两种沼虾溶菌酶基因ORF的克隆和罗氏沼虾溶菌酶基因的组织表达(英文)

分别提取罗氏沼虾和日本沼虾血细胞总RNA,RT-PCR扩增获得特异性cDNA片段,纯化后克隆到T载体上。序列测定表明所克隆的两种沼虾溶菌酶基因的开放阅读框(ORF)为477bp,共编码158个氨基酸,包括溶菌酶成熟肽140个氨基酸残基和信号肽18个氨基酸残基。同源性分析表明,罗氏沼虾和日本沼虾溶菌酶基因的碱基序列及推测氨基酸序列高度同源,分别为99.4%和98.1%。两种沼虾溶菌酶基因的碱基序列和推测氨基酸序列与Gen-Bank上其他对虾溶菌酶的同源性达83.0%和80.0%以上。两种沼虾溶菌酶都具有c-型溶菌酶典型的两个酶活性位点(Glu51)和(Asp68),以及8个保守结构氨基酸残基Cys,且在101、106和107位上缺少Asp,因而推测本实验所克隆的两种沼虾溶菌酶基因属c-型溶菌酶基因的非钙结合亚型。以PCR法制备罗氏沼虾溶菌酶基因的生物素标记探针,斑点杂交检测感染弧菌后溶菌酶基因mRNA在各组织中的转录水平,结果表明受感染6h后在眼、肌肉、鳃、肝胰腺、肠管中的表达量均有升高,其中在肝胰腺中的表达量最高,约为对照组的560%。在不同感染时间里,肝胰腺中该基因表达量有较大的变化:感染后3h表达量最低,24h后表达量升至最高,大约为对照组的430%,48h时的表达量又有所下降,但仍明显高于对照组(约为330%)。受弧菌感染后罗氏沼虾溶菌酶基因转录的上调证明溶菌酶基因在非特异性免疫中的直接作用,同时表明肝胰腺可能在沼虾的免疫防御过程起重要作用。       

三丁基锡(TBT)对罗氏沼虾的毒性效应

将罗氏沼虾(Macrobrachium rosenbergii)幼虾和成虾暴露在不同浓度的三丁基锡(tributyhin,TBT)溶液中,进行急性和慢性毒性实验,通过组织病理学观察研究TBT对罗氏沼虾的毒性效应。结果表明:TBT对罗氏沼虾幼虾和成虾的96h半致死浓度(96h LC50)分别为51和376μg.L-1,安全浓度(SC)分别为5.1和37.6μg.L-1;罗氏沼虾幼虾暴露在浓度分别为1.25、2.5和5μg.L-1的TBT溶液中30d后,其体重和体长均极显著低于对照组(P<0.05);罗氏沼虾成虾暴露在浓度分别为2、8和32μg.L-1的TBT溶液中30d后,生长速度虽未见明显差异,但鳃和肝胰脏组织结构均出现异常;TBT浓度高于8μg.L-1时,处理组鳃上皮细胞肿胀,支持细胞中的细胞核萎缩,鳃血窦中血细胞聚集,肝胰脏细胞肿大,出现空泡化,细胞中出现细小颗粒;TBT浓度达32μg.L-1时,罗氏沼虾肝胰脏细胞空泡化程度严重,细胞核消失,部分细胞解体并出现坏死区;表明一定剂量的TBT对罗氏沼虾的鳃和肝胰脏有明显的毒性效应。     

罗氏沼虾与克氏原螯虾血细胞的比较研究

对罗氏沼虾与克氏原螫虾血细胞的分类与组成进行了染色观察比较研究。根据染色后光镜下血细胞的核质比、颗粒的大小和数量等来对血细胞进行分类,罗氏沼虾与克氏原螯虾的血细胞均可分为透明细胞、半颗粒细胞和颗粒细胞三类;其血细胞浓度分别为1.02±0.21×10⁷ Ind·ml^-1和0.85±0.15×10⁷Ind·ml^-1。2种虾血细胞的颗粒形态存在显著差异;透明细胞、半颗粒细胞和颗粒细胞占血细胞总量的百分比在罗氏沼虾为21.3±6.3%,45.7±2.5%,33.0±6.8%:在克氏原螯虾为12.0±5.8%、49.5±5.1%和38.5±9.5%。     

