日本沼虾蜕皮过程中头胸甲外骨骼超微结构的改变

采用HE染色及石蜡切片的扫描电镜观察方法,描述了日本沼虾(Macrobrachium nipponense)蜕皮间期(C期,intermolt stage)及新旧表皮更替变化剧烈的蜕皮前晚期(D4期,late premolt stage)和蜕皮后期(postmolt stage)A-B期头胸甲外骨骼的形态结构变化特点。结果显示,日本沼虾外骨骼分为上表皮、外表皮、内表皮三层,D4期出现新的上表皮和外表皮,B期出现新内表皮,外表皮强嗜碱性而内表皮弱嗜酸性。扫描电镜观察发现,C期头胸甲内、外表皮均为几丁质-蛋白质纤维构成的平行板层结构且板层内有发达的孔道系统(pore canals,pc),但内外表皮板层间排列紧密程度不同,外表皮板层切面边缘较整齐,结构致密,板层内pc大小均一、近似圆形,内表皮板层切面边缘粗糙,结构较疏松,板层内pc大小不等、多为梭形。在蜕皮前后,新外表皮结构变化显著,与C期相比,D4期新外表皮超微结构与旧内表皮结构相似,而在A期,外表皮超微结构由疏松变为致密,这可能与蜕皮后表皮的钙化相关。本研究旨为阐明沼虾的蜕皮机制提供基础资料。     

KK-42对蜕皮后期日本沼虾头胸甲超微结构的影响

为了深入探究KK-42对表皮结构的影响,本实验以外表皮形成趋于结束而内表皮开始出现的蜕皮后期幼虾为材料,通过扫描电镜观察石蜡切片法,分析了KK-42处理前后头胸甲外表皮,尤其是内表皮结构的变化。将处于蜕皮间期的健康幼虾[体长(3.5±0.1)cm]随机分为2组,分别用1.95×10^-4 mol/L的KK-42溶液和不含KK-42的溶液处理,取蜕皮后1.5、3.0、6.0及12.0 h幼虾头胸甲,观察其超微结构。另取蜕皮后6.0 h头胸甲,用解剖刀轻轻刮去内面组织,通过扫描电镜观察内表皮的表面结构。结果显示,蜕皮后1.5和3.0 h,头胸甲只包含上表皮和外表皮,KK-42处理组外表皮的板层数量显著增加,外表皮厚度分别比相应对照组提高了72.07%和38.67%;蜕皮后6.0~12.0 h,外表皮中疏松的板层趋于致密,其厚度在二组间无统计学差异。蜕皮后6.0 h,只有1层板层的内表皮出现,到蜕皮后12.0 h,板层数增至2层(对照组)和3层(处理组),且板层结构均疏松。此外,内表皮表面分布着大小不等、头尾走向的梭形孔道(pore canals);KK-42处理组内表皮结构未见明显变化。实验表明,KK-42对蜕皮后期日本沼虾幼虾头胸甲的超微结构有显著影响,能加快外表皮及内表皮板层的形成速率。     

中华绒螯蟹蜕壳过程中螯足肌肉组织学及主要蛋白质含量变化

为探讨中华绒鳌蟹(Eriocheir sinensis)蜕皮过程中肌肉生长机制,采用石蜡切片、电镜技术及生物化学方法,以中华绒鳌蟹幼蟹为对象,研究了其蜕壳过程中螯足闭螯肌肌肉的显微结构、超微结构及主要蛋白质含量的变化。结果显示,在蜕皮间期,螯足肌纤维充分伸展,多核,横截面呈不规则圆形或者多边形;肌原纤维排列有序,具有甲壳动物骨骼肌的典型特征,A带、I带及肌质网、二联体、线粒体等细胞器清晰可见。蜕皮前期,肌纤维降解以致其横截面大小不一;肌原纤维内部降解,出现腐蚀性空洞。蜕皮之后,在肌纤维末端的肌节处于超收缩状态,这些肌节长度缩短至正常长度的50%,肌纤维典型结构消失,A带、I带模糊不易区分,但肌质网、二联体、线粒体等结构仍然完整;肌纤维中间部分的肌节结构正常如蜕皮间期。生物化学研究发现,蜕皮前后螯足肌肉中肌原纤维蛋白和可溶性蛋白含量的变化同其结构特征的变化一致。本研究结果表明,中华绒螯蟹蜕皮后螯足肌肉可能的一种生长机制是通过增加新的肌节来伸长,动物刚蜕壳后,这些新形成的肌节以超收缩的形式存在,随着动物吸水身体膨大,外骨骼伸展、硬化,这些较短的肌节由于被拉伸而达到正常肌节的长度,从而完成肌肉的生长。     

