Age‐dependence of cultured pearl grade and colour in the black‐lipped pearl oyster Pinctada margaritifera

Pinctada margaritifera is an economically important marine bivalve species for cultured pearl production in French Polynesian aquaculture. In order to evaluate the influence of donor oyster age on pearl quality traits, experiments were conducted over 6 years using both grafts and surgreffe operations. At harvest, six pearl quality traits were recorded and compared: surface defects, lustre, grade, darkness level and visual colour. Analysing the quality traits of pearls harvested in the initial graft process and those of pearls obtained from surgreffe experiments allowed a comparison of the influence of pearl sac cells originating from the initial mantle graft, which aged together with their recipient oysters. The results demonstrated a significant decrease between these successive grafts in lustre, grade (A‐B‐C), darkness level, and green colour – traits that are of major importance in the pearl market. The duplicated graft experiment allowed the comparison of donor oyster families at 2 and 5 years old, where a mantle graft was inserted into recipient oysters aged 2.5 years. The results showed the same tendencies to a lesser extent, with (i) an improved pearl grade, predominantly through a most important rate of 0 surface defect category, and (ii) a green/grey ratio in favour of the younger donor. A comparison between the graft‐surgreffe and the duplicated graft experiments also highlighted: (i) the indirect role played by the younger recipient oysters, which must be optimized for optimal pearl quality realization, and (ii) the complex interplay between the donor and recipient oysters.     

三角帆蚌珍珠囊细胞的分泌活动

本文对三角帆蚌珍珠囊细胞的分泌活动进行了亚显微结构的研究。发现珍珠囊表皮细胞能积极地进行物质合成 ,这些物质主要是蛋白质、硫酸化粘多糖和中性粘多糖 ,并将这些物质以微绒毛分泌、块状物分泌、细胞间隙分泌和大颗粒分泌等多种方式分泌到珍珠囊腔 ;而位于珍珠囊表皮细胞之间的、来源于外套膜结缔组织的腺细胞则含有丰富的中性粘多糖 ,并通过开口式分泌的方式释放到珍珠囊腔 ,同珍珠囊表皮细胞分泌的物质一起 ,共同参与珍珠结晶层的形成 ;珍珠囊细胞分泌的多样性与珍珠组成的复杂有关 ;珍珠囊细胞的分泌活动具有节律性和区段性的特点 ,这种区段性和节律性的分泌与珍珠多层结晶纤层的形成相适应。     

How cultured pearls acquire their colour

Round nucleated pearls are produced through a surgical operation, where a round nucleus and a mantle tissue ‘saibo’ from donor oyster are inserted into the gonad of the host oyster. The epithelial cells in the mantle tissue proliferate around the nucleus, and thus, the pearl sac is formed. Pearl sac secrets nacre and forms a pearl. The quality and economic value of pearls are assessed by pearl features such as colour, brightness, lustre and shape. Among all these features, colour has been reported as an important economic indicator and has been widely studied by researchers. Generally, pearl colour is affected by the donor oyster which is determined genetically and biological pigments (melanin and carotenoid). Organic matrices, metal ions and other factors have also been reported to influence the colour of a cultured pearl. Recently, multi‐omics methods have been used to study the colour formation of pearl, and some key genes and signal pathways related to the colour formation of pearls have been identified. Nevertheless, the specific mechanism of pearl formation needs further research. The review combines both fresh and sea water pearls focusing on Hyriopsis cumingii and pearl oysters to provide a general overview and understanding for pearl colour formation.     

Donor effect on cultured pearl nacre development and shell matrix gene expression in Pinctada margaritifera reared in different field sites

Understanding the respective roles played by donor and recipient pearl oysters in pearl quality determination in relation to the environment is a challenge for the pearl industry. In most Pinctada species, pearl size is mainly related to recipient oyster growth performance but also relies to some extent on the biomineralisation activity of the pearl sac, a tissue that originates from the donor oyster mantle. We examined donor effect on pearl size in response to culture in the lagoons on Arutua and Apataki atolls. Overall, nacre weight and thickness were greater in Arutua than in Apataki, but sensitivity to the environment differed between donors. Some donors were associated with significantly heavier and thicker nacre in Arutua (I group), while others had similar results at the two sites (NI group). On average, up to 20% of the pearl size could be attributed to the donor but, in group I, donor effect was responsible for up to 36% of nacre weight determination. Additionally, a real‐time PCR expression study of eight matrix protein genes related to biomineralisation in the pearl sac showed that MSI60, pearlin and pif177 were significantly and positively correlated with nacre weight and thickness, with the latter two genes explaining the larger pearl size observed in Arutua. Donor oysters in P. margaritifera therefore play a key role in pearl size improvement, related to the role of the shell matrix protein genes. Understanding such contributions could help in the design of genetic selection plans for specific and adapted donor oyster lines.     