亚硝酸盐胁迫对罗氏沼虾血细胞及其抗氧化酶活力的影响

【背景】亚硝酸盐是虾类集约化养殖过程中最常见的毒性污染物之一,研究亚硝酸盐胁迫对罗氏沼虾血细胞的毒性以及抗氧化酶在抗胁迫防御中的作用,能够为罗氏沼虾养殖过程中的亚硝酸盐中毒防治提供理论参考。【方法】以不同浓度(0、1、5和10 mg·L~(-1))的亚硝态氮(NO_2~--N)对罗氏沼虾进行胁迫,于胁迫后的0、6、12、24和48 h取样,应用流式细胞术检测血细胞活性氧(ROS)含量和细胞凋亡率,同时测定血细胞总数(THC)和胞内抗氧化酶活力。【结果】1 mg·L~(-1)NO_2~--N在48 h内对血细胞ROS含量、凋亡率和THC均无显著影响。5 mg·L~(-1)NO_2~--N胁迫24 h,血细胞ROS含量显著上升,THC显著下降,胁迫48 h凋亡率显著提高。10 mg·L~(-1)NO_2~--N胁迫6 h,血细胞ROS含量和凋亡率均显著上升,胁迫12 h THC显著下降。血细胞的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPx)的活力均不同程度地被NO_2~--N胁迫所诱导,CAT活力主要在胁迫前期提高,而GPx活力在胁迫后期提高。【结论与意义】亚硝酸盐存在浓度和时间毒性效应,一定浓度的亚硝酸盐会诱导虾血细胞产生ROS,这些ROS的过量产生诱导了血细胞发生凋亡,继而导致THC下降,这一氧化胁迫过程可能是亚硝酸盐对罗氏沼虾产生细胞毒性的重要机制之一。抗氧化酶活力的诱导表明抗氧化酶在亚硝酸盐胁迫过程中发挥防御作用。     

罗氏沼虾缅甸野生原种rDNA-ITS区序列特征

对罗氏沼虾(Macrobrachium rosenbergii)内转录间隔区(internal transcribed spacer,ITS)全序列特征进行了分析.罗氏沼虾ITS1长度为1 070～1 150 bp,GC含量为51.4%～52.7%;5.8S长度一致为163bp,GC含量为55.2%;ITS2长度为48...     

三种沼虾的COI基因序列变异及其分类地位探讨(英文)

罗氏沼虾(Macrobrachium rosenbergii)和日本沼虾(M.nipponense)经在我国得到广泛的养殖,产生巨大的经济效益.祁连沼虾(M. qilianensis)是自然分布在我国甘肃省的土著虾种,因其外部形态符合沼虾属的特征,而被前人归入沼虾属.为了从分子生物学的角度理解罗氏沼虾、日本沼虾与祁连沼虾的遗传差异,为合理开发和利用沼虾资源提供理论基础,作者对这3种沼虾的线粒体COI基因序列进行研究.从甘肃、浙江等地分别采集这三种沼虾的样本各10尾,共30尾,其中祁连沼虾是野生样本,而罗氏沼虾和日本沼虾都是养殖样本.通过PCR方法扩增线粒体COI基因,并测序.通过比对,获得一致序列649 bp.在30个样本中共检测到169个变异位点,占总变异的26.04%;共检测到7种单倍型.3种沼虾的核苷酸多态性分别为:罗氏沼虾0.411%、日本沼虾0.092%、祁连沼虾0.031%.野生的祁连沼虾遗传多样性远远低于养殖的罗氏沼虾和日本沼虾.三种沼虾单倍型之间的Kimura双参数遗传距离在19.87%～23.84%,三者之间的遗传距离较大,提示三者均为有效种.为进一步确定这三种沼虾在长臂虾科的分类地位, 我们从NCBI数据库中下载了长臂虾科的其它种类的COI序列进行系统发生分析.用NJ法构建的分子系统树显示:日本沼虾和罗氏沼虾与沼虾属的其它种类聚成一枝,而祁连沼虾与同亚科的脊尾白虾(Exopalaemon carinicauda)和长角长臂虾(Palaemon debilis)较沼虾属另10种虾的遗传距离近,即祁连沼虾与白虾属及长臂虾属聚成另一枝.凶此,COI序列的结果不支持祁连沼虾归入沼虾属.但其分类地位应该综合多方面证据重新进行分析确定.     

