中国对虾精子入卵过程的观察

利用光镜和电镜对中国对虾精子入卵过程进行了观察。结果表明，精子的核物质籍胞吐作用穿过卵黄膜到达卵膜，其余部分则留在卵黄膜外。只有在卵子激活前到达卵膜的精子才能参与受精作用，其余的精子将被卵子激活所形成的胶质层推离卵子。中国对虾精子激活过程，没有发现顶体丝的形成。精子的入卵开始于卵子的皮层反应后期，此时精卵的接触逐渐密切，首先接触部位的卵膜稍有突起，然后开始下陷，同时这一部位的卵膜变得粗糙不平，随后卵膜解体，精子由此进入卵子。中国对虾精子的入卵位置是随机的，有多精入卵的现象。     

Valine requirement of postlarval tiger shrimp, Penaeus monodon Fabricius

The valine requirement of juvenile tiger shrimp, Penaeus monodon Fabricius, was determined. Shrimp postlarvae, PL20, with a mean weight of 14 mg, were randomly distributed in 36 oval 40-L capacity fibreglass tanks at 10 shrimp per tank in a flow-through seawater system and reared for 8 weeks. Postlarvae were fed amino acid test diets containing 400 g kg^?1 protein with casein and gelatine as intact sources of protein. Crystalline L-amino acids were supplemented to simulate the amino acid profile of the shrimp muscle except valine. Valine was added in graded levels to obtain 7, 10, 13, 16, 19 and 22 g kg^?1 of the diet or 18, 25, 33, 40, 48 and 55 g kg^?1 of dietary protein. At termination of the feeding experiment, growth and survival were determined and nutritional deficiency signs noted. The relationship between weight gain and dietary valine level was analysed by the broken-line regression method to derive the valine requirement. The dietary valine requirement of Penaeus monodon postlarvae was found to be 13.5 g kg^?1 of the diet or 34 g kg^?1 of dietary protein. This value was lower than the level found in the shrimp tissue.     

斑节对虾基因组微卫星分离及其序列特征研究

采用(CA)_(12)(AG)_(12)及(TA)_(16)生物素标记探针及磁珠富集法构建了斑节对虾Penaeus monodon基因组微卫星富集文库。随机挑选254个克隆进行PCR筛选,得到51个候选克隆(20．1%)。其中,32个克隆来源于CA-文库,另19个克隆来源于AG-文库。测序发现48个克隆含有微卫星重复单元,通过序列比对,最终获得40个具有特异微卫星序列的阳性克隆。微卫星(GA/CT)_N及(CA/GT)_n 2碱基重复序列分别占所有分离的微卫星数目的20．7%及60．4%。此外,还检测到其它多种微卫星重复类型,如(AT)_n、(GC)_n、(TGG)_n、(AAG)_n、(AAT)_n、(GAA)_n、(GTGC)_n、(GCGT)_n、(GGTTA)_n、(GTGCGT)_n,占检测到的微卫星数目的18．9%。获得的微卫星序列中属于完全型序列的有76条(68．5%),不完全型序列的有22条(19．8%),另有13条属于复合型序列(11．7%)。微卫星(GT/CA)_n 2碱基重复次数(3～52次)要远大于(GA/CT)_n 2碱基次数(3～27次)。获得的微卫星序列长度大小范围为129～601 bp,平均为286 bp。研究为进一步开展斑节对虾分子育种及资源评价分析提供了基础资料。     

Dietary potassium requirement of juvenile grass shrimp Penaeus monodon

ABSTRACT: A feeding trial was conducted to estimate the minimal dietary potassium (K) requirement for juvenile grass shrimp Penaeus monodon . Purified diets with seven levels (0, 0.3, 0.6, 0.9, 1.2, 1.5, 2.0 g/100 g) of supplemental potassium were fed to P. monodon (mean initial weight 0.75 ± 0.01 g) for 8 weeks. Each diet was fed to three replicate groups of shrimp. The rearing water contained 360 mg/L potassium. Weight gain and protein efficiency ratio (PER) of shrimp improved as dietary potassium supplementation level increased up to 1.5 g K/100 g diet and thereafter declined significantly ( P < 0.05). Feed efficiency (FE) of shrimp also showed a similar trend. However, whole body K concentration of the shrimp kept an increasing trend up to 2.0 g K/100 g diet. Analysis of the weight gain (percent) and PER of the shrimp by polynomial regression indicate that the minimal dietary potassium requirement in growing P. monodon is approximately 1.2 g/100 g.     

