马氏珠母贝激光育苗的研究

马氏珠母贝是培育海水珍珠的主要贝类。近年来,由于近亲繁殖,种群退化,贝体越来越小,致使培育的人工珍珠颗粒也越来越细。为了改良品种,培育出数量多、质量好的马氏珠母贝苗,我们从1982年起,进行激光育苗研究,并于1987年结合生产试     

微胶囊饲料对马氏珠母贝育珠性能、生长基因和矿化基因表达的影响

本研究分析了自主研发的微胶囊饲料替代微藻对马氏珠母贝(Pinctada fucata martensii)育珠性能、生长相关基因和矿化相关基因表达的影响。共设置了3个实验组,其中,EG1组投喂亚心形扁藻(Platymonas subcordiformis),EG2组投喂微胶囊饲料+亚心形扁藻,EG3组投喂微胶囊饲料。养殖170 d后,比较马氏珠母贝的育珠性能、闭壳肌生长相关基因EGFR、FGF18、GHITM和TβRI以及外套膜的中央膜和边缘膜矿化相关基因pearlin、DPT、pif177和N19的表达。结果表明:(1)各组间马氏珠母贝育珠贝的存活率、留核率不存在显著性差异(P0.05),所采收珍珠的珍珠层厚度及平均质量也不存在显著性差异(P0.05);(2)各实验组间马氏珠母贝闭壳肌中EGFR、FGF18、GHITM和TβR I的相对表达量不存在显著性差异(P0.05);(3)各实验组间马氏珠母贝中央膜中DPT和N19的相对表达量差异不显著(P0.05),EG2和EG3组pearlin的表达量显著性高于EG1组(P0.05),pif177在EG3组中的表达量显著性高于EG1和EG2组(P0.05);(4)各实验组间马氏珠母贝边缘膜中pearlin、DPT和N19的相对表达量差异不显著(P0.05),EG2和EG3组pif177的表达量显著高于EG1组(P0.05)。研究结果说明,该微胶囊饲料可以替代部分微藻进行育珠生产,为进一步研发马氏珠母贝的人工配合饲料及开展工厂化育珠积累了参考资料。     

马氏珠母贝人工育苗中的技术问题

近年来不少单位在马氏珠母贝育苗过程中因技术问题而使育苗效果差、经济效益低。为此笔者根据育苗实践谈几个技术问题。 1.亲贝和幼虫的优选 优选亲贝的标准是:3～4龄,活力强,外形完整,贝体厚大;生殖腺丰满,在足基处的两个乳头状突起较高,呈乳白色或橙红色,不透明,吸出的卵子放在载玻片上不易分散,卵子大小整齐,呈圆形或椭圆形,精子游动活泼。这样的亲贝育苗效果最好。幼虫的优选     

马氏珠母贝术前处理的研究

实验结果表明:经术前处理后的插核贝休养期存活率为95 2%,育珠期存活率达85 5%,每贝只插1个核,休养期留核率为0 86,育珠期留核率达0 71。珍珠的质量明显提高,优质珍珠的生成率高达34 6%,比对照组高出27 5%。     

马氏珠母贝激光育苗机理的探讨

马氏珠母贝激光育苗，其产量比常规育苗增加１０倍以上，激光能增强精子的活力，提高精液品质，可影响卵子的通透性，使卵子新陈代谢中的某些反应发生良性变化。激光能抑制中致病菌及霉菌的生长，使受精卵发育成健壮的胚胎、幼虫、幼苗。所以，激光育苗是一种投产少，效益极高的育苗方法。     

马氏珠母贝EST微卫星的筛选

构建了马氏珠母贝的血液、足、鳃、胃、肝、心脏、外套膜、珍珠囊和感染多毛虫马氏珠母贝的血液(血感)等9个组织的cDNA文库,测序获得了6979个EST序列,从中查找到了268个重复序列,隶属于243个EST,含微卫星的EST数占EST总数的3.48%.珍珠囊cDNA文库含微卫星的序列所占比例最高,为6.16%,血感的最低,为1.65%.双碱基重复序列130个,占48.5%,(AT/AT)n类型最常见;三碱基类型的83个,占约31%,其中(AAT/ATT)n和(AAG/CTT)n较多;四碱基重复的有30个,占11.2%,(AAAT/ATTT)n占了该类型的约50%.一共能够设计151对微卫星引物,有130对可以扩增,占合成引物总数的86.09%,其中,多态性引物45对.多态性EST-SSR的筛选将为马氏珠母贝的分子遗传学研究、种质鉴定和野生资源保护等提供可靠的工具.     