复方中草药对罗氏沼虾碱性磷酸酶（AKP）和血清蛋白的影响

为了探究中草药添加剂对罗氏沼虾(Macrobrachium rosenbergii)免疫功能的影响,以罗氏沼虾为对象,在水温20～30℃、pH 7～8的试验条件下,往饲料中分别添加质量分数为0%、1.0%、1.5%、2.0%、2.5%、3.0%的复方中草药制剂,连续饲喂25 d。结果显示:随着饲料中复方中草药质量分数的提高,罗氏沼虾的生长指标、血清中碱性磷酸酶(AKP)活性、血清蛋白含量均呈现先上升后下降的趋势,试验组相较于对照组有很大提高;中草药质量分数的提高引起的血清中蛋白含量的提高远远大于肌肉中蛋白的含量,在各试验组中2.5%的复合中草药制剂的添加量效果最好。试验证明,添加复方中草药制剂对罗氏沼虾的免疫活性、生长性能均有明显的促进作用。该结果能够为罗氏沼虾的健康养殖提供基础资料。     

三种沼虾的COI基因序列变异及其分类地位探讨

罗氏沼虾(Macrobrachium rosenbergii)和日本沼虾(M. nipponense)已经在我国得到广泛的养殖,产生巨大的经济效益。祁连沼虾（M. qilianensis）是自然分布在我国甘肃省的土著虾种,因其外部形态符合沼虾属的特征,而被前人归入沼虾属。为了从分子生物学的角度理解罗氏沼虾、日本沼虾与祁连沼虾的遗传差异,为合理开发和利用沼虾资源提供理论基础,作者对这3种沼虾的线粒体COI基因序列进行研究。从甘肃、浙江等地分别采集这三种沼虾的样本各10尾,共30尾,其中祁连沼虾是野生样本,而罗氏沼虾和日本沼虾都是养殖样本。通过PCR方法扩增线粒体COI基因,并测序。通过比对,获得一致序列649 bp。在30个样本中共检测到169个变异位点,占总变异的26.04%;共检测到7种单倍型。 3种沼虾的核苷酸多态性分别为：罗氏沼虾0.411%、日本沼虾0.092%、祁连沼虾0.031%。野生的祁连沼虾遗传多样性远远低于养殖的罗氏沼虾和日本沼虾。三种沼虾单倍型之间的Kimura双参数遗传距离在19.87%~23.84%,三者之间的遗传距离较大,提示三者均为有效种。为进一步确定这三种沼虾在长臂虾科的分类地位, 我们从NCBI数据库中下载了长臂虾科的其它种类的COI序列进行系统发生分析。用NJ法构建的分子系统树显示：日本沼虾和罗氏沼虾与沼虾属的其它种类聚成一枝,而祁连沼虾与同亚科的脊尾白虾（Exopalaemon carinicauda）和长角长臂虾（Palaemon debilis）较沼虾属另10种虾的遗传距离近,即祁连沼虾与白虾属及长臂虾属聚成另一枝。因此,COI序列的结果不支持祁连沼虾归入沼虾属。但其分类地位应该综合多方面证据重新进行分析确定。     

罗氏沼虾Rab11基因cDNA克隆及其组织表达分析

为了发掘罗氏沼虾(Macrobrachium rosenbergii)免疫相关基因, 研究Rab蛋白(Ras-related proteins inbrain)在罗氏沼虾免疫应答中发挥的作用, 研究采用RACE-PCR技术克隆了罗氏沼虾Rab11基因全长cDNA序列, 记为MrRab11。全长1381 bp, 包括226 bp的5'UTR, 511 bp的3'UTR和645 bp的开放阅读框, 编码214个氨基酸, 含有一个Rab结构域。氨基酸序列比对显示, 罗氏沼虾与冈比亚按蚊(Anopheles gambiae)、蚤状溞(Daphniapulex)、叶蝉(Homalodisca vitripennis) Rab11一致性分别为82%、83%和82%。软件预测, MrRab11编码的蛋白分子量约为23.75 kD, 等电点约为5.34。实时荧光定量表达分析表明, MrRab11基因在罗氏沼虾各组织中都有表达, 肝胰腺中的表达量最高, 其次是肌肉和肠道。在阴沟肠杆菌(Enterobacter cloacae)感染12h后, 罗氏沼虾肝胰腺中MrRab11的表达量上升, 显著高于对照组(P0.05), 推测这是MrRab11对阴沟肠杆菌的应激表达,MrRab11在肝胰腺中参与了罗氏沼虾免疫应答过程。     

