Artificial interspecific hybridization between Macrobrachium species

Viable F-1 hybrids were obtained from crosses of female Macrobrachium nipponense and male Macrobrachium hainanense involving spermatophore transfer and artificial insemination. This represents the first successful known case of hybridization of two Macrobrachium species by means of artificial insemination. The hatching rate was over 90%. About 20-60% of newly hatched larvae metamorphosed to postlarvae. The morphological characteristics of the hybrids resembled a combination of features of both parents. Malate dehydrogenase (MDH) and esterase (EST) isozyme electrophoresis indicated parents and F-1 hybrids showed co-dominant expression of the paternal and maternal alleles controlling the isozymes and confirmed the hybridization. (C) 2004 Elsevier B.V. All rights reserved.

RAPD and AFLP techniques for the analysis of genetic relationships in two genera of Decapoda

RAPD was used fur analysing three (sub-)species of mitten crabs (Eriocheir sinensis, E. japonicus, and E. japonicus hepuensis) and three populations of E. sinensis. The results show that their relationships on DNA level are similar to the classical taxonomic hypotheses (Dai, 1991). No diagnostic RAPD marker could be found, but there were statistically significant genetic differences among these taxa (P < 0.001) or populations (P < 0.001). That is, the intraspecific similarities were larger than the interspecific similarities; the intrasubspecific similarities were larger than the intraspecific similarities; and the intrapopulational similarities were larger than the interpopulational similarities. In AFLP analysis, no significant genetic difference has been found between E. sinensis and E. japonicus, but AFLP markers among four species of Macrobrachium (M. rosenbergii. M. nipponense, M. hainanense, and M. asperulum) were found. The DNA similarities among these four species of Macrobrachium are in accordance with morphological similarities.

A preliminary phylogenetic analysis of Metapenaeopsis (Decapoda : Penaeidae) based on mitochondrial dna sequences of selected species from the Indo-West Pacific

Phylogenetic relationships within Metapenaeopsis remain largely unknown. The modern revision of the genus suggests that the shape of the petasma, followed by the presence of a stidulating organ, are the most important distinguishing taxonomic features. In the present study, phylogenetic relationships were studied among seven Metapenaeopsis species from the Indo-West Pacific based on partial sequences of mitochondrial 16S rRNA and cytochrome c oxidase I (COI) genes. Mean sequence divergence was 6.4% for 16S and 15.8% for COI. A strikingly large nucleotide distance (10.0% for 16S and 16.9% for COI) was recorded between M. commensalis, the only Indo-West Pacific species with a one-valved petasma, and the other species with a two-valved petasma. Phylogenetic analyses using neighbor-joining, maximum parsimony, and maximum likelihood generated mostly identical tree topologies in which M. commensalis is distantly related to the other species. Two clades were resolved for the remaining species, one with and the other without a stridulating organ, supporting the main groupings of the recent taxonomic revision. Results of the present study also indicate that the deep-water forms represent a relatively recent radiation in Metapenaeopsis.