三角帆蚌生长性状和内壳色与所产无核珍珠质量的相关性分析

为了研究供片蚌和育珠蚌对无核珍珠质量的影响,以三角帆蚌紫色选育系F₅为材料,在插植无核珍珠前,测量了供片蚌和育珠蚌的壳长、壳高、壳宽、体质量等生长性状,检测了供片蚌内壳色颜色参数L、a、b、dE。经过18个月育珠,测量了育珠蚌的壳长、壳高、壳宽、体质量,计算其特定生长率,检测了育珠蚌内壳色颜色参数L、a、b、dE,测量了育珠蚌所产无核珍珠的颜色、大小、圆度、光泽和产珠量。结果发现,供片蚌生长性状与所产无核珍珠大小、圆度和产珠量相关性不显著(P>0.05)。供片蚌内壳色与无核珍珠颜色相关性极显著(r=-0.400~0.376,P<0.01),供片蚌dE值越大所产紫色珍珠比例越高、dE值越小所产白色珍珠比例越高。育珠蚌特定生长率与所产无核珍珠大小、光泽和产珠量相关性极显著(r=0.237~0.516,P<0.01),相关程度为体重> 壳长> 壳宽> 壳高,育珠蚌特定生长率与所产无核珍珠圆度相关性极显著(r=-0.284~-0.256,P<0.01),相关程度为壳长>体质量>壳宽>壳高。育珠蚌内壳色与所产无核珍珠颜色、大小、圆度、光泽和产珠量相关性不显著(P>0.05)。综合各性状相关性分析,改良供片蚌内壳色可以改良珍珠颜色,改良育珠蚌的生长性状可以改良珍珠大小、光泽、圆度和产珠量。     

三角帆蚌内脏团培育圆形有核珍珠的试验

报导了三角帆蚌(Hyriopsis cumingii)内脏团培育圆形有核珍珠的试验结果。结果显示:插植珠核的7362只手术蚌植核后一个月内成活率99.2%。经23个月养殖后,随机抽取50只。结果留核率达94%,成珠率为86%,其中中高档珠占37.2%。有核珍珠直径在9.4~11.6 mm之间,其中10 mm以上占88.4%。11.6%的珍珠表面有小于3 mm的凸起,未发现带扁而长尾巴的珍珠。试验表明:三角帆蚌内脏团内能培育出高品质的有核珍珠。此外还发现,脱核或核片分离的细胞小片仍可在内脏团内形成珍珠。     

体外培养三角帆蚌外套膜细胞的生物学特性及有核珍珠培育

通过光学显微镜、透射电镜、流式细胞仪等方法对体外培养的三角帆蚌(Hyriopsis cumingiiLea)外套膜细胞进行观察和检测,证明其具有与蚌体内细胞相同的生物学结构、分裂增殖能力和分泌珍珠质的生物活性。在此基础上,对三角帆蚌有核珍珠培育技术进行探讨。将培养细胞黏附在表面经过促黏壁物质处理的珠核表面,再插入育珠蚌体内培育,120 d后,蚌体内可形成完备的珍珠囊,并在珠核表面有明显的珍珠质沉积;6个月后,珍珠质可将整个珠核包裹起来形成有核珍珠。本研究初步证明了细胞法培育有核珍珠的可行性。[中国水产科学,2007,14(1):149-154]     