马氏珠母贝精子的超低温保存

通过比较不同pH值（5．5-9．5）、不同盐度的海水等作为基础液对马氏珠母贝精子保存的影响,选择其中无激活精子作用又对其生理特性（活力,寿命,受精能力等）无影响者,加入二甲亚砜抗冻剂配制成超低温保存的抗冻保护液,精液与保护液按1：10和1：20的比例混合,样品按4组不同的降温程序平衡后转入液氮冷冻,比较其超低温冻存的效果。结果表明：以海水（pH7．5—8．0）为基础液,配制10％DMSO作为抗冻保护液,精液与保护液比例为1：20,在低温（0-4℃）中平衡约30min,在距液氮面15cm、5cm处分别停留5min、10min后转入液氮（-196℃）,冻存精子效果良好。冷冻24h、48h及5个月后,在38—40℃下水浴解冻复苏后,用终浓度0．25‰的氨海水刺激,精子存活率可超过60％,受精率可达80％。     

马氏珠母贝不同组织同工酶的比较

采用聚丙烯酰酰胺凝胶水平平板电泳技术,研究了马氏珠母贝的８种组织７种同工酶的酶谱表型,结果表明：所测组织中未检出α－磷酸甘油脱氢酶（α－ＧＰＤＨ）和醇脱氢酶（ＡＤＨ）,乳酸脱氢酶（ＬＤＨ）只在肝脏一条微弱谱带,苹果酸脱氢酶（ＭＤＨ）、苹果酸酶（ＭＥ）和超氧化物岐化酶（ＳＯＤ）部分组织间有显著差异,而酯酶（ＥＳＴ）则具有明显的组织特异性。     

马氏珠母贝鳃组织抗菌活性的研究

用纸片琼脂扩散法和微量测定法对制备的马氏珠母贝鳃组织匀浆物进行抗菌活性分析.结果表明,马氏珠母贝鳃组织匀浆物具有抗革兰氏阴性菌-副溶血弧菌、河流弧菌、嗜水气单胞菌、大肠杆菌、绿脓杆菌活性;对革兰氏阳性菌-金黄葡萄球菌具一定抑制活性,但对枯草芽孢杆菌无抑制活性.利用胰蛋白酶对马氏珠母贝鳃组织匀浆物水解后,其抗菌活性消失,表明其中抗菌成分为蛋白质.将马氏珠母贝鳃组织匀浆物在不同温度(37～95 ℃)和不同pH(2.5～5)保温,发现其抗菌活性成分对热及酸耐受性较强.马氏珠母贝鳃组织匀浆物在低浓度无溶血活性.该抗菌组分在马氏珠母贝的免疫防御中具有一定的作用.     

马氏珠母贝附着生物及防除方法研究

论文主要对马氏珠母贝附着敌害生物的种类、附着方式、危害及防治等进行了综述。     

Genetic linkage map of the pearl oyster,Pinctada martensii (Dunker)

Genetic linkage maps were constructed with amplified fragment length polymorphism (AFLP) and microsatellite markers for the pearl oyster, Pinctada martensii (Dunker), the main bivalve used for marine pearl production in Asia. Twenty‐four AFLP and 84 microsatellite primer pairs were used for linkage analysis in a full‐sib family with two parents and 78 offspring. Of the 2357 AFLP fragments generated, 394 (16.7%) were polymorphic and segregating. Most (340 or 86.2%) of the markers segregated according to expected Mendelian ratios. Female and male linkage maps were constructed using 230 and 189 markers, including 15 and 10 microsatellites respectively. The female map consisted of 110 markers in 15 linkage groups, covering 1415.9 cM, with an average interval of 14.9 cM. The male map consisted of 98 markers in 16 linkage groups, with a total length of 1323.2 cM and an average interval of 16.1 cM. When unlinked doublets were considered, genome coverages were 78.5% for the female and 73.5% for the male map. Although preliminary, the genetic maps constructed here should be useful for future linkage and quantitative trait loci mapping efforts.     