凤鹛属鸟类（Yuhina, Aves）的系统发育研究

本研究以凤鹛属（Yuhina）鸟类为研究对象，分别使用分子生物学性状和形 态学性状来重建凤鹛属种间的系统发育关系，为东南亚地区鹛类的起源与进化研 究提出新的见解。分子生物学研究的内群不仅包括了凤鹛属（11 种中的10 种）， 而且还包括了菲律宾特有穗鹛属（Stachyris）和绣眼鸟属（Zosterops）等近缘种。 通过测定线粒体上两个蛋白编码基因Cyt b、ND3 的全长序列和两个RNA 编码 基因 12S, 16S 的部分序列，得到长度为2379bp 的联合序列，使用最大简约法、 最大似然法和贝叶斯法进行系统发育分析的结果表明，菲律宾岛特有的穗鹛（金 冠穗鹛S. dennistouni、纹穗鹛S. striata、怀氏穗鹛S. whiteheadi）与绣眼鸟属（暗 绿绣眼Z. japonicus、红胁绣眼Z. erythropleurus、灰腹绣眼Z. palpebrosus）聚为 一支，位于凤鹛属支系内部。通过凤鹛、菲律宾穗鹛、绣眼鸟三个类群羽冠性状 演化趋势的分析，发现羽冠性状在菲律宾特有穗鹛和绣眼鸟支系中发生丢失。与 前人的研究结果相比，新发现纹喉凤鹛Y. gularis 与棕肛凤鹛Y. occipitalis 是姐妹 群，黄颈凤鹛Y. flavicollis 和白项凤鹛Y. bakeri 是姐妹群，这两个姐妹群又聚为 一大支的关系。 在分类问题上，暗绿绣眼、红胁绣眼和灰腹绣眼在本研究中与鹛类表现出近 缘关系，与前人使用绣眼鸟属其它物种进行的相关研究显示出高度一致性，说明 绣眼鸟属的系统学地位存在很大疑问，应该扩大取样进行深入研究。栗冠凤鹛 Y. everetti 与栗耳凤鹛Y. castaniceps 聚为姐妹群的关系，但是二者之间较小的遗 传距离和形态学差别显示二者之间应该为亚种关系，而不是种级关系。依据栗耳 凤鹛特有的尾羽形态提出的Staphiada 属没有得到本分子生物学研究结果的支持 （Harrison, 1986a, b）。在白腹凤鹛Y. zantholeuca 不属于凤鹛属支系这一结果的 提示下，发现白腹凤鹛独有的形态学特征，例如羽冠由冠羽形成、鼻孔半裸露、 飞羽外翈没有异色羽缘，这些独有的特征与其特殊的系统学地位相对应。因此， 我们支持前人提出的将白腹凤鹛从凤鹛属中分出去，单独成Erpornis 属的建议 （Cibois et al., 2002）。 在历史生物地理学方面，根据菲律宾特有穗鹛和绣眼鸟的近缘关系，认为菲 律宾特有穗鹛的祖先具有与现在绣眼鸟一样的跨海迁移的能力，在中新世末期（5.74Mya）由喜马拉雅地区或印度支那起源地经大巽他地区扩散到菲律宾岛屿 上，经过长期独立进化后，形成现在高度特有的穗鹛支系。更新世冰期时，大巽 他地区海平面下降，使栗冠凤鹛得以扩散至婆罗州（1.66Mya）。在上新世初期台 湾岛成陆后，由于台湾岛与中国大陆相连，褐头凤鹛Y. brunneiceps 的祖先扩散 至台湾岛屿上（5.05Mya）。高黎贡山为第四纪冰期时凤鹛属鸟类的避难场所，因 此形成了现在凤鹛属鸟类在高黎贡山地区密集分布的格局。喜马拉雅山和青藏高 原的隆起导致青藏高原上植被带的变化和喜马拉雅山南麓在第四纪冰期中避难 所的作用，是纹喉凤鹛、棕肛凤鹛、黄颈凤鹛、白项凤鹛等种类现今在喜马拉雅 山南麓密集分布的可能原因。 在凤鹛属分子系统学的研究基础上，从形态学角度探讨凤鹛属除白领凤鹛外 其它物种之间的系统发育关系。使用最先分化出来的白领凤鹛为外群以外群比较 法进行51 个形态学特征的性状极化，使用最大简约法分析后，结果不仅支持一 些分子系统学揭示的物种之间的亲缘关系，例如，栗耳凤鹛与栗冠凤鹛姐妹群的 关系，黑颏凤鹛和褐头凤鹛姐妹群的关系，三种绣眼鸟聚为一支，再与怀氏穗鹛 聚为一个大支；而且发现了新的系统关系，黄颈凤鹛与缅甸凤鹛Y. humilis 是姐 妹群，具有浅色下体的凤鹛属物种具有较近的系统关系。分子数据和形态数据对 凤鹛属系统发育树中部节点解决能力的差异，说明凤鹛属物种之间分化间隔时间 很短。 通过综述近年来核基因在鸟类分子系统发育研究中的应用情况，建议未来研 究中增加核基因的内含子序列，例如肌球素基因内含子2（ myoglobin intron Ⅱ） 或β纤维蛋白原基因内含子7（β-fibrinogen intron7, β-fibint 7）来解决本研究 中未确定的末端分枝分歧顺序。

