不同养殖模式克氏原螯虾(Procambarus clarkii)游离氨基酸的主成分分析及综合评价

为研究不同养殖模式的克氏原螯虾(Procambarus clarkii)肉中游离氨基酸(free amino acids,FAA)的综合品质差异,以江苏兴化地区具有代表性的3大类养殖模式的克氏原螯虾尾肉为研究对象,测定FAA并结合滋味活性值(taste active value,TAV)分析各氨基酸在克氏原螯虾肉中的呈味特性,然后运用主成分分析(principal componentanalysis,PCA)及聚类分析法综合评定克氏原螯虾肉的FAA品质。结果表明:克氏原螯虾肉中均含有17种FAAs,总量在21.80~27.11 mg/g之间。精氨酸(Arg)在所有克氏原螯虾肉中均为含量最高的FAA,远高于其他氨基酸,占总FAA含量的55.64%~67.76%,其对虾肉呈味贡献也最大。此外,还有丙氨酸(Ala)和组氨酸(His)在所有克氏原螯虾肉中的TAV都大于1,对呈味有贡献。谷氨酸(Glu)作为鲜味最强的氨基酸,只在稻虾一稻两虾模式(RC2)的克氏原螯虾肉中TAV大于1。17种氨基酸利用PCA提取到3个PC,累计方差贡献率为89.937%,可以代表氨基酸的综合信息。根据综合分析,排名最高的是一稻两虾养殖模式RC2,排名最低的是虾蟹混养模式CC1。聚类分析将所采集的克氏原螯虾虾肉分为3类,该结果与PCA的结果相似,表明稻虾模式养殖的克氏原螯虾肉的FAA综合品质相对较好。

Bacillus clarkii 7364 γ-环糊精葡萄糖基转移酶的可溶性表达及其催化特性分析

按照大肠杆菌偏爱密码子对Bacillus clarkii 7364来源的γ-环糊精葡萄糖基转移酶(γ-CGTase)进行优化,构建γ-CGTase原核表达菌株,摸索γ-CGTase的可溶性表达条件及纯化条件,并对其催化特性进行研究。结果表明:在28℃条件下实现了γ-CGTase的高效可溶性表达,可溶性蛋白占总蛋白表达量的63%,酶活可达3 830 U/mL。经(NH4)2SO4沉淀和α-CD-Sepharose 6B亲和柱纯化后,酶蛋白纯化了12.97倍,酶收率20.31%。使用该酶对木薯淀粉进行转化,转化产物中γ-环糊精(γ-CD)的比例可达90.9%,几乎无α-环糊精(α-CD),与天然酶的79%相比提高了15%。将该基因工程菌在20 L发酵罐中发酵,10 h后酶活达到4 375 U/mL,证实了其工业化放大的可能。该酶具有非常高的转化专一性,有非常好的工业化前景。

饲料中不同蛋白质和脂肪水平对克氏螯虾(Procambarus clarkii)生长性能、体组成和消化酶活性的影响

采用3×3因子方法,进行了饲料中不同蛋白质和脂肪水平对克氏螯虾[均重(7.03±0.05)g]生长性能、体组成和消化酶活性的影响研究。试验设3个蛋白质水平(24%、27%和30%)和3个脂肪水平(4%、7%和10%),共9组,每组4个重复,每个重复10尾虾,为期8周。结果表明,蛋白质和脂肪水平对成活率无显著影响(P>0.05),增重率和特定生长率显著受脂肪水平影响(P<0.05)。10%脂肪组的增重率及特定生长率显著低于4%和7%脂肪组(P<0.05)。饲料中蛋白质水平对全虾的干物质、蛋白和灰分含量,肌肉蛋白和脂肪含量有显著影响(P<0.05)。饲料中脂肪水平对全虾和肌肉脂肪含量有显著影响(P<0.05)。饲料蛋白水平和脂肪水平分别显著影响肠道及肝胰脏中蛋白酶活性和脂肪酶活性(P<0.05)。综上所述,建议克氏螯虾成虾前期饲料的蛋白质水平以27%左右,脂肪水平以4%—7%为宜。

克氏原螯虾(Procambius clarkii)卵黄蛋白的部分生化性质

通过电泳回收的方法对克氏原螯虾(Procambius clarkii)的卵黄蛋白进行了纯化,鉴定结果显示总分子质量为481 ku,具有6个亚基(198,176,132,111,92,82 ku);染色分析表明该蛋白是一种糖脂磷类胡萝卜素蛋白,亚基之间有1个二硫键交联.

