三倍体皱纹盘鲍活体倍性检测

以皱纹盘鲍的血细胞为材料，采用植物血球凝集素(PHA)体外浸泡法，运用正交实验设计，探讨PHA处理时间、PHA浓度、抽血时间对细胞分裂频度的影响。根据正交实验直观分析结果，得出用血细胞直接制备染色体标本进行三倍体皱纹盘鲍活体倍性检测的最优组合：PHA处理时间18h、PHA浓度0．04％，抽血时间22：00-24：00，3因素的主次顺序：PHA处理时间→PHA浓度→抽血时间。并可获得大量图象清晰、长度适中、分散良好的中期分裂相，是皱纹盘鲍活体倍性检测的最佳方法。同时，通过流式细胞仪测定皱纹盘鲍血细胞DNA相对含量进行三倍体皱纹盘鲍活体倍性检测，该方法制样简单，测试速度快。     

鲍活体倍性检测技术的研究

在生物有机体中,一个细胞核中所含的DNA总量对于某种类有机体是一定的。利用流式细胞仪(FCM)测量细胞中的DNA含量,可以用来鉴别有机体的倍性。本研究分别取杂交鲍[即皱纹盘鲍(Haliotis discus hannai)×盘鲍(Haliotis.discus discus)]的一小块触手、足肌,设计不同的振荡时间、不同的DAPI染色时间、不同的取样部位,制备成不同的细胞悬液检查样品;用PA-Ⅱ型倍体分析仪测定其倍性,以探讨其对倍性检测效果的影响。试验结果表明:振荡的最适时间为1.5～2.0 min,染色的最适时间为4.0～5.0 min;取材部位的不同对检测结果无显著影响。     

皱纹盘鲍幼鲍溃烂病病原菌的ELISA检测法

皱纹盘鲍幼鲍溃烂病病原菌的ＥＬＩＳＡ检测法Ａｅｌｉｓａｍｅｔｈｏｄｆｏｒｄｅｔｅｃｔｉｎｇｔｈｅｐａｔｈｏｇｅｎｉｃｂａｃｔｅｒｉａｏｆｆｅｓｔｅｒｄｉｓｅａｓｅｉｎｃｕｌｔｕｒｅｄｊｕｖｅｎｉｌｅａｂａｌｏｎｅ叶林俞开康王如才（青岛海洋大学水产学院...     

关于玛瑙鲍的探索与研究

选择皱纹盘鲍（Ｈａｌｉｏｔｉｓ ｄｉｓｃｕｓ ｈａｎｎａｉ）的变异个体－－贝壳为红色的玛瑙鲍（Ａｇａｔｅ Ａｂａｌｏｎｅ）作亲体,进行人工繁殖的培育。发现在性产物排放诱导率、孵化率、幼虫附着率和稚鲍生长等方面,玛瑙鲍与皱纹盘鲍大致相同。但稚鲍对不良环境（如赤潮）的抗性,玛瑙鲍明显强于皱纹盘鲍,３２ｄ存活率,前者为６３．１％,后者为３４．３％。ＬＤＨ电泳图谱显示,玛瑙鲍内脏组织中ＬＤＨ活性较高,而被     

虹鳟鱼核型及DNA含量分析

采用植物血球凝素（ＰＨＡ）体内注射、肾组织细胞短期培养、常规空气干燥法制备了虹鳟的染色体，并进行了核型分析。虹鳟二倍体的染色体为６０条，其核型为２２ｍ＋１ｓｔ＋７ｔ，ＮＦ＝１０４。以外周血红血球为样本，鸡红血细胞为对照，用美国产的ＦＡＣＳｔａｒｐｌｕｓ流式细胞仪测定了虹鳟的二倍体细胞核的ＤＮＡ含量，其ＤＮＡ含量为鸡血对照的１．５１倍，绝对含量为３．４７±０．２１ｐｇ／Ｎ。     