Fruit anatomy,seed germination and seedling development in the Japanese seagrass phyllospadix

Phyllospadix iwatensis Makino and phyllospadix japonicus Makino have similar frunt morphology and anatomy.The rhomboid fruit of Japanese phyllospadix is dark brown in colour and is characterized by two arms bearing stiff inflected bristles which can act as an anchoring system. The fruit covering consists of a thin cuticular seed coat and pericarp remains mainly fibrous endocarp. In the groove region of the fruit.the cuticular seed coat and endocarp are replaced by nucellus cells with wall in growths and crushed pigment strands with lignified walls.these tissues appera to control the transfer of nutrients to developing seed.the seed is oval with a small embryo and a large hypocotyl. the embryo is straight and simple,with the plumule containing three leaf primordia and a pair of root primordia surrounded by a cotyledon.the hypocotyl has large vontral lobe containing central provascular tissue and two small dorsal lobes.the hypocotyl contains starch.lipid and protein.and acts as a nutrient store.the seed of P.iwatensis has a dormancy period of 2-6 weeks and germination eventually reaches-65%.but is not synchronized.during germination the leaves emerge first.and then after at least three young leaves have formed and abseised.the roots emerge,usually?6 months after the commencement of germination.Utilizaton of the nutrient reserves is initially from the perihpery of the hypocotyl and then progressively towards its centre.

The mitochondrial manganese superoxide dismutase gene in Chinese shrimp Fenneropenaeus chinensis: Cloning, distribution and expression

Manganese superoxide dismutase (MnSOD) plays an important role in crustacean immune defense reaction by eliminating oxidative stress. Knowledge on MnSOD at molecular level allows us to understand its regulatory mechanism in crustacean immune system. A novel mitochondrial manganese superoxide dismutase (mMnSOD) was cloned from hepatopancreas of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1185 bp with a 660 bp open reading frame, encoding 220 amino acids. The deduced amino acid sequence contains a putative signal peptide of 20 amino acids. Sequence comparison showed that the mMnSOD of F. chinensis shares 88% and 82% identity with that of giant freshwater prawn Macrobrachium rosenbergii and blue crab Callinectes sapidus, respectively. mMnSOD transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by Northern blotting. RT-PCR analysis indicated that mMnSOD showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with while spot syndrome virus (WSSV). In addition, a fusion protein containing mMnSOD was produced in vitro. LC-ESI-MS analysis showed that two peptide fragments (-GDVNTVISLAPALK- and -NVRPDYVNAIWK-) of the recombinant protein were identical to the corresponding sequence of M. rosenbergii mMnSOD, and the enzyme activity of the refolded recombinant protein was also measured. (c) 2006 Elsevier Ltd. All rights reserved.

A Dicer-1 gene from white shrimp Litopenaeus vannamei: Expression pattern in the processes of immune response and larval development

Dicer is a member of the RNAase III family which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro RNAs, and then directs sequence-specific gene silencing. In this paper, the full-length cDNA of Dicer-1 was cloned from white shrimp Litopenaeus vannamei (designated as LvDcr1). It was of 7636 bp, including a poly A tail, a 5' UTR of 136 bp, a 3' UTR of 78 bp, and an open reading frame (ORF) of 7422 bp encoding a putative protein of 2473 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other Dicer-1 homologues and showed the highest (97.7%) similarity to the Dicer-1 from tiger shrimp Penaeus mondon. Quantitative real-time PCR was employed to investigate the tissue distribution of LvDcr1 mRNA, and its expression in shrimps under virus challenge and larvae at different developmental stages. The LvDcr1 mRNA could be detected in all examined tissues with the highest expression level in hemocyte, and was up-regulated in hemocytes and gills after virus injection. These results indicated that LvDcr1 was involved in antiviral defense in adult shrimp. During the developmental stages from fertilized egg to postlarva VII, LvDcr1 was constitutively expressed at all examined development stages, but the expression level varied significantly. The highest expression level was observed in fertilized eggs and followed a decrease from fertilized egg to nauplius I stage. Then, the higher levels of expression were detected at nauplius V and postlarva stages. LvDcr1 expression regularly increased at the upper phase of nauplius, zoea and mysis stages than their prophase. The different expression of LvDcr1 in the larval stages could provide clues for understanding the early innate immunity in the process of shrimp larval development. (C) 2010 Elsevier Ltd. All rights reserved.