三角帆蚌EFCB1基因cDNA序列克隆及表达研究

为了发掘更多三角帆蚌具有EF-hand结构域的功能基因及其蛋白质,本研究运用RACE-PCR技术,克隆得到了三角帆蚌包含EF-hand结构域钙结合蛋白1基因(EF-hand calcium-binding domain-containing protein 1,EFCB1)的cDNA全长并进行了生物信息学分析;通过real-time Q-PCR技术,分析了EFCB1基因在三角帆蚌10个组织,以及内脏团、外套膜插核后不同时间点的时空表达特点。结果表明三角帆蚌EFCB1基因cDNA序列全长981 bp,ORF为531 bp,编码176个氨基酸残基,5'-UTR 239 bp和3'-UTR 211 bp。EFCB1分子式为C₈₇₇H₁₃₄₈N₂₃₈O₂₇₀S₁₀,分子量约19.9 ku,等电点为4.70,不稳定系数为62.65,属亲水蛋白。其序列无信号肽序列,存在1个跨膜区域和2个EF-hand结构域,EF-hand模块分别为DLNDDKLISPEE(98-109)和DTNGDDKLDGEE(129-140)。荧光定量结果显示三角帆蚌EFCB1基因在各组织中均有表达,其中在肠和鳃中表达量最高(P<0.05),外套膜中表达量显著高于内脏团(P<0.05)。EFCB1基因在插核后不同时期的外套膜和内脏团育珠部位组织中表达具有显著差异(P<0.05),在外套膜中的表达量均显著高于内脏团(P<0.05),在插核后第20 天时表达量显著高于各时期(P<0.05)。研究表明,EFCB1在三角帆蚌Ca²⁺的吸收过程中发挥调节作用,在珍珠囊形成过程中以及珍珠形成初期具有重要功能。     

Contribution of donor and host oysters to the cultured pearl colour in Pinctada martensii

Cultured free round pearls are produced by implanting a spherical nucleus with a small piece of nacre‐secreting mantle graft from a donor oyster into the gonad of a recipient oyster (host). To examine the possible contribution of host and donor oysters to the colouration of the harvested pearls, the CSE‐1 Imaging and Color‐Measuring System were used to quantitatively measure the L*a*b* values of donor and host shells and the produced pearls in Pinctada martensii. Results showed that the colour of pearls had significant positive correlation (r = 0.1–0.22, P = 0.00) with that of donors, but had no correlation with that of host oysters, thus convincingly confirmed the contribution of nacre colour of donor to the realization of pearl quality of colour. To further clarify the relationship between the donor and the pearl colour, the donors from pearls of good and poor colour quality were further compared and the results demonstrated the significant difference in L* values (P < 0.05) and insignificant difference in a* and b* values, suggesting the necessity of selecting donors with bright and lustrous nacre in pearl production.     

转录组测序研究三角帆蚌珍珠颜色相关基因

为筛选影响三角帆蚌珍珠颜色的候选基因,采用转录组测序平台Illumina Hi Seq TM2500对蚌壳珍珠层颜色为紫色和白色的三角帆蚌中央膜组织进行高通量测序。所得序列经质控、组装后比对到NR、Swiss-Prot、COG、KOG、KEGG、GO和Pfam数据库中注释,并进行差异基因聚类分析。结果显示,获得干净数据共72 800 581 bp,拼接获得了89 529个Unigene,差异表达基因2644个,其中上调表达基因1323个,下调表达基因1321个。根据GO功能分类可分为分子功能、细胞组分、生物过程等3大类50分支;根据KEGG代谢通路分析可以分为187类。对差异表达基因分析发现,部分基因与色素合成(眼黄素、黑色素、喋啶色素)及金属离子转运相关,还有6个细胞色素P450,3个细胞色素b561和1个细胞色素b560。研究表明,这些基因可能在珍珠颜色形成中发挥作用。     

应用激光共聚焦显微技术研究Ca2+在三角帆蚌组织内的积累与分布

为了探讨淡水贝类Ca2+吸收和转运信号传递机理,采用激光共聚焦技术研究了三角帆蚌不同组织细胞在静息状态下细胞内游离Ca2+浓度,以及水体Ca2+浓度对外套膜组织细胞内钙沉积的影响。试验选取10只健康的三角帆蚌,分别获得外套膜外膜、内膜、斧足、腮及内脏团上表皮组织细胞,短期培养后用Fluo-3/AM荧光探针孵育细胞1 h,观察细胞内Ca2+荧光强度。研究结果表明,蚌不同组织细胞内Ca2+荧光强度存在显著差异,外套膜外膜细胞内Ca2+荧光强度最高,内脏团上表皮细胞的最低(P<0.05);育珠三角帆蚌在相同Ca2+浓度的孵育水平下,外套膜细胞内Ca2+荧光强度比非育珠蚌都有增加的趋势,其中在1.25 mmol/L添加组中,两组外套膜内膜细胞内Ca2+荧光强度差异达到显著水平(P<0.05);不同Ca2+孵育水平对细胞内Ca2+荧光强度有显著影响(P<0.05),随着Ca2+在孵育液中浓度的升高,外套膜细胞内Ca2+荧光强度显著增强(P<0.05),表明外套膜是从外界吸收Ca2+的主要组织,蚌体珍珠的培育增强了其对Ca2+的沉积,并且水体Ca2+浓度在1.25～3.00 mmol/L有助于蚌体内Ca2+的贮藏,这对进...     

