Gametogenic cycle of the transplanted-cultured pearl oyster, Pinctada fucata martensii (Bivalvia: Pteriidae) in Korea

The gonadal development and gametogenic cycle of transplanted-cultured pearl oyster, Pinctada fucata martensii, were investigated using individuals collected monthly from Tong-Young along the south coast of Korea from October 2000 to September 2001. The result of monthly change of condition index was similar to tissue weight rate. The highest value was observed in June and the lowest value was observed in November. The gonad of the pearl oyster was located around the digestive diverticula. The ripe testis was milky white while the ovary was light brown. The spawning period of the pearl oyster extended from April to August, with a peak between June and July. The gametogenic cycle could be classified into five successive stages: multiplicative stage (November to February), growing stage (January to March), mature stage (March and April), spawning stage (April to August) and resting stage (September to November).     

Family by environment interactions in shell size of 43-day old silver-lip pearl oyster (Pinctada maxima), five families reared under different nursery conditions

To understand the influence the environment and associated genotype by environment interactions will exert on future silver-lip pearl oyster (Pinctada maxima) selective breeding programs, this study assessed the relative performance in four shell growth traits of spat from five full-sib families, when spat were communally reared at different salinities (29, 34 and 40 ppt), food availability (high, medium and low), food quality (high, medium and low), and in a hatchery vs. ocean environment for 43 days.Rearing environment was found to influence growth expression, with significant differences evident when oysters were grown at different salinities in the ocean instead of hatchery, or when fed algae of differing nutritional quality. As indicated by MANOVA, family comparative growth performances were also altered when the environment changed, with significant environment by family interactions apparent in the food quality, food availability and hatchery vs. ocean rearing treatments. Changes in salinity, however, did not affect relevant family performances.These results indicate that growth and relative family performance in P. maxima may change dependent on local environmental conditions and that genotype by environment effects may need to be considered in breeding programs for this species.     

合浦珠母贝双列杂交家系的建立与遗传分析

以采自广西北海(B)、广东徐闻(X)、海南三亚(S)的3个不同养殖群体的合浦珠母贝为亲贝,按照完全双列杂交的方式,建立了9个交配组合,每个组合10个家系,共90个家系。出苗经60d养殖后,比较各家系的育种值,并对家系进行综合评价。结果表明:壳长的Kung育种值大于20的家系有8个,分别为BX2(表示北海♂×徐闻♀的2号家系,余依此类推)、BX9、BB4、BB5、BB8、BS9、XS3、SB8,其中XS3的壳长及壳宽的Kung育种值和综合评定值均最大,XX3最小。BB4和XS3的综合评定值大于1。壳长的一般配合力为三亚群体17.40,徐闻17.45,北海18.27;壳高为三亚14.74,徐闻14.79,北海15.54。壳长的特殊配合力为三亚—徐闻17.75,三亚—北海18.27,徐闻—北海18.12;壳高为三亚—徐闻14.98,三亚—北海15.51,徐闻—北海15.46。壳长的平均杂种优势为三亚—徐闻1.41,三亚—北海0.96,徐闻—北海0.67;壳高为三亚—徐闻1.13,三亚—北海1.60,徐闻—北海1.19。表明北海的亲本较好。     

Effects of different substrates on settlement and growth of pearl oyster (Pinctada maxima) larvae in hatcheries

Metamorphosis of pearl oyster Pinctada maxima pediveliger is affected by physical and chemical characteristics of the collectors. In the present study, we conducted four experiments to evaluate the effects of collector characteristics on the settlement and growth of hatchery-reared pearl oyster. In the first experiment, black, red, yellow, and white plastic sheets were used as collectors. Settlement of P. maxima larvae in black and red plastic sheets was significantly higher than that in yellow and white plastic sheets (P < 0.05). Mean shell length was not significantly different among the four colored sheets (P > 0.05). In the second experiment, pediveliger larvae were settled onto palm rope and polypropylene rope collectors. The number of spat in the palm rope collector was significantly higher than that of spat in the polypropylene rope collector (P < 0.05). Mean shell length was not significantly different between palm rope and polypropylene rope collectors (P > 0.05). In the third experiment, settlement and subsequent growth were compared between plastic sheets with and without biofilm. The number of spat in the plastic sheets with biofilm was significantly higher than that in the plastic sheets without biofilm (P < 0.05). Mean shell length after settlement were not significantly affected (P > 0.05). In the fourth experiment, pediveliger larvae were stored in tanks with (experimental group) and without collectors (control group). The settlement rate of larvae on the wall was not significantly different between the two groups(P > 0.05). The settlement rates of the larvae on the wall and collectors in the experimental group were significantly larger than those on the wall in the control group(P < 0.05). Mean shell length was not significantly different between the groups (P > 0.05). Our results suggest that appropriate selection of collector types, color, and conditioning can improve the settlement of P. maxima larvae in hatcheries.     