中华绒螯蟹外骨骼矿化期肝胰腺脂代谢与钙离子的相关性

外骨骼矿化不仅是甲壳动物蜕壳过程中的关键环节,而且还关系到甲壳动物蜕壳后的成活和生产效益。虽然已经知道外骨骼矿化期虾蟹类体内钙离子及营养物质发生了变化,但外骨骼矿化期虾蟹类脂代谢特点及与钙离子的关系还未知。因此,本研究以中华绒螯蟹为研究对象,选择蜕壳前期(Premolt stage, D期)和蜕壳后期(Postmolt stage, A-B期)实验蟹各6只,比较两个时期不同组织离子和营养物质变化情况,并进一步通过转录组学分析肝胰腺中营养物质代谢特点。结果表明,蜕壳后期肝胰腺和血淋巴钙离子含量显著降低(P < 0.05),而蜕壳后期附肢肌肉中钙离子的含量则显著升高(P < 0.05),蜕壳后期肝胰腺中钙磷比极显著降低(P < 0.001),附肢肌肉钙磷比与之呈相反的趋势。与蜕壳前期相比,蜕壳后期肝胰腺蛋白质和葡萄糖含量均无显著变化(P > 0.05),而肝胰腺中甘油三酯含量则极显著降低(P < 0.001),肝胰腺甘油三酯和钙离子呈极显著的相关性(P < 0.001)。通过转录组学分析,发现肝胰腺脂代谢相关差异基因113个,89个为上调基因,24个为下调基因。这些差异基因主要富集在类固醇激素合成、甘油酯代谢、脂肪酸分解代谢、甘油磷脂代谢和亚油酸代谢。综上可知,中华绒螯蟹外骨骼矿化过程中,肝胰腺中储存的钙离子被运送至肌肉组织用于新表皮的矿化,该过程和肝胰腺甘油三酯相关;甘油三酯可能被分解为外骨骼矿化提供了能量或者物质基础,以及参与类固醇类激素的合成作用于外骨骼的矿化。     

在体条件下miR-305-5p对日本沼虾MnCHT3A的靶向调控作用

为了探索miRNA对日本沼虾几丁质酶3A(Macrobrachium nipponense chitinase 3A,MnCHT3A)基因的调控作用,实验采用生物信息学方法预测并筛选与MnCHT3A靶向结合的miRNA—miR-305-5p;利用qRT-PCR、生物化学以及组织学方法,研究了在体条件下,miR-305-5p对靶基因的调控作用。结果显示,一个蜕皮周期中miR-305-5p与MnCHT3A的表达量呈负相关,前者C期最高、A期最低;而MnCHT3A的表达趋势则相反。注射miR-305-5p mimics和miR-305-5p inhibitor后,与对照组相比,MnCHT3A转录水平分别降低60%和升高166%;MnCHT酶活性分别降低39.53%和升高133%。组织学观察发现,C期的头胸甲表皮为3层结构,由外向内依次为上表皮、外表皮和内表皮(H.E染色);几丁质荧光染色结果显示,几丁质分布在内、外表皮;扫描电镜结果清晰显示了内、外表皮的板层结构。与对照组相比,miR-305-5p mimics组内表皮的板层厚度增加,呈蓝色荧光的几丁质条带有增厚趋势;而miR-305-5p inhibitor组表皮结构紊乱,几丁质荧光分布不均且部分区域明显减弱。研究表明,miR-305-5p的靶基因为MnCHT3A,在体条件下,miR-305-5p能够靶向抑制MnCHT3A的转录。     

