Environmental concentration of ammonia nitrogen induced marked changes in proteome of clam Ruditapes philippinarum in dose-and time-dependent manner

Previous studies have revealed that ammonia nitrogen has several adverse effects on clam Ruditapes philippinarum.However,knowledge is lacking regarding the related proteins involved in the toxicological responses,which is vital to elucidate the underlying mechanism of ammonia nitrogen.In this study,clams R.philippinarum were exposed to ammonia nitrogen for 21 d at two environmentally relevant concentrations.The tandem mass tags approach(TMT)was applied to assay the differentially expressed proteins(DEPs)in clam gill tissues on the 3 rd and 21 st day.Finally,a total of 7263 proteins were identified.Bioinformatics analyses revealed that clam protein profiles changed in dose-and time dependent manner after ammonia nitrogen exposure.We inferred that the clams may face heavy challenges after ammonia exposure,such as unbalanced gender ratio,lysosomal disease,energy lack,neurological disorders,altered glutamine metabolism,increased lipid synthesis,and impaired immunity.Variation profiles of enzyme activities of glutaminase and glutamine synthase provided direct evidence to verify the related inference from proteome data.Most of the inferred toxic effects merit further study.This study identified important proteins related to ammonia nitrogen toxicity in the clam and indicated the severe stress of marine ammonia pollution on the healthy development of mollusc aquaculture.

AFLP analysis revealed differences in genetic diversity of four natural populations of Manila clam (Ruditapes philippinarum) in China

The amplified fragment length polymorphism （AFLP） technology was used to analyze the genetic diversities in four natural populations of Manila clam （ Ruditapes philippinarum）, distributed in four sea areas of China, i.e. , the Bohai Sea, the Huanghai Sea, the East China Sea and the South China Sea. Two hundred and sixty-four AFLP loci were analysed in 195 individuals and revealed high levels of genetic diversity. The percentage of polymorphic loci ranged from 92.13% to 96.06% and the Shannon＇ s information index was from 0.256 8 to 0. 275 6. By analyzing molecular variance （ AMOVA）, it was found that there were high levels of genetic differentiation between populations of Qingdao and the other three sea areas. Cluster analysis by Nei＇ s pairwise distances grouped specimens by geographical origin, except the population of Qingdao. A conclusion can be drawn that there are high genetic diversities in the four natural populations of Manila clam in China and some distinct differences existed among and between the four populations. The results also indicated that human cultivation activities will have great influence on the genetic structure of the population of Qingdao.

Inbreeding Depression on Growth and Survival of Full-Sib Family of Manila Clam(Ruditapes philippinarum)

In present study, the inbreeding depression(ID) of growth and survival of Manila clam(Ruditapes philippinarum) was investigated at larval and juvenile stages. Nine inbred families(A_2, B_2, C_2, D_2, E_2, F_2, G_2, H_2 and I_2) were established by mating within nine full-sib families with expected inbreeding coefficient of 0.25. Inbred families showed significant differences in shell length and hatching rate of D-larvae(straight-hinged larvae). The larvae of the nine inbred families grew slower than those of control group(CG), and their ID value ranged from 0.81% ± 6.09% to 16.10% ± 1.49%. The ID value of larval survival rate varied between 27.47% ± 9.36% and 70.50% ± 13.66%. The ID was also detected for juvenile growth in A_2, B_2, C_2, and D_2, which ranged from 4.60 ± 2.21 to 17.71 ± 7.73. The A_2 family maintained the highest juvenile survival rate, whereas the other inbred families exhibited ID values varying between 62.79% ± 4.54% and 96.14% ± 0.87%. The linear relationship of estimated ID between growth and survival was negatively correlated(R =-0.434, P < 0.05). The results of this study suggested that the ID of growth was common at the larval stage but was less prevalent at juvenile stage. In contrast, the ID of survival increased from larval to juvenile stage. A better understanding of the effect of inbreeding may aid to selective breeding of Manila clam.