Freshwater nucleated pearl quality is influenced by host mussel growth traits in Hyriopsis cumingii

Hyriopsis cumingii is one of the most important freshwater pearl mussels in China. Recently, this species can produce freshwater nucleated pearls of high quality. Here, we investigated whether nucleated pearl quality is influenced by the growth traits of the host mussel or other factors like cultivation period. We implanted host mussels with a spherical nucleus consisting of a small piece of mantle tissue from donor mussels. After 24 and 36 months of culture, host mussel growth traits including body weight and shell length, height, width and weight were recorded. These factors were then correlated with the quality traits of the pearls they produced, such as nacre thickness, size, weight, lustre and colour. Results indicated pearls obtained at 36 months after seeding were significantly larger in terms of nacre thickness, size and weight compared to those harvested at 24 months. In particular, nacre thickness (r = 0.33–0.48, P = 0.00), pearl size (r = 0.39–0.43, P = 0.00) and pearl weight (r = 0.35–0.47, P = 0.00) were showed to be significantly correlated with host mussels shell length, body weight and shell weight at 24 or 36 months. Larger and heavier host mussels tended to produce bigger pearls. In contrast, host mussels did not affect pearl colour. Cumulatively, our results suggest that longer culturing times and a larger host mussel may help produce better quality nucleated pearl. This information can help guide selective breeding programs designed to improve pearl quality produced by H. cumingii.     

Phenotypic indicators for cultured pearl size improvement in the black‐lipped pearl oyster (Pinctada margaritifera): towards selection for the recipient growth performance

The black‐lipped pearl oyster, Pinctada margaritifera, is the most important farmed species in French Polynesia and the basis of the most valuable export industry. Mass production of black pearls relies on a surgical operation requiring tissue from a donor pearl oyster to be grafted, together with a nucleus made of shell, into the gonad of a recipient oyster. Improving pearl size through family selection remains one of the main challenges for future aquaculture development. This study analyses the relative contribution of donor and recipient oysters to pearl size. To this end, hatchery‐produced donor oysters of two batches, large and small (based on shell height), were used to supply grafts for recipients, which were then monitored individually for their growth performance by recording shell height, width, and thickness, and total live weight (flesh + shells) every 6 months (four biometric measurement times) over 20 months of culture. Pearls issued from the two batches of donors showed no significant differences in nacre weight or thickness. In contrast, recipient oyster shell height and total weight were increasingly positively correlated with these pearl size parameters over the culture period, becoming significant at 8 months post‐grafting. Potential therefore exists to use shell height and oyster weight as phenotypic indicators for selective breeding of recipient oysters with high growth performance to increase pearl size in P. margaritifera.     

Comparison of harvested rate and nacre deposition parameters between cultured pearls issued from initial graft and second nucleus insertion in P. margaritifera

Cultured pearls produced with Pinctada margaritifera, using the surgreffe method (implantation of a second nucleus following pearl harvest) were studied for the first time to: (1) examine family effect on nacre thickness, nacre weight and nacre deposition speed and (2) compare variation in these three traits with that obtained from the cultured pearls previously harvested after the corresponding initial grafts. A surgreffe experiment using 783 recipient oysters was realized in Rangiroa atoll (French Polynesia). After 24 months of culture, 389 cultured pearls were harvested. Significant donor family effect was found for the harvested pearl rate from surgreffe (P = 0.046). Highly significant donor family effect was recorded for nacre thickness (P = 0.004). Very highly significant donor family effects were recorded for nacre weight and nacre deposition speed (P < 0.0001). Comparison between surgreffe and initial graft showed: (1) no significant effect for the average cultured pearl rate harvested (P = 0.052) and average cultured pearl nacre deposition speed (P = 0.622) and (2) very highly significant differences (P < 0.0001) for the average cultured pearl nacre thickness and nacre weight. This study highlighted three major implications for pearl industry management: (1) donor family effect was maintained from initial graft to surgreffe, for nacre thickness, weight and deposition speed, (2) the persistence of the pearl sac metabolic activity over three years of culture and (3) the relation between harvested pearl rate and the size of the nucleus inserted in the pearl sac.     

