利用实时荧光定量PCR法测算红螯螯虾实体组织的细胞数量

螯虾是重要的水产养殖动物,也是研究甲壳动物疾病与免疫的一个良好模型.在研究不同组织的功能及受病原感染的影响时,需要了解组织内的细胞数量.但是,目前常用的细胞计数方法仅适用于单细胞悬液,无法用于实体组织.本研究以红螯螯虾( Cherax quadricarinatus)为对象,建立了基于基因拷贝数定量分析的实体组织细胞数...     

红螯螯虾鳃细胞与血细胞分子标记的鉴定

甲壳动物的鳃中富集了大量血细胞,在循环血细胞稳态调节和免疫防御中发挥重要作用,因此获得区分鳃细胞和血细胞的分子标记将有助于研究鳃血细胞的功能。本研究采用SDS-PAGE电泳法比较了红螯螯虾(Cherax quadricarinatus)鳃细胞和血细胞的蛋白表达谱,获得3条组织特异性蛋白条带,并通过质谱技术鉴定了8个候选蛋白。实时荧光定量PCR(qRT-PCR)分析表明,空泡型-H⁺-ATP合酶C亚基(vacuolar-H⁺-ATPase subunit C, V-H⁺-ATPase)在鳃细胞中的转录水平是在血细胞中的53.3倍;而α-2-巨球蛋白(alpha-2-macroglobulin, α2M)和卵黄膜外层蛋白(vitelline membrane outer layer protein I-like protein, VMO-Ia)在血细胞中的转录水平分别是在鳃细胞中的10.3倍和10.9倍。Western-blot和免疫荧光分析表明,V-H⁺-ATPase和VMO-Ia蛋白分别在鳃细胞和血...     

红螯螯虾网格蛋白轻链的克隆及制备抗体的检测

网格蛋白为三脚蛋白复合体,其介导的内吞作用是病毒侵染细胞的常用途径之一.而对虾白斑综合症病毒(white spot syndrome virus,WSSV)是对虾养殖业的主要病原,为了研究分析WSSV的侵染机制及进入宿主细胞的途径,在本研究中,克隆得到红螯螯虾(Cherax quadricarinatus)网格蛋白轻链的c DNA全长,并将其命名为Cq CLC(Genbank登录号为KR075008).进化树分析表明,其与黑腹果蝇(Drosophila melanogaster)的网格蛋白轻链进化距离较近.此外,在其蛋白序列中选择抗原性较好的片段合成多肽,并以此制备单克隆抗体,而后经免疫荧光和western blot检测从中筛选出具有特异性的抗体,为进一步对WSSV侵染虾细胞机制及入胞途径的研究奠定了基础.     

中华绒螯蟹受精卵离体培育的初步研究

用实验生态学的方法对培养中华绒螯蟹离体胚胎的条件进行了研究。结果表明,温度不仅直接影响胚胎的发育速度,而且通过影响水中真菌的繁殖间接但明显地影响胚胎的存活率。光照对胚胎发育的影响不明显,用０．０５￣０．１ｍｇ／Ｌ的孔雀石绿、２￣８ｍｇ／Ｌ的亚甲基蓝,２０￣５０ｍｇ／Ｌ的福尔马林或３￣５ｍｌ／Ｌ的制霉茵素都能有效地抑制水中真菌的生长,在无真菌感染的情况下,能有效地在４￣８℃保存离体河蟹胚胎６０ｄ,孵     

早熟中华绒螯蟹眼柄视神经节对卵巢发育的生殖调控作用

采用组织切片技术于光镜下观察了早熟中华绒螯蟹（Eriocheir sinensis）眼柄视神经节的组织形态结构,以体外培养法研究早熟蟹眼柄视神经节对同种甲壳动物中华绒螯蟹和异种甲壳动物克氏原螯虾卵巢发育的影响,并以放射化学测定法测定早熟蟹眼柄粗提物及单侧或双侧眼柄镊烫后对大颚器生物合成甲基法尼酯（MF）的影响．结果表明,早熟蟹眼柄视神经节呈乳白色,椭圆柱形,外包一层黑色视网膜,由外髓、内髓、端髓3部分构成;端髓区域可明显区分3种类型的神经分泌细胞;早熟蟹眼柄视神经节对同种和异种甲壳动物的卵巢发育均有抑制作用;早熟蟹眼柄粗提物对MF生物合成的抑制作用随浓度的增加而增强．研究证实早熟蟹眼柄视神经节对卵巢的抑制作用较正常发育蟹弱,且对同种甲壳动物卵巢发育的抑制作用强于对异种动物．     

