珠母贝人工培育黑珍珠的影响因素分析

采用不同术前处理方法、不同贝龄和不同珠核规格的实验条件,探讨珠母贝(Pinctada margaritifera)育珠效果的影响因素.术前处理方法包括温差促排和阴干促排,贝龄设为1.5龄、2.0龄、2.5龄和3.0龄,珠核直径设为5.5 mm、6.0mm、6.5 mm和7.0mm.经1个月的休养和21个月的育珠,结果显示,休养与育珠期间,温差促排、阴干促排和未处理组间的成活率没有显著性差异(P>0.05),但留核率、收珠率和优珠率存在显著性差异(P<0.05),其中温差促排组和未处理组分别具有最高和最低的留核率、收珠率和优珠率;4个贝龄组的成活率、留核率和收珠率存在显著性差异(P<0.05),其中1.5龄组具有最高的成活率,2龄组具有最高的留核率和收珠率,3龄组具有最低的成活率、留核率和收珠率;4个珠核规格组间的成活率、留核率和收珠率存在显著性差异(P<0.05),其中6.0 mm组和 7.0 mm组分别具有最高和最低的留核率和收珠率.研究结果说明,术前处理具有明显提高珠母贝植核贝的成活率、留核率和收珠率;珠母贝第一次植核的最适贝龄应是1.5～2龄:珠母贝第一次植核的珠核适宜规格为6.0 mm.本研究旨在为优质珍珠的培育提供理论参考.     

Feeding the pearl oyster Pinctada margaritifera during reproductive conditioning

This study aimed to model the food intake of P. margaritifera to examine the relationship between food level and reproductive activity. The effect of microalgae concentration on ingestion rate and assimilation efficiency was studied over a broad concentration range, using a mixture of Isochrysis galbana and Chaetoceros gracilis. Reproductive effort was assessed using three microalgae concentrations of 0.5, 7 and 18 cell μL^?1. Reproductive status was assessed by gonad development index (GDI) – the ratio of the gonad surface to the visceral mass surface – and histological analysis of the gonad based on the presence (continuous or discontinuous) or the absence of gonial cells (GC). Ingestion is a saturating function of seston concentration for bivalves modelled with an adapted Michaelis‐Menten function. The maximum ingestion rate of P. margaritifera adults was 193.50 × 10⁶ cell h^?1 g^?1 dw and the half saturation coefficient was 15 cell μL^?1. The concentration of 18 cell μL^?1, supplied for 45 days, induced a significantly higher GDI than the other treatments. GC decreased significantly and even stopped when pearl oysters were under‐fed, suggesting that the mitotic process of the germinal stem cells was altered. Differentiation of germinal stem cells therefore appears to be controlled by food availability.     

Quantitative measurement of reproductive output in the American oyster, Crassostrea virginica (Gmelin), using an enzyme-linked immunosorbent assay (ELISA)

Abstract. A quantitative gonadal index was developed for oysters, Crassostrea virginica (Gmelin. 1791), using polyclonal antibodies from eggs and sperm. Percoll used in the purification of oyster eggs and sperm greatly improved the purity of antigens compared to filtering the egg or sperm through a fine mesh only. The antigen-antibody reaction was tested with indirect sandwich ELISA using alkaline phosphatase-conjugated goat anti-rabbit igG as a secondary antibody. Rabbit anti-oyster egg IgG and anti-oyster sperm IgG initially exhibited a weak cross-reactivity over somatic tissue. Absoring with acetone-dried oyster tissue powder removed this cross-reactivity. Both antisera exhibited strong specific immunological reactions to oyster eggs or sperm respectively. The quantity of eggs or sperm was measured using ELISA and a quantitative gonadosomatic index (dry wt of egg or sperm/dry wt oyster) (GSI) calculated. GSI from ELISA correlated with gonadal stage measured histologically. Monthly mean GSI of female oysters was highest during late spring to early summer (0·157–0·201) and lowest during early winter to early spring (0·002-0·000). Maximum GSI observed during the study was 0·422 for female oysters and 0·446 for male oysters. Female oysters produce 3·7–65·4 million eggs, with an average of 21·1 million during each spawning. A positive correlation was observed between the number of eggs produced and oyster size; the number of eggs in the gonad increased as oyster size (i.e. total dry wt) increased (r= 0·67); however, the relationship was non-linear. Large oysters contained proportionally fewer eggs. Prevalence of Perkinsus marinus parasitism was high, 90–100%, during the study, as was weighted incidence, 1·33 to 2·67, No statistically significant correlation was observed between infection intensity and the per cent weight of oyster eggs or egg number.     