Thermal challenge of the Indian shrimp Penaeus indicus

This study investigated short‐term effects of increasing water temperature from 27 to 41°C on survival and feed consumption of Penaeus indicus at three different ages: PL₂₅ (postlarvae 25 days old), PL₅₀ and PL₉₀. For each age group, water temperature was maintained at 27°C in the control, but increased to 32, 35, 38 and 41°C at a rate of 1°C every eight hours. The temperature was then kept stable until the end of the 7‐day experiment. Results showed that increasing water temperature affected both survival and feed consumption of the experimental shrimps (p < .01). Survival was highest at 32 and 35°C ranging from 93.8% to 100%, but significantly reduced to 40.0%–81.6% at 38°C. No shrimp survived the 41°C treatment. PL₂₅ were more tolerant to 38–41°C than PL₅₀ and PL₉₀ in terms of survival. Increasing water temperature had no effects on feed consumption of PL₂₅ (p > .05). For PL₅₀ and PL_90, feed consumption significantly increased at 38 and 41°C (p < .01) and was similar within the range of 27–35°C. This study suggests that P. indicus in tropical areas can tolerate water temperatures of at least 35°C and should be considered for farming during the summer time.     

中国对虾主要过敏原的鉴定及理化性质

以中国对虾为研究对象,旨在获得有关中国对虾过敏原的基础数据,为水产品中过敏原的控制提供依据.首先利用聚丙烯酰胺凝胶电泳对中国对虾过敏原组分进行分析,经免疫印迹鉴定其主要过敏原.然后利用高效液相色谱法纯化中国对虾的主要过敏原,并用β消去、紫外扫描、肽质量指纹图谱和红外光谱等方法对其理化性质进行研究.表明中国对虾主要过敏原为分子量36 ku的糖蛋白,糖含量为4.4%,存在O-型糖肽键,其二级结构主要为α-螺旋,与其他甲壳类过敏原之间存在很高的同源性.     

环境条件对中国对虾交尾影响的试验观察↑（*）

本文报告了在室内条件下中国对虾雄性生殖系统不同部位精子形态、激活反应及外界环境对对虾交尾影响等的试验观察。雄性生殖系统被分为３部分：精巢（Ｔ）、输精管和精荚（ＴＡ）;输精管又分为前、中、后３段,其中的前（ＡＶＤ）、中（ＭＶＤ）、后（ＰＶＤ）３部分以及精巢和精荚被分别匀浆取样镜检,观察精子形态。结果表明：中国对虾雄性精巢、输精管、精荚及雌虾纳精囊内精子的形态、对卵水的激活反应能力有所差异,是一逐渐成熟过程;雄虾具有多次交尾能力,不同环境条件对中国对虾的交尾影响明显;当雌虾比例较高时交尾率也高;不同对虾密度对交尾影响不明显     

Spermatophore cryopreservation and artificial insemination of black tiger shrimp, Penaeus monodon (Fabricius)

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post‐thaw spermatophores. Spermatophores were collected during consecutive regenerations (15‐day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post‐thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen‐thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning.     

南美白对虾血蓝蛋白对酚氧化酶活性的影响

以采自厦门的南美白对虾(Penaeus vannamei)为研究对象，运用病原菌人工感染、血蓝蛋白浓度和酚氧化酶(phenoloxidase，PO)活性测定等方法探索南美白对虾血蓝蛋白在体内、外对其血清酚氧化酶活性的影响。结果表明，南美白对虾人工感染副溶血弧菌后，其血淋巴中血蓝蛋白浓度和PO活性的变化趋势基本一致。在体外实验中将一定量的血蓝蛋白重组蛋白添加至血清中，其PO活性可明显升高；但同时添加血蓝蛋白重组蛋白和E．coli K12超声破碎液后，其PO活力反而降低。研究提示，血蓝蛋白在一定范围内确实可以正反馈调节PO活性，但在细菌等干扰之下，其又可表现出一定的负反馈抑制活性。     

Effect of immunostimulants on the haemolymph haemagglutinins of tiger shrimp Penaeus monodon