克氏原螯虾(Procambarus clarkii)血细胞染色方法的研究

甲壳类血细胞的形态和分类是甲壳类免疫学研究的基础。本文选择三种甲壳类血细胞的常用染料:瑞氏染液、姬姆萨染液和瑞氏-姬姆萨混合染液,研究它们对于克氏原螯虾血细胞的染色效果。通过改变染色时间、染色温度、分色方式以及分色时间等染色条件,观察、比较不同染色条件下血细胞内的颗粒、细胞核和细胞质的着色情况,以及细胞整体轮廓清晰程度,确定适用于克氏原螯虾血细胞染色的理想染色方法,并建立相应的操作程序。

Cu^(2+)对克氏原螯虾(Procambarus clarkii)肝胰腺的蛋白质氧化损伤效应

克氏原螯虾(Procambarus clarkii)是一种潜在的水质环境污染的指示生物,为研究水体中不同浓度的重金属Cu2+对其肝胰腺组织的蛋白质氧化损伤作用,在测得Cu2+致克氏原螯虾96 hLC50为30 mg·L-1后,将螯虾分别暴露于0、0.5、1.0、3.0、5.0、10.0 mg·L-1的Cu2+溶液中,并在暴露24、48、72、96 h后测定各处理组肝胰腺组织的活性氧自由基(ROS)、总抗氧化能力(T-AOC)、谷胱甘肽过氧化物酶(GSH-PX)活性、蛋白质羰基(PCO)含量以及DNA-蛋白质交联(DPC)系数。结果表明,在暴露24h时,随着Cu2+浓度的升高,ROS、T-AOC和GSH-PX水平的变化规律相似,均为先上升后下降,PCO含量和DPC系数随暴露浓度的升高而增加;在暴露48、72、96 h后,ROS、T-AOC、PCO水平和DPC系数均表现为先升后降的趋势,GSH-PX活性在染毒96 h后,随着浓度的升高而下降。PCO含量的变化具有一定的剂量-效应关系。结果提示,肝胰腺的蛋白质氧化损伤可能是重金属Cu2+致克氏原螯虾毒性效应的主要机制之一。

Tropomyosin基因在克氏原螯虾(Procambarus clarkii)卵巢发育过程中的表达

原肌球蛋白Tropomyosin是虾蟹类的主要致敏物质,除此以外在其他方面功能的研究非常少。本研究克隆了克氏原螯虾Tropomyosin基因,进行了同源序列比对,并分析了Tropomyosin基因在克氏原螯虾卵巢发育过程中的转录水平表达模式。研究结果显示,Tropomyosin蛋白序列高度保守,在虾蟹类甲壳动物中,8个抗原决定簇序列几乎完全一致。本研究克隆的基因存在3个核苷酸位置(c61—t,g118—a,c359—t)的变化,但是均不处于抗原决定簇内,免疫原性不受影响。Tropomyosin基因mR NA在克氏原螯虾卵巢组织中表达量很低,但是随着卵巢的发育存在表达差异,即呈现一个先提高再降低的峰型特征。本研究暗示Tropomyosin除过敏原特性外,可能也受性腺发育的部分调控影响。

Growth Response and Fatty Acid Composition of Juvenile Procambarus clarkii Fed Different Sources of Dietary Lipid

An experiment was conducted to evaluate the effects of feeding various sources of dietary lipid on weight gain, feed conversion, survival and fatty acid composition of juvenile red swamp crawfish, Procam-barus clarkii. Six semi-purified diets containing vitamin-free casein, defatted soybean meal, 0. 5% choline chloride, 0. 5% glycine and 0. 5% cholesterol were supplemented with 6. 0% of either anchovy oil, linseed oil, soybean oil, rapeseed oil, safflower oil or pork lard. Each diet was fed to crawfish (3. 07±0. 21 g average weight) in three replicate aquaria for 60 days. Survival rate, weight gain and feed conversion were best for crawfish fed the diet containing anchovy oil. Crawfish fed the linseed oil diet had the second highest weight gain, followed by crawfish on soybean oil, repaseed oil, safflower oil and pork lard diets, respectively. Feed conversion values were a reflection of weight gain. Results showed that both n-6 and n-3 fatty acids are dietary essential for juvenile Procambarus clarkii, although n-3 fatty acids promoted faster growth than n-6. However, highly unsaturated fatty acids (HUFA) (20 : 5n-3 and 22 :6n-3) had better growth-promoting effect than 18 : 3n-3, due probably to the limited ability of crawfish to bioconvert fatty acids to polyenoic forms of longer chain length. The fatty acid composition of the crawfish generally reflected that of the dietary lipids, especially for the diets containing unsaturated fatty acids.