日本沼虾表皮几丁质合成酶基因克隆及表达分析

为了解日本沼虾几丁质合成酶（Chs）基因在蜕皮过程中的作用,本实验采用RACE技术首次从表皮中克隆了几丁质合成酶基因（MnChs）cDNA全长,并用生物软件对其序列进行生物信息学分析,RT - PCR技术检测该基因的时空表达模式。结果表明,其cDNA全长5133 bp,5′UTR为 283 bp,3′UTR为 159 bp,开放阅读框（ORF）长度为4701 bp,编码1566个氨基酸,分子量为179.57 KDa,理论等电点为6.09,包含两个几丁质合成酶的标签序列EDR和QRRRW及Chitin-synth-C结构域。经BLAST比对,与日本仿长额虾、淡水枝角水蚤Chs相似性分别为89 %和63 %。RT - PCR结果显示,在蜕皮周期各阶段,不同组织MnChs的表达量差异显著：头胸甲在A期达到最高,胃肠在D0和D4期均较高,尾扇和肌肉在D4最高,肝胰腺则普遍较低。结果表明,MnChs基因转录物不仅存在于表皮,在其它组织也有分布,且mRNA水平的变化与蜕皮周期有关,作为几丁质生物合成的关键酶,推测该基因的表达在新外表皮和内表皮的形成中发挥重要作用。     

蟹类原肌球蛋白基因的克隆与表达

原肌球蛋白是甲壳类动物的主要过敏原之一。本文通过分子生物学技术方法,分别克隆得到锯缘青蟹(Scylla serrata)、中华绒鳌蟹(Eriocheir sinensis)和三疣梭子蟹(Portunus trituberculatus)的原肌球蛋白基因序列。测序结果表明,三个基因的序列长度均为855 bp,编码284个氨基酸残基,三者氨基酸序列同源性为99.3%。三种蟹的原肌球蛋白基因序列与GenBank中其他甲壳类动物的原肌球蛋白具有很高的同源性。将锯缘青蟹的原肌球蛋白基因与pGEX-4T-3载体连接后,经IPTG诱导得到分子量约为61 kDa的融合表达蛋白。通过与甲壳类过敏患者血清的免疫印迹反应,证实融合表达的原肌球蛋白具有过敏原性,表明原肌球蛋白是蟹类的主要过敏原之一。该融合蛋白有望用于甲壳类食物过敏诊断试剂的开发与应用。     

中华绒螯蟹种质研究进展

总结我国在中华绒螯蟹形态学、养殖性能、细胞遗传学、生化遗传学及分子遗传学方面的种质研究的主要成果 ,归纳中华绒螯蟹不同种群在形态、细胞、生化、分子等层次上的遗传差异。在形态学研究方面 ,运用形态多元分析 ,建立不同种群绒螯蟹从幼蟹到成蟹的量化判别函数 ;在生化遗传和分子遗传研究方面 ,找到中华绒螯蟹区别于其他绒螯蟹的生化遗传标记和分子遗传标记 ,以及中华绒螯蟹不同种群由南到北的分子遗传渐变标记 ;在养殖性能方面 ,阐明中华绒螯蟹不同种群在幼蟹和成蟹阶段的养殖性能 (包括蜕壳、生长、性腺发育、性成熟及生殖洄游等 )差异和特点     

Identification of SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene and their association with precocity of the Chinese mitten crab Eriocheir sinensis