The levels of haemagglutinins in Penaeus monodon , following administration of immunostimulants, β-glucans and/or vibrio bacterin either orally or by immersion, were studied. The freshly drawn haemolymph was incubated with microbial materials like β-glucans/vibrio bacterin and serum obtained after coagulation was administered to naïve animals. The immunostimulant treatments either via immersion, feeding or injection were found to cause an increase ( P <0.006) in haemagglutination activity (HA) of the haemolymph against mouse erythrocytes. Injection of saline or heterologous haemolymph caused an increase in the HA, but injection of haemolymph serum obtained by clotting haemolymph in the presence of vibrio bacterin or glucan did not bring about an increase in HA. There was no change in the haemolymph protein profile of the groups receiving immunostimulants through immersion or feed. Two protein bands (27 and 30 kDa), which were present in the uninjected group, were found to be overexpressed in the haemolymph-injected groups. Three bands of 17, 21 and 23 kDa, which were absent in control or saline-injected groups, were present in all the haemolymph serum-injected groups. The study indicates that modulation of HA may partly account for the immunomodulatory activity of immunostimulants like β-glucan or vibrio bacterins.     

Acrosome reaction in the sperm of the black tiger shrimp Penaeus monodon (Decapoda, Penaeidae)

Owing to the problem of male infertility in the domesticated shrimp Penaeus monodon, this study was conducted to reveal the morphological events of an acrosome reaction (AR) of sperm of this highly valuable species. The AR observed in an in vitro incubation of sperm with egg water (EW) and that during actual spawning was compared. Under transmission and scanning electron microscopy, sperm taken from the female thelycum was composed of a posterior main body, a central cap and an anterior single spike. Upon contact with EW, the sperm underwent two phases of AR: acrosomal exocytosis and spherical mass formation. The former was composed of a degeneration of the spike, swelling of the cap region and rupture of the acrosomal pouch. The latter began with polymerization of materials within the subacrosomal region and ended with re‐configuration of the subacrosomal region into an electron‐dense spherical mass. The AR of the sperm observed during spawning revealed similar morphological events, with degeneration of the spike upon contact with the vitelline envelope and formation of the spherical mass while penetrating into jelly material produced by protruding cortical rods. The results suggest the presence of AR inducers derived from the vitelline envelope and cortical rods of the egg. This study forms the basis for an evaluation of infertility regarding to AR in the domesticated P. monodon male.     

EM在水泥池封闭、半封闭式生态养虾中的应用

本实验以EM作为饲料添加剂和水质净化剂,采用全封闭、半封闭与传统养殖方式—对照组在实验室水泥池(118d)养殖实验。结果表明:EM能改良水质条件,促进对虾生长。全封闭和半封闭实验组成活率分别比对照组提高了48 7%,24 1%;饵料系数则降低了21 2%,14 1%;全封闭和半封闭实验组DO含量与对照组相比分别提高了9 4%,12 6%;COD含量与对照组相比分别降低了0 25%,0 48%;NH 2-N含量与对照4-N含量与对照组相比分别降低了19 2%,19 2%;NO 组相比分别降低了15 2%,21 1%。     

中国对虾白黑斑病组织病理学研究↑（*）

报道了中国对虾白黑斑病的肝胰腺、鳃、血液、胃、肠和肌肉等组织的压制片、显微切片及亚显微切片的观察结果。病虾鳃上常见肉芽肿和菌团（３７％）,肝胰腺有细小样病毒（ＨＰＶ）包涵体感染（４１％）,但这些组织均未出现有规律的因生物性病原侵入而引起的病理组织变化。肉眼所见的白斑和黑斑是由一些大小不等、形状不一的棕黄色和褐色团块状物质沉积而成。超微结构显示这些类似脂褐质物质内没有包被细菌等生物性病原。据观察结果分析,该病为生理性疾病,而非感染性疾病。还讨论了白斑、黑斑的形成与病虾的生理代谢、酚氧化酶作用之间的关系     

Cu2+对南美蓝对虾无节幼体的急性致毒研究

研究了Cu2+对南美蓝对虾(Penaeus stylirostris)无节幼体的急性毒性影响。当Cu2+浓度不同时,无节幼体的活性和死亡率均有较大变化,其中12h和24h的半致死浓度(LC50)分别为0.132mg/L和0.102mg/L。实验结果及与其他种类毒性实验的比较表明,Cu2+对南美蓝对虾无节幼体毒性作用明显,并产生畸形现象及对变态率的影响。从而得到Cu2+对南美蓝对虾无节幼体的24h LC50和安全浓度,所得值高于国家海水水质标准值。     

Mycoflora of the 'green water' culture system of tiger shrimp Penaeus monodon Fabricius