流式细胞术在水生生物DNA含量和倍性分析中的应用

利用流式细胞仪对鲤鱼、银鲫、黄颡、牙鲆等淡水和海水鱼类进行了DNA含量测定或倍性分析。通过几百个样品的测定和分析，认为流式细胞仪在DNA相对含量测定和倍性分析上结果比较稳定一致，可以用于大量样品的测定分析。同时对待测样品的保存、处理及上样量等进行了一系列比较研究，找出最好的保存方法、最简便的样品处理方法和最少的上样量。流式细胞仪在水生生物的遗传育种、种群分化等方面有很重要的应用价值。     

广西沿海文蛤及其生境中细菌菌群的初步调查

对广西沿海文蛤及其养殖场的细菌菌群用ＶＩＴＥＫ微生物自动分析仪进行了初步检测，发现文蛤体内有腐败假单胞菌（Ａｌｔｅｒｏｍｏｎａｓｐｕｔｒｅｆａｃｉｅｎｓ）、河弧菌（Ｖｉｂｒｉｏｆｌｕｖｉａｌｉｓ）、副溶血弧菌（Ｖ．ｐａｒａｈａｅｍｏｌｙｔｉｃｕｓ）、溶藻弧菌（Ｖ．ａｌｇｉｎｏｌｙｔｉｃｕｓ）、非发酵性革兰氏阴性芽胞菌（ＮｏｎｆｅｒｍｅｎｔｉｎｇＧｒａｍ－ｎｅｇａｔｉｖｅＢａｃｉｌｌｕｓ）、假单胞菌属（Ｐｓｅｕｄｏｍｏｎａｓｓｐｐ．）以及一些未知菌。从海水中亦经常检测到腐败假单胞菌、溶藻弧菌、河弧菌、温和气单胞菌（Ａｅｒｏｍｏｎａｓｓｏｂｒｉａ）等细菌。分析表明，副溶血弧菌可能为文蛤的一种病原菌。药敏试验显示．上述所有细菌几乎都对环丙沙星、庆大霉素和氯霉素敏感。     

应用双抗夹心ELISA法检测皱纹盘鲍致病病原—创伤弧菌的研究

以皱纹盘鲍脓足病致病病原－ － 创伤弧菌为抗原,制备兔抗血清,抗体纯化后以辣根过氧化物酶标记,建立检测创伤弧菌的双抗夹心ＥＬＩＳＡ 检测法。结果表明,双抗夹心ＥＬＩＳＡ 法有较高的灵敏度,可检出含菌１０４ 个／ｍｌ 的菌悬液。与创伤弧菌对照菌株有明显的阳性反应不与副溶血弧菌、溶藻胶弧菌、河流弧菌等８ 株对照菌株产生影响检测结果的交叉反应。应用该法检测３０ 份病鲍样品,阳性检出率为６６ ．７ ％ 。     

皱纹盘鲍淀粉酶和褐藻酸酶的研究

利用分光光度计比色法测定了２～３ｃｍ的皱纹盘鲍（ＨａｌｉｏｔｉｓｄｉｓｃｕｓｈａｎｎａｉＩｎｏ）的淀粉酶和褐藻酸酶的最适温度和最适ｐＨ以及１１种金属离子对其的影响。结果表明：最适温度分别为３０℃、３５℃,最适ｐＨ分别为６．２４、７．１９,在最适温度下的活化能分别为５．０３×１０４Ｊ／ｍｏｌ和１．３３×１０４Ｊ／ｍｏｌ;皱纹盘鲍的三种主要消化酶的活性依次为：褐藻酸酶＞蛋白酶＞淀粉酶。Ｃｕ２＋、Ｈｇ２＋、Ａｇ＋、Ｐｂ２＋对淀粉酶活性具有显著的抑制作用,其他离子则具有促进作用,其中又以Ｍｎ２＋、Ｂａ２＋、Ｃａ２＋三种金属离子尤为突出,其促进作用比对照组高一倍以上。而对褐藻酸酶,Ｚｎ２＋、Ａｇ＋、Ｈｇ２＋、Ｌｉ２＋、Ｂａ２＋具显著的抑制作用,其他离子则没有作用。     