Moult‐inhibiting hormone (MIH), an important regulator of steroidogenesis, inhibits the synthesis of ecdysteroid in Y‐organ (YO) and plays a significant role in the regulation of moulting and post‐embryonic development of crustacean. Because unsuccessful moulting have been widely observed in precocious crabs, we investigated whether genetic variants in the 5′‐flanking region and 3′‐untranslated region (3′‐UTR) of the MIH gene are associated with precocity of the Chinese mitten crab. Thirty individual DNA samples were sequenced to search for SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene. Five SNPs (g.196 T>A, g.230 C>T, g.305 T>C, g.323 C>A and g.372 C>T) in the 5′‐flanking region and 6 SNPs (g.2677 C>T, g.2759 T>A, g.2807 T>C, g.3042 A>G, g.3088 T>G and g.3295 T>G) in the 3′‐UTR of the MIH gene were selected for the individual genotyping in a two‐stage association study. We found that a SNP g.3088 T>G in the 3′‐UTR of MIH gene was consistently associated with precocity of the Chinese mitten crab in stage 1 and stage 2, with a per‐allele OR (Odds Ratio) of 1.469 (95% CI: 1.169–1.844) after two stages combined (P = 0.001). However, no significant associations were observed between the other 10 SNPs and precocity of the Chinese mitten crab. To our best knowledge, this is the first association study between various SNP genotypes and phenotype attributes in Chinese mitten crab. Our findings suggest that the SNP g.3088 T>G may be a candidate marker for effective marker‐assisted selection to decrease the precocity of the Chinese mitten crab in future studies.     

Advances in precocity research of the Chinese mitten crab <Emphasis Type="Italic">Eriocheir sinensis</Emphasis>

Precocity has been observed in fishes and crustaceans. However, mechanisms underlying precocity have not been well documented in crustaceans and are thought to be influenced by both genetic and environmental factors. Also, precocity is generally considered to have negative effects on crustaceans. The Chinese mitten crab Eriocheir sinensis, a catadromous species endemic to China, is a high valued commodity and in the recent past is being extensively cultured to meet the growing demand by the restaurant trade. The mitten crab is an ideal candidate for precocity studies because of their large size, distinct secondary sex characters, wide distribution and abundant availability from commercial farms. In this article, progress in several aspects of precocity of E. sinensis is reviewed, including the phenomena of precocity and its effect, identification of precocious crabs, factors related to precocity (temperature, salinity, light, nutrition, stocking density, and germplasm), relationships between precocity and neuro-endocrine system, steroid hormones or hepatopancreas, prevention and control methods of precocity. In addition, possible future directions for the study of precocity are suggested.     

Mitochondrial DNA variation and population structure of the Japanese mitten crab Eriocheir japonica

ABSTRACT:   The Japanese mitten crab Eriocheir japonica is a common grapsid species found throughout freshwater and estuarine regions in Japan. In order to obtain information on the genetic variation and population structure of this species, a polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis was conducted on the cytochrome oxidase subunit I (COI) of mitochondrial DNA, on 666 individuals from 19 sample sites covering the three main geographic regions of Japan (Main Islands, Okinawa, and Ogasawara). Genetic analysis using seven restriction enzymes produced an array of 61 composite haplotypes. Three regional groups corresponding to the three geographic regions were clearly identified by cluster and molecular variance model ( amova ) analyses. Each of the three groups showed dominant haplotypes that were almost completely absent in populations from the other geographic areas. Comparison with published information for other species indicates that the degree of genetic divergence between these three main groups is equivalent to the genetic distance between congeneric species. Thus, the population structure of the Japanese mitten crab, as inferred from mtDNA analysis, is formed by genetically distinct groups that closely reflect their geographic distribution in the Japanese archipelago as well as restricted gene flow.     