This study was conducted to quantify and characterize the mycoflora associated with the ‘green water’ culture system of Penaeus monodon. Samples of water, tilapia gut and mucus, and shrimp hepatopancreas from three shrimp farms were collected during 15, 30, 45 and 60 days of culture (DOC). Results showed that high fungal loads were observed in tilapia gut (total: 117–1352 colony forming unit (CFU) 5 cm hind gut^?1; yeasts: 0–136 CFU 5 cm hind gut^?1) and mucus (total: 12–311 CFU (5 cm²)^?1; yeasts: 0–88 CFU (5 cm²)^?1), while minimal fungal populations were observed in water samples (total: 0–110CFU mL^?1; yeasts: 0–5 CFU ml^?1). Shrimp hepatopancreas harboured a very low number of filamentous fungi (0–27 CFU 0.1 g^?1) and yeasts (0–7CFU 0.1 g^?1) especially at 60 DOC. The filamentous fungal isolates were dominated by Penicillium and Aspergillus species, while the yeast populations were dominated by Rhodotorula and Saccharomyces species. The dominance of these fungi on tilapia mucus and gut and their presence in the rearing water might play an important role in the overall mechanisms involved in the control of luminous Vibrio in the ‘green water’ grow‐out culture of P. monodon.     

Hatching envelope formation in the egg of the black tiger shrimp,Penaeus monodon (Decapoda,Penaeidae)

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)‐labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti‐FITC‐conjugated gold‐lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar‐associated components of the HE. This revealed that both Con A and WGA‐binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold‐labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N‐acetyl‐β‐d ‐glucosamine (sugar affinity to WGA).     

中国对虾肝胰腺_卵巢及血淋巴中的_省略_雌二醇含量的生殖周期变化

首次证实了孕酮和雌二醇这两种类固醇激素在中国对虾体内的存在.在性腺未发育阶段,肝胰腺、卵巢和血液中两种激素的含量均很低氐,难以检测到.而在卵黄发生前期(核仁周边期),三种组织中孕酮和雌二醇含量迅速上升,卵巢的雌二醇含量达到高峰(450.1±86.7).进入初级卵黄发生阶段,三种组织中,两种激素均具较高含量,卵巢和肝胰腺的孕酮含量(分别为1975 1±175.2和902.6±130.5pg/g)以及血淋巴中的雌二醇含量(451.3±73.7)达到高峰.到了次级卵黄发生阶段,孕酮和雌二醇的含量迅速下降,肝胰腺等组织中儿乎检测不到.对虾性腺指数(GSl)的增长既显著且有规律性,每一期增长幅度都达到或超过l00%.肝胰腺指数(HSI)从性腺未发育期(3.4±04)到卵黄发生前期(4.9±0 7)以及从卵黄发生前期到初级卵H黄发生期(6.3±1.0)有显著的增长,而从初级卵黄发生期到次级卵黄发生期HSI增长不显著(6.7±1.2).肝胰腺指数的增长与两种类固醇激素含量的变化具相似的趋势.上述结果显示,孕酮和雌二醇可能具有刺激和凋控中国对虾性腺发育的作用,肝胰腺可能是卵黄蛋白原的合成场所.     

斑节对虾增殖细胞核抗原基因克隆及表达分析

为研究增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在斑节对虾(Penaeus monodon)卵巢发育中的功能,利用RACE技术克隆得到了斑节对虾PCNA基因(PmPCNA)的cDNA全长序列。该序列全长978 bp,包括135 bp的5′非编码区(5′UTR)、60 bp的3′UTR和编码260个氨基酸的783 bp的开放阅读框(ORF)。同源性分析结果表明,PmPCNA与其他物种具有很高的蛋白同源性。组织表达模式分析表明,PmPCNA在所监测的各组织中为组成性表达,其中在卵巢和脑中表达量较高;卵巢发育不同阶段基因的表达分析则表明PmPCNA在Ⅲ期表达量最高,是其他各期表达量的2倍;注射五羟色胺(5-hydroxy tryptamine,5-HT)后,PmPCNA在卵巢中表达量明显上升,其中在48 h表达量升高幅度最大,是对照组的5倍左右。利用原核表达技术获得了PCNA的体外重组蛋白,Western Blot分析证实该重组蛋白为PCNA蛋白。以上研究结果表明,PmPCNA蛋白是斑节对虾卵巢发育过程中的重要调节因子,本研究为进一步认识斑节对虾卵巢发育机制提供了基础资料。