马氏珠母贝丝裂原活化蛋白激酶p38的克隆与表达

丝裂原活化蛋白激酶（MAPK）信号通路在细胞对细胞外刺激的反应中起着重要作用。它是一种丝氨酸/苏氨酸蛋白激酶,通过磷酸化级联将细胞外信号传递给细胞。本研究利用cDNA 末端快速扩增(RACE)技术克隆获得PmMAKP p38基因cDNA 全长序列并对其序列进行生物信息学分析;利用实时荧光定量 PCR（qPCR）技术分析了PmMAKP p38在马氏珠母贝不同组织以及不同免疫刺激后的表达水平。结果显示PmMAKP p38 cDNA全长为1516 bp,开放阅读框长度为1071 bp,共编码356个氨基酸,分子量为 40.88 kDa;结构域预测分析表明PmMAPK p38含有MAPK家族典型的S_TKc结构域;多序列比对、进化树构建以及MatGAT计算结果显示PmMAPK p38与其他物种的相似度、保守程度较高;荧光定量分析结果表明该基因在马氏珠母贝中存在广泛表达,在肝胰腺中表达量最高,其次为外套膜,最低是闭壳肌。机体在受到LPS刺激后,相对表达量在2 h达到最高,12 h降到最低,最高约为最低的5倍;哈维氏弧菌刺激后,相对表达量在2 h达到最高,8 h降到最低,最高约为最低的4倍。研究表明,PmMAPK p38可能在马氏珠母贝的免疫反应中起着重要的作用。     

我国热带海水大型珍珠贝类的珍珠养殖技术现状及对策建议

为改善马氏珠母贝 (Pinctada martensii) 外套膜组织和细胞体外培养的效果,探究了添加不同浓度的天然提取物茶多酚对外套膜组织和细胞培养液的优化效果。结果显示,当茶多酚的添加量为0.5%~2.0% (体积分数) 时,马氏珠母贝外套膜细胞的生物活性显著高于对照组 (P<0.05);当添加量为1.0%时,相对细胞生物活性最高。5组完全培养液 (0%、0.5%、1.0%、1.5%、2.0%组) 均能较好地促进细胞的生长增殖,且细胞的生长曲线均呈“S”型;当培养液中茶多酚的添加量为1.0%时,外套膜细胞增殖效果最优。研究表明,马氏珠母贝外套膜组织和细胞在最适培养液中均能大量增殖游离细胞,且正常分泌珍珠质,其中培养液中的钙离子 (Ca²⁺) 浓度随着组织和细胞增殖过程呈先减后增的趋势,但始终低于初始浓度。研究结果进一步优化了马氏珠母贝外套膜组织和细胞培养液,为推动马氏珠母贝无核珍珠的培育提供了理论基础。

     

Towards development of large-scale hatchery cultivation of larvae and spat of the pearl oyster Pinctada mazatlanica in Mexico