珠母贝幼虫运动、自然沉降及摄食行为

主要通过分析幼虫形态变化、幼虫运动、自然沉降及摄食行为等生物学信息,为珠母贝(Pinctada margaritifera)幼虫培育设施及饵料投喂策略提供优化思路。在显微镜下观察不同日龄幼虫形态变化、幼虫运动状态、测量幼虫运动速率,观察幼虫静置下自然沉降及幼虫对单胞藻摄食过程。结果显示,幼虫依靠面盘外周纤毛摆动维持运动并完成摄食,运动时幼虫开壳朝上,壳顶朝下,面盘伸出壳外牵引幼虫运动。俯视观幼虫绕一定半径的圆顺时针方向运动,垂直方向螺旋上升或下降。水平运动速率范围246.88~3641.94μm/s,运动速率v(μm/s)与壳长l(μm)之间存在的关系为v=-0.0841l2+37.2690l-2149.1031(R~2=0.9707),运动速率与环绕半径显著相关(P0.05)。随着幼虫生长,体重增加,面盘逐渐退化,面盘纤毛摆动难以维持幼虫悬浮,静置时幼虫沉降率增加,13 dph(days post hatching,孵化后天数)后沉降至底部的幼虫壳长显著(P0.05)大于悬浮幼虫。幼虫饵料投喂过多,导致饵料不消化或不完全消化就排出体外,以致摄食的饵料不能完全消化利用。     

Abnormal periostracum secretion during the mineralization process of the pearl in the blacklip pearl oyster Pinctada margaritifera

Whitish parasite-like bodies (PLBs), 0.5–2 μm in size, were detected within the pearl bag of some grafted pearl oysters, Pinctada margaritifera, from Takapoto island (French Polynesia). Histological and ultrastructural investigations have revealed that these bodies consisted of lamellar organic material locally secreted by some epithelial cells of the pearl bag. The fine structure and the formation of these bodies are described. The causes of this abnormal secretion of organic mater, which actually corresponds to periostracum, are not presently known. No parasite nor infectious micro-organism was found associated with the PLBs.     

Changes in fatty acid and sterol composition during oogenesis in the pearl oyster Pinctada margaritifera

The fatty acid and sterol composition of the oyster Pinctada margaritifera during oogenesis and in eggs was analysed. No major differences were observed during oogenesis, but the egg composition was significantly different from that of gonads. The amount of saturated fatty acids was the highest in eggs and the C16:0 predominant (P<5%); by contrast, the amount of 22:6(n‐3) was significantly lower (P<5%) than in gonads. No major differences were observed for the polar lipid (PL) composition during oogenesis. The main free sterols in gonads and eggs were cholesterol and brassicasterol. Among free sterols, the proportion of cholesterol diminished continuously from the beginning to the end of gonad maturation, and this decrease persisted in eggs after spawning. Cholesterol represented 40% to 55% of the sterol ester encountered in gonad and eggs. This study allowed us to investigate the fatty acid and sterol composition during oogenesis of the pearl oyster P. margaritifera, leading to a clearer understanding of the nutritional requirements of pearl oyster during the reproduction process.     

Influence of nacre deposition rate on cultured pearl grade and colour in the black-lipped pearl oyster Pinctada margaritifera using farmed donor families

Cultured pearl nacre deposition rate in Pinctada margaritifera oysters was studied to examine its influence on Tahitian classification grade and colour and to further our understanding of using selected donor oysters produced in a hatchery system for the improvement of these traits. A large-scale grafting experiment using 150 farmed donor oysters was realised in Rangiroa atoll (French Polynesia). After 18 months of culture, 874 pearls were harvested and 7 pearls quality traits were recorded: (1) nacre deposition rate assessed by nacre thickness and weight, (2) classification grade, lustre and surface defects, and (3) colour assessed by pearl colour darkness level and visual colour categories. Nacre deposition rate significantly influence pearl grade, surface defects, darkness level and colour categories, but not the lustre. Grade A pearls had the thickest and heaviest nacre on average, compared with grade D pearls and rejects. In addition, pearls without any surface defect had thicker and heavier nacre. Pearls with low darkness were the thinnest and the lightest. Pearls of aubergine–peacock secondary colours were the thickest and the heaviest. These results highlight the importance of rapid nacre deposition rate for increasing the production of grade A pearls, with less surface defects and dominance of a black tone colour. Fast nacre deposition may, however, have a negative impact on other pearl quality traits, such as pearl lustre. This study demonstrates the importance of examining relationships between pearl quality traits in a way that provides solid information that can be directly used for breeding and genetic selection programmes.     

