利用AFLP技术分析海扇贝与三种扇贝的亲缘关系

本研究采用AFLP技术,对2005年引自加拿大的海扇贝(Placopecten magellanicus)以及中国北方主要养殖的虾夷扇贝(Patinopecten yessoensis)、栉孔扇贝(Chlamys farreri)和海湾扇贝(Argopecten irradians)进行了遗传学比较分析。结果表明,共用5对AFLP引物进行了扩增,共扩增出501个位点,多态性位点为392个,多态位点比例为78.24%。平均Nei氏基因多样性指数H分别为0.2139、0.1684、0.1441和0.1670,平均Shannon氏指数I分别为0.3356、0.2681、0.2439和0.2656。海扇贝多态位点比例、Nei氏基因多样性指数和Shannon氏指数均处在较高水平,说明海扇贝遗传多样性较高。UPGMA聚类结果表明,海扇贝与虾夷扇贝亲缘关系最近。

采用16S rRNA基因序列分析海扇贝与3种扇贝的亲缘关系

采用16S rRNA基因序列分析技术,对2005年引自加拿大的海扇贝Placopecten magellanicus以及中国现有种类——虾夷扇贝P.yessoensis、栉孔扇贝Chlamys farreri和海湾扇贝Argopecten irradians进行了遗传学比较分析。每个扇贝种群随机抽取2个个体,利用通用引物进行16S rRNA基因片段扩增并双向测序。所得序列去掉不可信位点后,得到433 bp的基因片段。序列比对分析得到306个变异位点,其中转换位点为124个,颠换位点为111个,既有转换又有颠换的位点为45个,插入和缺失为26个。核苷酸变异值及分子系统树结果表明,海扇贝与虾夷扇贝的亲缘关系较近。

Screening microsatellite markers from EST sequences of bay scallop Argopecten irradians

Seventy-five simple sequence repeats （SSRs） were identified by the bioinformatic analysis from 5008 expressed sequence tags （ESTs） of Argopecten irradians. Among the SSRs, the number of repeat nucleotide varied from 2 to 6. Dinucleotide and trinueleotide repeat motifs were dominant in EST-SSRs of bay scallop, with a proportion of 80% over the total screened SSRs. Twenty-nine pairs of primer were designed based on the flank sequences of the selected ESTs using the software of Primet 5, and verified under the given PCR reaction condition. Eighteen of the 29 primer pairs resulted in the expected products, while the remaining either failed to produce any fragments or yielded products over expected size. Thirteen of the 18 SSRs, accounting for 72%, were detected to show polymorphism in the examined scallop samples. A preliminary test in this study indicated that the majority of the identified SSRs were informative in the cultured bay scallops, making them suitable for the population and other genetic analysis. EST-SSR markers have more advantages than the traditional genomic-derived SSRs and there is a wide range of application in comparative mapping, functional gene cloning and marker assisted selection. This research provides a reference to the identification of EST-SSRs with relative bioinformatic analysis from aquaculture species, as well as to those with a large number of ESTs.

Yield trait improvement of bay scallops following complete diallel crosses between different scallop stocks

This study describes the complete diaUel hybridization between newly introduced bay scallop stock （W） from Canada and local commercial stock （D） grown under laboratory conditions, in China. Larval survival and growth during all life stages （larvae, spat, and adult） were compared among hybrid （DW, WD） and purebred （DD, WW） populations. Significant heterosis was detected for survival during the larval stage （〉 1% of the mid-parent values）. The mean heterosis （Hm） varied in growth throughout the life span. More than 50% of the Hm values were positive and negative in the DW and WD groups, respectively. The influence of maternal effects and mating types （intrapopulation vs. interpopulation crosses） on growth for all life stages was not consistent. Larval survival did not differ significantly （P〉0.05） with maternal effect or mating type. In the harvest stage, shell length （SL）, shell height （SH）, shell width （SW）, and total weight （TW） were larger in the hybrid compared with the inbred groups. Positive Hm values were observed in SL （1.5%）, SW （5.8%）, and TW （12.3%）, and were more significant in the DW groups （6.1%, 4.5%, 6.8%, and 27.2%）. These results suggest that hybridization between two geographic populations is a good tool for improving bay scallop growth. However, unstable heterosis between the two populations requires further study.

Immunotoxicity Effect of Benzo[α]Pyrene on Scallop Chlamys farreri

The toxic effects of benzo[α]pyrene （B[α]P） at different concentrations （0.1, 0.5, 1, 2.5 and 7.5 μgL^-1） on scallop （Chlamys farreri） immune system were studied. The results showed that B[α]P had significant toxic effects on the haemocyte counts, neutral red uptake, phagocytosis, bacteriolytic and antibacterial activity （P〈0.05）, while the seawater control and acetone control had no significant differences. The haemocyte counts, neutral red uptake, phagocytosis and bacteriolytic activity in all B[α]P treatment groups as well as antibacterial activity in groups of 0.5, 1, 2.5 and 7.5 μgL^-1·B[α]P decreased significantly （P〈0.05）. Some of these indices tended to be stable on the sixth day and others on the ninth day, and the indices showed clear time- and concentration-response to B[α]E Bactenolytic activity in 0.1 μgL^-1 B[α]P treatment group and antibacterial activity in 0.1 ggLl and 0.5 μgL^-1 B[α]P treatment groups increased at the beginning of exposure and reached their peaks on day 1 and day 6, respectively. Following that, both activities decreased gradually and became stable after day 9. When all the indices reached stability, they were significantly lower than those in control group （P〈0.05）, except for antibacterial activity in 0.1 μgLl B[α]P treatment group （P〉0.05）. Thus, B[α]P has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and lmmunomodulation in aquatic organisms.

Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.

Variations in retention efficiency of bivalves to different concentrations and organic content of suspended particles

Retention efficiencies (RE) of scallop (Chlamys farreri),oyster (Crassostrea gigas) and mussel (Mytilus edulis) in a flow-through system were measured to understand the short-term response to various particle and organic matter concentrations.By comparing the RE of C.farreri with that of C.gigas and M.edulis,we gained further knowledge on the feeding physiological characteristics of C.farreri and ascertained the possible cause of high summer mortalities of this species.The experimental feeding conditions included natural differences in the abundance and composition of suspended seston,as well as conditions in which seston abundance and composition were manipulated by adding natural silt or cultured microalgae.The results show that in natural sea water,the minimum particle size for maximal retention in M.edulis,C.gigas and C.farreri was approximately 4,6,and 8 μm,respectively;the RE of 2-μm (equivalent spherical diameter) particles was 17%,19%,and 8%,respectively;and the relative standardized RE was 58%,49%,and 18%,respectively.In C.gigas and M.edulis,the minimal particle size for maximal retention did not change with food quality (organic content).C.farreri was more sensitive to fluctuations in particle concentration and organic content.With particle concentration increase,the minimal particle size for maximal retention in scallop shifted to large particles and the RE for 2-μm particle decreased from 8% in natural seawater to 1.6%-6% in silt-enriched groups.With organic content increase,the minimal particle size for maximal retention shifted from 8 to 5 μm in natural seawater.Variation in RE of C.farreri with food conditions and the relationship between lower RE and smaller particle size may hamper C.farreri from food taking due to the decrease in the size distribution of phytoplankton in Sungo Bay.

Responses to two-way selection on growth in mass-spawned F_1 progeny of Argopecten irradians concentricus(Say)

In the present study, the effect of one-generation divergent selection on the growth and survival of the bay scallop （Argopecten irradians concentricus） was examined to evaluate the efficacy of a selection program currently being carried out in Beibu Bay in the South China Sea. A total of 146 adult scallops were randomly selected from the same cultured population ofA. i. concentricus, and divided into two groups in shell length （anterior-posterior measurement）： large （4.91-6.02 cm, n=74） and small （3.31-4.18 cm, n=72）. At the same time, a control group was also randomly sampled （4.21-4.88 cm, n=80）. Mass-spawned F1 progenies from the three size groups were obtained and reared under identical conditions at all growth phases. The effects of two-way （or upward-downward） selection on fertilization rate, hatching rate, survival rate, daily growth in shell length and body weight were assessed in the three size groups. Results show that significant differences （P〈0.01） were found in hatching rate, survival rate and daily growth of F~ progenies, but not in fertilization rate （P〉0.05）, among the three groups. The hatching rate, survival rate and daily growth of the progeny of large-sized parents were greater than those of the control group （P〈0.05）, which in turn were larger than those of small-sized group （P〈0.05）. Responses to selection by shell length and body weight were 0.32±0.04 cm and 2.18±0.05 g, respectively, for the upward selection, and -0.14±0.03 cm and -2.77±0.06 g, respectively, for the downward selection. The realized heritability estimates of shell length and body weight were 0.38±0.06 cm and 0.22±0.07 g for the upward selection, and 0.24±0.06 cm and 0.37±0.09 g for the downward selection, respectively. The change in growth by bidirectional selection suggests that high genetic variation may be present in the cultured bay scallop population in China.

Survival,growth and immune activity of scallop Chlamys farreri cultured at different depths in Haizhou Bay(Yellow Sea,China) during hot season

Survival, growth and immune response of the scallop, Chlamys farreri, cultured in lantern nets at five different depths (2, 5, 10, 15, and 20 m below the sea surface) were studied in Haizhou Bay during the hot season (summer and autumn) of 2007. Survival and growth rates were quantified bimonthly. Immune activities in hemolymph (superoxide dismutase (SOD) and acid phosphatase (ACP)) were measured to evaluate the health of scallops at the end of the study. Environmental parameters at the five depths were also monitored during the experiment. Mortalities mainly occurred during summer. Survival of scallops suspended at 15 m (78.0%) and 20 m (86.7%) was significantly higher than at 2 m (62.9%), 5 m (60.8%) or 10 m (66.8%) at the end of the study. Mean shell height grew significantly faster at 10 m (205.0 μm/d) and 20 m (236.9 μm/d) than at 2, 5 or 15 m in summer (July 9 to September 1); however, shell growth rate at 20 m was significantly lower than at the other four depths in autumn (September 2 to November 6). In contrast to summer, scallops at 5 m grew faster (262.9 μm/d) during autumn. The growth of soft tissue at different depths showed a similar trend to the shell. Growth rates of shell height and soft tissue were faster in autumn than in summer, with the exception of shell height at 20 m. SOD activity of scallops increased with depth, and ACP activity was significantly higher at 15 and 20 m than at other depths, which suggests that scallops were healthier near the bottom. Factors explaining the depth-related mortality and growth of scallops are also discussed. We conclude that the mass mortality of scallop, C. farreri, during summer can be prevented by moving the culture area to deeper water and yield can be maximized by suspending the scallops in deep water during summer and then transferring them to shallow water in autumn.