The influence of salinity on toxicological effects of arsenic in digestive gland of clam Ruditapes philippinarum using metabolomics

Ruditapes philippinarum,a clam that thrives in intertidal zones of various salinities,is a useful biomonitor to marine contaminants.We investigated the influence of dilution to 75% and 50% of normal seawater salinity(31.1) on the responses of the digestive gland of R.philippinarum to arsenic exposure(20 μg/L),using nuclear magnetic resonance(NMR)-based metabolomics.After acute arsenic exposure for 48 h,salinity-dependent differential metabolic responses were detected.In normal seawater,arsenic exposure increased the concentrations of branched-chain amino acids,and of threonine,proline,phosphocholine and adenosine,and it decreased the levels of alanine,hypotaurine,glucose,glycogen and ATP in the digestive glands.Differential changes in metabolic biomarkers observed at lower salinity(～23.3) included elevation of succinate,taurine and ATP,and depletion of branched-chain amino acids,threonine and glutamine.Unique effects of arsenic at the lowest salinity(～15.6) included down-regulation of glutamate,succinate and ADP,and up-regulation of phosphocholine.We conclude that salinity influences the metabolic responses of this clam to arsenic.

Mass Selection for Fast Growth in the Third Generation of the Orange Line of the Manila Clam Ruditapes philippinarum

The Manila clam Ruditapes philippinarum is natively distributed along the Pacific coast of Asia,where it is one of the most important bivalve species for local fisheries.This species has been intentionally introduced due to its high adaptability to various coastal environments and is currently widely cultivated in Europe and North America.We have initiated a selective breeding program to improve the growth rate of the Manila clam since 2007,and a full-sib family with orange shell color was selected.This family features high survival but slow growth.Thereafter,two generations of mass selection were conducted in this family by 10% upward selection for faster growth.In 2011,three types of lines were produced by selecting the breeder from the second-generation of selected lines.These lines are SS(subject to three generations of selection for growth in shell length),SC(selected for the second but not the third generation),and C(randomly sampled individuals from the national population).The genetic parameters of the shell length including hR^2(realized heritability),SR(selection response),and GG(genetic gain)were analyzed.Results showed that the shell length of the SS line was significantly larger than those of the SC and C lines at all developmental stages(P<0.05).For the larval stage,the values of hR^2,SR,and GG were 0.42,0.73,and 6.66,respectively.For the juvenile stage,the values of hR^2,SR,and GG were 0.40,0.69,21.76,respectively.For grow-out stage,the values of hR^2,SR,and GG were 0.48,0.83,and 18.22,respectively.The results of hR^2 indicated the good potential of the SS line in selective breeding.The level of GG improvement was encouraging and consistent with the previous expectations.No inbreeding depressions in the shell length were observed in the SS and SC lines at all sampling days.All of the results indicate that maintaining selection pressure in successive generations may be effective in the selective breeding program of this family.

Use of antioxidant enzymes of clam Ruditapesphilippinarum as biomarker to polycyclic aromatic hydrocarbon pollution

The typical organic pollutant polycyclic aromatic hydrocarbon(PAH) anthracene was selected as a contaminant to investigate its eff ects on the activities of superoxide dismutase(SOD), catalase(CAT) and glutathione peroxidase(GSH-Px) in the clam Ruditapes philippinarum. The results show that SOD, CAT and GSH-Px had diff erent induction and inhibition reactions to anthracene stress, and that three diff erent organs in R. philippinarum(visceral mass, muscle tissue and mantle) had diff erent sensitivities to anthracene stress. This study suggest that SOD activities of the visceral mass, CAT activitities of the mantle and the visceral mass, and GSH-Px activity of the muscle tissue could be used as sensitive indicators of anthracene stress in R. philippinarum.

