日本囊对虾(Marsupenaeus japonicus)3个野生种群和1个养殖种群的形态差异与判别分析

采用3种多元分析方法,对3个野生种群和1个养殖种群的13个形态比例参数进行比较研究。聚类分析结果表明,广东湛江和海南临高的种群形态最接近,福建厦门种群的趋异程度最大。主成分分析构建了6个主成分,其贡献率分别为:第1主成分23.084%,第2主成分17.976%,第3主成分15.321%,第4主成分13.84%,第5主成分12.707%,第6主成分7.770%,累积贡献率为90.699%。逐步判别选入6个贡献率较大的性状进行判别分析,F检验结果显示,4个种群形态差异显著(P<0.01);同时建立了4个种群的判别函数,其判别准确率P1、P2均为100%。综合判别率为100%。3种多元分析结果均认为,4个种群日本囊对虾在形态上已产生一定程度的差异,且集中表现在体重、头胸甲的性状上。

养殖密度、饵料种类和丰度对日本囊对虾(Marsupenaeus japonicus)争胜行为的影响

本研究设置10、25、50尾/m2 3个养殖密度,人工饵料、鲜菲律宾蛤仔肉两种饵料,不喂(N)、少喂(L)、饱喂(F)3个处理,分别于投喂前1 h、投喂时、投喂后1 h测定日本囊对虾(Marsupenaeus japonicus)的争斗次数、平均优势指数等指标,分析养殖密度、饵料种类及丰度对日本囊对虾争胜行为的影响。结果显示,随着养殖密度的增加,对虾的争胜行为逐渐增强,投喂时与投喂前、投喂后对虾的争胜行为差异显著(P<0.05);投喂时对虾的争斗次数最多;饵料种类和丰度显著影响日本囊对虾的争胜行为,投喂菲律宾蛤仔肉的争胜行为强于投喂人工饵料(P<0.05),少喂与不喂、饱喂处理间差异显著(P<0.05)。研究表明,养殖密度、饵料种类及丰度均显著影响日本囊对虾的争胜行为。

多因子对日本囊对虾Marsupenaeus japonicus氮磷代谢的影响

对日本囊对虾Marsupenaeus japonicus在不同条件下的无机磷、亚硝酸氮、硝酸氮、氨氮的代谢率进行了测定。结果表明:温度、体质量、盐度、pH值和摄食状态对其代谢存在影响。无机磷、硝酸氮、氨氮的代谢率与体质量呈负相关,而亚硝酸氮的代谢率与体质量随体质量的上升而增大;在20～30℃的温度范围内,随着温度的上升,日本囊对虾磷、亚硝酸氮、硝酸氮、氨氮的代谢率均上升;在10‰～31‰的盐度范围内,随着盐度的升高,日本囊对虾(6.739±0.023)g磷、亚硝酸氮的代谢率升高,当盐度为31‰盐度时,磷代谢率达到(0.736±0.002)μg.g-.1h-1,亚硝酸氮代谢率为(0.0778±0.011)μg.g-.1h-1,而硝酸氮、氨氮的代谢率与盐度呈负相关,当盐度为31‰时,硝酸氮的代谢率降为(0.257±0.207)μg.g-.1h-1,氨氮代谢率降为(4.445±0.259)μg.g-.1h-1;在7.5～9.0的pH值范围内,随着pH值的升高,日本囊对虾(6.749±0.013)g磷的代谢率明显下降,氨氮、硝酸氮的代谢率提高,亚硝酸氮的代谢率在pH值为8.5时达到最大值,后又呈下降趋势;日本囊对虾(6.729±0.028)g摄食配合饲料,饱食状态下磷、亚硝酸氮、硝酸氮、氨氮的代谢率比饥饿状态下分别提高了272.02%、91.67%、795.38%、98.54%。