Cloning and Sequence Analysis of Prophenoloxidase from Haemocytes of the Red Swamp Crayfish,Procambarus clarkii

The full-length cDNA sequence of prophenoloxidase was obtained through RACE technology. The complete cDNA sequence is 3 721-bp long, containing an open reading frame （ORF） of 1 881 bp, a 154-bp 5′-untranslated region, and a 1 686- bp 3′-untranslated region with three potential functional poly（A） signals （AATAAA）. The molecular mass of the deduced amino acid sequence （627 aa） was 72.3 kDa with an estimatedpI of 5.88. It contained putative copper-binding sites （copper A： 131, 135, 167 and copper B： 301,305, 341）, and a tentative complement-like motif （GCGWPDHL）. Eight potential N-linked glycosylation sites were predicted to be present in P. clarkii prophenoloxidase. Similar to those in other arthropod prophenoloxidases reported so far, no signal peptide was detected in the crayfish prophenoloxidase. The phylogenetic trees confirmed that P. clarkii prophenoloxidase was most closely related to that of freshwater crayfish P. leniusculus and more closely related to other crustacean prophenoloxidases from shrimp, prawn, and lobster than to the insect prophenoloxidases. Besides, two putative introns were found in this sequence of genomic DNA.

Starvation on changes in growth and fatty acid composition of juvenile red swamp crawfish, Procambarus clarkii

The effect of starvation on physiological and biochemical indicators of body, muscle and hepatopancreas were investigated for juvenile red swamp crawfish （Procambarus clarkii）, and the results showed the effect was obvious. In a 70-day starvation experiment, mean body weight, exuvial rate, abdominal muscle ratio and hepatosomatic index decreased significantly. The percentage of protein, fat, ash and moisture were also significantly affected. The protein content decreased sharply at Day 30 onward and the percentage of fat decreased rapidly from Day 50 and on. The percentages of ash and moisture started to increase significantly at Day 40 and 30 respectively. After the starvation experiment, the hepatopancreatic total and neutral lipid content decreased slightly from 23.90% to 20.39% and 73.75% to 70.35%, while the hepatopancreatic polar lipid content increased from 23.81% to 27.32%. On the other hand, all the muscular total, neutral and polar lipid contents, and the muscular fatty acid compositions did not change significantly except for the hepatopancreatic fatty acid composition. The relative percentages of 14：0, 16：0, 16：1n-7 and 18：1n-9 were decreased by 55.17%, 11.11%, 8.5% and 6.70% from the initial values, and the relative percentages of 18：2n-6, 18：3n-3, 20：4n-6, 20：5n-3 and 22：6n-3 increased obviously from 5.2%, 3.9%,4, 2.4%, 10.6%, and 13.7% to 7.1%, 5.0%, 2.8%, 11.5%, and 14.4% respectively. Preference of utilization of fatty acids from hepatopancreas during starvation was as follows： 14：0〉16：0〉16：1n-7〉18：1n-9, and the conservation of highly unsaturated fatty acids was in the order of： 18：2n-6 〉18：3n-3〉20：4n-6〉20：5n-3〉22：6n-3.