利用流式细胞仪鉴别转基因鲤鲫杂交回交子代的倍性

本文报道三倍体回交杂种的流式细胞仪测试方法和结果。取正常二倍体鲤鲫杂交鱼的体细胞液进样所得直方图为标准,通过对36尾鲤鲫杂交回交种和其雌核发育子代的细胞核DNA相对含量的测定,观察到杂种F1雌性回交子代中产生的三倍体和非整倍三倍体（2－3倍体之间）现象,测试结果以直方图表示。     

Triploidy induction in Australian greenlip abalone Haliotis laevigata (Donovan) with cytochalasin B

Triploid induction in Australian greenlip abalone, Haliotis laevigata (Donovan), was conducted by blocking the formation of the second polar body using cytochalasin B (CB). Twenty minutes after fertilization, the zygotes of greenlip abalone were treated with four CB concentrations (0, 0.25, 0.5 and 0.75 mg L⁻¹) for 10, 15 and 20 min. The ploidy of resultant larvae was determined using flow cytometry at 72-h post fertilization. Our study showed that fertilization, hatching, survival and induced triploidy of abalone larvae were significantly affected by the CB concentration and treatment duration. The effective range of CB concentration for triploid induction on greenlip abalone was 0.5–0.75 mg L⁻¹ with an induction duration of 10–15 min. The results indicate that the most effective treatment combination for triploid induction in greenlip abalone is 0.5 mg CB L⁻¹ for 15 min starting at 20-min post fertilization.     

Growth and feeding in juvenile triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814), at two temperatures

Growth and feeding of juvenile triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were investigated at two temperatures of 17 and 21 °C over a 50‐day period. There were no differences in growth between triploid and diploid abalone as measured by shell length and body weight. Both triploid and diploid abalone increased in length but not in weight at 21 °C. Condition indices were similar for triploid abalone maintained at both temperatures; however, those for diploid abalone were significantly higher at 17 °C than at 21 °C. Food intake was significantly greater yet feed conversion efficiency was significantly lower in triploid than in diploid abalone. Both the feeding variables were independent of temperature. On average, diploid abalone were able to convert 1 g of dry food ingested to 0.58 g of body weight, but triploid abalone only 0.44 g.     

三倍体九孔鲍的育种和养成技术研究

当 5 0 %九孔鲍受精卵出现第一极体后 ,以 6 -DMAP为诱导剂 ,用不同的浓度和不同的持续时间抑制第二极体的释放诱导三倍体 ;并进行了生产性苗种培育和养成试验。结果表明 ,6 -DMAP的诱导浓度为30 0μmol·dm-3 、诱导持续时间为 10min时 ,其胚胎的三倍体率为 90 %以上 ,并且胚胎的孵化率高达 85 % ,幼体的畸形率较低 ,为 5 0 %～ 5 5 % ;经过 7个月培育 ,试验组养成鲍的三倍体率为 6 5 %以上 ;试验组养成鲍的平均壳长比对照组增长 10 % ,平均体重比对照组增重 30 %。与二倍体鲍相比较 ,三倍体鲍在壳长增长和体重增重等方面 ,显示了显著的优势 [t>t0 .0 1( 58) ]。     

Growth and reproductive performance of triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814)