Impact of seawater acidification on shell property of the Manila clam Ruditapes philippinarum grown within and without sediment

Although the impact of ocean acidification on marine bivalves has been previously investigated under mainly controlled laboratory conditions,it is still unclear whether the impact of acidification on sediment-burrowing species differs between those within or without sediment.In order to fill this gap in our knowledge,we compared shell properties of the infaunal Manila clam(Ruditapes philippinarum)exposed to three pH concentrations(7.4,7.7,and 8.0),within and without sediments.In the first experiment(140 d),clams were exposed to seawater in an acidification system without sediment.A decrease in shell weight corresponding to the increase in dissolution rate was observed in the group ofpH 7.4,at which shell color disappeared or whitened.SEM observations confirmed the changes of the external shell surface.In the second experiment(170 d),sediment was placed at the bottom of each exposure chamber.The effects were found obvious in shell dissolution rate and shell color in the shell specimens exposed to overlying seawater but not found in the shell specimens exposed to sediment.Although the experimental period was longer in the second experiment,shell specimens in the first experiment were more seriously damaged than those in the second experiment under acidic seawater conditions.Our results,in relation to the defense function of the shell,show that marine bivalves in burrowing behavior are more adaptable to seawater acidification than those who do not burrow into sediment.

Annual Variations of Biogenic Element Contents of Manila Clam(Ruditapes philippinarum) Bottom-Cultivated in Jiaozhou Bay, China

Manila clam （Ruditapes philippinarum） was monthly sampled from its benthic aquaculture area in Jiaozhou Bay from May 2009 to June 2010.The annual variations of major elemental composition,organic content,fatness and element ratio of Manila clam were examined.The element removal effect of clam farming in Jiaozhou Bay was analyzed based on natural mortality and clam harvest.The results indicated that the variation trend of carbon content in shell （Cshell） was similar to that in clam （Cclam）.Such a variation was higher in summer and autumn than in other seasons,which ranged from 9.10±0.13 to 10.38 ± 0.09 mmol g^-1 and from 11.28 ±0.29 to 12.36±0.06 mmol g 1,respectively.Carbon content of flesh （Cflnesh） showed an opposite variation trend to that of shell in most months,varying from 29.42 ± 0.05 to 33.64 ± 0.62 mmol g^-1.Nitrogen content of shell （Nshell） and flesh （Nflesh） changed seasonally,which was relatively low in spring and summer.Nshell and Nflesh varied from 0.07 ±0.009 to 0.14±0.009 mmol g^-1 and from 5.46 ± 0.12 to 7.39 ± 0.43 mmol g^-1,respectively.Total nitrogen content of clam ranged from 0.50 ± 0.003 to 0.76 ± 0.10 mmol g^-1 with a falling tend except for a high value in March 2010.Phosphorus content of clam （Nclam） fluctuated largely,while phosphorus content of shell （Pshell） was less varied than that of flesh （Pflesh）.Pshell varied from 0.006±0.001 to 0.016±0.001 mmol g^-1; while Pflesh fluctuated between 0.058±0.017 and 0.293±0.029mmolg^-1.Pclam ranged from 0.015±0.002 to 0.041 ±0.006mmolg^-1.Carbon and nitrogen content were slightly affected by shell length,width or height.Elemental contents were closely related to the reproduction cycle.The removal amounts of carbon,nitrogen and phosphorus from clam harvest and natural death in Jiaozhou Bay were 2.92× 10^4t,1420t and 145 t,respectively.The nutrient removal may aid to reduce the concentrations of nitrogen and phosphorus,the main causes of eutrophication,and to maintain the ecosystem health of Jiaozhou Bay.

