牡蛎中糖蛋白成分的分离纯化及其性质研究

本文以青岛产牡蛎为原料,通过低温水提取工艺得到牡蛎糖蛋白粗提物,然后用凝胶柱层析(Sephacryls-100 HR)进行纯化,最后用高效液相色谱制备得到纯度较高的物质F22.由SDS-聚丙烯酰胺凝胶电泳证实F22是纯度较高的单一组分;分子量为34.2 kDa;等电聚焦的结果显示等电点为5.5;红外色谱呈现出典型的糖的特征吸收峰;气相色谱分析结果显示F22的中性单糖是由葡萄糖这一种单糖组成的同多糖;β-消去反应表明F22的糖肽键应为N-型糖苷键.     

Echinoderms (Echinodermata) from the Gulf of California, Mexico

A systematic list of the echinoderms of the Gulf of California, based on museum specimens of the Colección Nacional de Equinodermos, Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autónoma de México and the National Museum of Natural History, Smithsonian Institution, Washington, D.C. is presented. A total of 193 echinoderm species is recorded, distributed in 108 genera, 51 families and 19 orders. 12 new records for the Gulf of California are presented: Asteroidea (four), Ophiuroidea (three) and Holothuroidea (five).     

Comparison of quantitative gonad maturation scales in a temperate oyster (Crassostrea gigas) and a sub-tropical oyster (Crassostrea corteziensis)

Quantitative scales to evaluate maturity of female gonads were compared for a temperate oyster, Crassostrea gigas and a tropical oyster, Crassostrea corteziensis grown in the same locality. C. gigas had well-defined maturation and spawning periods and a resting phase in winter; in C. corteziensis mature individuals occurred most of the year and there were several spawning peaks. Of the quantitative scales used here, average oocyte diameter and gonad coverage area, much used for C. gigas, and ovary maturity index, less used, were inadequate to distinguish the reproductive pattern of C. corteziensis, since they both skewed the degree of maturation in vitellogenic stages in favor of C. gigas. Maximum oocyte diameter and maximum cytoplasm area were different among species at vitellogenic stages and also in previtellogenic stages. Nucleus:cytoplasm ratio was significantly different in previtellogenic and spawned stages between C. gigas and C. corteziensis. The Index of gonadal development was skewed in favor of C. gigas in postvitellogenic stage. The only scale that was comparable between species was reproductive potential, but it also was one of the most laborious. Other ordinal scales commonly used, such as a visual external evaluation of the gonad, only classified correctly a quarter of the stages.     

Sporulation of Minchinia sp. (Haplosporida, Haplosporidiidae) in the Pacific Oyster Crassostrea gigas (Thunberg) from the Republic of Korea

SYNOPSIS. A haplosporidan parasite belonging to the genus Minchinia was found in 4 of 1,438 oysters, Crassostrea gigas , collected from the Republic of Korea. Multinucleated vegetative stages were present in the infected oysters. Spores found in one of these oysters were acid-fast and had operculate characteristics of the genus Minchinia .     

Deep-water polychaetes (Annelida) from the southeastern Gulf of California, Mexico

Polychaetes inhabiting deep-sea soft bottoms from the southeastern Gulf of California were collected during four oceanographic cruises during 2000 and 2001. Sampling of benthic organisms was performed with a benthic sledge to collect epifauna and a Karling dredge for epifauna and infauna, in a depth range from 732 to 2 250 m. A list of the polychaetes that were collected and their distribution are presented here. A total of 73 species (distributed among 33 families) were identified. Moreover, 11 species were identified only to genus level and 20 species only to family level. With the exception of Ancistrosyllis hartmanae and Melinnampharete eoa, all identified species have been previously reported in soft bottoms of the Gulf of California or in adjacent areas. Additional previously unreported information is provided herein regarding depth ranges, geographical distribution, morphology and tubes inhabited by the organisms. The morphology of the ampharetids Amage sp. and Samytha sp. does not coincide with that of other species in these genera reported for the Gulf of California, which suggests that they are probably undescribed species.     

Survival,Growth and Reproduction of Cryopreserved Larvae from a Marine Invertebrate,the Pacific Oyster (Crassostrea gigas)

This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13±2 hours post fertilization: hpf), early D-larvae (24±2 hpf) and late D-larvae (43±2 hpf). From the beginning (88 days) at the end of the ongrowing phase (195 days), no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days), survival of the oysters cryopreserved at 13±2 hpf and at 43±2 hpf was significantly higher (P<0.001) than those of the control (non cryopreserved larvae). Only the batches cryopreserved at 24±2 hpf showed lower survival than the control. Reproductive integrity of the mature oysters, formely cryopreserved at 13±2 hpf and 24±2 hpf, was estimated by the sperm movement and the larval development of their offspring in 13 crosses gamete pools (five males and five females in each pool). In all but two crosses out of 13 tested (P<0.001), development rates of the offspring were not significantly different between frozen and unfrozen parents. In all, the growth and reproductive performances of oysters formerly cryopreserved at larval stages are close to those of controls. Furthermore, these performances did not differ between the three initial larval stages of cryopreservation. The utility of larvae cryopreservation is discussed and compared with the cryopreservation of gametes as a technique for selection programs and shellfish cryobanking.     