Growth and reproduction of triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were compared in a 30-month study. Triploidy was induced by inhibition of the second polar body formation using 6-dimethylaminopurine (6-DMAP) or cytochalasin B (CB). There were no significant differences in growth and survivorship between triploid and diploid abalone. However, triploid abalone had a more elongated shell and greater foot muscles than diploid abalone. A slightly curvilinear growth in shell length was conformed to all treatments. While diploid abalone had reached sexual maturity and spawned during the study, gonadal development and gamete maturation were abnormal in triploids. Female triploids lacked an apparent gonad at the macroscopic level but microscopic examination revealed that they had a thin layer of oogonia development. In contrast, male triploids were able to form similar-sized gonads to diploids during most of the reproductive period, but with brown-yellow discolouration and stalled gametogenesis at spermatocyte formation. Sex ratio of triploid abalone did not deviate from 1:1. With the onset of sexual maturation, growth and gonadal maturation occurred concurrently in diploid abalone, and there was no indication that growth of (diploid) abalone was reduced.     

二倍体和三倍体皱纹盘鲍精子发生过程的超微结构

比较了二倍体和三倍体皱纹盘鲍精子发生过程中细胞和细胞器的超微结构变化。结果表明,二倍体皱纹盘鲍的精子发生经历了精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子5个阶段。其形态结构发生了一系列变化,主要包括：核染色质浓缩、线粒体的发达与融合、顶体形成和胞质的减少。三倍体皱纹盘鲍各种生精细胞的直径和核径均大于二倍体,精原细胞结构与二倍体相似;初级、次级精母细胞的胞质中,除溶酶体外,线粒体、内质网等细胞器少于二倍体,线粒体大小与二倍体没有差别,但形态不典型,层状嵴不发达;三倍体皱纹盘鲍的精子发生停滞在精子细胞阶段,表现出各种畸形状态,很多趋于解体,没有发现成熟精子。     

鲍遗传育种研究进展

Abalones are important farming species with a high economic value. They have already been farmed for more than 50 years. As problems and new requirements rose continuously in culture industry of abalone, studies on genetic and breeding techniques are needed to improve characteristics and to gain new traits. This review concentrates on advances in genetics and breeding techniques in abalone. As for genetic studies, karyological analyses, allozyme, DNA markers and genetic diversity were reviewed. So far, karyological analyses in abalone have been performed in 12 species that can be divided into three groups according to the chromosome number. In some economically important species, loci of allozymes and. microsatellites have been isolated and applied to investigate the genetic structure of natural and hatchery populations and to identify the result of chromosome set manipulation, but the related reports are only a few yet. The resultsof investigation with DNA markers and allozymes showed that the genetic structure of natural populations presents two characteristics: excessive homozygosity and subdivision. Advances of various breeding techniques, including introduction, selection,hybridization, polyploidy, gynogenesis and gene manipulation, were reviewed in the other part. Although Haliotis discus discus, introduced from Japan, has become one of the most important culture species in China, the economic, social and environmental effects of introduction have been rarely studied. Selection is one of the most important and basic breeding techniques, but the studies on selection are only a few and preliminary, referring to the relations between genetic characteristics and the traits of growth and resistance, genetic diversity and heritability of quantitative traits, and the effect of selection. Interspecific hybridization was the first breeding program carded out in abalone. Experimental hybridization have been carded out for about 20 crosses. Heterosis,such as faster growth and high survival rate, has been observed in some crosses. Triploids have been successfully induced in many species of abalone with physical or chemical shock, e.g.H, discus hannai, H. rufescens, H.diversicolor diversicolor and H. rnidae. Field experiments were conducted in some species of triploid abalone. In comparison with triploid, the research on tetraploid is still in quest stage. The progress of induction of gynogenesis in abalone is quite slow. Conditions of sperm inactivation, diploid restoration and nuclear behavior of gynogenetically activated eggs have been researched on in H. discus han nai. Notwithstanding the gene transfer technology in abalone is in the quest stage, the research have already involved preparation of exogenic DNA,means of gene transfer, identification integration and expression of target gene, etc. Three research directions in these topics were proposed : to investigate the germplasm resources of abalone deeply and widely, to make use of traditional breeding methods and modem biotechnique synthetically, and to combine the science research with production practice.     