图们江水系绒螯蟹的形态差异与遗传混杂

为了研究图们江水系中华绒螯蟹的种群现状,以图们江水系绒螯蟹为研究材料,将我国黄河和辽河水系的中华绒螯蟹(Eriocheir sinensis),以及日本本土的日本绒螯蟹(Eriocheir japonicus)作为参照对象,应用三种形态多元分析方法与STRUCTURE聚类分析方法,对它们的32个外部形态性状进行分析。判别分析显示,图们江群体的判别准确率最低(83.30%);主成分分析显示图们江群体的12个差异最大的表型性状均位于中华绒螯蟹与日本绒螯蟹之间,呈现为中间类型;传统聚类显示图们江水系绒螯蟹与日本绒螯蟹的形态差异最小;STRUCTURE聚类显示,图们江有60%的个体聚入日本绒螯蟹群体,而只有10%的个体聚入中华绒螯蟹群体,其余30%个体为中华绒螯蟹与日本绒螯蟹的中间类型。形态研究结果表明,图们江水系绒螯蟹为中华绒螯蟹与日本绒螯蟹分布的重叠区与混杂区,但其形态偏向日本绒螯蟹。     

5-羟色胺对中华绒螯蟹仔蟹生长及存活率的影响

为弄清5-HT在中华绒螯蟹生长过程中对其生长、退壳和存活率的影响。采用腹腔注射的方法,分别给予浓度为0、1.0、5.0和15.0μg/g的5-HT溶液,以生理盐水为对照组,观察蟹由仔蟹Ⅲ蜕壳到仔蟹IV的时间和存活率,及仔蟹IV的体长、体宽、体重。结果表明:体长、体宽、体重以15.0μg/g浓度组的最高,但与其他各组间没有显著差异。退壳时间以5.0μg/g浓度组的最短,正常养殖组最长,但各组间没有显著差异。存活率以正常养殖组的最低,其他各组都比正常养殖组的成活率高,但各组间没有显著差异。退壳率以1.0μg/g浓度组的最高,5.0μg/g浓度组的最低,但在各组之间没有显著差异。5-HT对蟹的生长和存活有一定的促进作用,但作用不是很明显,有待于进一步的研究。     

中华绒螯蟹Y-器官在蜕皮周期中的超微结构

以光镜和电镜观察处于蜕皮周期不同阶段的中华绒螯蟹(Eriocheirsinensis),结果显示,中华绒螯蟹具Y-器官(YO)1对,卵圆形,直径1 5～2 0mm,苍黄色,位于头胸部鳃腔前端,大颚外侧内收肌腹缘,邻近头胸甲内侧上皮。YO细胞排列成索状,细胞索之间为血窦,YO的细胞索由一种细胞构成,细胞核质比高。YO细胞具有类似脊椎动物合成类固醇激素的典型超微结构特征,蜕皮前期YO细胞的光面内质网、管嵴状线粒体和游离核糖体丰富;蜕皮后期和蜕皮间期的内质网、线粒体和游离核糖体明显减少;青春蜕皮后的YO出现大面积细胞凋亡,YO趋向退化。     

蜕壳周期内中华绒螯蟹钙含量、组织结构及相关基因表达变化

运用生化分析、组织切片和分子生物学等技术,测定蜕壳期(蜕下旧壳后0.5 h内)和蜕壳后期(蜕下旧壳0.5 h后至新壳未完全硬化之前)平均体质量(5.63±2.35)g中华绒螯蟹肝胰腺和鳃中的钙含量,观察肝胰腺和鳃组织结构变化,用荧光定量PCR技术分析肝胰腺和鳃中的钙网蛋白(CRT)基因的表达情况.试验结果表明,蜕壳后期...     