中华绒螯蟹与日本绒螯蟹线粒体COI基因片段的序列比较研究

以相应引物 PCR扩增了黄河口中华绒螯蟹线粒体细胞色素氧化酶 I亚基基因 (COI)片段 ,PCR产物经 T载体连接之后进行克隆、测序 ,得到 70 9bp的碱基序列 ,其 A,T,G,C含量分别为 34.4 1% ,2 7.93% ,2 0 .0 3%和 17.6 3%。并比较它与珠江流域中华绒螯蟹 COI序列和日本绒螯蟹 COI序列的差异 ,发现黄河口中华绒螯蟹与珠江流域中华绒螯蟹 COI序列完全相同 ,而与日本绒螯蟹差异非常明显 ,70 9或 6 5 8(不计引物 )位点中核苷酸差异数为 32 ,核苷酸差异率为 4 .5 1%或 4 .86 % (不计引物 ) ,其中 2 5个位点为转换 ,7个位点为颠换。作者倾向于支持存在中华绒螯蟹和日本绒螯蟹 ,或它们为同一种的两个地理亚种的观点     

背景色对红螯螯虾生长、存活、体色及栖息行为的影响

本研究通过设置5种不同背景色(白色、红色、绿色、蓝色、黑色),以探究背景色差异对红螯螯虾(Cherax quadricarinatus)生长、存活、体色及栖息行为的影响。结果表明养殖56d后黑色背景色组红螯螯虾体重增长量、特定生长率和成活率最高,分别为32.66 g、1.74%和76.4%,其次为绿色、蓝色和红色背景色组,白色组最差(P<0.05);黑色和红色背景色红螯螯虾饵料系数最小(3.73～3.80);黑色背景色条件下红螯螯虾体色偏蓝, ImageJ分析表明,黑色背景色组虾壳RGB值中R和G值显著低于其他各组,黑色背景色虾壳虾青素含20.23 mg/kg,亦显著低于其他各组(69.34～82.15 mg/kg)(P<0.05);黑色背景色条件下红螯螯虾分布于遮蔽物外的几率显著高于其他各组(P<0.05)。综上所述,背景色能够影响红螯螯虾生长存活、体色及行为分布,养殖中采用黑色作为背景色能够显著提高红螯螯虾生长存活,改善虾体颜色。     

摘除眼柄对中华绒螯蟹肝脏结构及其氨基酸含量影响的研究

报道摘除中华绒螯蟹眼柄对其肝脏结构及其氨基酸含量的影响。实验分成两组,即摘除眼柄和对照组。     

四脊滑螯虾造血组织细胞培养条件的优化

稳定的体外细胞培养体系,可以为基因功能和宿主 病原相互作用研究提供平台。本研究通过对四脊滑螯虾(Cherax quadricarinatus)造血组织(hematopoietic tissue, HPT)细胞的体外培养条件进行优化,以期获得状态稳定、活力良好的造血组织细胞培养物。我们从四脊滑螯虾中分离提取造血组织细胞后,通过对培养基种类(L 15、Express Five、DMEM),谷氨酰胺浓度(0、1%、5%、10%),胎牛血清浓度(0、1%、5%、10%),以及体外培养温度(20、27 ℃)等培养条件进行优化。细胞形态观察和细胞活力分析结果表明,HPT细胞在添加1%青霉素 链霉素、10%L 谷氨酰胺、1%胎牛血清的L 15培养基中状态最佳,在20 ℃培养温度下能存活30 d左右。这些培养条件将有利于四脊滑螯虾造血组织细胞的体外培养。     

中华绒螯蟹(Eriocheir sinensis)野生和养殖蟹种对池塘养殖成蟹可食率和营养品质的影响研究

长江水系野生中华绒螯蟹(以下简称河蟹)蟹种比经过多代人工养殖的池塘群体蟹种具有更好的养殖性能,尚不清楚两种蟹种养成后成蟹的可食率及其营养品质是否存在差异。本研究采用养殖实验和生化分析方法,比较了长江水系野生和养殖蟹种经池塘养成成蟹的可食率、肥满度、常规生化成分、脂肪酸和氨基酸组成。结果显示:(1)在相似的体重条件下,野生群体成蟹的肝胰腺指数极显著高于养殖群体(P0.01),性腺指数显著低于养殖群体(P0.05),两群体间的出肉率、总可食率及肥满度无显著差异(P0.05);(2)两群体成蟹肝胰腺和性腺中的常规营养组成接近,但野生群体雄体肝胰腺和性腺中的碳水化合物含量显著高于养殖群体雄体(P0.05);无论雌体还是雄体,野生群体肌肉中的粗蛋白含量显著低于养殖群体,但野生雄体肌肉中的粗脂肪含量和雌体肌肉中的碳水化合物显著高于后者(P0.05);(3)就野生雌体脂肪酸组成而言,野生群体成蟹肝胰腺、卵巢和肌肉中的C18:1n9含量显著高于养殖群体雌体,但其C20:5n3、C22:6n3、ΣPUFA和ΣHUFA显著低于后者;就雄体而言,野生群体成蟹三种可食组织中的C18:2n6高于养殖群体,但其C20:5n3和C20:4n6低于养殖群体;整体上,肌肉中的HUFA含量显著高于肝胰腺和性腺;(4)就氨基酸组成而言,两群体雌体和雄体性腺中的氨基酸含量接近,均无显著差异,仅精巢中组氨酸和精氨酸含量存在显著差异;无论雌体还是雄体,养殖群体肌肉中的主要氨基酸和总氨基酸含量略高于野生群体,仅异亮氨酸和赖氨酸含量差异显著;此外,野生群体雄蟹肌肉中的亮氨酸、天冬氨酸、丝氨酸和组氨酸含量也显著高于养殖群体。综上,野生群体和养殖群体蟹种养成后成蟹的性腺指数和肝胰腺指数存在显著差异,但是两者总可食率和肥满度接近,整体上两群体脂肪酸组成存在较大差异,但是氨基酸组成较为接近。     