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.     

An enzyme-linked immunosorbent assay (ELISA) for detecting penaeid baculovirus

Abstract. A technique for detecting shrimp haculovirus based upon the sandwich indirect enzyme-linked immunosorbent assay ( elisa ) was developed. Baculovirus antiserums were developed in rabbits and guinea-pigs. Wells of microagglutination plates were coated with guinea-pig antiserum. Various dilutions of the purified virus preparation suspended either in Hanks' balanced salt solution (BSS) or in 30% hepatopancreas tissue in BSS were placed in wells. After washing, secondary anti-serum (rabbit antibaculovirus serum) was added. Goat horseradish peroxidase-labelled anti-rabbit globulin was used to detect reactions between baculovirus and rabbit antiserum. At least 10 ng viral protein could be detected by the technique.     

Growth and gonad development of the tropical black-lip pearl oyster, Pinctada margaritifera (L.), in the Gambier archipelago (French Polynesia)

The growth and reproductive cycle of cultured black-lipped pearl oysters, Pinctada margaritifera (L.), were studied in the Gambier Islands (134°52′ W, 23°07′ S) from September 2002 to August 2003. Temperatures were recorded throughout the year, revealing seasonal temperature variations between 22.3 and 27.8°C. The mean annual chlorophyll a value, as computed from satellite data, was 0.188 ± 0.075 μg L⁻¹. To study growth and reproduction, 720 two-year-old individuals were ear hung on long-lines suspended at a depth of 7 m. Samples were taken twice a month to obtain the following measurements: shell height; wet weight of flesh and total oyster; dry weight of adductor muscle, mantle and visceral mass; and glycogen content. Gonad development was also studied by histology on parallel samples. Growth was relatively fast during the first 6 months of the study: average shell height increased from 89.1 ± 9.1 to 119.7 ± 10.8 mm and total weight from 93.4 ± 24.5 to 155.1 ± 33.6 g, between September and the end of March. Subsequently, from April to August, no significant growth was observed for shell and flesh, while the muscle weight decreased significantly. Condition index (CI), defined as the ratio of wet weight of the visceral mass to shell weight, and histological changes in the gonad revealed 3 significant reproductive events of different intensities. The analysis of correlations revealed a specific effect of the chlorophyll a concentration on the growth of shell and soma, and one of the temperature on tissue glycogen content. This study also showed also that CI could be an efficient indicator of reproductive events in pearl oyster. It thus appears that the development of gonads goes on throughout the year in the Gambier Islands, without any detectable phase of sexual rest.     

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.     

迟缓爱德华氏菌间接ELISA快速检测法

以迟缓爱德华氏菌(Edwardsiella tarda)WY28作为抗原,免疫新西兰兔,获得效价为1:2 048的多克隆抗体;以此多克隆抗体作为-抗,山羊抗兔IgG-HRP作为酶标二抗,建立迟缓爱德华氏菌的间接EIJSA快速检测法.采用棋盘滴定法确定抗原与一抗的最佳工作浓度分别为106 CFU·mL-1和1:10 000;酶标二抗的最适工作浓度为1:1 000.病原菌检测灵敏度为每孔103 CFU.该方法标准化后具有快速、灵敏等特性,与肠杆菌科其他细菌参考菌株无交叉反应,具良好的特异性.对养殖场发病大菱鲆(Scophthalmus maximus)和半滑舌鳎中分离的细菌菌株进行检测,从56株分离菌株中检测出27株迟缓爱德华氏菌,阳性检出率为48.2%.该方法的建立有助于快速准确地诊断由迟缓爱德华氏菌引起的养殖鱼类病害.     

Impact of season and grafter skill on nucleus retention and pearl oyster mortality rate in Pinctada margaritifera aquaculture

