Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation

To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis (F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection. (C) 2010 Elsevier Ltd. All rights reserved.

Comparative growth and viability of hybrids between two populations of Chinese shrimp (Fennropenaeus chinensis)

Fenneropenaeus chinensis is confined to the Yellow Sea and Bohai Sea in China and the West Coast of the Korean Peninsula. Intra- and intercross populations were produced between Rushany (YP) and Korean (KN) populations. Seven traits were recorded. The heterosis of hybrids was computed and comparison between treatments was performed by ANOVA. At the fourth month after post-larvae, six indexes of growth trait and viability showed a range of heterosis, ranging from 0.514% to 14.950%. At the fifth month after post-larvae, six indexes of growth trait and viability ranged from -9.000% to 19.090%. The negative heterosis was observed in CL, HST and viability. The heterosis of KN female xYP male tended generally to increase as the age of the Chinese shrimp increased while the heterosis of YP female xKN male tended to decrease. The results indicated that the viability of reciprocal hybrids were not significantly different (P > 0.05) from their parents during the experiment. The result of ANOVA indicated that the F1 hybrids were significantly different (P < 0.05) in WST and TW at the fourth month. The multiple comparisons of LSD test indicated that the hybrids of YP female xKN male were significantly different (P < 0.05) from their parents in TW. The hybrids of YP female xKN male were significantly different (P < 0.05) from the other three combinations in WST. At the fifth months, the F1 hybrids had significant difference (P < 0.05) in body weight while other traits showed no significant differences (P > 0.05) from their parents. The multiple comparisons of LSD test indicated that the hybrids of KN female xYP male were significantly different (P < 0.05) from the KN parents in TW. The results indicate that in experimental conditions, the F-1 hybrids created from two populations of Chinese shrimp showed a certain level of heterosis for growth performance and viability. The crossing scheme may improve growth performance and viability in Chinese shrimp, but the improvement may be limited because effective crossbreeding requires the maintenance of pure, preferably inbred, lines and possibly involves specialized sire and dam lines. Therefore, the exploitation of heterosis through single crossbreeding in Chinese shrimp is of limited utility in practical commercial shrimp aquaculture in spite of the potential of significant heterosis. The crossbreeding of different populations can be applied in the establishment of base populations.

Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH2-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.

Evaluation of induced triploid shrimp Penaeus (Fenneropenaeus) chinensis cultured under laboratory conditions

Triploid Penaeus (Fenneropenaeus) chinensis was successfully produced by heat shock. Their metamorphosis, the relationship between body weight and length and difference in appearance between triploids and their diploid siblings under laboratory culture were studied. Hematological studies showed a smaller number of haemocytes, but larger cell volume, in triploids than in diploids. Triploid shrimp did not show higher growth during the immature stage, but exhibited superior growth during the maturation stage. Characteristics of reproductive organs indicated that triploid shrimp may be sterile and sex ratio can be changed through triploidization of shrimp. This paper summarizes the progress made in triploid shrimp research which would be helpful in understanding more about triploids of crustaceans. (c) 2006 Elsevier B.V. All rights reserved.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis

Ferritin, the iron storage protein, plays a key role in iron metabolism. A cDNA encoding ferritin (FcFer) was cloned from hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The predicted protein contains 170 amino acid residues with a predicted molecular weight (MW) about 19, 422.89 Da and theoretical isoelectric point (PI) of 4.73. Amino acid alignment of FcFer revealed 97% homology with Litopenaeus vannamei ferritin. Results of the RT-PCR showed that the expression of FcFer mRNA was up-regulated after shrimp was challenged with either white spot syndrome virus (WSSV) or heavy metal ions (Zn2+ and Cu2+) in the laboratory. A fusion protein containing FcFer was produced and the purified recombinant protein exhibited similar function of iron uptake in vitro. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments (-DDVALPGFAK- and -LLEDEYLEEQVDS1KK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei ferritin. The recombinant FcFer protein will be proved useful for study on the structure and function of ferritin in F chinensis. (c) 2006 Elsevier B.V. All rights reserved.

Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis

A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fenneropenaeus chinensis by 3' and 5' RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12.3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus (Litopenaeas) setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary electrophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.