Importance of kelp-derived organic carbon to the scallop Chlamys farreri in an integrated multi-trophic aquaculture system

Bivalves and seaweeds are important cleaners that are widely used in integrated multi-trophic aquaculture （IMTA） systems. A beneficial relationship between seaweed and bivalve in the seaweed- based IMTA system has been confirmed, but the trophic importance of seaweed-derived particulate organic materials to the co-cultured bivalve is still unclear. We evaluated the trophic importance of the kelp Saccharinajaponica to the co-cultured scallop Chlamysfarreri in a typical IMTA farm in Sungo Bay （Weihai, North China）. The dynamics of detritus carbon in the water were monitored during the culturing period. The proportion of kelp-derived organic matter in the diet of the co-cultured scallop was assessed via the stable carbon isotope method. Results showed that the detritus carbon in the water ranged from 75.52 to 265.19 ~tg/L, which was 25.6% to 73.8% of total particulate organic carbon （TPOC） during the study period. The amount of detritus carbon and its proportion in the TPOC changed throughout the culture cycle of the kelp. Stable carbon isotope analysis showed that the cultured scallop obtained 14.1% to 42.8% of its tissue carbon from the kelp, and that the percentages were closely correlated with the proportion of detritus carbon in the water （F=0.993, P=0.003）. Evaluation showed that for 17 000 tons （wet weight） of annual scallop production, the kelp contributed about 139.3 tons of carbon （535.8 tons of dry mass）. This confirms that cultured kelp plays a similar trophic role in IMTA systems as it does in a natural kelp bed. It is a major contributor to the detritus pool and supplies a vital food source to filter-feeding scallops in the IMTA system, especially during winter and early spring when phytoplankton are scarce.

Ecological effects of co-culturing sea cucumber Apostichopus japonicus (Selenka) with scallop Chlamys farreri in earthen ponds

Monthly changes in sedimentation and sediment properties were studied for three different culture treatments： sea cucumber monoculture （Mc）, sea cucumber and scallop polyculture （Ps-c） and scallop monoculture （Ms）. Results indicated that the survival rate of sea cucumber was significantly higher in Ps-c cultures than in Mc cultures. Sea cucumber yield was 69.6% higher in Ps-c culture than in Mc culture. No significant differences in body weight and scallop shell length were found between Ps-c and Ms cultures. The mean sedimentation rate of total particulate matter （TPM） was 72.2 g/（m^2.d） in Ps-c cultures, with a maximum of 119.7 g/（mE.d）, which was markedly higher than that of Mc （mean value）. Sedimentation rates of organic matter （OM）, total organic carbon （TOC）, total nitrogen （TN） and total phosphorus （TP） in Ps-c cultures were also significantly higher than those in Mc cultures. TOC and TN contents of sediment increased rapidly in the first 5 months in Ms cultures and remained at a high level. TOC and TN contents in Mc and Ps-c cultures decreased during sea cucumber feeding seasons and increased during sea cucumber dormancy periods （summer and winter）. The study demonstrates that co-culture of sea cucumber and scallop in earthen ponds is an alternative way to alleviate nutrient loads and improve water quality in coastal aquaculture systems. Moreover, it provides the additional benefit of an increased sea cucumber yield.

Development of 101 Novel EST-Derived Single Nucleotide Polymorphism Markers for Zhikong Scallop (Chlamys farreri)

Zhikong scallop(Chlamys farreri) is an important maricultured species in China.Many researches on this species,such as population genetics and QTL fine-mapping,need a large number of molecular markers.In this study,based on the expressed sequence tags(EST),a total of 300 putative single nucleotide polymorphisms(SNPs) were selected and validated using high resolution melting(HRM) technology with unlabeled probe.Of them,101(33.7%) were found to be polymorphic in 48 individuals from 4 populations.Further evaluation with 48 individuals from Qingdao population showed that all the polymorphic loci had two alleles with the minor allele frequency ranged from 0.046 to 0.500.The observed and expected heterozygosities ranged from 0.000 to 0.925 and from 0.089 to 0.505,respectively.Fifteen loci deviated significantly from Hardy-Weinberg equilibrium and significant linkage disequilibrate was detected in one pair of markers.BLASTx gave significant hits for 72 of the 101 polymorphic SNP-containing ESTs.Thirty four polymorphic SNP loci were predicted to be non-synonymous substitutions as they caused either the change of codons(33 SNPs) or pretermination of translation(1 SNP).The markers developed can be used for the population studies and genetic improvement on Zhikong scallop.