The mollusc Pinctada margaritifera is the top economic aquaculture species in French Polynesia (export value) and forms the basis of the black pearl industry. Mass production of this unique gem, produced by a living organism, 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 receiving pearl oyster. This technique is performed by expert grafters, whose work constitutes the first step influencing pearl farm production yield. This study makes the first report of effects mediated by individual grafters and by the season of grafting on three rates scored at 45 days post operation: nucleus retention, nucleus rejection and pearl oyster mortality. These results were obtained in a very large scale grafting experiment, designed and conducted in a single culture site in the atoll of Arutua (Tuamotu Archipelago) and involving a total of 52,910 grafts performed by ten professional grafters, during two contrasting seasons: autumn (four grafting experiments) and spring (three grafting experiments). Statistical analysis using linear mixed-effect model for both univariate and multivariate analysis was performed. The results show a very highly significant season effect (p < 0.001), with a higher rate of nucleus retention in autumn than spring: 91.1 versus 79.4 %, respectively. A very highly significant season effect was also recorded for nucleus rejection rate (p < 0.001), which was greater in spring than in autumn: 12.6 versus 7.7 %. Oyster mortality rate was up to six times higher in spring than in autumn (p < 0.001). Within the two seasons, there was no significant difference between the grafting experiments for any of the three variables, adding robustness to the inter-season results. Taking into account this seasonal variation effect, grafter skill had a significant impact on the three variables (p < 0.001), making it possible to rank grafters from most to least efficient. Aside from skill differences between grafters, grafting in autumn may significantly increase graft success. Water temperature and its indirect consequences could be one of the most important environmental factors implicated in overall efficiency. These findings may help the potential development of an annual grafting schedule according to season/ lagoon temperature as a way to maximize production yield for the black-lipped pearl oyster industry in French Polynesia.     

Development of an enzyme-linked immunosorbent assay (ELISA) for vitellogenin measurement in greenback flounder Rhombosolea tapirina

Vitellogenin (Vtg) was purified from the plasma of 17-estradiol (E₂)-injected male greenback flounder,Rhombosolea tapirina. The molecular weight of the native Vtg was estimated by gel filtration as 540 kD. SDS-PAGE and Western blotting analyses indicated that this protein consisted of three bands with molecular weights of 155, 104, 79 kD, respectively. A polyclonal antibody against the highest molecular weight band of putative Vtg was generated in sheep and an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA) was developed. The assay was validatedfor plasma Vtg measurement in greenback flounder. Serial dilutions of plasma from vitellogenic females parallelled the standard Vtg curve, whereas no cross-reaction was observed with the plasma of males in the ELISA. The Vtg ELISA was used to assess the induction of Vtg by E₂ in vivo in males. The induction of Vtg in greenback flounder showed a time- and dose-dependent response as in other species. In E₂-treated fish, detectable levels of Vtg were first found at 48 h, and reached a peak at 96 h post-injection. Plasma levels of Vtg increased as the E2 dose increased with a threshold of 0.1 mg kg^–1.     

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.     

The combined effects of temperature and salinity on embryos and larvae of the black-lip pearl oyster, Pinctada margaritifera (L.)

This paper reports on a 4 × 4 factorial design experiment conducted to examine the combined effects of temperature and salinity on embryonic development and growth and survival of black-lip pearl oyster, Pinctada margaritifera (L.) larvae. The temperatures used were 20 °C, 25 °C, 30 °C and 35 °C, and the salinities were 25°/_oo, 30°/_oo, 35°/_oo and 40°/_oo. Response surface contour diagrams were generated from the survival and growth data to estimate optimal conditions. Normal development of embryos occurred only from 25 °C to 30 °C. The optimal conditions for maximum survival and growth were 26–29 °C and 28–32°/_oo. Temperatures of 35 °C or greater were lethal for larvae and, at all temperatures tested, larval growth and survival were lowest at a salinity of 40°/_oo.     

Variation in clearance and ingestion rates by larvae of the black-lip pearl oyster (Pinctada margaritifera, L.) feeding on various microalgae

Clearance rate (CR) and ingestion rate (IR) of different sizes (89, 125 and 188 μm shell length) of Pinctada margaritifera larvae were determined when feeding on various microalgae. The microalgae tested were the diatoms, Chaetoceros muelleri and C. simplex, and flagellates, Tahitian Isochrysis aff. galbana, Pavlova lutheri and P. salina at 5 or 10 cells μL^–1. Both CR and IR of microalgae tested in this study increased with increasing larval size; but at all larval sizes, diatoms resulted in lower CR and IR. Of the microalgae tested, P. margaritifera larvae showed greatest CR and IR with the two Pavlova spp. Maximum CR for P. salina was 10.5, 21.2 and 29.7 μL h^–1 for larvae with shell lengths of 89, 125 and 188 μm, respectively. The highest IR values for P. margaritifera larvae with shell lengths of 89, 125 and 188 μm were 8.7, 81.0 and 165.7 cells·larva^–1 h^–1, respectively. CR and IR of P. salina were approximately five times higher than those recorded for C. muelleri and C. simplex.