Studies on sperm of diploid and triploid tench, Tinca tinca (L.)

The tench Tinca tinca is an interesting fish from the viewpoint of polyploidy and related atypical reproduction aspects. Triploid tench were produced artificially. Studies of spermiation as well as of sperm motility and structure were performed on several triploid and diploid males simultaneously with individual experimental crosses with diploid females to define their reproductive capacities. The testes of triploids visually looked less developed in the most of cases with lower sperm production (0.05 cm³ sperm per male), GSI and weight of testes compared to diploids (0.58 cm³ sperm per male). Analysis of variance showed significant influence of ploidy level on the percentage of motile spermatozoa. Triploidy did not change percentage of live spermatozoa and velocity of spermatozoa at the first time of sperm movement. The study of sperm structure by scanning electron microscopy revealed that most sperm cells were of normal structure with some anomalies. Sperm heads of triploid and diploid males were mostly round-shaped, 1.86±0.2 and 1.6±0.18 μm in diameter. The midpiece of triploid spermatozoa was slightly narrower than that of diploid ones with typical cylindrical shape. Flow cytometry revealed sperm cells of triploids to be largely aneuploid (1.47 n) with high mosaic DNA, oscillating from haploid DNA content (1.0 n) to diploid DNA content (1.9 n). Experimental crosses between triploid males and diploid females revealed that these males were capable to stimulate effective development with relatively high level of fertilization and hatching rates from 0 to 70%. In conclusion, triploidization does not seem to guarantee sterility of tench.     

微卫星分离模式显示雄性三倍体鲫产生非整倍体精子

三倍体鲫(Carassius auratus)行天然雌核发育,其自然种群中却有较高比例的雄性个体,这些雄性个体的性腺发育正常,能产生有活力的精子。而且其精子的DNA含量约为体细胞的一半,显示三倍体鲫精子发生过程中可能经历了均等的减数分裂。流式细胞术虽然能够较准确地测定细胞群的平均DNA含量,但是却很难检测到单个精子中的个别染色体增减,要明确回答雄性三倍体鲫产生的精子是否为整倍体需要定量地检测单个精子的遗传组成。本研究用微卫星标记检测以雌性鲤(Cyprinus carpio)与雄性三倍体鲫为亲本构建的杂种家系的基因型,结果发现母本鲤的多态位点在子代中呈孟德尔分离,父本三倍体鲫具有三套鲫基因组,其等位基因在子代中呈随机分离。上述研究结果提示:三倍体鲫起源于二倍体鲫的同源加倍,而非二倍体鲫和鲤的种间杂交;三倍体鲫通过染色体的随机分离产生非整倍体的精子,其精子发生过程中没有均等的减数分裂。三倍体鲫行雌核发育生殖,却可能并非起源于种间杂交且群体中的雄性个体可育,因而是单性生殖鱼类中的一个特例。     

Highly efficient induction of triploid Pacific abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> by caffeine treatment

ABSTRACT:   In order to develop a highly efficient method for mass production of triploid Pacific abalone Haliotis discus hannai , caffeine treatment that is safe and inexpensive was optimized. To suppress the first meiotic division, fertilized eggs were exposed to either a 10- or 15-mM caffeine solution for 24 min beginning at 12 min after fertilization. In most treated batches, the rates of cleaved eggs showed no significant difference from the control batches. However, in most treated batches, the rates of occurrence of normal larvae and the survival rates of the early juveniles were significantly lower than those of the controls. The triploid rates at 6 days to 11 months after settlement in all the treated batches were extremely high (91–100%). There was no significant difference in the mean triploid rates between 10- and 15-mM caffeine treatments. These results suggest that both treatment conditions were conducive to triploid abalone production. One live 2n/3n mosaic specimen was found in the treated batches. However, since the frequency of mosaic was extremely low, the mosaicism would probably not have an adverse effect on the stable production of triploid abalones.