The results of a series of pilot-scale runs with P. mazatlanica larvae from 2004 through 2006 are reported. Preliminary runs in 2004 and 2005 used broodstock collected in summer, when massive spawning of wild populations naturally occurs. However, results of larval development were very poor and failed to produce spat in both years. In 2006, ripe broodstock were still collected in summer, but also in the spring time, based on the hypothesis that the gonads in this season were in better reproductive condition that in summer. Three larval runs were conducted in 2006: two in spring and one in summer. Larvae growth and survival greatly increased in both spring runs, ending with two successful productions of spat (∼ 20 × 10³ and ∼ 100 × 10³ juveniles). The summer larval run in 2006 failed again to produce spat. Additionally, the first run of April 2006 refers to an experiment that evaluated two different larval culture conditions: constant temperature (27 °C) and low stocking density (3-4 larvae ml^− 1) versus variable temperature (24-28 °C) and high stocking density (8-9 larvae ml^− 1). The first trial significantly increased larval survival and growth, which in turn resulted in greater numbers of settled spat, in comparison of the second trial, where survival, growth, and settlement of spat were significantly lower. Also in 2006, the quality of seawater used at the hatchery was evaluated with microbiological and chemical tests. The implication of these tests, together with results from all experiments are analyzed and discussed in terms of the potential development of large-scale hatchery cultivation of P. mazatlanica larvae in Mexico.     

利用马氏珠母贝4个壳色系F3培育厚层优质珍珠

为了评价红、金黄、白和黑壳色系F3育珠性状,设立两个实验,分别利用马氏珠母贝红、金黄、白和黑壳色系F3作为植核(受体)贝和小片(供体)贝进行植核育珠。实验Ⅰ:利用红、金黄、白和黑壳色系F3为插核贝和小片贝建立16个组合,另外利用以普通养殖群体为插核贝、金黄壳色系F3为小片贝建立了1个组合,共建立17个组合,育珠期为24个月;实验Ⅱ:分别利用红、金黄、白和黑壳色系F3为植核贝和小片贝建立16个组合,育珠期为18个月。结果表明,育珠期结束后实验Ⅰ和Ⅱ的4个壳色系F3平均壳高和成活率均存在显著性差异(P<0.05),实验Ⅰ和Ⅱ的黑壳色系F3具有最大的平均壳高,实验Ⅰ金黄壳色系F3具有最高的成活率,实验Ⅱ黑壳色系F3具有最高的成活率。实验Ⅰ:各组合间的留核率、商品珠率、优良珠率和育珠绩效均存在显著性差异(P<0.05),珍珠层厚度差异不显著(P>0.05);BW组具有最大的留核率、商品珠率、珠层厚度和育珠绩效值,RB组具有最大的优良珠率;实验Ⅱ:各组合的留核率、商品珠率、优良珠率、珍珠层厚度和育珠绩效均存在显著性差异(P<0.05)。黑壳色系F3作为插核贝具有较好的育珠效果,经进一步测试其育珠性能,可在珍珠生产中推广应用。     

Studies on aneuploid pearl oyster (Pinctada martensii Dunker) produced by crossing triploid females and a diploid male following the inhibition of PB1

Viable aneuploid embryos of pearl oyster, Pinctada martensii Dunker, were produced by inhibiting the first polar body (PB1) with cytochalasin B treatment in eggs from triploids fertilized with haploid sperm. During the period of growth measurement, aneuploid showed the slowest growth compared with diploid and triploid groups. The body size and weight measurement data showed that there were no differences between aneuploids (as a group) and diploids in body size and weight (P>0.10), but that they were significantly different from triploids (P<0.01). There were no differences between aneuploids within diploid condition (2n±n) and diploids in SL (P>0.1), but significant differences in BW were found (P<0.05). Aneuploids within triploid condition (3n±n) were significantly smaller than triploids in BW (P<0.05), but not different from triploids in SL (P>0.05), and almost identical to diploids in both (P>0.1). These dada indicated that some aneuploids might be associated with growth retardation. Karyotype analysis revealed that there were metacentric, submetacentric or subtelocentric chromosomes losses or gains; aneuploid pearl oysters with the same chromosome numbers had different chromosome composition. Aneuploids are valuable research materials for genetic analysis.     