日本囊对虾(Marsupenaeus japonicus)组织蛋白酶D基因克隆及表达分析

组织蛋白酶D是溶酶体天冬氨酸蛋白酶家族的主要成员,广泛参与动物机体细胞内蛋白质的降解过程,对维持细胞稳态和正常代谢具有重要的作用。为研究组织蛋白酶D在甲壳动物非特异性免疫和幼体发育过程中的作用,本研究采用RACE技术首次克隆得到日本囊对虾组织蛋白酶D基因cDNA序列,命名为MjCatD,其中开放阅读框长为1 161bp,编码386个氨基酸残基。序列分析和同源建模显示该基因编码的蛋白含有保守的N-糖基化位点、天冬氨酸蛋白酶签名序列、酶活化位点和非消化性组织蛋白酶D的特征序列,并且呈保守的双叶形结构。同源性比较和系统进化分析发现,MjCatD与斑节对虾、美洲螯龙虾和脊尾白虾相似性较高,并且与它们紧密聚为一支。实时荧光定量PCR结果显示,MjCatD基因在日本囊对虾多个组织中均有表达,其中肝胰腺中表达量最高。在白斑综合征病毒（white spot syndrome virus,WSSV）感染后3～24h日本囊对虾肝胰腺中MjCatD的表达量逐渐下降,而在48h急剧上调至最高表达量并且与对照组差异极显著（P〈0.01）。此外,MjCatD基因在幼体发育不同阶段中也表现出明显的变化趋势。以上研究表明,MjCatD基因可能参与日本囊对虾先天免疫反应和幼体发育过程。

日本囊对虾(Marsupenaeus japonicus)视神经节、脑和胸神经节5-羟色胺的定位研究

采用兔抗5-羟色胺抗体和SABC免疫细胞化学方法,对日本囊对虾视神经节、脑和胸神经节进行定位研究。结果表明,视神经节内的5-羟色胺免疫阳性细胞分布于视外髓、视内髓和视端髓。前脑前中群,前脑中部前端神经髓质、中央体,中脑嗅叶内侧细胞群,后脑后中群、侧位群和后侧群呈5-羟色胺免疫阳性反应。胸神经节5-羟色胺阳性细胞较少,第4对胸神经节检出1对阳性细胞,第5对胸神经节检出2对阳性细胞,神经髓质不呈阳性反应。5-羟色胺的特异性分布,为日本囊对虾体内的生理功能和甲壳动物腹神经索演化提供了形态学依据。

多重PCR技术分析日本对虾Marsupenaeus japonicus精巢和卵巢差异表达基因

通过多重PCR方法 ,对采用SSH(抑制消减杂交 )技术制备日本对虾卵巢特异探针并筛选卵巢cDNA全长文库所获得的 8个阳性克隆进行分析 ,研究这些新克隆的基因在精巢和卵巢的差异表达情况。这 8个基因可分为二类 ,一类为卵巢特异表达的基因 。

Effects of the Dietary Probiotic Clostridium butyricum on Intestine Digestive and Metabolic Capacities, SCFA Content and Body Composition in Marsupenaeus japonicus

A 56-day feeding trial was performed to evaluate the effects of dietary probiotic Clostridium butyricum(CB) on intestine digestive and metabolic capacities, intestine short-chain fatty acids(SCFA) content and body composition of kuruma shrimp Marsupenaeus japonicus. Shrimp s were randomly allocated into 9 tanks, 30 each, and f ed with diets containing different levels of C. butyricum(1×10~9 cfug^(-1)): 0 mgg^(-1) feed(Control), 100 mgg^(-1) feed(CB-100), 200 mgg^(-1) feed(CB-200), while each level was triplicated. The results indicated that compared with the control group, the intestine pepsin(Pep) activity and 5-hydroxytryptamine(5-HT) concentration of two C. butyricum group s were both increased. Amylase(AM Y) and lipase(LPS) activities wer e only induced in CB-200 group. Alanine aminotransferase(ALT) and aspartate aminotransferase(A ST) activities of two C. butyricum group s showed no significant change. The α-amylase(AM Y) gene expression was induced in CB-200 group, and tryp sin gene expression of two C. butyricum treated group s were both induced. Intestine SCFA content and body composition analysis showed that the contents of propionic acid, butyric acid and the crude protein of two C. butyricum group s were all higher than those of control. These results revealed that C. butyricum can modulate intestine digestive and metabolic capacities, improve intestine SCFA content and body crude protein content in M. japonicus.