Estimating the heritability for growth-related traits in the pearl oyster,Pinctada fucata martensii (Dunker)

Twelve paternal half-sib families (or 36 full-sib families) of the pearl oyster, Pinctada fucata martensii (Dunker), were produced according to the requirements of hierarchical genetic mating design. A total of 4320 individuals, aged 15 months, were measured for seven growth-related traits. Predicated upon the additive-dominance genetic analysis model, varying genetic variance components and then heritabilities of the growth-related traits of interest were estimated using analysis of variance. Results showed that seven growth-related traits had larger additive genetic variances (P<0.05); the dominance genetic variance of shell weight (SW) was smaller (P>0.05), the dominance genetic variances of other six traits were all larger (P<0.05). Narrow- and broad-sense heritabilities for the seven traits were, respectively, 0.64 ± 0.10 and 0.78 ± 0.12 for shell length, 0.49 ± 0.06 and 0.63 ± 0.09 for shell height, 0.38 ± 0.14 and 0.54 ± 0.16 for shell breadth, 0.41 ± 0.17 and 0.56 ± 0.11 for hinge length, 0.53 ± 0.11 and 0.68 ± 0.08 for body weight, 0.35 ± 0.07 and 0.55 ± 0.08 for tissue weight and 0.67 ± 0.10 and 0.75 ± 0.16 for SW. All heritability estimates were statistically significant (P<0.05). According to these results, the mass selection procedure is suggested for the breeding of P. martensii.     

Interactive effects of temperature and diet on the growth and biochemical composition of juveniles of the pearl oyster Pinctada mazatlanica

The combined effects of temperature and diet on the growth and biochemical composition of juveniles of the pearl oyster Pinctada mazatlanica at the hatchery were investigated. Specimens were subjected to a combination of four temperatures (20, 23, 26 and 29 °C) and five microalgal diets ( Isochrysis galbana alone, I. galbana + Pavlova salina, I. galbana + Chaetoceros muelleri, C. muelleri + P. salina and I. galbana + P. salina + C. muelleri ). An increase in shell height occurred in a linear pattern, while wet weight gain fitted a power law. Temperature, more than diet, exerted a stronger influence on the growth and condition of the specimens. The combination of 29 °C with P. salina+C. muelleri led to the fastest growth in shell height, while the combination of 29 °C with I. galbana + C. muelleri led to the highest wet and dry weight biomass. In contrast, specimens grew significantly less in shell height and wet weight at 20 °C, regardless of the diet. At all temperatures, the monoalgal diet of I. galbana led to the slowest growth of specimens, but in turn favoured the highest levels of protein, carbohydrate and lipid reserves as a possible strategy to store more energy reserves under stressful events.     

日本珍珠贝人工繁殖苗在热带海区的养殖生长观察

日本珍珠贝引进海南陵水热带海区养殖,人工繁殖幼贝苗1·5~2·0mm下海养殖。经1年的养殖生长观察,结果为:平均壳长59·2mm、壳高59·7mm、壳宽20·7mm,平均个体重34·8g;养殖90d,生长速度较快,壳长平均增长值为11·9mm,相对增长率为55·4%;壳高平均增长值11·2mm,相对增长率61·2%;壳宽平均增长值5·9mm,相对增长率为190·3%。壳长、壳高和体重增长之间具有非常显著的相关关系。研究表明,该贝在海南热带海区养殖的生长速度快,是适宜引进的养殖对象。     