罗氏沼虾幼体精氨酸激酶基因的cDNA克隆及其在双顺反子病毒感染后的表达特征

为探求罗氏沼虾精氨酸激酶（Macrobrechium rosenbergii arginine kinase,MrAK）基因特征和与病毒感染的相关性,研究通过AK基因mRNA全长克隆,并采用QPCR检测病毒感染前后不同时间点罗氏沼虾幼体AK基因的转录差异。最终克隆得到的AK序列全长为1740 bp（GenBank:KT970484）,其开放阅读框包含1068个碱基,编码356个氨基酸;同源性分析显示MrAK基因编码区蛋白与多齿新米虾、南极磷虾、鲑鱼海虱和安氏伪镖水蚤的同源性分别为97%、82%、83%和79%;系统进化树分析表明,该基因与多齿新米虾的AK聚在同一分支簇,而蟹、对虾和螯虾聚在另一分支;氨基酸结构分析表明,其存在一个可能的ATP:胍基磷酸转移酶的活性位点（Cys286-Pro287-Thr288-Asn289-Leu290-Gly291-Thr292）;病毒感染罗氏沼虾幼体后,其AK基因表达在第9h开始出现显著性上调,在12h时达到峰值,随后开始降低。以上研究结果表明,AK基因在无脊椎甲壳动物中存在多样性,其蛋白结构存在保守性,并且其在罗氏沼虾病毒感染过程中起到潜在的能量供给调节作用。     

Hemocytes of the blowfly Calliphora vicina and their dynamics during larval development and metamorphosis

On the basis of in vitro observation of live cells and examination of stained slides of larval and prepupal Calliphora vicina hemolymph, seven types of hemocytes have been detected: prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes, and plasmatocytes I-IV, a. The last representing sequential stages of one cell line differentiation. Prohemocytes are basic cells, from which other forms of hemocytes derive outside the hemopoietic tissue, i.e. in free hemolymph. At the last larval instar, three waves of hemopoiesis occur. Either wave tends to increase the general number of cells and to change the quality of hemocyte population. The first wave occurs at the close of larva feeding and is accompanied by increase in the number of hyaline hemocytes, thrombocytoids and larval plasmatocytes. The second wave of hemopoiesis occurs after the larva's crop emptying. In this period the main increase of hemocyte population occurs at the expense of prohemocytes and plasmatocytes I. The most significant (five-fold) explosion of the population of free hemocytes takes place at the onset of pupariation and correlates with the rise of ecdysone titer. At the first stage of this peak, the amount of plasmatocytes I sharply increases. Further on these are rapidly differentiated into plasmatocytes II and III. After the puparium formation, hemocytes are reduced in number. Plasmatocytes III phagocytose fragments of destroyed larval tissues, pass to the stage of plasmatocytes IV (macrophages), and partially settle on tissues.     

Agglutinin-mediated phagocytosis-associated generation of superoxide anion and nitric oxide by the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii

Hemocyte mediated phagocytosis is one of the vital components of innate defence mechanisms in crustaceans and this phagocytic process is aided by serum agglutinins. However, literature on agglutinin mediated opsono-phagocytosis is unclear in the case of Macrobrachium rosenbergii hemocytes. Further, very few studies in the case of superoxide anion generation and none with regard to nitric oxide generation during phagocytosis exist among crustaceans. We investigated the occurrence of agglutinins in the serum and the role of serum agglutinins in mediating phagocytosis by the hemocytes. We show that the prawn serum possesses agglutinins that function as opsonins during phagocytosis of HB RBC by the hemocytes. Hemagglutination-inhibition assays revealed the specificity of serum agglutinins for N-acetylated hexoses, namely GalNAc, GlcNAc and ManNAc, with a higher affinity for ManNAc. In addition, ManNAc was able to inhibit the phagocytic response (by about 60%) of the hemocytes against serum pretreated HB RBC, wherein the serum was previously treated with ManNAc. We next investigated the ability of the hemocytes to generate superoxide anion and nitric oxide during HB RBC phagocytosis and results show generation of both these free radicals. In addition, there was an enhancement in generation (75% increase) of these free radicals during agglutinin mediated opsonophagocytosis, when compared to buffer treated targets and interestingly this enhanced generation was inhibited by ManNAc (27% for superoxide anion and 36% for nitric oxide), an inhibitory sugar for phagocytosis. Inhibition of phagocytosis induced superoxide anion generation by DPI (53%), sodium azide (56%) and tropolone (61%), reveals the possible involvement of NADPH-oxidases, peroxidases and probably phenoloxidases, respectively, in the generation of superoxide anion. Similarly, decrease in nitric oxide generation in the presence of l-NIO (47%) during phagocytosis lends support to the role of nitric oxide generation during cellular immune processes. These findings thus suggest a role for superoxide anion and nitric oxide in the innate defense mechanism, namely phagocytosis, in Macrobrachium rosenbergii.     

Hepatopancreas is the likely organ of vitellogenin synthesis in the freshwater prawn, Macrobrachium rosenbergii

The objective of the present study was to investigate the source of vitellogenin in the freshwater prawn, Macrobrachium rosenbergii. Ovarian development of M. rosenbergii was classified into five stages (stage I-V). Vitellin/vitellogenin was detected in the ovary and the hepatopancreas in different stages by native-PAGE and Western blotting. Two and three subunits of vitellin were observed in the ovary at the early- (I-II), mid- and late- (III-V) stages, respectively. The subunit of vitellogenin was not detected in the hepatopancreas at different stages of prawns. Hepatopancreas had positive immunocytological staining (against vitellin antibody) in different ovarian stages of prawn. Only vitellogenic oocyte but not previtellogenic oocytes and follicle cells had a positive immunocytological staining. Hepatopancreas could synthesize radiolabeled immunoreactive proteins after incubation with radiolabeled glycine on the basis of immunoprecipitation (against vitellin antiserum). Therefore, it is concluded that hepatopancreas is the most likely organ to synthesize vitellogenin in the freshwater prawn, M. rosenbergii.     

Sialylation is modulated through maturation in hemocytes from Macrobrachium rosenbergii

In this work we identified in adult and juvenile freshwater prawn, Macrobrachium rosenbergii, three major type of circulating hemocytes: fusiform; rounded; and large ovoid hemocytes. Rounded and large hemocytes represent the first defense line, since this type of cells exerts phagocytic activity as well as lectin synthesis. Considering that glycosylation plays important roles in cell communication and as a target for pathogenic microorganisms, in this report was also described the main glycosidic modifications that occur in the large and rounded hemocytes from the freshwater prawn during maturation as determined with lectins. Neu5Acalpha2,6Gal, was identified homogeneously distributed in the membrane in 90% of hemocytes from juvenile organisms. Maturation of the freshwater prawn induced a decrease or complete loss of Neu5Acalpha2,6Gal residues that were replaced with Neu5Acalpha2,3 molecules in practically all hemocytes from adult organisms. This change was paralleled by a diminution in 9-O-acetyl-neuraminic acid (Neu5,9Ac(2)) expression. T and Tn antigens (Galbetal,3 GalNAcalpha1-0-Ser/Thr or GalNAcalpha1-0-Ser/Thr, respectively), as well as N-glycosidically linked glycans, seem to be highly conserved throughout maturation. Our results show that sialylation of freshwater prawn hemocytes is modulated throughout the maturation process.     