Primary Structure of Myostracal Prism Soluble Protein (MPSP) in Oyster Shell, Crassostrea gigas

Soluble protein (MPSP, myostracal prism soluble protein) obtained from myostracum in oyster shell (Crassostrea gigas) was characterized using biochemical and molecular biological techniques. From an analysis of secondary protein structure, it was shown that β-structure was predominant in MPSP. And via in vitro assays, the relation of MPSP to biomineral phase and morphology was studied. SDS-PAGE revealed one major protein band of 20 kDa. An amino acid sequence of 160 amino acids was deduced for myostracum by characterization of the complementary DNA encoding the protein. The deduced protein was composed of a high proportion of Gly and Asp, typifying a calcium-binding protein for shell formation, and a relatively high proportion of Val, Ala and Ile, typifying an adhesive protein. In contrast to prevailing expectations, (Gly–Asp)n-type sequence motifs exist in MPSP, demanding a revision of previous theories of protein–mineral interactions. The cDNA sequence of myostracum is elucidated for the first time.     

Development of Novel Microsatellite DNA Markers from the Pacific Oyster Crassostrea gigas

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2–20; observed heterozygosity, 0.119–0.925; unbiased expected heterozygosity, 0.139–0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.     

Reactive Oxygen Species in Unstimulated Hemocytes of the Pacific Oyster Crassostrea gigas: A Mitochondrial Involvement

The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ_m) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ_m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ_m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.     

Analysis of Stomach and Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) from Coastal Louisiana,USA

We used high throughput pyrosequencing to characterize stomach and gut content microbiomes of Crassostrea virginica, the Easter oyster, obtained from two sites, one in Barataria Bay (Hackberry Bay) and the other in Terrebonne Bay (Lake Caillou), Louisiana, USA. Stomach microbiomes in oysters from Hackberry Bay were overwhelmingly dominated by Mollicutes most closely related to Mycoplasma; a more rich community dominated by Planctomyctes occurred in Lake Caillou oyster stomachs. Gut communities for oysters from both sites differed from stomach communities, and harbored a relatively diverse assemblage of phylotypes. Phylotypes most closely related to Shewanella and a Chloroflexi strain dominated the Lake Caillou and Hackberry Bay gut microbiota, respectively. While many members of the stomach and gut microbiomes appeared to be transients or opportunists, a putative core microbiome was identified based on phylotypes that occurred in all stomach or gut samples only. The putative core stomach microbiome comprised 5 OTUs in 3 phyla, while the putative core gut microbiome contained 44 OTUs in 12 phyla. These results collectively revealed novel microbial communities within the oyster digestive system, the functions of the oyster microbiome are largely unknown. A comparison of microbiomes from Louisiana oysters with bacterial communities reported for other marine invertebrates and fish indicated that molluscan microbiomes were more similar to each other than to microbiomes of polychaetes, decapods and fish.     

太平洋牡蛎i型溶菌酶基因的克隆与重组高效表达

在双壳类软体动物牡蛎体内,溶菌酶(Lysozyme)在实现宿主免疫防御,破坏和消除侵入体内的病原中发挥着重要的作用.根据GenBank已有的太平洋牡蛎溶菌酶的全长cDNA序列(GenBank:AB179775),通过RT-PCR技术,从太平洋牡蛎(Crassostrea gigas)中克隆得到溶菌酶(简称为CgLys)基因的开放阅读框(open reading frame,ORF)序列.生物信息软件分析表明,其ORF为414 bp,编码137个氨基酸(aa),前20个aa为信号肽,成熟肽由117个aa组成,其分子量为13.2 kD.通过构建分子系统发育树对其同源性进行分析比较,初步推断该CgLys属于i型溶菌酶.将该CgLys基因的成熟肽亚克隆进原核表达载体pET-32a(+)中,构建重组质粒pET32a(+)-CgLys,再转化至大肠杆菌BL21(DE3)pLysS.该基因工程菌经IPTG诱导发酵后,成功高效地表达了重组CgLys蛋白,其分子质量约为18 kD.该重组CgLya蛋白主要存在于细菌裂解液的上清液中,即以可溶性蛋白形式存在.上述结果将显著简化后续的蛋白纯化过程,为今后扩大规模生产牡蛎溶菌酶提供参考.     

Effects of Dietary Cystine and Tyrosine Supplementation on Melanin Synthesis in the Pacific Oyster (Crassostrea gigas)

Marine Biotechnology - Melanogenesis is a multistep process to produce melanin for dark pigmentation in skin coloration. Previous studies in vertebrates demonstrated that cystine and tyrosine amino...     