Seawater acidification affects the immune enzyme activities of the Manila clam Ruditapes philippinarum

Ocean acidification leads to changes in physiological and immune responses of bivalves, but the eff ect on the immune enzyme activities of the Manila clam, R uditapes philippinarum, when the pH is lower than the normal value has not been studied in detail. In this study, experiments were conducted to determine how pH(7.4, 7.7, 8.0) affects the immune enzyme activities in the gill and hemocytes of the Manila clam. Membrane stability and phagocytosis increased with decrease of pH from 8.0 to 7.7 and then decreased at pH 7.4. The total protein content in the hemocytes and gills decreased with decreasing pH. Lysozyme content in the hemocytes increased with decreasing pH, and the diff erences were significant among the different pH groups(P < 0.05). Adenosine triphosphatase activity at pH 7.4 was significantly higher than in the other two groups, but no significant diff erence was observed between pH 7.7 and 8.0. Catalase activity decreased from pH 8.0 to 7.7 and then increased at pH 7.4, and the dif ferences were significant among the experimental groups( P < 0.05). These findings provide valuable information about the immune response of R. philippinarum to reduced water pH and insights for future research investigating exposure of bivalves to elevated CO_2 conditions.

荧蒽、菲、芘对菲律宾蛤仔(Ruditapes philippinarum)超氧化物歧化酶的影响

以菲律宾蛤仔为研究对象，在实验生态条件下采用不同浓度的三－四环多环芳烃（荧蒽、菲和芘）的混合物进行染毒实验，研究菲律宾蛤仔体内超氧化物歧化酶（ＳＯＤ）活性随污染物浓度和作用时间的变化趋势．结果表明，此三种混合污染物对ＳＯＤ活性的影响为一动态过程，基本上可以分为两个阶段：诱导反应和抑制反应阶段．

菲律宾蛤仔(Ruditapes philippinarum)大连群体家系建立及生长比较

于2008年6月,以大连野生型蛤仔为材料,采用巢式设计即1个雄性亲本与3个雌性亲本交配的方式建立了菲律宾蛤仔15个半同胞家系(A,B,…,O)和45个全同胞家系(A1,A2,A3,…,O1,O2,O3)。对各家系的壳长、壳宽、壳高、鲜重等各项指标进行了方差分析和多重比较。结果表明,3、4、5月龄,菲律宾蛤仔45个全同胞家系的表型性状具有显著差异,且各月龄的差异趋势基本一致。5月龄时,从其生长性状上看,家系F2、G2、G3表现出明显的生长优势,壳长分别提高28.43%、24.72%、30.53%,壳宽分别提高21.14%、22.3%、25.58%,壳高分别提高30.88%、26.96%、25.25%,鲜重分别提高50.00%、68.42%、44.12%。从其存活上看,家系E3的四个表型性状在全同胞家系中最小,但存活数量最多为2269粒,表现出很强的抗逆性;具有生长优势的家系F2存活个数最少仅为43粒,抗逆性相对最差。从其产量上看,家系N3、I1、E3、C1、N1、G2产量较高,分别比平均产量提高了77.8%、75.61%、75%、69.45%、53.96%、50.19%。综合以上各项指标家系G2、G3、N3、E3、I1、C1、N1可作为优良的家系育种材料,在下一步育种工作中可以利用家系选择及家系杂交的方法对7个家系进行遗传改良。