三角帆蚌不同蚌龄外套膜细胞的增殖能力及其矿化功能

研究了淡水珍珠贝——三角帆蚌(Hyriopsis cumingii)在不同蚌龄下外套膜组织的细胞增殖及生物矿化相关因子活性。选择5组不同蚌龄三角帆蚌(0.5龄、1龄、2龄、3龄、4龄),通过流式细胞技术分析了外套膜细胞的增殖指数(proliferation index,PrI)和胞内Ca~(2+)浓度,并应用实时荧光定量PCR检测了与生物矿化相关的碳酸酐酶(carbonic anhydrase, CA)、碱性磷酸酶(alkaline phosphatase, ALP)基因的表达并测定其酶活性。结果表明, 2龄蚌的外套膜细胞PrI及胞内Ca~(2+)浓度显著高于其他蚌龄(P0.05);生物矿化相关基因在不同蚌龄的外套膜组织中表达量不同, 1龄三角帆蚌CA基因表达量和CA酶活性最高(P0.05), ALP基因表达量和ALP酶活在0.5龄三角帆蚌中显著高于其他蚌龄(P0.05)。本研究旨为深入探讨三角帆蚌外套膜细胞增殖能力及人工育珠过程中供体蚌的选择奠定基础。     

诸暨养殖池塘内水环境、三角帆蚌生长与珍珠产量

2006.06.03-11.08研究了浙江省诸暨市3口蚌养殖池塘内的水温、透明度(SD)、溶氧(DO)、pH等理化因子以及三角帆蚌(Hyriopsis cumingii)生长和珍珠产量。结果显示:实验期间池塘内水温平均为28℃,透明度为24~31 cm,pH为8.5~8.8,DO为6.6~7.8 mg/L,盐度为0.1,Ca2+含量为19.4~37.0 mg/L,总硬度为64.0~123.3 mg CaCO3/L,总碱度为58.4~109.4 mg CaCO3/L,TN为0.76~1.12 mg/L,TP为0.14~0.33 mg/L,TN/TP为4~8,CODMn为9.99~15.5 mg/L。经过158 d生长,池塘内育无核珠或有核珠的3龄蚌蚌壳长增加0.16~0.94 cm,蚌重增加21~61 g;育无核珠的5龄蚌蚌壳长增加0.1~0.25 cm,蚌重增加27~76 g。蚌壳和蚌重生长速度受蚌龄影响,蚌重生长速度的变化幅度往往比蚌壳大。育无核珠的3龄蚌和5龄蚌内珍珠增重分别为每蚌2.03~2.87 g和3.52~5.23 g。3龄蚌蚌壳长和蚌重生长比5龄蚌快,但珍珠产量低于后者,表明三角帆蚌珍珠产量与其蚌壳和蚌重生长速度并不完全一致。     

Freshwater Pearl Culture Technology Development in India

ABSTRACT

Development of science and technology of cultured pearl production in a freshwater environment in India is described. Distribution of Indian pearl mussels, pond mussel Lamellidens marginalis, paddy field mussel L. corrianus, and riverine mussel Parreysia corrugata in relation to environmental variables such as type of soil, nature of sediment substratum, presence or absence of macrophytes (Eichhornia sp., Nechamandra sp., andNymphaea sp.) is described. Food and feeding of the mussels, together with density aspects in culture conditions are discussed. Mussel breeding including specificity in fish hosts, and mussel larval parasitic relationships are discussed. Basic steps involved in indigenous freshwater pearl culture technology are summarized. Pearl culture grafting procedures such as mantle cavity, mantle tissue and gonadal implantation along with different pearl products are also described.     

马氏珠母贝转录组数据的重新组装

对本实验室和其他学者已经发表的马氏珠母贝外套膜和珍珠囊的454焦磷酸测序转录组数据以及NCBI中的EST数据进行了重组装、同源序列比对和GO注释分析。重组装获得了30266个contig,平均长度522 bp,其中最长的contig为4144 bp;NR数据库BLAST分析获取了2310个相似性contig(E≤10?5),其中1902个在模式动物(人、小鼠、海胆、线虫、斑马鱼、果蝇或者太平洋牡蛎和珠母贝)中可以查找到同源序列。基因注释(GO)结果表明,注释到生物学过程的3846个contig可分为23个亚类,其中代谢过程蛋白、细胞过程蛋白和生物学调控蛋白为contig数量最多的3个亚类;注释到分子功能的4601个contig共分为11个亚类,其中结合蛋白的contig数量最多,其次是具有催化活性的蛋白和结构分子活性蛋白;注释到细胞成分的2992个contig分为17个亚类,其中细胞和细胞组成部分的contig数量最多,其次分别是细胞器和大分子复合体contig数量。本研究结果有助于开展外套膜和珍珠囊特异性表达基因的大规模筛选。