Broader pattern of tandem repeats in the mitochondrial control region of Perciformes

Perciformes, the largest order of vertebrates with 20 suborders, is the most diverse fish order that dominates vertebrate ocean life. The complete mitochondrial control region (CR) of Trichiurus japonicus (Trichiuridae, Scombroidei) and Pampus sp. (Stromateidae, Stromateoidei) were amplified and sequenced. Together with data from GenBank, the tandem repeats in the mitochondrial CR from 48 species, which covered nine suborders of Perciformes, are reported in this study. The tandem repeats tend to be long in the suborder Percoidei and Stromateoidei. The identical repeats in 21 species of Cichlidae suggest a common origin and have existed before species divergence. Larimichthys crocea shows tandem repeats instead of the typical structure of the central conserved sequence blocks, which was first reported in Perciformes and vertebrates. This might have resulted from interruption of the polymerase activity during the H-strand synthesis. The four broader patterns presented here for the tandem repeats, including those in both the 5' and 3' ends, only in the either 5' or 3' end, and in the central conserved domain of the control region, will be useful for understanding the evolution of species.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. 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.

中国对虾对微量元素硒的吸收、代谢及硒对其生长的影响

中国对虾（Penaeus orientalis）对硒的需要量为20ppm或高于此值，饵料中添加的硒可能通过促进谷胱苷肽过氧化物酶（GSH-Px）的活性而间接提高对虾的增重率和抗病能力；GSH-Px活性是对虾重要的生理指标，可用来衡量对虾对硒的营养状态。对虾可以直接吸收海水中的硒，但能力较低，由于海水中的硒含量很低，所以，有必要在饵料中添加适量的硒。肝胰脏是对虾体内各种来源的硒的储存库；肝胰脏中的硒除了少量与小分子的氨基酸、肽等结合外，大部分与大分子的蛋白质（部分是酶）结合。对虾体内的硒可以通过甲壳和鳃排出体外。