Integrative analysis of transcriptomic profile reveals potential roles of miRNAs in regulating development of Marsupenaeus japonicas

Regulation of microRNAs(miRNAs)on various biological processes has been a surprising and exciting field.Identification of miRNAs is the first step to comprehensively understand their functions.However,attempts on global identification and functional verification of miRNAs are very limited in penaeid shrimp Marsupenaeus japonicus,an economically important aquatic species.By performing an integrated analysis of transcriptomic profile from gastrula embryos of M.japonicus,21conserved miRNAs in M.japonicas(mja-miRNAs),belonging to 19 miRNA families,were identified and characterized.Of the 21 mja-miRNAs,15 miRNAs were successfully verified to be predominantly expressed in gastrula stage,where they displayed dynamic expression patterns compared with those in naupliuin stage.Based on perfect or near-perfect match to target region,120 target genes were predicted at transcriptome-wide level.Noteworthy,gene ontology(GO)classification and metabolic pathway annotation revealed eight targets that were actively involved in developmental processes.Of the predicted miRNA-mRNA pairs,six targets were then randomly selected and experimentally validated by dual luciferase reporter assay,where three pairs were proved with potential targeting activity.Overall,to search for conserved miRNAs potentially involved in early development of M.japonicus,we combined in silico and experimental methods,which can be applied in other organisms as well.Our data implied important roles of miRNAs in the early embryonic development and also suggested the presence of complex miRNA-mRNA functional networks in M.japonicus.

Molecular characteristics of three thymosin-repeat proteins from Marsupenaeus japonicus and their responses to WSSV infection

β-thymosins, a family of highly conserved peptides, play a vital role in wound-healing, angiogenesis,antimicrobial process and antiviral immunity. Three novel β-thymosin-repeat proteins, named mjthm4, mjthm3 and mjthm2, were cloned from Marsupenaeus japonicus using expressed sequence tags（EST） from suppression subtractive hybridization. The mjthm4, mjthm3 and mjthm2 c DNAs possessed open reading frames that encoded166, 128 and 90 amino acid residue polypeptides and contained four, three and two β-thymosin actin binding modules, respectively. Blast analysis demonstrated that mjthm4, mjthm3 and mjthm2 shared high homology with known invertebrate multi-repeat β-thymosins. These proteins are ubiquitously expressed in all of the examined tissues, and the transcriptional levels were highest in the intestine. Further investigation revealed that mjthm4,mjthm3 and mjthm2 were remarkably up-regulated 6 h after WSSV infection. Moreover, while mjthm4 transcriptional levels displayed no changes, mjthm3 and mjthm2 levels decreased in the virus-resistant shrimps.The results indicate that mjthm4, mjthm3 and mjthm2 are novel multi-repeat β-thymosin homologues, have a close relationship with WSSV infection, and might contribute to a better understanding of host defense and/or virus invasion interactions in shrimps.

Molecular characterization and expression analysis of a novel dual-CRD C-type lectin in kuruma shrimp(Marsupenaeus japonicus)