Immune role of MrNFκBI-α, an IκB family member characterized in prawn M. rosenbergii

NF kappa B inhibitor alpha (MrNFκBI-α) was sequenced from a freshwater prawn Macrobrachium rosenbergii. The MrNFκBI-α protein contains a long ankyrin repeat region circular domain between 193 and 413 along with its 6 repeats (ankyrin repeat 1,2,3,4,5 and 6). An IκB degradation motif and a putative PEST motif is present at 37-64 and 418-471 of the N- and C-terminal regions of MrNFκBI-α respectively. The gene expressions of MrNFκBI-α in healthy and infectious hematopoietic and hypodermal necrosis virus (IHHNV), poly I:C, Aeromonas hydrophila and Enterococcus faecium injected M.?rosenbergii were examined using quantitative real time PCR. The MrNFκBI-α is expressed in all the tissues taken for examination and the highest is observed in hemocytes. The MrNFκBI-α gene expression is strongly up-regulated in hemocytes of prawn after IHHNV, poly I:C, A.?hydrophila and E.?faecium infection. This result indicates an important role of MrNFκBI-α in M.?rosenbergii immune system. This, however, remains to be verified by further studies.     

Responses of giant freshwater prawn (Macrobrachium rosenbergii) to challenge by two strains of Aeromonas spp

The virulence of two Aeromonas strains (A. veronii and A. caviae) isolated from the hepatopancreas of apparently healthy giant freshwater prawns (Macrobrachium rosenbergii) was compared using a challenge by injections. For the A. veronii strain, challenge with 3.7 x 10(5) cells/g of body weight led to 100% mortality; for the A. caviae strain, 3.8 x 10(6) cells/g produced 100% mortality. The 50% lethal doses (LD50) were 2.0 x 10(3) cells/g for A. veronii and 51.2 x 10(3) cells/g for A. caviae. Use of different culture media (trypticase soy broth vs prawn muscle extract) did not significantly affect the virulence of A. veronii. Injection of a sublethal dose (1 x 10(3) cells/g) of A. veronii led to a significant decrease in the total hemocyte count (THC) between 4 and 24 h after injection. Saline injections also caused a similar though less decrease in THC. In the first 24 h after injection of A. veronii (1 x 10(3) cells/g), the change in the percentages of granulocytes (both granular cells and semigranular cells) in the hemolymph was significantly different. After a significant initial increase, the percentage of hyaline cells fell by a factor of 4, from 9 to 2%. Phenoloxidase activity increased fourfold immediately after injection and returned to preinjection levels at 24 h.     

The ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) differentially affects cells mediating the immune response of its flesh fly host,Sarcophaga bullata Parker (Diptera: Sarcophagidae)

In this study, we examined cellular immune responses in the flesh fly, Sarcophaga bullata, when parasitized by the ectoparasitoid Nasonia vitripennis. In unparasitized, young pharate adults and third instar, wandering larvae of S. bullata, four main hemocyte types were identified by light microscopy: plasmatocytes, granular cells, oenocytoids, and pro-hemocytes. Parasitism of young pharate adults had a differential effect on host hemocytes; oenocytoids and pro-hemocytes appeared to be unaltered by parasitism, whereas adhesion and spreading behavior were completely inhibited in plasmatocytes and granular cells by 60 min after oviposition. The suppression of spreading behavior in granular cells lasted the duration of parasitism. Plasmatocytes were found to decline significantly during the first hour after parasitism and this drop was attributed to cell death. Melanization and clotting of host hemolymph did not occur in parasitized flies, or the onset of both events was retarded by several hours in comparison to unparasitized pharate adults. Hemocytes from envenomated flies were altered in nearly identical fashion to that observed for natural parasitism; the total number of circulating hemocytes declined sharply by 60 min post-envenomation, the number of plasmatocytes declined but not granular cells, and the ability of plasmatocytes and granular cells to spread when cultured in vitro was abolished within 1 h. As with parasitized hosts, the decrease in plasmatocytes was due to cell death, and inhibition of spreading lasted until the host died. Isolated crude venom also blocked adhesion and spreading of these hemocyte types in vitro. Thus, it appears that maternally derived venom disrupts host immune responses almost immediately following oviposition and the inhibition is permanent. The possibility that this ectoparasite disables host defenses to afford protection to feeding larvae and adult females is discussed.     