Blooms of Cochlodinium polykrikoides (Gymnodiniaceae) in the Gulf of California, Mexico

Cochlodinium polykrikoides was the species responsible for the discoloration that occurred between September 15th and 27th, 2000 in a shallow coastal lagoon located in the southern part of the Bahia de La Paz, on the west side of the Gulf of California. Blooms of C. polykrikoides were observed four days after two rainy days with a seawater temperature of 29 to 31 degrees C. Nutrient concentration ranges during the bloom were 0.165-0.897 microM NO2+NO3, 0.16-3.25 microM PO4, and 1.0-35.36 microM SiO4. Abundance of C. polykrikoides ranged from 360 x 10(3) to 7.05 x 10(6)/cells l(-1). Biomass expressed in terms of chlorophyll a was high, ranging from 2.7 to 56.8 mg/m3. A typical dinoflagellate pigment profile (chlorophyll a and c, peridinin, diadinoxantin, and beta-carotene) was recorded. In this study, the red tide occurred in front of several fish and shrimp-culture ponds. No PST toxins were found in the samples. However, 180 fish were found dead in the infected fish-pond; the gills were the most affected part. C. polykrikoides is a cyst-forming species that recurs in this area. New blooms were observed in November 2000 and September-November 2001 in the same area. Anthropogenic activities, such as eutrophication caused by water discharge in this shallow lagoon, and nutrient enrichment in the culture ponds, as well as effects from precipitation and wind stress, could have favored the outbreak of this dinoflagellate.     

Population demography and genetic characteristics of the Pacific Oyster Crassostrea gigas in Japan

The Pacific oyster Crassostrea gigas is a commercially important bivalve distributed along the northwest Pacific coast. Here C. gigas in Japan was investigated using mtDNA and microsatellite markers to elucidate its genetic structure and phylogeny. On the basis of mtDNA all populations showed high genetic diversity with limited genetic differentiation among populations. The pattern of MtDNA diversity suggested that C. gigas had experienced population expansion about 112 Kya, prior to the last glacial maximum (LGM), which accorded well with other marine organisms. For microsatellites, a Bayesian-based assignment test demonstrated that C. gigas is nearly panmictic. However, on the basis of estimates of F_ST, Kumano populations differed significantly from other populations, a recent occurrence based on low R_ST. Irrespective of geographical distance, genetic similarity was observed in the main aquaculture regions with large-scale transportation of cultured spat. Unlike in the Yellow Sea, a genetic bottleneck was not detected in Japanese populations. These results imply, contrary to the prevailing view, that C. gigas in Japan was demographically stable during the LGM. Gene flow by larval dispersal seems to be regionally restricted to localities of congenital areas by ocean currents, while genetic homogenization by cultivated oysters might have occurred in aquaculture areas.     

Age-specific birth rates of California sea lions (Zalophus californianus) in the Gulf of California, Mexico

Estimates of demographic parameters are essential for assessing the status of populations and assigning conservation priority. In light of the difficulties associated with obtaining such estimates, vital rates are rarely available even for well-studied species. We present the first estimates of age-specific birth rates for female California sea lions ( Zalophus californianus ) >10 yr of age. These rates were estimated from the reproductive histories of five cohorts of animals branded as pups between 1980 and 1984 at Los Islotes colony in the Gulf of California, Mexico. Age-specific birth rates varied among age classes and ranged between 0.06 and 0.80. The highest birth rates were observed for females between 10 and 15 yr of age, with decreased birth rates among older females. The effect of age, year, and resighting effort were explored using logistic regression analysis. Based on Akaike Information Criteria, birth rates were best explained by female age, while year and resighting effort did not have a significant effect. The odds ratio of producing a pup decreased with age but did not change significantly for middle-aged females. Our estimates of age-specific birth rates are consistent with general patterns observed for other large vertebrates.     

Isolation of sperm bindin from the oyster (Crassostrea gigas)

A method was devised for isolating the insoluble content of the acrosome granule of sperm of the oyster Crassostrea gigas. The method involves the dissolution of the entire cell, except for the acrosome granule, in the detergent sodium lauroyl sarcosinate (sarcosy I). The isolated acrosome granule content is ring-shaped and is 84% protein by weight. SDS-polyacrylamide gel electro-phoresis of this material yields from 1 to 4 bands of 65,000; 53,000; 43,000 and 34,000 apparent molecular weight, all of which stain with the PAS reaction indicating the material is a glycoprotein. The 65,000 molecular weight component is always present, but the presence of the other three bands varies with each preparation. The isolated acrosome granules agglutinate formaldehyde-fixed oyster eggs. A trypsin-generated glycopeptide digest of oyster egg surfaces inhibits the agglutinin activity of the isolated acrosome granules. We propose that the acrosomal glyco-protein material is oyster sperm bindin which functions as the adhesive substance bonding the sperm to the egg during fertilizaion.