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。

南美白对虾（Penaeus vannamei Boone,1931)卵子质量的形态学评估

1）本文描述了南美白对虾正常的卵子形态及发生特点，以及受精卵和非受精卵的形态学鉴别方法。2）从生产的角度出发提出了评估卵子质量的五项指标：同批卵子规格的一致程度，卵子的沉降速度，孵化膜的抗变形能力，4－8细胞期的分裂特点，扩膜卵在同批卵中所占的比例。除此外，对于亲虾持续产卵与所产卵子质量的关系等也作了相应的探讨。

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。

中国对虾Penaeus orientalis 精子形态结构与生理生化功能的研究

本文报导中国对虾Penaeus orientalis成熟精子的亚显微结构，此工作是进一步研究配子激活、超低温保存技术的前提。通常纯形态学的方法虽然可以正确地描述生物细胞的形态、结构，并能从结构推测功能，但不能直接证实其功能。相反，细胞化学则可从功能方面推测结构。由于细胞内部大分子物质的鉴定，常需依赖于酶的细胞化学研究。本实验应用近十种细胞化学方法对中国对虾P．orientalis精子进行观察，并对这些细胞化学反应的机能意义加以简单的讨论。目前，受精率的高低作为精子质量分析的指标已得到广泛应用，但该方法具有较大局限性，不仅受到精子，还受到卵子质量的影响，特别是受精时外界条件的影响，而且实验条件严格，复杂，时间持续长，Anchordojuy (1988)曾报道把单肢虾Sicyonia ingentis精子的双相顶体反应作为精子质量评析的标准，在精子活力判断中取得了新的突破，很值得我们借鉴。这一方法具有准确度高和时间短的优点。为此，作者对中国对虾P．orientalis精子的顶体反应进行初步的研究，并探索出一种更方便更可行的鉴别手段，通过多次重复实验，发现应用活体染色法来判断中国对虾P．orientalis精子的活动，是简便有效的。活体染色法曾广泛应用于支植物细胞死活的判定，但在十足甲壳类精子中却未见有所研究报道。总之，十足类无鞭毛的精子形态的独特和顶体结构的复杂无疑揭示其顶体反应必然具有一定的特殊性。中国对虾是我国的重要经济虾类，搞清其精子顶体反应对于弄清其受精机制，促进中国对虾人工受精工作开展有很大的意义。

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。

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l(-1) ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W > NW > N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l(-1) ammonia-N reduced the immunocompetence of P japonicus and may have decreased the virulence of WSSV (C) 2004 Elsevier B.V. All rights reserved.

Effects of different light sources and illumination methods on growth and body color of shrimp Litopenaeus vannamei

Shrimps Litopenaeus vannamei with initial body weight of 2.108 +/- 0.036 g were sampled for specific growth rates (SGR) and body color measurements for 50 days under different light sources (incandescent lamp, IL; cool-white fluorescent lamp, FL; metal halide lamp, MHL; and control without lamp) and different illumination methods (illumination only in day, IOD, and illumination day and night, IDN). Body color of L. vannamei was measured according to the free astaxanthin concentration (FAC) of shrimp. The SGR, food intake (FI), feed conversion efficiency (FCE) and FAC of shrimps showed significant differences among the experimental treatment groups (P < 0.05). Maximum and minimum SGR occurred under IOD by MHL and IDN by FL, respectively (difference 56.34%). The FI of shrimp for the control group did not rank lowest among treatments, confirming that shrimp primarily use scent, not vision, to search for food. FI and FCE of shrimps were both the lowest among treatment groups under IDN by FL and growth was slow, thus FL is not a preferred light source for shrimp culture. Under IOD by MHL, shrimps had the highest FCE and the third highest FI among treatment groups ensuring rapid growth. FAC of shrimp were about 3.31 +/- 0.20 mg/kg. When under IOD by MHL and IDN by FL, FAC was significantly higher than the other treatments (P < 0.05). To summarize, when illuminated by MHL, L. vannamei had not only vivid body color due to high astaxanthin concentration but also rapid growth. Therefore, MHL is an appropriate indoor light source for shrimp super-intensive culture. SGR of shrimp was in significantly negative correlation to FAC of shrimp (P < 0.05). Thus, when FAC increased, SGR did not always follow, suggesting that the purpose of astaxanthin accumulation was not for growth promotion but for protection against intense light. (c) 2005 Elsevier B.V. All rights reserved.

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections. (C) 2006 Elsevier Ltd. 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.