Effects of benzene and dimethylnaphthalene on homeostatic processes in two species of crabs

Long-term exposure of animals to sub-lethal doses of toxicants such as benzene (B) and dimethylnaphthalene (DMN) may result in subtle changes in their physiology and biochemistry. In crustaceans such changes include decreased rate of limb regeneration, extended time to molt and decreased growth increment at molt.^1,2 These processes depend upon an adequate supply of stored nutrients in the tissues and appropriate release of neuroendocrine substances from the central nervous system.^3,4 We are examining the effects of sub-lethal doses of B and DMN on osmotic and ionic regulation and on nutrient storage in the blue crab, Callinectes sapidus, and the green crab, Carcinus maenas. Disturbances in these physiological processes may reflect alterations in neuroendocrine functions brought about by the presence of toxicants. We find that exposure of C. maenas to B or DMN, followed by transfer to a dilute medium, results in impaired osmoregulatory capacity, while addition of these compounds to a dilute medium to which the animal is already acclimated does not affect their regulatory ability. Storage of carbohydrate in the midgut gland (hepatopancreas) is decreased by B or DMN, while the accessory storage tissues (gill, muscle) are not significantly affected.     

两种大眼蟹线粒体16S rRNA基因序列分析

测定了宽身大眼蟹和日本大眼蟹线粒体16SrRNA基因部分片段的序列,其长度均为517bp。二者的核苷酸序列A,T,G,C的含量相似:宽身大眼蟹分别为33.3%,35.6%,19.9%,11.2%;日本大眼蟹分别为35.2%,35.4%,18.4%,11.0%。不包括6处插入/缺失位点,两序列间有63个变异位点,核苷酸差异率为12.26%,其中转换32个、颠换31个,转换与颠换比约为1.0。对国内外大眼蟹的24条长度为415bp的16SrRNA基因同源序列进行分析,A+T的平均含量为71.7%,明显高于G+C的平均含量,且存有变异位点171个,简约信息位点137个。中国和日本的日本大眼蟹之间的核苷酸差异率为4.42%,表明二者已有明显的遗传分化;中国的日本大眼蟹与日本的万岁大眼蟹之间的核苷酸差异率仅为0.21%,表明二者有可能为同一物种。上述结果得到了系统发生树拓扑结构的支持。     

A comparison of genetic structure in two low-dispersal crabs from the Wild Coast,South Africa

The Wild Coast in south-eastern South Africa is strongly influenced by the warm, southward-flowing Agulhas Current. This current has a significant impact on dispersal in the coastal biota of the region, and facilitates high levels of connectivity among populations. However, it is not known how the region's high-velocity hydrology affects genetic population structure in endemic estuarine species, populations of which are frequently isolated from the sea. Here, we compared genetic structure in two estuarine crabs of the family Hymenosomatidae. Both are presumed to have low dispersal potential, but they differ in terms of their life histories. Hymenosoma longicrure has abbreviated larval development and can complete its entire life cycle within estuaries, whereas Neorhynchoplax bovis is a direct developer that lacks planktonic larvae. Using DNA sequence data from the mitochondrial COI gene and the intron of the nuclear ANT gene, we found that levels of genetic structure differ considerably between the species. Depending on the genetic marker used, H. longicrure is genetically homogeneous (COI) or displays low levels of genetic structure and minor evidence of recruitment near natal sites (ANT). In contrast, connectivity in N. bovis is much lower, as this species has a unique combination of alleles at each site, indicating that recruitment is mostly local. These results support previous findings suggesting that even a short larval dispersal phase is sufficient to maintain high levels of connectivity and prevent genetic divergence among populations.     