利用微卫星标记进行马氏珠母贝家系遗传结构分析与系谱认证

为改良马氏珠母贝养殖群体性状,2010年4月,从马氏珠母贝选育系F3选择性腺成熟个体为亲本,建立了36个家系,按照常规技术进行幼体培育和海区养成.2010年11月,从36个家系中随机选取4个家系,每个家系取样30个个体,利用13对微卫星引物进行家系遗传结构和系谱鉴别分析.结果显示,(1) 13个微卫星引物在4个家系中共检测到39个等位基因,每个位点的等位基因数为2～5,4个家系的平均观测杂合度(Ho)为0.531 ～0.597,平均期望杂合度(He)为0.474 ～0.507;(2)根据子代基因型成功地推断出4个家系的亲本基因型,据此鉴别各个家系;用UPGMA法对120个样本进行聚类分析,98.3％的同一家系的子代个体能够聚到一起,分类结果与系谱来源基本一致.结果说明,这4个家系具有较高的遗传多样性和家系间具有明显的遗传分化;微卫星标记能有效地为马氏珠母贝群体的系谱分析提供技术支持.     

Realized heritability and response to selection for shell height in the pearl oyster Pinctada fucata (Gould)

The common pearl oyster, Pinctada fucata (Gould), is the most important species that is cultured for production of marine pearls in China. Heavy mortality and the decline of pearl quality have resulted in a breeding programme being established in recent years. In this study, we conducted selective breeding for the second generation of pearl oyster P. fucata (JCS-2) by mass selection for shell height (SH) with a selection intensity of 1.614, and analysed the growth of the selected line (JCS-2) and the non-selected control line (JCC) during a 1-year grow-out period. The results show that the selected line grew faster than the control one in the SH and total weight (TW) ( P <0.05), and there were higher proportion of larger sized oysters. Coefficient of variation for SH of JCS-2 was smaller than that of JCC. The current genetic gains and realized heritability for JCS-2 averaged 16.03 ± 4.79% and 0.713 ± 0.208 at 3–15 months of age respectively. The findings indicated the selection response to faster growth for SH is markedly effective in the second generation, and there was a high correlated response of TW when selecting for SH.     

Chemical cues promote settlement in larvae of the green-lipped mussel, Perna canaliculus

The large (70,000 tonnes) mussel aquaculture industry for the endemic New Zealand green-lipped mussel, Perna canaliculus Gmelin, currently relies almost entirely (80%) on wild caught seed that is often harvested attached to great quantities of floating algae. Natural vagaries in catches of wild mussel seed frequently result in shortages of mussel seed that, at times, have severely affected commercial aquaculture production. Both field and laboratory experiments were used to establish that chemical cues derived from algae significantly increase settlement of mussel larvae on artificial substrates. This is the first time an algal chemical cue has been implicated in the settlement behavior of P. canaliculus larvae. These results have potential commercial implications for improving mussel seed supply through inducing higher or more reliable levels of mussel settlement for aquaculture seed collection.     

合浦珠母贝C-型凝集素基因的序列特征和功能分析

通过EST筛选结合重测序法获得合浦珠母贝一种C-型凝集素cDNA的全长序列(命名为PoLEC1)。PoLEC1全长988 bp,5′-UTR为33 bp,3′-UTR为101 bp,开放阅读框为864 bp,编码287个氨基酸,分子量为31.68 ku,理论等电点约5.83。预测的氨基酸序列中含有信号肽(Met¹-Ser¹⁹)和糖结合位点。同源性分析结果表明,PoLEC1与其它物种C-型凝集素的的糖识别结构域序列的同源性在18.8%~28.6%,相似性在28.9%~50.0%之间。进化树分析结果表明,PoLEC1与栉孔扇贝聚为一支。组织表达分析表明,PoLEC1 mRNA在消化腺、外套膜、性腺、闭壳肌、肠、鳃和血淋巴组织均有表达,在消化腺中的表达分析表明,经溶藻弧菌刺激后2 h表达显著下调,而在4和24 h后表达显著上调。利用PoLEC1成熟肽构建原核表达载体,在大肠杆菌中进行重组表达。制备包涵体后,用IDA HisBind 树脂纯化得到单一的重组蛋白,凝菌试验表明该蛋白对大肠杆菌有明显的凝集作用。     

