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.

Phylogenetic relationships of 12 Penaeoidea shrimp species deduced from mitochondrial DNA sequences

DNA sequences of an 847 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) gene and a 514 bp fragment of 16s rRNA gene were determined to examine the phylogenetic relationships of 12 Penaeoidea shrimp species (Penaeus chinensis, Penaeus japon

原位杂交研究对虾白斑杆状病毒在虾体内感染过程

应用地高辛标记的对虾白斑杆状病毒 (whitespotsyndromebaculovirus,WSSV)核酸探针 ,与人工感染后不同时间采集的对虾组织样品进行原位杂交 ,以动态研究病毒从侵染至对虾发病死亡的过程。将典型感染WSSV的病虾组织投喂健康对虾 ,结果显示 :WSSV首先通过侵染消化道上皮进入虾体内增殖 ,此后随着细胞裂解、病毒粒子释放 ,游离的病毒粒子伴随血淋巴循环进而侵染其它靶组织 ,直至对虾发病死亡。

Phylogenetic relationships among the genera of the Penaeidae (Crustacea : Decapoda) revealed by mitochondrial 16S rRNA gene sequences

The phylogenetic relationships within the family Penaeidae are examined based on mitochondrial 16S rRNA gene sequence analysis of 30 species from 20 genera. The analysis generally supports the three- tribe scheme proposed by Burkenroad ( 1983) but it is not consistent with the five- group classification of Kubo ( 1949). Three clades are resolved: ( Penaeus sensu stricto + Fenneropenaeus + Litopenaeus + Farfantepenaeus + Marsupenaeus + Melicertus + Funchalia + Heteropenaeus), ( Metapenaeus + Parapenaeopsis + Xiphopenaeus + Rimapenaeus + Megokris + Trachysalambria) and ( Metapenaeopsis + Penaeopsis + Parapenaeus), corresponding to the Penaeini, Trachypenaeini and Parapenaeini respectively, while the affinities of Atypopenaeus and Trachypenaeopsis are obscure. The molecular data support that Miyadiella represents the juvenile stage of Atypopenaeus. Within the Trachypenaeini, Trachypenaeus sensu lato is clearly paraphyletic, while the monophyly of Penaeus sensu lato in the Penaeini is questionable.

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.

Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

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.

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。