山东半岛近岸海域蟹类群落结构特征的研究

根据2014年8月和10月、2015年2月和5月使用单船底拖网进行的4个航次调查数据,对山东半岛近岸海区的莱州湾及渤海南部、山东半岛北部和山东半岛南部等3个海域蟹类种类组成、时空分布、优势种、群落结构稳定性等群落结构特征进行了研究。结果显示,4次调查共捕获蟹类20种,隶属于10科17属,其中日本蟳（Charybdis japonica）和三疣梭子蟹（Portunus trituberculatus）是主要经济种类,其余均为小型饵料型蟹类;海区内主要的优势种为双斑蟳（Charybdis bimaculata）、日本蟳（Charybdis japonica）和三疣梭子蟹（Portunus trituberculatus）,3个海域优势种季节更替均十分明显;莱州湾及渤海南部和山东半岛南部2个海域生物量季节变化明显,春季较低,在夏季达到峰值,夏季到冬季呈降低趋势,山东半岛北部海域生物量季节变化不明显。3个海域蟹类群落物种多样性指数（H'）、丰富度指数（D）及均匀度指数（J'）均处于较低水平,并有一定的季节变化;水深、水温和盐度是影响蟹类群落结构的重要因子,渤莱沿岸流、黄海暖流和黄海冷水团通过影响海域内水温与盐度影响蟹类群落结构。研究表明山东半岛近岸海区蟹类以小型饵料型蟹类为主,蟹类多样性水平较低,优势种更替明显,群落季节更替指数较高,季节间迁移频繁,蟹类群落不稳定。     

厦门港湾蟹类的分布

本文根据我所1980年8月至1981年10月厦门港湾海洋环境综合调查,选取其季度月(11,2,5,8月)资料,研究该港湾蟹类的种类组成、数量分布与季节变化。     

Hitching a ride on a hermit crabs home: Movement of gastropod shells inhabited by hermit crabs

There is little information on the movement and connectivity patterns for many species. The movement by shells occupied by the hermit crab Pagurus bernhardus and the organisms encrusting these shells was investigated on the south coast of Wales (UK). Hermit crabs shells moved considerable distances along the shore over 1 month periods, moving a minimum mean distance (±SE) of 148 (±8) and 174 (±9) m from release sites in the January and August respectively. Hermit crab-inhabited shells were also found to travel across habitats (sandy areas) that are unsuitable for both hermit crabs and epibionts. Through the examination of 150 of the most abundant shells used by P. bernhardus (Nucella lapillus), twelve epibiont species were found (10 animal and 2 algal species) and choice experiments demonstrated that hermit crabs preferred epibiont covered shells (84%), compared with bare shells (16%). The distance that shells were moved and the preference of hermit crabs for epibiont encrusted shells, coupled with the ability for epibionts to cross unsuitable habitats, may provide a dispersal advantage for epibiont species.     

厦门地区潮间带蟹类的种类组成与分布

本文根据厦门地区三个岛屿潮间带调查所获的资料，研究了蟹类的种类组成与分布，并应用多样性指数分析了蟹类的结构特征。经鉴定的蟹类有１４种５５属９１种，其中软相５７种，岩相４４种。软相的蟹类组成与岩相的明显不同。     

Interpreting odours in hermit crabs: A comparative study

Odours of different sources can indicate to hermit crabs the availability of empty shells, crucial resources for the life cycle of almost all of them. Here, we compared Clibanarius erythropus and Pagurus bernhardus for the intensity of investigative behaviour exhibited towards an empty, well-fitting shell in the presence of (1) plain seawater as control and seawater conditioned by (2) dead and live snails, (3) dead and live conspecifics, (4) live predators, and (5) food. During 10 min of observation, we recorded latency (the time until the first contact with the shell), and the number and duration of shell investigation bouts. The two species behaved similarly when exposed to the odours of food, live snails, and predators, while a more intense shell investigation was induced by dead snail odour in C. erythropus and by dead or live conspecific odour in P. bernhardus. Further studies should investigate the influence of phylogeny and ecology on this interspecific difference.     

Domoic acid excretion in dungeness crabs, razor clams and mussels

Domoic acid (DA) is a neurotoxic amino acid produced by several marine algal species of the Pseudo-nitzschia (PN) genus. We studied the elimination of DA from hemolymph after intravascular (IV) injection in razor clams (Siliqua patula), mussels (Mytilus edulis) and Dungeness crabs (Cancer magister). Crabs were also injected with two other organic acids, dichloroacetic acid (DCAA) and kainic acid (KA). For IV dosing, hemolymph was repetitively sampled and DA concentrations measured by HPLC-UV. Toxicokinetic analysis of DA in crabs suggested most of the injected dose remained within hemolymph compartment with little extravascular distribution. This observation is in sharp contrast to results obtained from clams and mussels which exhibited similarly large apparent volumes of distribution despite large differences in overall clearance. These findings suggest fundamentally different storage and elimination processes are occurring for DA between bivalves and crabs.