16S rRNA gene sequencing analysis on changes in the intestinal flora of Procambarus clarkii with“Black May”disease

The morbidity and mortality peak of farmed Procambarus clarkii occurs around May every year,a phenomenon known as"Black May"disease(BMD).Increasing evidence shows that the intestinal flora is closely related to host health.We analyzed and compared the microbiota of healthy and BMDaffected P.clarkii intestines.The results show that there was no significant difference in bacterial a-diversity(richness P=0.59;evenness P=0.43;and diversity P=0.052)between the diseased group and the control group.Four dominant phyla in the intestines of crayfish in the control group,namely Tenericutes(30.86%),Bacteroidetes(29.99%),Firmicutes(22.23%),and Proteobacteria(15.23%),were identified.However,a striking shift in the microbial compo sition were found in the intestines of P.clarkii with BMD.Bacteroidetes was a dominant phylum in healthy P.clarkii,whereas the prevalence was low in diseased P.clarkii(1.87%).By contrast,the prevalence of Proteobacteria was significantly higher(P<0.05)in P.clarkii with BMD than in P.clarkii without BMD.Candidatus Bacilloplasma,Bacteroides,Vibrio,and Aeromonas showed significant differences(P<0.05)at the genus level.Tax4 Fun function prediction indicated that the relative abundance of genes involved in energy metabolism in the intestinal flora of P.clarkii with BMD was significantly reduced(P<0.05).Therefore,BMD can change the composition of the intestinal microbiota of P.clarkii.This study contributes to the understanding of the relationship between intestinal flora and host especially in aquatic animals.

Effect of Hydroponic Salix saposhnikovii on Cultivation of Procambarus clarkii

[ Objective] To provide basic research parameters for the laboratory culture of Procambarus clarkii using Salix saposhnikovii and the breeding materials for the clean environment-friendly aquaculture. [Method] The de-aerated water was used to culture the post larvae and P. clarkii juvenile. The effects of S. saposhnikovii roots on the survival rate, body length increase and body length variation coefficient of P. clarkii were in- vestigated. The survival rate of juvenile P. clarkii in the V class wastewater at 2 ammonia nitrogen concentrations were examined. [ Result] The post larvae of P. clarkii cultivated with S. saposhnikovii in the de-aerated water had significantly higher survival rate and longer body length than the control （ P 〈 0.05）, but there was no significant difference between the 2 treatments in the variation coefficient of body length. In the wastewater containing 2 mg/L ammonia nitrogen, the survival rate and ammonia nitrogen wastewater purification rate of the experimental juvenile P. clarkii were higher than the control. When the concentration of ammonia nitrogen increased to 10 mg/L, the survival rate of the experimental juvenile P. clarkii was still higher than the control, but there was no significant difference between the 2 groups in the purification rate of ammonia nitrogen. [ Conclu- sien] S. saposhnikovii has significant effect on the survival rate and growth of juvenile P. clarloTand can purify the wastewater containing 2 -10 mg/L ammonia nitrogen.

Longitudinal genetic analysis of growth-related traits in red swamp crayfish Procambarus clarkii (Girard)

The red swamp crayfish,Procambarus clarkii,is an economically important species especially in China.Their exoskeleton places serious constraints on growth and culture management.Their growth is achieved through intermittent molting/ecdysis.The longitudinal genetic dynamics for growth-related traits at different ecdysial points in P.clarkii has been unclear to date.In this study,conditional genetic analysis was carried out for growth-related traits(body weight,body length,chela length,and cephalothorax length)based upon a mixed genetic model with conditional additive,dominance,and genotype by environment effects in P.clarkii.A complete diallel cross was made among three geographic populations of P.clarkii for the genetic mating design.Results of the conditional genetic analysis showed that from 4 th molt to 9 th molt the conditional additive variations were increased significantly whereas the conditional non-additive genetic variations(dominance and genotype by environment interaction)were decreased significantly for these growth-related traits.This indicated that lots of new expression of additive effect genes for body weight,body length,chela length,and cephalothorax length occurred during ontogeny,and environment played a signifi cant role in the expression of genes affecting these growth-related traits.Growth of the four traits was mainly affected by non-additive genetic effects in early developmental stage(prior to 4 th molt).The cumulative conditional additive variation for the growth-related traits from 4 th molt to 9 th molt accounted for a large majority of the total conditional additive variations from 2 nd molt to 9 th molt,indicating that this period was very important for the growth of this species.Using the conditional analysis method,dynamics of growth-related traits during an important ontogenetic phase of red swamp crayfish was uncovered.Our results provide valuable insights into refining production of this species.