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

Genetic improvement on Chinese shrimp (Fenneropenaeus chinensis): growth and viability performance in F1 hybrids of different populations

Fenneropenaeus chinensis distributed in the Yellow Sea and Bohai Sea of China and the west coast of the Korean Peninsula. Different geographical populations represent potentially different genetic resources. To learn further the characteristics of different geographical population, crosses among two wild and three farmed populations were produced. The two wild populations were from the Yellow Sea and Bohai Sea (WYP), and the west coast of the Korean Peninsula and coast (WKN). The three farmed populations included the offspring of first generation of wild shrimp from coast in Korea (FKN), the Huang Hai (the Yellow Sea in Chinese) No.1 (HH1), and JK98. The phenotypes growth and survival rates of these populations were compared to confirm the feasibility for crossbreeding. The body length (BL), carapace length (CL), carapace width (CW), height of the second and third abdominal segment (HST), width of the second and third abdominal segment (WST), length of the first abdominal segment (LF), length of the last abdominal segment (LL), live body weight (BW), and survival rate were measured. Different combinations were statistically performed with ANOVA and Duncan's Multiple Range Test. The results show that the survival rate of JK98(a (TM) Euro)xWKN(a (TM),) was the highest, followed by WYP(a (TM) Euro)xWKN(a (TM),), FKN(a (TM) Euro)xWYP(a (TM),), FKN(a (TM) Euro)xHH1(a (TM),) and WYP(a (TM) Euro)xFKN(a (TM),); the body weight of FKN(a (TM) Euro)sxHH1(a (TM),) was the highest, followed by FKN(a (TM) Euro)xWYP(a (TM),), WYP(a (TM) Euro)xWKN(a (TM),), WYP(a (TM) Euro)xFKN(a (TM),) and JK98(a (TM) Euro)xWKN(a (TM),); the total length had the same ranking as the body weight. All growth traits in hybrids JK98(a (TM) Euro)xWKN(a (TM),) were the lowest among all combinations. F1 hybrids had significant difference (P < 0.05) in BL, CL, HST, LL, and BW; and insignificant difference (P > 0.05) in other growth traits and survival rate. The results of Duncan's Multiple Range Test are that BL and CL of JK98(a (TM) Euro)xWKN(a (TM),) were significantly different from the other combinations; HST different from the combination of FKN(a (TM) Euro)xWYP(a (TM),), FKN(a (TM) Euro)xHH1(a (TM),) and WYP(a (TM) Euro)xWKN(a (TM),); and BW different from FKN(a (TM) Euro)xWYP(a (TM),) and FKN(a (TM) Euro)xHH1(a (TM),). As a whole, the results indicate that the FKN(a (TM) Euro)xHH1(a (TM),) was the best combination in all growth traits. Therefore, hybridization can introduce the variation to base populations. The systematic selection program based on additive genetic performance may be more effective than crossbreeding.

滇东南发现新元古代岩浆岩：SHRIMP锆石U-Pb年代学和岩石地球化学证据

滇东南老君山地区出露了一套穹隆状变形-变质岩系,新近完成的1：5万都龙幅、麻栗坡县幅区域地质矿产调查报告,将其分解为晚志留纪南温河花岗岩、新元古界新寨岩组和古元古界猛硐岩群.首次对猛硐岩群的石英角闪斜长片麻岩进行了SHRIMP锆石U-Pb定年,结合阴极发光图像分析,获得两组岩浆锆石的结晶年龄分别为761±12Ma和829±10Ma,指示滇东南老君山地区存在新元古代岩浆活动,同时从同位素年代学上,表明猛硐岩群是一套前寒武纪的变质沉积-岩浆杂岩.此外,还获得一些年龄为≈1.83Ga的残留锆石,表明该区存在古元古代的结晶基底.石英角闪斜长片麻岩及共存的斜长角闪岩,它们的原岩具有大陆裂谷背景下玄武质-玄武安山质岩浆系列的地球化学特征,可能与新元古代Rodinia超大陆聚合-裂解过程中的岩浆活动有关,本区在新元古代可能位于南华裂谷与康滇裂谷的交汇部位。

滇西北中旬松诺含矿斑岩的锆石SHRIMP U-Pb年龄及地质意义

位于义敦岛弧南端的中甸岛弧中广泛发育印支期斑岩及斑岩型和矽卡岩型铜矿床。松诺（或称松诺力赞）复式岩体位于东斑岩带中部，由石英闪长玢岩、黑云石英二长斑岩、闪长玢岩和含矿石英二长斑岩组成，其南部为普朗超大型斑岩铜矿床，北部为地苏嘎铜矿点。本文对含矿石英二长斑岩进行了岩相学和锆石SHRIMPU-Pb定年研究，结果表明所有锆石颗粒自形较好且均发育规则的韵律环带，Th含量为180-854μg／g，U含量为270-709μg／g，Th／U比值为0．77-1．24，为典型岩浆成因锆石。获得了含矿石英二长斑岩的侵位年龄为220．9±3．5Ma（n=9，MSWD=1．6），这与中甸岛弧洋壳俯冲造山作用的时限（210-235Ma）相吻合。

SHRIMP锆石年代学对西藏玉龙斑岩铜矿成矿年龄的制约

锆石SHRIMP U-Pb定年结果表明,玉龙岩体侵入时限至少为2.6Ma,其中成矿前石英二长斑岩的年龄为43.6±0.8Ma,成矿期黑云母二长花岗斑岩的年龄为41.0Ma±1.0Ma,代表了两幕较大的岩浆活动.玉龙斑岩铜矿主体成矿年龄约为40Ma,长时限多期幕式岩浆侵入和成矿期物理化学条件的剧变,是形成玉龙超大型斑岩铜矿的主要原因.玉龙斑岩铜矿与金沙江-红河成矿带众多新生代斑岩铜矿一样,属于印度-亚欧大陆45Ma陆陆主体碰撞之后第一次大规模应力释放的产物.