溶藻弧菌诱导马氏珠母贝血淋巴cDNA差减文库的构建及分析

为了探讨马氏珠母贝免疫防御机制,筛选免疫防御相关功能基因,以致病性溶藻弧菌人工感染的马氏珠母贝血淋巴为材料,采用抑制性差减杂交(SSH)技术,构建了溶藻弧菌诱导的马氏珠母贝血淋巴cDNA差减文库;以马氏珠母贝管家基因β-actin作为差减指标检测该文库的差减效率;对选取的600个阳性克隆进行测序及生物信息学分析。结果显示,该文库的差减效率可达210倍;PCR阳性检测显示差减片段为100~750 bp,对随机挑取600个克隆的测序结果显示,共获得414个有效EST序列;通过BLAST同源性比对,有167个EST序列(占40.34%)与NCBI数据库中已知功能蛋白质具较高同源性,其中7个EST序列(占1.69%)与免疫防御相关基因同源;此外,204个EST(占49.28%)在数据库中未发现同源序列。研究表明,SSH技术能有效富集马氏珠母贝血淋巴差异表达基因,研究结果为探索马氏珠母贝免疫基因作用机理和调控机制奠定了基础。     

合浦珠母贝matrilin-1基因的克隆和表达分析

以合浦珠母贝(Pinctada fucata)转录组中的matrilin-1基因片段为基础,开展了matrilin-1的c DNA全长(Pfmatrilin-1)克隆和定量表达分析。Pfmatrilin-1基因c DNA全长2 036 bp,开放阅读框(open reading frame,ORF)1 194 bp,编码397个氨基酸,包括1个信号肽序列和2个血管性血友病因子A(von Willebrand factor A,VWA)样结构域。系统进化分析显示无脊椎动物的matrilins与脊椎动物的matrilins进化关系较远。荧光定量PCR表达分析结果显示,Pfmatrilin-1 mRNA在合浦珠母贝各个组织中均有表达,其中在血液中表达量最高(P0.05);Pfmatrilin-1从担轮幼虫期至变态期均有表达,眼点期表达量最高(P0.05),而贝类幼虫发育到眼点期就会开始附着,眼点期幼虫原壳生长停滞,次生壳形成,因此推测其可能与次生壳的形成有关。这些结果表明Pfmatrilin-1在合浦珠母贝生物矿化中发挥重要作用。     

合浦珠母贝章鱼胺受体(OAR)基因的克隆与表达分析

章鱼胺受体(octopamine receptor,OAR)广泛存在于无脊椎动物中,具有调节神经肌源性节奏收缩、调节c AMP水平、参与磷酸化途径等多种重要功能,但其在贝类中的研究却很少。该研究克隆了合浦珠母贝(Pinctada fucata)OAR基因(pf OAR)的c DNA全长序列,比较分析了氨基酸序列的同源性,并采用荧光定量PCR技术检测了该基因在不同组织和幼体不同发育阶段的表达差异以及贝壳损伤后该基因在外套膜中表达水平的变化。结果显示:pf OAR的c DNA全长1 890 bp,开放阅读框1 659 bp,编码552个氨基酸,有7个跨膜结构域,具有G蛋白偶联受体的共性。pf OAR在6个被检测的组织中均有表达,且在外套膜中的表达水平显著高于其他组织;幼体发育过程中pf OAR表达水平逐渐升高,变态期的表达水平显著高于其他时期;人为损伤贝壳36 h后,pf OAR的表达水平显著升高。这些结果表明pf OAR在合浦珠母贝中可能具有多样化的功能,参与了贝壳的形成和修复,为进一步开展pf OAR的功能研究奠定了重要基础。     

Ingestion and digestion of 10 species of microalgae by winged pearl oyster Pteria sterna (Gould, 1851) larvae

Ten species of microalgae were tested for ingestion and digestion in Pteria sterna larvae using epifluorescence microscopy to choose an appropriate diet. An experiment was conducted using 2, 4, 5 (straight-hinge) 10 and 22 (umbo stage) day old larvae. Larvae were stocked in 150 ml flasks at 30 ml⁻¹ and fed 100,000 algal cells ml⁻¹ of each species individually. Larvae were fed for 1 h and then were observed under the microscope to detect ingestion; larvae were then sieved and placed in flasks containing filtered seawater and were observed after 1 and 2 h to analyse digestion for the microalgae ingested. Out of the 10 species administered, only Nannochloris sp., Pavlova lutheri and Isochrysis aff. galbana (T-ISO) were ingested, and only the last two species were digested. No ingestion of Phaeodactylum tricornutum, Chaetoceros muelleri, Ch. calcitrans, Thalassiosira weisflogii, Dunaliella salina, Tetraselmis tetrathele and T. suecica was evident at any stage of larval development tested. Only T-ISO and P. lutheri should be used for larval rearing of P. sterna until other species are identified as suitable (ingested and digested) for this species.     