Accumulation of Copper, Lead, Chrome, Cadmium in Some Tissues of <i>Procambarus clarkii</i>in Rharb Region in Morocco

Different concentrations of four metals namely copper, lead, chrome and cadmium were evaluated in crayfish Procambarus clarkii tissues (carapace, flesh) for a six-month period, from March to August 2013 in the Rharb of Morocco (Lagoon Merja Zerga and Nador canal). The determination of heavy metals was carried using atomic absorption spectroscopy Varian VV20. The results obtained revealed a metal’s contamination of Procambarus clarkii in both sites. Concentrations of heavy metals examined Procambarus clarkii carapace ranged as follows: Merja Zerga (Pb 2.07 - 6.7 μg/g;Cu 2.9 - 9.97 μg/g;Cr 0.89 - 14.22 μg/g dry weight) and Nador canal: (Pb 0.08 - 7.8 μg/g;Cu 3.40 - 9.3 μg/g;Cr 0.87 - 3.5 μg/g dry weight). Concentrations of heavy metals found in flesh were as follows: Merja Zerga (Pb 0.02 - 5.25 μg/g;Cu 9.58 - 23.59 μg/g;Cr 0 - 2.71 μg/g dry weight) and Nador canal (Pb 0.06 - 6.81 μg/g;Cu 9.5 - 37.20 μg/g;Cr 0 - 3.23 μg/g dry weight). The distribution of those metals in Procambarus clarkii shows high levels of contamination for lead, copper and chrome with an absence of cadmium in both sites (Merja Zerga and Nador canal). The concentrations differed in carapace and flesh.

Study on the Spread of Procambarus clarkii at Gharb(Morocco)and Its Impact on Rice Growing

The red swamp crayfish(Procambarus clarkii)is a species from the northeast of Mexico and the central south of the United States(Louisiana),actually is the dominant macroinvertebrate in several countries.It was introduced to several countries,generally for gastronomic reasons or to repopulate destroyed areas by a disease.While the first introduction of this species into the world is well-documented,little is known about its pathways of invasion and the reason for its rapid spread in several countries.P.clarkii is an aggressive crustacean that multiplies very fast and adapts to features of the invaded location.Adults of the species can disperse up to 1.6 km on land.Also,they dig deep holes which allows them to establish easily in wetlands and adjacent waterbodies that are hydrologically disconnected.This study takes place in the Gharb area(northwestern of Morocco)where several carcasses of P.clarkii have been found in different places.In the present study,it is a question of understanding how this species was introduced in this area,to establish a map of propagation of the species,and to evaluate the damage that it would have caused in the rice growing of Gharb.In Morocco,it was first sighted in the Gharb area in 2008 in the commune of Sidi Allal Tazi.Following this discovery,possible ways of the species introduction and the dispersal modes were explored that P.clarkii could use to invade the Gharb area.The results of this study can be used by natural resource managers,rice farmers,fishermen and the local population,to help them to locate the spread of P.clarkii,to identify vectors that move P.clarkii across political boundaries and illustrate the importance of limiting and prohibiting the movement of this invasive species across boundaries or into new ecosystems.

高效氯氰菊酯对克氏原螯虾(Procambarus clarkii)抗氧化酶活性的影响

为研究水体中不同浓度的高效氯氰菊酯对克氏原螯虾抗氧化酶活性的影响,在本实验室测得高效氯氰菊酯致克氏原螯虾的96hLC50为0.2μg·L-1的基础上,将克氏原螯虾分别暴露于0、0.005、0.01、0.02、0.04μg·L-1的高效氯氰菊酯溶液中,在24、48、72h和96h后分别测定其鳃、肝胰腺和血清中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活性以及丙二醛(MDA)的含量。结果表明,在染毒96h期间各组织中的SOD活性变化规律相似,均为先上升后下降;肝胰腺中的CAT活性先下降后上升再下降,而鳃和血清中的CAT活性表现为先上升后下降;丙二醛(MDA)含量则一直高于对照组,随着暴露时间和暴露浓度的增加而增加。染毒96h后,0.04μg·L-1处理组与空白对照组相比,肝胰腺、鳃和血清中的SOD活性分别下降了28.3%、44.1%和27.2%,CAT活性分别下降了38.1%、39.4%和12.3%,而MDA含量分别较空白对照组提高了0.72、1.09倍和0.46倍。研究结果提示,高效氯氰菊酯对克氏原螯虾的抗氧化酶活性具有显著的抑制效应。