中国对虾Penaeus Chinensis Osbeck卵母细胞发育的初步研究

本文描述了中国对虾Panaeus chinensis (Osbeck)卵母细胞的发育及其内部结构变化和卵巢的周年变化。根据卵母细胞的大小，核及核仁的形态，以及卵黄粒的有无，皮质棒的出现及卵母细胞与滤泡细胞的关系，将中国对虾卵母细胞的发育分为七个主要时期：（1）中央核仁期，（2）核仁染色质期，（3）周边核仁期，（4）滤泡期，（5）卵黄粒期，（6）皮质特化期和（7）长成期。卵原细胞含有酸性粘多糖。从滤泡期始，卵母细胞核外周出现中性粘多糖，并逐渐充满细胞质。根据卵母细胞的发育，内部结构及卵巢的周年变化特征，将卵巢发育分为五期：卵黄未发生期，卵黄发生早期，卵黄发生后期，皮质棒期和产后期。本文还讨论了卵黄的形成，卵巢发育的分期及亲虾产卵少，即孵化率低等问题。

中国对虾 Penaeus chinensis 血细胞及细胞免疫的研究

无脊椎动物免疫是一个新兴的研究领域。本文研究了中国对虾的血细胞形态，亚显微结构以及定量测定细胞吞噬活性的方法。中国对虾血细胞分三种：透明细胞，呈现球形或梭形，内部不含颗粒，半颗粒细胞及颗粒细胞卵球形椭球形，内部含有电子致密颗粒，颗粒又分均匀和非均匀颗粒。半颗粒细胞富含线粒体，可能与它对异物敏感易发生胞吐作用有关，半颗粒细胞是识别外源异物的关键细胞。可以观察到高尔基器，它可能与酶原颗粒的形成有关。姬姆萨染色细胞为紫红色，细胞核与颗粒染色较深，呈现蓝紫色与人白细胞中的粒细胞染色结果相近，这或许与两者功能相似有关。应用医学上常用的化学发光法（CL）定量测定中国对虾血细胞的吞噬活性，加入脂多糖及弧菌悬液的血细胞的发光值较只另菌液未加脂多糖的血细胞和对照组的发光值高。实验表明，脂多糖（<10μg/ml）对中国对虾血细胞的吞噬活性具有激活加强作用，而酵母聚糖不能引发血细胞的吞噬作用。上述测定方法可为监测对虾免疫机能的强弱提供定量指标，在对虾养殖生产实战中具有潜在的应用前景。

Ecdysteroid-responsive genes: E75 and Retinoid X Receptor (RXR), essential molt-regulating genes in the shrimp, Fenneropenaeus chinensis

本论文主要研究两种重要的调节蜕皮过程的基因—蜕皮激素效应基因E75和RXR在中国明对虾蜕皮中的作用。利用RT-PCR和RACE技术获得了编码FcE75和FcRXR的全长cDNA序列。FcRXR包含7个内含子，在对虾中存在不同的异形体，命名为RXR-1和RXR-2。应用荧光实时定量PCR分析表明FcE75和FcRXR基因在中国明对虾蜕皮前期（D3）其转录表达量明显上调。另外，FcE75和FcRXR基因在不同组织中的转录表达存在明显的差异。利用FcE75和FcRXR基因的双链RNA注射对虾能有效降低FcE75和FcRXR的表达水平。FcE75和FcRXR的体内沉默完全抑制了对虾的蜕皮过程，并且引起对虾的死亡。对不能正常蜕皮个体进行观察的结果表明，FcE75沉默的对虾，其上皮的收缩、新的刚毛及新表皮的形成均收到限制。在FcE75双链RNA沉默后的对虾中，我们检测了与蜕皮相关的一些效应因子，如chitinase等的转录，发现这些效应因子的转录明显受到抑制，说明FcE75和FcRXR在蜕皮过程中起到非常重要的作用。本论文首次阐明了这些基因在十足目甲壳动物蜕皮过程中的功能。

中国明对虾（Fenneropenaeus chinensis）几种免疫相关因子的分离纯化、基因克隆及相互关系研究

中国明对虾（Fenneropenaeus chinensis）是我国重要的海水养殖动物，但是近年来在其养殖过程中深受病害的影响，细菌性和病毒性疾病给对虾养殖业造成了巨大的经济损失，因此对中国明对虾免疫机理的研究对于对虾的疾病控制以及海水养殖业的持续发展具有重要意义。甲壳动物只具有天然免疫系统。天然免疫系统对于异己物质的识别是通过有限数量的模式识别蛋白（PRPs）来实现的。C型凝集素（C-type lectin）作为宿主防御系统中的一种模式识别受体，在病原识别和细胞间的相互作用上具有重要作用，此外它们还参与止血、凝固、物质运输和创伤修复等一系列作用；酚氧化酶（phenoloxidase）又称为酪氨酸酶(tyrosinase,EC1.14.18.1)。它是一种含铜的酶,能够催化单酚羟化成二酚(如多巴)，把二酚氧化成醌；醌在非酶促条件下形成最终的反应产物黑色素。血蓝蛋白(hemocyanins，Hc)是在软体动物和节肢动物血淋巴中发现的存在于细胞外、负责运输氧的一类巨大蛋白质。本论文从中国明对虾血清中纯化了利用亲和层析纯化了一种凝集素FC-L，并对FC-L的理化性质、多糖结合和细菌结合特性进行了研究。从中国明对虾血细胞中克隆了一种酚氧化酶原(prophenoloxidase,proPO)基因FCproPO，从肝胰腺中克隆了一种血蓝蛋白基因FCHc，分析了它们的分子结构特征，预测了其可能的作用，并对它们的组织分布、应答不同病原感染的表达变化模式以及相互关系进行了研究。