刺参急性口围肿胀病和皱纹盘鲍肌肉萎缩症的病理学研究

本论文以大连沿海养殖刺参劾皱纹盘鲍为研究对象，利用组织学、超微病理学、病理生理学的方法，开展了患病病理学研究，并对可能诱发刺参和皱纹盘鲍病害的原因，以及病变过程进行了初步探讨。 中国北方养殖刺参[Apostichopus japonicus (Selenka)]幼参和成参的急性口围肿胀病。急性口围肿胀病是爆发于中国北方辽宁省和山东省沿岸养殖刺参群体中的一种新发现的疾病，并且从2004年开始已经引起刺参的大面积死亡现象，经济损失重大。 本文报道在养殖患病刺参的肠上皮细胞内发现大量的病毒样颗粒。这是首次报道病毒样粒子感染刺参。组织学检查表明该病毒具有包涵体结构，寄生于肠上皮细胞中。电镜检查的结果表明，该病毒粒子呈球形，直径80-100纳米，螺旋状核衣壳，具有囊膜结构且囊膜表面具有纤突结构。 进一步的形态学和病理学分析发现该病毒具有许多报道的关于冠状病毒的特征。细胞质内的 病毒颗粒大部分以团聚方式存在于一个完整的包膜内，形成典型的病毒包涵体结构。最明显的细胞病理学特征是细胞质内大面积的粒状物质的存在，该区域相对缺少相应的细胞器。在病毒包涵体内的管状结构，核衣壳包涵体以及双层膜囊泡也在病变细胞内发现。 在病参体内未发现立克次氏体，衣原体，细菌以及其他寄生生物。 中国北方养殖皱纹盘鲍(Haliotis discus hannai)的肌肉萎缩症病理研究 。 在2004年和2005年的中国北方海区，养殖皱纹盘鲍幼体和成体群体中爆发了严重的肌肉萎缩症。肌电图监测显示病鲍的肌肉萎缩是肌源性的而不是神经源性的。 与正常对照组对比结果显示，病鲍的肌纤维数量和肌纤维直径都显著降低。 肌酶谱测定结果显示，病鲍血清中的肌酸激酶水平，肌酸激酶—肌同工酶水平和乳酸脱氢酶水平，与正常血清对比，呈显著升高状态，说明病鲍体内发生了肌细胞损伤。超薄切片电镜检测结果显示在发生病变的肌细胞内存在双螺旋丝状结构的包涵体（Paired helical filaments, PHFs）, 该结构是人类患包涵体肌炎的主要的病理特征。 本文首次报道在无脊椎动物中发现双螺旋丝包涵体结构。 对损伤的肌纤维的进一步研究发现了异常增生的小圆柱体结构（Small Cylinder Structure, SCS）和致密小体(Dense colored particles, DCP),同时观察到这两种异常结构与肌纤维的损伤直接相关。本文还对该病的感染机制作了探讨。 中国北方皱纹盘鲍(Haliotis discus hannai)肌肉萎缩的病理生理研究。严重的足肌肉萎缩症是鲍的一种慢性致死性疾病。在中国北方的养殖皱纹盘鲍群体中第一次发现是在2000年，随后在各养殖海区，自然海区及实验室养殖过程中都发现了该病的暴发。 超微结构电镜检测显示肌肉纤维损伤严重，其内的大部分肌原纤维断裂或消失。 大量的花瓣状的糖原颗粒聚集在断裂的肌原纤维中，还有一些被包裹在包膜内。 这说明在病鲍体内的糖原代谢处于被抑制状态（或停止状态），随后的血清学检测表明病鲍血清中的葡萄糖含量与正常对照的含量显著降低，也证明了糖代谢途径的终止。病鲍血清中胆固醇和甘油三酯的量显著降低，伴随着高密度脂蛋白的水平显著升高，说明脂肪代谢途径的亢进状态。病鲍血清中的二氧化碳水平的显著降低说明出现了酸碱平衡紊乱，通过病因学分析可知该酸碱平衡紊乱应属于代谢性酸中毒。病鲍血清中的钠离子和钾离子浓度显著升高，无机磷和镁离子浓度显著降低说明出现了患病皱纹盘鲍体内出现了电解质紊乱。病鲍血清中蛋白质含量的检测表明患病皱纹盘鲍已出现低蛋白血症，说明病鲍出现了严重的营养不良。病鲍血清中的尿酸含量，胆碱酯酶水平和γ-转肽酶水平与正常对照组相比显著降低。各项指标检测的结果说明糖代谢途径的终止造成了病鲍一系列的调节体系的不平衡和各种生理功能的紊乱，甚至造成了鲍的最终死亡。

有机污染对水环境生态效应及中国对虾内环境的影响

以人工配合饵料作为有机污染源，选定试验池、对照池各1个，分别放养5cm左右的中国对虾12尾；除了试验池的投饵量为对照池的2倍以及对照池每天吸底外，其他养殖条件均保持一致。目的在于研究有机污染对水环境生态效应和对中国对虾内环境的影响，进而探讨有机污染对虾病的诱发作用。结果表明，有机污染影响水环境，且改变中国对虾内环境，降低其免疫水平，使虾病易于发生。同时，温度在有机污染对水环境的影响中也具有重要的作用。