Determination of mortality in exploited freshwater pearl mussel (Margaritifera margaritifera) populations

Estimates of mortality, based on relative numbers of live mussels and empty shells observed were obtained for a sample of seven exploited Scottish freshwater pearl mussel (Margaritifera margaritifera) populations. Total mortality (Z) estimates ranged from 0.058 to 0.189 year⁻¹ and fishing mortality (F) estimates ranged from 0.005 to 0.129 year⁻¹. General trends of increasing Z and F with age were apparent. Pearl fishers generally targeted larger, older mussels. In one heavily exploited population, the Z and F estimates converged, indicating that most of the mortality in older mussels (>50 years) was due to the effects of destructive pearl fishing. Since a complete ban on pearl fishing was introduced in 1998, illegal pearl fishing has continued albeit at a reduced level.     

马氏珠母贝Bcl-2-like基因克隆及其功能初探

B淋巴细胞瘤-2(B-cell lymphoma, Bcl-2)基因作为一种重要的细胞凋亡调控基因,在内源性细胞凋亡通路中发挥着重要的调控作用。实验利用c DNA末端快速扩增(RACE)技术克隆获得PmBcl-2-like基因cDNA全长序列,并对其序列进行生物信息学分析;利用实时定量PCR(qPCR)技术分析了PmBcl-2-like在马氏珠母贝不同组织、不同发育时期以及不同免疫刺激后的表达水平。结果显示,PmBcl-2-like cDNA全长为2 180 bp,开放阅读框长度为1 650 bp,共编码549个氨基酸,分子量为21.62 ku;结构域预测分析表明PmBcl-2-like含有Bcl-2家族典型的BH1-4结构域;多序列比对以及进化树构建结果表明,PmBcl-2-like与其他物种的相似度较高,保守性较强;荧光定量结果表明PmBcl-2-like在马氏珠母贝8个组织中均有表达,在鳃中表达量最高,其次为性腺,表达量最低为中央膜区;在胚胎期表达量较高,受精卵时期表达量最高。机体受到脂多糖(LPS)刺激后,相对表达量在24 h达到最高,72 h降到最低,最高约为最低的2.5倍;肽聚糖(PGN)刺激后,相对表达量在6 h达到最高,48 h降到最低,最高约为最低的7.28倍;聚肌胞苷酸(Poly:IC)刺激后,相对表达量在3 h达到最高,12 h降到最低,最高约为最低的6.49倍。研究表明,PmBcl-2-like可能在马氏珠母贝发育和免疫防御反应中担任着重要的角色。     

北部湾钦州港近江牡蛎重金属污染分析

为近江牡蛎的食用安全及养殖环境监督管理提供科学依据,2015年7—11月在北部湾钦州港采集近江牡蛎样品,利用原子荧光光度计和原子吸收光谱仪测定近江牡蛎体内重金属含量,分析其污染程度。结果显示,调查海域牡蛎体内重金属平均含量从高到低依次为ZnCuCdCrAsPbHg,其中Zn、Cu含量较高,平均值分别为532.03、126.96 mg/kg。Cu参照《无公害食品水产品有毒有害物质限量》(NY 5073-2006),发现除了7月2号站样品未超标,其余样品均超标,超标率达89%。As参照《食品安全国家标准食品中污染物限量》(GB 2762-2012),发现总超标率为11%,仅7月3号站样品存在超标。Pb、Hg含量较低,其平均值分别为0.02、0.009 mg/kg。重金属富集系数揭示近江牡蛎对Zn、Cu、Cd、Cr富集严重。膳食暴露评估方法显示,重金属的膳食摄入量低于联合国粮食和农业组织的食品添加剂联合专家委员会(JECFA)推荐的暂定每周摄入量,钦州湾近江牡蛎属于安全食用范围。