The effect of sugars and free amino acids from the freshwater prawn Macrobrachium rosenbergii hemolymph on lectin activity and on oxidative burst

We determined the effect of low molecular weight components (LMWC) from healthy juvenile and adult Macrobrachium rosenbergii hemolymph on lectin activity and oxidative burst (OB) in hemocytes. In an attempt to identify the LMWC that affect the lectin's hemagglutinating activity or oxidative burst, we determined the hemolymph carbohydrates and free amino acids (FAA) concentration. The LMWC (<2000 Da) were obtained after dialysis of the hemolymph. Our results showed that LMWC from juveniles exerted a greater inhibition on lectin than LMWC from adult hemolymph. Production of superoxide radicals by hemocytes was lower in the presence of juvenile (p<0.05) as compared to adult LMWC. FAA composition of the hemolymph and of LMWC from adults showed higher proportion of alanine (which corresponded to 25% of total FAA) and proline (>20%); whereas, in juveniles, the main FAA identified were glycine (>40%) and alanine (26%). N-Acetyl-D-glucosamine (GlcNAc) was the main sugar residue in the hemolymph and LMWC from juveniles; its concentration was 2.4 times higher than glucose (Glc), whereas, in adults, Glc was the main free sugar residue. Our results suggest that the proportion of FAA and carbohydrates in the hemolymph of M. rosenbergii seems to be correlated with the maturation process; furthermore, the high proportion of free GlcNAc and glycine regulate, in the juvenile stage, lectin activity and cellular oxidative mechanisms, respectively.     

Effect of hypoxia on the immune response of giant freshwater prawn Macrobrachium rosenbergii and its susceptibility to pathogen Enterococcus

Giant freshwater prawns Macrobrachium rosenbergii (14-19 g) were challenged with Enterococcus (3 x 10(5) cfu prawn(-1)) previously incubated in TSB medium for 24 h, then placed in water having concentrations of dissolved oxygen (DO) at 7.75, 4.75, 2.75 and 1.75 mg l(-1). Onset of mortality occurred after 6 h exposure to 1.75 mg l(-1) DO, and after 12 h exposure to 2.75 mg 1(-1) DO. Cumulative mortality of prawns at 1.75 mg l(-1) DO was significantly higher than that at 4.75 and 2.75 mg l(-1) DO, and cumulative mortality of prawns at 4.75 and 2.75 mg l(-1) DO was significantly higher than that at 7.75 mg l(-1) DO after 96 h. The prawns (20-30 g) which had been placed in water for 0 to 120 h at 7.75, 4.75, 2.75 and 1.75 mg l(-1) DO were examined for the THC (total haemocyte counts), DHC (differential haemocyte counts), phenoloxidase activity, respiratory burst, percentage phagocytosis and clearance efficiency. No significant difference in semi-granular cells and granular cells of prawns was observed among four treatments. The prawns following 120 h exposure to 2.75 mg l(-1) DO decreased significantly the hyaline cells and THC by 39% and 36%, respectively. Phenoloxidase activity and respiratory burst decreased significantly by 33% and 11% when the prawns were exposed to 2.75 mg l(-1) DO after 24 h, respectively. Percentage phagocytosis and clearance efficiency to Enterococcus decreased significantly by 44% and 54% for the prawns following 12 h exposure to 2.75 mg l(-1) DO, respectively. It is concluded that DO as low as 2.75 mg l(-1) and 4.75 mg l(-1) causes depression in immune system of M. rosenbergii, and increases its susceptibility to Enterococcus infection.     

对虾白斑综合征杆状病毒体内增殖模型的建立

应用对虾白斑综合征杆状病毒（WSSV）,对淡水克氏螯虾、罗氏沼虾、日本沼虾和两种淡水蟹（中华绒螯蟹、长江华溪蟹）进行人工感染实验。结果除淡水克氏螯虾之外,其它受试的虾蟹均不能感染WSSV。克氏螯虾3个不同剂量级感染至12d平均死亡率为94％。从发病或死亡个体采集血淋巴,经电镜负染色可观察到完整的病毒粒子,其形态大小、靶细胞组织病理均与从中国对虾中分离的WSSV相似或相同。同时,通过原位杂交技术进一步证明该实验的可靠性。克氏螯虾重复感染效果良好,有可能成为研究WSSV的一种理想的病毒体内增殖模型。