中国对虾(Penaeus chinensis) 与鹰爪虾(Trachypenaeus curvirostris)生殖生物学及早期发育的比较研究

本文主要对中国对虾及鹰的生殖生物学及早期胚胎的发育进行了较为系统的比较研究。在前人工作的基础上对中国对虾及鹰爪虾的精子及精荚形成，精子的超微结构，纳精囊的形态及结构，卵子的激活，受精卵的卵裂等进行了比较研究。使用激光扫描共聚焦显微镜对中国对虾精管的结构及精子的形成过程进行研究。中国对虾的输精管可分为近端、中段、远端输精管及壶腹四部分。输精管壁的本结构相似，分泌细胞的结构及分泌活动有较大的差别。精管自中段膨大部开始管腔逐渐被一隔膜分为大小不等的两部分，较大的腔内充满了很多精子，输精管较小的腔内充满胶体状物质。中国对精子细胞在精巢内产生，在两条输精管内渐发育成熟，经过细胞质的变化并与精子外部进行物质交换，形成顶体、亚顶体、棘突。核的变化则经历了染色质的解凝和核膜的消失，最后形成成熟的精子。对鹰爪虾精子的形成过程及精子的形态结构特征进行，结果表明鹰爪虾精子细胞产生于精巢的生精小管，在输精管内进一步发育，逐渐形成成熟的精子，许多精子在输精管内聚集成团外包胶质膜，形成许多大小不一的精子囊。精子由体 及棘突两部分组成，入海水中不运动。电镜下可见体部包括核区及细胞质区两部分。核区位于精子中部，核内由均质解凝的细胞核，外包一薄层细胞质带。细胞质区比核区略小，末端变尖，细胞质区内含有大量囊泡及膜层体。棘突从体部前端斜向伸出，由许多纤丝平行紧密排列组成，外包质膜。雄虾精子囊中的精子与已交配雌虾纳精囊内的精子在形态结构上没有明显差别。对中国对虾及鹰爪虾的纳精囊及精子的贮存特征进行研究。中国对虾及鹰爪虾的纳精囊都是封闭型纳精囊，中国对虾的纳精囊腔，表面为两个对称的半圆形甲壳版所覆盖，囊内由前向后伸出三个锥状突起。鹰爪纳精表面为前后两个甲壳版所覆盖。纳精囊中部为单一的腔，向后部两侧呈管状延伸，末端膨大形成两个袋状囊，内部贮存精子囊。纳精囊从功能上分为三个部分，即纳精囊中部的开口，交配时为胶体物质所充塞；内部的囊腔为贮存精子的部位；前端的开口为产卵时精释放口。中国对虾及鹰卵子具有相似的激活和卵裂过程。卵子可以不受精子的激活，只受海水刺激即可发生皮层反应；先排出皮质棒或皮质囊泡，形成胶质层，进而释放出皮层颗粒，形成孵化膜。中国对虾的孵化膜在卵子表面形成以后再举起；鹰爪虾的孵化膜远离卵子表面形成，然后进一步举起，鹰爪虾卵周隙较大。发生皮层反应的过程中，减数分裂重新启动，排出第一极体和第二极体。中国对虾与鹰爪虾卵子的皮层反应和减数分裂都可由海水刺激而进行，与受精与否无关，但未受精卵不能分裂或公分裂为大小不等的子细胞。中国对虾与鹰爪虾受精卵都具有原始的螺旋卵裂的特性。对海水中的Mg~(2+)、 Ca~(2+)、 K~+及胰蛋白酶和胰蛋白酶抑制剂对中国对虾卵子激活的影响进行研究。结果表明海水中的Mg~(2+)对中国虾卵子的激活是必须的，Ca~(2+)、 K~+是非必须的，而且它们对受精卵和非受精卵都具有相同的作用。胰蛋白酶（0．1％）在卵子产出后可以破坏胶质层及孵化膜的形成并影响受精卵的正常卵裂。胰蛋白酶抑制剂（0．01％）可以完全抑制卵子的激活及受精，也可以使发生皮层反应的卵子处于停滞状态。卵子产出后30min胰蛋白酶及胰蛋白酶抑制剂对卵子失去作用，表明胶膜及孵化膜完全形成后具有抵抗胰蛋白酶及胰蛋白酶抑制剂的作用。

Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection

The nitric oxide synthase (NOS) activity in the haemocytes of shrimps Fenneropenaeus chinensis (Osbeck) and Marsupenaeus japonicus (Bate) was Studied after white spot syndrome virus (WSSV) infection to determine its characteristics in response to virus infection. First, the NOS activity in haemocytes of shrimps was determined by the means of NBT reduction and changes in cell conformation. And the variations of NOS activity in shrimps after challenge with WSSV intramuscularly were evaluated through the analysis Of L-citrulline and total nitrite/nitrate (both as NO derivates) concentrations. The result showed that NOS activity in the haemocytes of F chinensis increased slightly from 0 to 12 h postchallenge, indicated by the variations Of L-Citrulline (from 11.15 +/- 0.10 to 12.08 +/- 0.64 mu M) and total nitrite/nitrate concentrations (from 10.45 +/- 0.65 to 12.67 +/- 0.52 mu M). Then it decreased sharply till the end of the experiment (84 h postchallenge), the concentrations Of L-Citrulline and total nitrite/nitrate at 84 It were 1.58 +/- 0.24 and 2.69 +/- 0.70 mu M, respectively. The LPS-stimulated NOS activity kept constant during the experiment. However, in M. japonicus, the NOS activity kept increasing during the first 72 It postchallenge, the concentrations Of L-Citrulline and total nitrite/nitrate increased from 7.82 +/- 0.77 at 0 h to 10.79 +/- 0.50 mu M at 72 h, and from 8.98 +/- 0.43 at 0 h to 11.20 +/- 0.37 mu M at 72 h, respectively. Then it decreased till the end of the experiment (216 h postchallenge), and the concentrations of L-Citrulline and total nitrite/nitrate at 216 h were 5.66 +/- 0.27 and 4.68 +/- 0.16 mu M, respectively. More importantly, an apparent increase of I-PS-stimulated NOS activity was observed in M japonicus at 48 h postchallenge, which was about 4 times higher than that in the control group of health shrimps. In correspondence with the difference of NOS activity between the two species of shrimps, the Cumulative mortalities of the shrimps were also different. All shrimps of F. chinensis in the mortality experiment died in 66 h, much more quickly than M. japonicus, Whose accumulative mortality reached 100% after 240 h. Data here reported let us hypothesize that NOS activity in the haemocytes of shrimps F chinensis and M. japonicus responses to WSSV infection differently, and this might be one of the reasons for the different susceptibility of F chinensis and M. japonicus to WSSV infection. (c) 2005 Elsevier Inc. All rights reserved.

The effect of acute salinity change on white spot syndrome (WSS) outbreaks in Fenneropenaeus chinensis

In order to observe the effect of salinity on disease resistance and white spot syndrome virus (WSSV) proliferation in Fenneropenaeus chinensis, shrimps with latent WSSV were subjected to two acute salinity changes from the original salinity of 22 ppt to 18 and 14 ppt in an hour, respectively. The total haemocyte count (THC) of the challenged group showed no evident change under salinity adjustments, but the phenoloxidase (PO) index declined significantly (P<0.05) corresponding to continuing acute salinity changes from the 24th to the 72nd hour. According to the WSSV load detected by quantitative real-time PCR method, it was found that WSSV carried by the challenged group and control group were significantly different (P<0.05); acute salinity change from 22 to 14 ppt led to the WSSV carried in the challenged group being significantly higher (P<0.05) than that of those surviving in 22 ppt, but salinity change from 22 to 18 ppt had no such effect. At the end of the 72-h experiment, the challenged group subjected to salinity change from 22 to 14 ppt had nearly 3 times the WSSV load as the control group with no salinity change. Therefore, salinity changes over a particular range could result in a decrease of immunocompetence and obvious WSSV proliferation in the shrimps, leading to white spot syndrome developing from a latent infection to an acute outbreak. (C) 2005 Elsevier B.V All rights reserved.

Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis

A natural lectin from the plasma of the shrimp Fenneropenaeus chinensis was purified by singlestep affinity chromatography using fetuin-coupled agarose. The purified plasma lectin showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC and chicken RBC. The hemagglutinating (HA) activity of the lectin was dependent on Ca2+ and reversibly sensitive to EDTA. This lectin was named FC-L and its inactive form had a molecular mass estimate of 168 kDa. Fifteen N-terminal amino acid sequences of this protein were determined. We performed HA-inhibition assays with several carbohydrates and glycoproteins. FC-L showed a distinct and unique specificity to N-acetylated sugars, particularly sialic acid and sialoproteins. The FC-L also has binding activity to some Gram-negative bacteria which caused disease in shrimp and fish. The activity of FC-L was inhibited at temperatures greater than 75 degrees C and at a pH less than 7 or greater than 11. These results suggest that FC-L may play a role as pattern recognition proteins in the reorganization and clearance of invaders in shrimp F. chinensis. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.