9个马氏珠母贝家系的中期生长性状评估

2008年3月以2006年构建的4个家系F1为亲本,采用不完全双列杂交法,成功构建了9个马氏珠母贝（Pinctadafacata）家系,在相同养殖条件下对其壳高、壳长、壳宽和体质量进行了周年测量,对不同遗传背景的家系和不同时期4个生长性状的生长速率进行了比较分析。结果显示,不同家系间及不同生长时期在各性状上存在显著差异（P〈0．05）。9个家系的壳高生长速率顺序为F11〉F4〉F9〉F3〉F6〉F12〉F2〉F7〉F5,壳长生长速率顺序为F11〉174〉F3〉F9〉F12〉F2〉F6〉F7〉F5,壳宽生长速率顺序为F6〉F11〉F3〉P9〉F4〉F12〉F2〉F5〉F7,体质量生长速率顺序为F11〉F12〉179〉F3〉F7〉F2〉F4〉F5〉F6。F11、附和F、9家系壳高生长较快,F11家系壳长生长较快,F11、F3和F9家系壳宽生长较快,F11、F12和F9家系体质量生长较快,彼此间差异显著（P〈0．05）。在不同的生长时期各生长指标差异显著性有所不同,10月～12月以壳高、壳长和壳宽增长较为显著,3月～5月以体质量增长较为显著;慢速生长期各生长性状指标增长速率差异均不显著,而快速增长时期各生长性状增长速率均存在显著性差异。筛选出生长性状最优的家系F11,为通过家系选择培育优良品种提供了遗传素材,同时为家系育种策略的制定提供了指导。     

Effects of replacing microalgae with an artificial diet on pearl production traits and mineralization‐related gene expression in pearl oyster Pinctada fucata martensii

The effects of replacing dietary microalgae with artificial diet on pearl production traits and biomineralization‐related gene expression were investigated in pearl oyster (Pinctada fucata martensii). Three experiment groups (EG1, EG2 and EG3) were set. EG1 and EG3 were separately fed Platymonas subcordiformis and artificial diet (D4), respectively, and EG2 was fed with mixed P. subcordiformis and D4. A control group (CG) was cultured in natural sea. All groups were continuously fed for 150 days. The results showed that the survival rates of EG1, EG2 and EG3 were significantly higher than that of CG. No significant differences in retention rate and pearl thickness were observed among the four groups, but CG had the lowest values of both parameters. The relative expression levels of EGFR and FGF18 mRNA did not significantly differ among EG1, EG2 and EG3. Moreover, the relative expression levels of GHITM and TβR I mRNA significantly varied among the groups and EG3, and EG1 had the highest and lowest relative expression levels of GHITM and TβR I mRNA respectively. The relative expression levels of nacrein and chitin‐binding mRNA significantly differed among the groups, and EG1 had the lowest expression levels of nacrein and chitin‐binding mRNA. No significant differences in the relative expression levels of MSI60 and TIMP mRNA were observed among the groups. The results suggested that D4 can replace part of microalgae, which is helpful for further studies on developing artificial diet of pearl oyster.     

CB抑制合浦珠母贝受精卵第一极体释放的染色体分离

对CB抑制合浦珠母贝受精卵 (3n♀× 2n ,2n× 2n)第一极体释放的染色体分离行为进行了细胞学观察。结果表明 ,CB抑制第一极体改变了正常的染色体分离行为 ,在第二次减数分裂过程中出现了多极分离 ,主要有二极分离 (2 8.6 7% )、三极分离 (40 .5 6 % )及四极分离 (2 3.78% )三种模式 ,不能确定的占 6 .99%。均等的二极分离可能导致四倍体的产生 ,而部分四极分离也能产生四倍体 ,三极分离主要产生非整倍体。未处理组的染色体仍按正常的二极形式进行分离 ,释放两个极体 ,但两极的染色体数目通常存在极大差异 ,导致大量的非整倍体产生。二倍体组 (抑制第一极体 )也存在同样的多极分离模式 ,二级分离占 2 5 .0 0 % ,三极分离占2 1.5 1% ,四极分离占 34.30 % ,不能确定的占 19.19%。另外 ,对各种染色体分离模式形成的机制进行了讨论