中国大陆沿海六水系绒螯蟹_中华绒_省略__群体亲缘关系_生化遗传差异分析

采用聚丙烯酰胺凝胶电泳分析比较了分布于我国大陆沿海 6个主要水系的两种绒螯蟹 (中华绒螯蟹、日本绒螯蟹 )群体间的生化遗传差异。结果发现 ,(1)辽河、黄河、长江和瓯江中华绒螯蟹群体间的生化遗传差异不显著 ,四个群体间的遗传距离D =0 .0 0 0 6～ 0 .0 0 53,属种内群体间差异。 (2 )珠江和南流江日本绒螯蟹群体间的生化遗传差异也不显著 ,两个群体间遗传距离D=0 .0 0 0 7,尚未达到亚种分化水平。 (3)辽河、黄河、长江和瓯江中华绒螯蟹与珠江和南流江日本绒螯蟹在SOD同工酶表型上有明显差异 ,SOD - 2位点仅在辽河、黄河、长江和瓯江中华绒螯蟹中表达 ,而SOD - 4位点仅在珠江和南流江日本绒螯蟹中表达 ,这些可作为中华绒螯蟹与日本绒螯蟹群体区分的生化遗传标志。辽河、黄河、长江和瓯江中华绒螯蟹群体同珠江和南流江日本绒螯蟹群体间的遗传距离较大 ,D =0 .0 72 1～ 0 .0 74 7。 (4 )聚类分析表明 ,在我国大陆沿海水系分布的绒螯蟹可分为两大类群 ,辽河、黄河、长江和瓯江水系的绒螯蟹属北方类群 ,即中华绒螯蟹 ;珠江和南流江的绒螯蟹属南方类群 ,即日本绒螯蟹。     

Carbonic anhydrase and Na/K-ATPase activities at different molting stages of the giant freshwater prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

ABSTRACT:   In the present study, the role of carbonic anhydrase (CA) and Na/K-ATPase in the gill and epidermal tissues in the giant freshwater prawn Macrobrachium rosenbergii was examined as a function of molting stage. CA activity levels in the front and back gills were low at the intermolt stage C₀, but increased significantly at premolt stage D₃, and then decreased after molting. In the epidermal tissue, activity levels decreased gradually towards premolt to a minimum level at stage D₃, but became elevated at postmolt stages A and B. Na/K-ATPase levels in the front and back gills did not change significantly during the molt cycle. CA in the gill is possibly involved in supplying counter-ions for ion uptake, while CA in the epidermal tissue may play a role in mineralizing the exoskeleton after ecdysis. Na/K-ATPase in the gills may function in ion uptake from the ambient medium; however, since its activity was not influenced by the molt cycle, it probably does not have a major role in osmoregulation in the freshwater environment.     

Practical Molt Staging of Penaeus setiferus and Penaeus stylirostris

Molt staging has many applications in penaeid aquaculture as a routine procedure. Using criteria developed for various crustaceans, a method of rapid molt staging was applied to Penaeus stylirostris and Penaeus setiferus adults. The most useful characters were the degree of epidermal retraction from the setal bases and epidermal retraction from the cuticle coupled with the development of new setae. The duration of the molt cycle of P. stylirostris was 11.5 ± 1.0 days ( N = 5) at 27–29 C. The early postmolt, late postmolt, intermolt, early premolt and late premolt stages were readily determined. For P. stylirostris , the duration of postmolt was 27% of the cycle; intermolt, 17%; and premolt, 56%. The length of the molt cycle of P. setiferus at 27–29 C was 13.6 ± 1.0 days ( N = 21) with postmolt occupying 22% of the cycle; intermolt, 19%; and premolt, 59%. Unilaterally eyestalk enucleated P. setiferus had a significantly shorter molt cycle than intact P. setiferus (11.7 ± 1.21 days vs. 13.6 ± 1.02 days, P < 0.05). There were no differences in molt cycle duration between the sexes, whether enucleated or intact ( P > 0.05). A series of illustrations identifies the key characteristics for determining stage of molt in these penaeids.     

白洋淀中华绒螯蟹种质资源形态判别与评价

对中华绒螯蟹外部形态32个测量点进行测量．利用形态判别法对白洋淀的中华绒螯蟹种质资源分析评价．建立了白洋淀中华绒鳌蟹形态判别式。结果表明．白洋淀河蟹种群属于北方蟹的辽河蟹．判别准确率达100％。结果有利于白洋淀中华绒螯蟹均质种群的维持，建议应加强白洋淀中华绒鳌蟹资源的管理。