C-type lectins are among the most significant pattern recognition receptors（PRRs） found in invertebrate. They are a class of carbohydrate-binding proteins that can recognize specific sugar moieties on the surface of pathogens. In the present study, a novel C-type lecitn（termed Mj Lectin） from kuruma shrimp Marsupenaeus japonicus was identified. The full-length c DNA of Mj Lectin was 1 245 bp with a 1 011 bp open reading frame（ORF） that encoded a polypeptide of 336 amino acid residues. Mj Lectin consisted of two tandemly arrayed carbohydrate-recognition domains（CRDs）, unlike other reported M. japonicus C-type lectins with only one CRD. It showed a high similarity to other shrimp dual-CRD lectins. Among the Ca2＋-binding Site 2, the tripeptide motif dictating the carbohydrate binding specificity was exhibited as a rare mutant LPN（Leu134-Pro135-Asn136） in CRD1 and a traditional EPN（Glu299-Pro300-Asn301） in CRD2, respectively. Mj Lectin showed a specific expression pattern in both tissue and cellular levels, for its m RNA transcript was mainly expressed in the F-cells of the hepatopancreas. After white spot syndrome virus（WSSV） challenge（3.6×108 virions/μL）, the expression of Mj Lectin in the hepatopancreas was up-regulated significantly at 48 h（P〈0.01） compared with the control group. These results suggested that Mj Lectin might be involved in the innate immune defense against WSSV infection.

日本囊对虾(Marsupenaeus japonicus)秋繁仔虾形态表型对氨氮耐受性能的影响效应

以体长7.643±0.639mm的日本囊对虾秋繁同生群仔虾为实验对象,以氨氮为胁迫因子,在水温18.0±1.0oC、盐度20、p H 8.1±0.2的条件下,经确认其96h成活率略低于5%的氨氮攻毒质量浓度为52.37mg/L后,以此为氨氮急性攻毒实验质量浓度,借助显微扫描像素测量技术和多元分析方法定量研究了A、B、C、D、E实验群体(依次为氨氮攻毒0—24h、24—48h、48—72h、72—96 h时段内的死亡群体和氨氮攻毒96 h时的存活群体)间形态表型特征的差异。结果表明:(1)在所涉15项形态性状中,各实验群体间均无显著差异(P>0.05)的性状仅为总长和第四腹节长;(2)在所涉17项形态比例指标中,各实验群体间均无显著差异(P>0.05)的指标共计3项,依次为第五腹节长/总长、额剑长/头胸甲长和尾节高/尾节长,各实验群体间的欧氏距离均达到显著水平(P<0.01),且均有随耐氨氮性能差异的增大而呈显著增大的趋势;(3)经主成分分析,提取到的5个特征值均大于1的主成分,累计贡献率达85.940%,其中第1主成分的贡献率高达46.121%,其载荷绝对值大于0.5的主要影响变量占形态比例指标总数的64.706%;(4)将氨氮急性攻毒处理所获E实验群体定义为选留群,其余实验群体统归为淘汰群,经判别分析,所建的Fisher分类函数方程组可较清晰地区分淘汰群和选留群个体,其中选留群和淘汰群个体的判别准确率P1分别为99%和86.75%,P2分别为88.20%和98.86%,两者综合判别准确率为92.88%。

日本囊对虾(Marsupenaeus japonicus)秋繁同生群仔虾不同耐寒性能群体间的形态表型差异

以水温16oC、p H 8.1±0.2、盐度20的砂滤海水为实验用水,以体长(7.643±0.639)mm的日本囊对虾(Marsupenaeus japonicus)秋繁同生群仔虾为实验对象,经确认其在水温由16oC瞬降至14oC后并保持96h恒温的最终成活率略低于5%后,以此为水温骤降胁迫处理方式,采取显微扫描像素测量技术和多元分析方法定量研究了A、B、C、D、E实验群体,依次为温降胁迫处理后(0—24)h、(24—48)h、(48—72)h、(72—96)h时段内的死亡群体和温降胁迫处理后96h时的存活群体间形态特征的差异。结果表明:(1)各实验群体在本研究所涉15项形态性状中均无显著差异(P>0.05)仅为第一触角柄长、额剑长和眼径,在所涉17项形态比例指标均无显著差异(P>0.05)仅为尾节高/尾节长;(2)随耐寒性能差异的增大,各实验群体间的欧氏距离也随之增大,且均达到极显著水平(P<0.01);(3)经主成分分析,提取到的4个特征值均大于1的主成分,累计贡献率达87.590%,其中载荷绝对值大于0.80的主要影响变量均仅存在于第1主成分中;(4)将E实验群体定义为耐寒处理选留群,其余实验群体统归为耐寒处理淘汰群,采用逐步导入判别法建立的Fisher分类函数方程组可较清晰地区分淘汰群和选留群个体,其中选留群和淘汰群个体的判别准确率P1分别为99%和88.75%,P2分别为89.80%和98.88%,两者综合判别准确率为93.88%。

