involvement in the regulation of reproduction during the period of rapid development in E. sinensis was confirmed.     

Phylogenetic relationships of catostomid fishes (Actinopterygii: Cypriniformes) based on mitochondrial ND4/ND5 gene sequences

Family Catostomidae is a diverse group of benthic freshwater fishes that are distributed across North America and in parts of East Asia. In this study, the phylogenetic relationships of Catostomidae is examined using 3436 nucleotides of mitochondrial ND4 and ND5 protein coding genes and intervening tRNAs. All 13 genera and 60 species of catostomids were sampled to represent diversity of the family. Catostomidae and its four subfamilies were found to be monophyletic; however, relationships of the subfamilies are not strongly supported with bootstrapping. The analysis provides strong support for recognizing four tribes in subfamily Catostominae.     

Development and characterization of new microsatellite loci from Chinese mitten crab (Eriocheir sinensis)

A set of new microsatellite loci were isolated from Chinese mitten crab and characterized in two Chinese mitten crab populations. The number of the alleles ranged from 3 to 8 and observed and expected heterozygosity varied from 0.0870 to 0.9565 and 0.3092 to 0.8367 in each population, respectively. Loci HLJEsin1, HLJEsin4, HLJEsin30 in CLN population and HLJEsin6 in CJS population presented significant deviation from Hardy–Weinberg equilibrium (PHWE < 0.001). These markers will be useful for population studies.     

饥饿胁迫对中华绒螯蟹(Eriocheir sinensis)仔蟹的影响

在26.2 ～28.4℃水温条件下,研究了饥饿对中华绒螯蟹(Eriocheir sinensis)仔蟹的形态、行为、存活及体重损失的影响,同时确定了仔Ⅱ的营养饱和储存点(PRS)和不可恢复点(PNR).结果表明:饥饿胁迫下中华绒螯蟹仔Ⅰ、仔Ⅱ、仔Ⅲ的初次死亡时间(T1)分别为8.0、14.0和20.3 d,50％死亡时间(Ts0)分别为11.4、16.0和25.5 d,100％死亡时间(T100)分别为15.0、22.0和32.3 d,耐饥饿能力为仔Ⅲ＞仔Ⅱ＞仔Ⅰ;饥饿期间中华绒螯蟹体内水分含量持续升高,干重量降低显著,干重损失速率也随时间延长逐渐减小;先饱食后饥饿的给饵模式中仔Ⅱ蜕皮率随初始饱食时间延长而升高,50％个体完成蜕皮所需饱食时间(PRS50)为2.10d,各处理组仔Ⅱ的蜕皮周期与持续饱食组均无显著差异(P＞0.05),但只有饱食3d以上才能达到持续饱食饲养蟹的增重率;先饥饿后饱食的给饵模式中,仔Ⅱ的蜕皮率随着初始饥饿时间的延长而下降,其中仔Ⅱ50％不能蜕皮的初始饥饿时间(PNR50)和100％不能蜕皮的初始饥饿时间(PNR100)分别为10 d和14 d,并且蜕皮周期相对延长,延长时间约等于初始饥饿时间,不存在额外的摄食时间来弥补饥饿期间损失的能量,各处理组蜕皮后仔蟹与对照组体重无显著差异(P＞0.05).     

Microsatellite analysis of genetic diversity and population structure of Chinese mitten crab （Eriocheir sinensis）

Chinese mitten crab (Eriocheir sinensis) has higher commercial value as food source than any other species of Eriocheir in China.To evaluate the germplasm resources and characterize the genetic diversity and population structure of the crabs in different water systems,two stocks and two farming populations were assessed with 25 polymorphic microsallite loci available in public GenBank.Basic statistics showed that the average observed heterozygosity (Ho) amongst populations ranged from 0.5789 to 0.6824.However,a remarkable presence of inbreeding and heterozygote deficiencies were observed.To analyze population structure,pairwise FST coefficients explained only ～10.3% variability from the subdivision of mitten crab populations,the remaining variability stems from the subdivision within subpopulations.Although the four populations had slight differentiation,different allelic frequencies resulted in distinct population structures.Two stocks and one farming population were clustered together to the phylogenetic branch of Yangtze crab,with an approximate membership of 95%.Whereas,another fanning population was clustered singly to the phylogenetic branch of the Liaohe crab,with a membership of 97.1%.The tests for individual admixture showed that Yangtze crab had probably been contaminated with individuals from other water systems.Genetic relationships between populations also supported the conclusion that Yangtze crab and Liaohe crab had different gene pools in spite of the origins of the same species.     

Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.