菲律宾蛤仔(Ruditapes philippinarum)蛋白聚糖的分离提取及其抗肿瘤活性的初步研究

采用凝胶过滤层析、SDS-PAGE电泳、Bradford法和苯酚-硫酸法等生物化学方法对菲律宾蛤仔中的蛋白聚糖(PG1和PG2)进行分离纯化,研究其理化性质。采用MTT方法研究蛋白聚糖对人肝癌细胞SMMC-7721和人正常肝细胞HL-7702生长的影响。通过小鼠体内实验研究蛋白聚糖的抗肿瘤活性和对荷瘤小鼠免疫功能的影响。结果表明,PG1分子量大约为20kDa,多糖含量为70.09%,蛋白质含量为15.4%。PG2的多糖含量为82.53%,蛋白质含量为7.26%。PG1在200μg/ml浓度时对SMMC-7721的生长抑制率为73.30%,随浓度的升高,抑制率并未发生明显改变。PG2对SMMC-7721的生长抑制作用呈剂量依赖性,在500μg/ml的浓度下,其抑制效果与PG1的抑制效果相当。在同样浓度下,PG1对人正常肝细胞的生长和功能没有明显影响。PG1呈剂量-效应关系明显增加荷瘤小鼠脾指数,促进荷瘤小鼠脾淋巴细胞转化,且没有降低体重及其他毒副作用。PG1对小鼠S180肉瘤的抑制作用可能是通过直接杀伤肿瘤细胞的细胞毒作用和提高小鼠细胞免疫水平来实现。

菲律宾蛤仔(Ruditapes philippinarum)养殖水体中石油烃安全限量的研究

石油烃污染是影响双壳贝类食用安全的重要因素.文中以菲律宾蛤仔（Ruditapes philippinarum）为研究对象,采用半静态动力学富集实验探讨了双壳贝类对养殖水体中石油烃的富集规律.结果表明,经过4 d,石油烃在贝体和水环境之间达到稳态平衡;生物富集系数（BCF）为545;石油烃浓度为0.068 mg/L的水体组,富集平衡后贝体内的石油烃含量高于菲律宾蛤仔的石油烃异味阈值（25～30 mg/kgg）,其它水体组则相反.参考USEPA、GB18421-2001等关于双壳贝类中石油烃安全限量的规定,经讨论贝体内石油多环芳烃的毒力代谢机理后,判定菲律宾蛤仔养殖水体中石油烃的安全限量定为0.05 mg/L较为合理.

菲律宾蛤仔(Ruditapes philippinarum)海洋橙品系两个世代的杂交与近交效应

于2009年8月以菲律宾蛤仔海洋橙品系的全同胞子一代和子二代上选10%的个体作为亲本,通过建立近交组合(F22、F33)、杂交组合(F23、F32)和设置对照组(C22、C33),研究了两个近交世代的杂交效应及近交效应,以提高菲律宾蛤仔海洋橙品系的表型性状。结果表明,杂交使幼虫和稚贝的生长性状得到了部分改良,但存活性状尚未得到提高。幼虫和稚贝表现出微弱的生长优势,正反交组单亲生长优势表现出正负不对称性。杂交效应主要受到母本效应的影响,母本效应贯穿于幼虫期及稚贝期。近交使世代F33的生长性状得到了改良,且两个世代的存活性状均有不同程度的提高。表型性状的近交效应在幼虫期主要受到交配方式的影响,而在稚贝期主要受到世代效应的影响。通过上选,杂交与近交的有机结合,有效改良了海洋橙品系的表型性状。

菲律宾蛤仔(Ruditapes philippinarum)低温保活方法的研究

本文在菲律宾蛤仔（Ｒｕｄｉｔａｐｅｓ ｐｈｉｌｉｐｐｉｎａｒｕｍ）生物学研究基础上，对菲律宾蛤仔在 ２７． ０～－１． ７℃范围内不同温度条件下的存活率，失重率及保活过程中的化学变化等进行了系统的分析研究，确定了菲律宾蛤仔的保活工艺和方法。结果表明，（１）菲律宾蛤仔的最肥季节为每年的 ３～ ７月，出肉率（干物计）最高可达 １０％；（２）最低保活温度为－１．７℃；（３）－１．０～－１．７℃可保活１３天，存活率９１％；（４）－１．０～１．７℃保活，保活时间最长，失重率也低，主要化学成分无显著变化。