舟山近海日本囊对虾(Marsupenaeus japonicus)野生群体与露天养殖越冬群体间的形质差异

鉴于舟山沿海日本囊对虾秋苗养殖成活率显著低于春季和夏季人工养殖苗种之实际,深入开展适合于本地沿海养殖的日本囊对虾秋苗的优质亲体的选种研究显得十分必要。为此,作者采用聚类分析、主成分分析和判别分析等方法定量研究了舟山近海日本囊对虾野生群体和露天池塘越冬养成群体间的形质特征差异。结果表明:两者在本研究所涉18项形质评价指标中的相似性指数仅为11.11%,欧氏距离为4.243(P<0.01);经主成分分析,提取到的5个特征值均大于1的主成分,累计贡献率达85.27%,其中第1主成分为以营养生理状况指数、额剑上缘锯齿数以及腹部肌肉发达程度为主导的体质强壮因子,第2主成分为以寻食视野与胸腔空间大小为主导的捕食运动功能因子,后3个主成分为以尾节肌肉发达程度为主导的避敌运动功能因子;采用逐步判别法,以判别贡献率较大的9项形质评价指标为自变量,所建Fisher分类函数方程组可清晰区分日本囊对虾野生群体和越冬养成群体,其中野生群体的判别准确率P1、P2分别为99.33%和96.13%,越冬养成群体的判别准确率P1、P2分别为96%和99.31%,两者综合判别准确率为97.67%。因此,日本囊对虾秋苗野生亲本形质特征的选择,应以秋苗越冬养成群体为参照。

Genome-wide mining, characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats(SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class(44.08%), followed by mononucleotides(29.67%), trinucleotides(18.96%), tetranucleotides(5.66%), hexanucleotides(1.07%), and pentanucleotides(0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci(90.0%) were successfully amplified with specific products and 24(80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17(with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.

Transcriptome of hepatopancreas in kuruma shrimp Marsupenaeus japonicus under low-salinity stress

The kuruma shrimp Marsupenaeus japonicus is one of the most commercially important shrimp species in the world.Low salinity would aff ect the penetration and immunity,and even led to its death of the shrimp.However,little is known about the molecular mechanism of the effect.Therefore,hepatopancreas of M.japonicus reared under low-salinity stress for 6,12,24,48,and 96 h was analyzed and the results were compared with that of the control group using transcriptomics.After removing reads containing adapters,88890960-1051300444 clean reads were generated from 10 libraries in the control group and experimental group.Compared with the control group,811,589,1095,745,and 875 diff erentially expressed genes were obtained in the five treatment groups.The N50 and N90 lengths of the transcripts were 1746 bp and 436 bp,respectively.The top 20 gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways associated with the diff erentially expressed genes were related mainly to osmotic regulation(ion exchange,lipid metabolism and carbohydrate metabolism),immune regulation(cellular and humoral immunity),chitin metabolism,and related functions.The diff erential expression patterns of nine randomly selected genes were confi rmed by quantitative real-time PCR.This is the fi rst report of osmotic regulation-related genes that are diff erentially expressed under low-salinity stress in the hepatopancreas of M.japonicus.Furthermore,we found that M.japonicus initiated its own immune regulation under low-salinity stress.These results will help elucidating the mechanism of osmotic regulation and immune responses in this shrimp species.

日本对虾(Penaeus japonicus)血细胞的培养

在日本对虾血细胞原代培养的基础上 ,测定培养细胞的RNA/DNA值进行不同 pH值培养基的比较 ,同时利用倒置显微镜和透射电子显微镜对血细胞的形态和结构进行观察 .发现血细胞于 pH 7.2时生长最好 .