Isolation and Identification of Aeromonas allosaccharophila from Procambarus clarkii

In order to provide a scientific basis for the control of Aeromonas allosaccharophila from Procambarus clarkii , a dominant strain was isolated from moribund P. clarkii in some farm in Hubei Province, and designated X1. It was preliminarily identified to be A. allosaccharophila through physiological-biochemical test. Further sequence analysis showed that 16S rDNA of this bacterium shared 90% identity with 16S rDNA of A. allosaccharophila , suggesting that it is A. allosaccharophila . Drug sensitivity test showed that isolate X1 was sensitive to doxycycline, cefotaxime, norfloxacin and gentamicin. In animal regression test, the bacterium the same as that from naturally-diseased P. clarkii could be isolated, with the same disease symptoms as well.

基于图像识别的5个不同产地克氏原螯虾(Procambarus clarkii)形态差异分析

为了解江苏、江西、湖北、上海、河南5个地区克氏原螯虾(Procambarus clarkii)的形态差异和获取快速、有效的形态鉴别方法,本研究采用传统形态测量法和地标点法来分析各产地形态差异。结果显示:(1)克氏原螯虾雌雄群体相对扭曲主成分分析,前三个主成分累计贡献率分别为79.96%、67.21%,传统形态测量法前三个主成分累计贡献率分别为76.77%、82.70%,两种方法均表明其形态差异主要体现在头胸甲及腹部部位;(2)聚类分析将克氏原螯虾5群体聚为两支,上海、河南、江西、湖北群体聚为一支,江苏群体单独聚为一支。(3)地标点法雌雄群体综合判别准确率分别为100%、94%,传统形态测量法综合判别准确率均为56%。以上研究结果表明不同产地间克氏原螯虾具有一定的形态差异,且地标点法区分不同产地克氏原螯虾群体差异性效果显著,这将有利于克氏原螯虾生产和选育过程中群体的鉴别及外形特征的快速获取。

Does Procambarus Clarkii （Girard, 1852） Represent a Threat for Estuarine Brackish Ecosystems of Northeastern Adriatic Coast （Italy）？

The ongoing expansion of P. clarkii in the aquatic environments of northern Italy, where it is very common and abundant in most of the streams, lakes and reservoirs, led us to worry about the possibility of invasion into the estuarine and lagoonal brackish environments of the Adriatic coast, which is important not only for their peculiar fauna and flora and high biodiversity, but also for fishing and aquaculture. We carried out laboratory tests to investigate whether this species could live, moult and breed in water with different salinity levels. Several stocks of adult individuals, acclimated before the laboratory experiments, were maintained and monitored daily for a long period （100 days） in glass aquaria with water salinity varying from 5 to 33 ppt. They not only survived during the whole experiment period in levels up to 25 ppt, but also they regularly moulted and mated. These results confirmed that the species could invade the estuarine and brackish environments of the Adriatic coast, with all the implied consequences for the native species of both vertebrates and invertebrates and for human livelihoods.

Cu^(2+)胁迫对克氏原螯虾(Procambarus clarkii)酚氧化酶原激活系统活性的影响

重金属铜是养殖水体的主要污染物,严重影响甲壳动物的免疫机能。以淡水克氏原螯虾(Procambarus clarkii)为实验对象,以亚致死浓度(1.0、3.0、5.0、10.0 mg·L^(-1),96-h LC50=22.14 mg·L^(-1))的Cu^(2+)为胁迫因子,采用静态水质接触染毒法,通过分析血细胞内酚氧化酶原(pro PO)、丝氨酸蛋白酶(SP)以及血淋巴中酚氧化酶(PO)、血蓝蛋白(Hc)和血细胞数(THC)的水平,探讨了Cu^(2+)胁迫对酚氧化酶原激活系统(pro PO-AS)活性的影响。结果表明,与未染毒的对照组相比,Cu^(2+)显著抑制proPO、PO的活性以及THC、Hc的含量(P<0.05);但仅在10.0 mg·L^(-1)Cu^(2+)浓度下,SP的活性显著降低(P<0.05)。研究结果提示,水体Cu^(2+)对螯虾具有免疫毒性效应,氧化应激导致的血细胞数、血蓝蛋白含量、丝氨酸蛋白酶活性抑制可能是影响pro PO-AS活性的主要机制之一。