菲律宾蛤仔(Ruditapes philippinarum)软体部位含氮物的组成及其分布

从食品原料学角度出发,为系统阐述菲律宾蛤仔的工艺学特性,以含氮物质为出发点,蛋白质为重点,对其各个软体解剖部位的质量组成、一般化学组成、含氮物分布及蛋白质组分等进行了分析讨论.结果表明：菲律宾蛤仔可食部分占原料20％左右,各个软体组织（足、闭壳肌、外套膜、水管、鳃、内脏团）含有水分74.01％～80.07％,其他化学组成为干基,粗蛋白29.19％～43.46％、总糖12.46％～30.75％、粗脂肪1.61％～6.84％和灰分6.69％～11.10％;各组织部位的含氮组分分布比例依次为：非蛋白氮14.7％～26.42％、水溶性蛋白12.55％～19.17％、盐溶性蛋白34.83％～50.4％、碱溶性蛋白17.49％～25.06％及基质蛋白2.19％～10.23％;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示：菲律宾蛤仔软体部位不同组织、不同组分的蛋白图谱有所差异.水溶性蛋白分子质量较小,在100kD以下;盐溶性蛋白种类丰富,在200、100、45、35、20 kD附近有明显条带;碱溶性蛋白仅在200、100、45 kD附近有4条明显条带;基质蛋白分子质量分布范围较宽,主要集中在200kD以上和45 kD区域.

蛤仔(Ruditapes philippinarum)养成期壳长遗传力与育种值估计

采用巢式设计构建蛤仔(Ruditapes philippinarum)家系,通过REML法估计了4种模型下蛤仔养成期90、300和330日龄壳长的遗传力,对不同模型进行比较并选出最优模型。通过最优模型估计育种值,并对表型值选择法和育种值选择法进行了比较。结果表明,移入土池时各家系平均壳长协变量是重要影响因子,共同环境效应影响较小,综合分析得出模型ABC为最优模型,其在90、300和330日龄的遗传力估计值分别为0.264、0.020和0.363,共同环境系数分别为0.116、0.376和0.098。对表型值选择法和育种值选择法比较的结果表明,表型值选择法均优于育种值选择法。对家系选择时,在90、300和330日龄,家系相同率分别为66.67%、75%和66.67%,表型值选择的家系平均壳长比育种值选择高7.30%、2.28%和5.81%。按10%的留种率对个体选择时,在90、300和330日龄,个体相同率分别为54.17%、59.72%和52.78%,表型值选择的个体平均壳长比育种值选择分别高27.08%、29.19%和16.27%;按1%的留种率,相同率则分别为12.50%、37.50%和37.50%,表型值选择比育种值选择的平均壳长高39.86%、39.81%和23.23%。

胶州湾菲律宾蛤仔(Ruditapes philippinarum)呼吸排泄作用的现场研究

于2006年2月、5月、8月和11月四个季度月采用呼吸瓶法现场研究了胶州湾菲律宾蛤仔的呼吸排泄作用。结果表明,胶州湾菲律宾蛤仔呼吸排泄速率具有明显的季节变化趋势,春、夏季高于秋、冬季。不同生物学规格的蛤仔呼吸排泄速率具有明显的差异,单位个体耗氧率、排氨率和排磷率均随着个体大小的增加而增加,而单位体重的耗氧率、排氨率和排磷率则随着个体大小的增加而降低;且耗氧率、排氨率和排磷率与软体干重(DW,g/ind)呈异速方程关系,方程式为:Rx=a×DWb。本实验中,耗氧率、排氨率和排磷率的a值变化范围分别为0.20—0.93、0.29—2.46和0.27—0.59;耗氧率、排氨率和排磷率的b值变化范围分别为0.52—0.83、0.35—0.62和0.28—0.88。据估算,胶州湾滩涂养殖的菲律宾蛤仔通过呼吸排泄作用在夏季将向水体中释放8.28t/d氨氮和0.86t/d无机磷,以满足浮游植物生长的需要,在胶州湾生态系统营养循环和能量流动中起着重要的作用。

菲律宾蛤仔(Ruditapes philippinarum)血淋巴中抑菌活性物质的活性影响因素

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