日本对虾(Penaeus japonicus)病原需钠弧菌(Vibrio natriegens)表型与分子特征及LAMP检测方法的建立

采用常规表观生物学特性及16S rRNA、gyrB及rpoA基因同源性检索与系统发育学分析等方法,对分离自江苏连云港某育苗场的大批死亡日本对虾蚤状幼体的优势生长菌进行了综合鉴定。结果表明,其形态和生理生化特征与弧菌属的需钠弧菌相近;16S rRNA、gyrB及rpoA基因同源性检索也均与需钠弧菌相似性最高,分别为98%、89%和95%,且三种基因的NJ系统发育树也均与需钠弧菌聚为一个分支;分离菌的致病性试验表明其半数致死量LD50为1.8×106CFU/ml。综合分离菌的致病性、形态与生理生化特征及基因同源性与系统发育分析结果,认为引起日本对虾蚤状幼体大批死亡的病原为需钠弧菌。基于gyrB基因序列设计1套LAMP特异性引物,建立了需钠弧菌的快速特异性检测方法,可用于由需钠弧菌引起的水产动物疾病的诊断及分子流行病学的调查研究。

低压静电场处理对竹节虾(Penaeus japonicus)微冻贮藏过程中品质的影响

为探明低压静电场对舟山竹节虾保鲜过程中品质的影响,以舟山竹节虾为研究对象,以-4℃微冻贮藏的竹节虾为对照组,以静电场(3000 V、50 Hz)-4℃微冻贮藏的竹节虾为实验组,进行对照试验。通过对0~28 d内竹节虾冰点、pH、汁液流失率、挥发性盐基总氮(TVB-N)、菌落总数(TVC)、硫代巴比妥酸值(TBA)、质构分析(TPA)等理化指标的测定与组织微观结构分析,探明低压静电场对舟山竹节虾贮藏保鲜过程中品质的影响。研究结果表明,未经电场处理的对照组竹节虾贮藏至28 d时,pH波动明显,汁液损失严重,TVB-N值达到30.42 mg/100 g,TVC值增长到了7.06 lg(CFU/g),TBA值高达0.82 mg/kg,肌肉纤维之间产生了明显断裂和无规律扩散,此时竹节虾处于不可食用状态;而经过电场处理的实验组竹节虾品质较好,pH波动范围≤0.04,竹节虾汁液流失率仅为5.01%,TVB-N、TVC值远低于对照组,脂肪氧化程度显著低于对照组(P<0.05),硬度和弹性下降缓慢,经H&E染色亦发现微观肌间结构完整,缝隙数量很少,无明显冰晶,竹节虾处于可食状态。实验结果表明低压静电场的添加可以有效延缓竹节虾贮藏过程中的品质变化,该项技术成果对改变传统水产品的保鲜方式具有重要的指导意义。

日本对虾(Penaeus japonicus)性腺差异表达基因GD13之cDNA5’端克隆

采用基于SMART(SwitchingMechanismAt5'endofRNATranscript)原理的cDNA末端快速扩增技术(RapidAmpli ficationofcDNAends:RACE),对日本对虾性腺差异表达基因cDNA5'端进行克隆,即在逆转录过程中通过SMARTⅡoligo的介导,在cDNA第1条链的3'端接上接头,然后根据已知序列设计的基因特异性引物(GSP1和GSP2)和接头引物(UPM和NUP)进行Nested PCR特异性扩增,并对特异产物进行克隆和序列分析,最终获得全长cDNA序列.测序结果表明,在卵巢内表达量高于精巢的GD13基因全长为656bp,编码164氨基酸,经NCBI检索,此基因与文昌鱼、家鼠的核糖体蛋白L24及黄牛的核糖体蛋白L30氨基酸序列同源性分别高达58%、56%和56%,可断定该基因为编码日本对虾核糖体蛋白L24亚基的基因.为进一步认识对虾生长发育和繁殖的调控机制奠定基础.