Shellfish In China Research Articles

Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa).

BackgroundThe veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in R. venosa for use as internal controls for qRT-PCR.MethodsIn this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α (EF-1α), α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1α subcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms.ResultsOf the 13 candidate genes tested, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α, RL5, and NDUFA7 for liver, GAPDH, PPIA, and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, PPIA, RS25, and RL28 for stage 1, RL5 and RL28 for stage 2 and 5, RL28 and NDUFA7 for stage 3, and PPIA and TUBB for stage 4.DiscussionOur results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development of R. venosa in the future.

Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro

The ark shell Scapharca broughtonii is a commercially important shellfish in China. Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS) are common anticoagulants used for shellfish blood. To observe the immune response mediated by its hemocytes, we challenged in vivo S. broughtonii hemolymph with Vibrio anguillarum and dealt with the following three anticoagulants in vitro: Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS). The methodologies we used were immunostimulation with V. anguillarum, Wright-Giemsa staining, micro-examination, and flow cytometric and hydrolyzing enzyme activity analysis. The results showed that all three types of anticoagulants effectively prevented blood clotting in ark shellfish. The morphology of hemocytes did not significantly change 30 h after anticoagulant treatment, except for the shrinking of hemocytes after administering HSS. The size and permeability of hemocytes changed when treated with the anticoagulants and when stimulated with V. anguillarum. Both alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemocytes and Plasma were measured at different times after they were stimulated with V. anguillarum in HSS and MAS. The AKP enzymatic activity in HSS was somewhat higher than in the MAS anticoagulant, but changes in response to V. anguillarum challenge of enzymatic activity were almost the same in HSS and MAS groups. In conclusion, all three types of anticoagulants may be used for ark shell blood preservation. They all changed the cell-surface characteristics of hemocytes to inhibit clot formation. The AS anticoagulant was appropriate for maintaining white and red cell shapes, while MAS was ideal for retaining throbus cell function. Lastly, HSS was appropriate for maintaining enzymatic activity in hemolymph and function of hemocytes. Following this investigation, we gained insight into the changes in hemolymph characteristic during immune response.

Effects of light intensity on metabolism and antioxidant defense in Haliotis discus hannai Ino

We examined the effects of light intensity on the metabolism, antioxidant enzymes, and expression of related genes in Haliotis discus hannai Ino, to determine the optimal lighting conditions for aquaculture of these animals. Succinate dehydrogenase activity was significantly lower after 48h in animals exposed to light intensities of 30 and 60μmol/m2/s, while lactate dehydrogenase activity and lactic acid content at 96h were significantly higher compared with animals in the dark or exposed to 5 or 15μmol/m2/s (both P<0.05). There was no significant difference in reactive oxygen species (ROS) contents between animals in the dark and those exposed to 5 or 15μmol/m2/s (P>0.05) throughout the experiment. However, ROS contents under light intensities of 30 and 60μmol/m2/s peaked at 12h, when expression levels of genes encoding catalase (CAT), thioredoxin peroxidase (TPX), sigma-glutathione-s-transferase (GSTS), and mu-glutathione-s-transferase (GSTm) also began to increase, and CAT and glutathione peroxidase (GPX) activities were significantly higher than in animals in the dark or exposed to 5 or 15μmol/m2/s (P<0.05). After a further 12h, the ROS content at 30μmol/m2/s began to decrease, and GPX activity and malondialdehyde (MDA) content also gradually decreased, followed by increases in ROS and MDA 48h later, to reach respective peaks at 96h. Gene expression levels of TPX, GSTS, GSTm, heat shock protein 70 (HSP70), and CAT began to decrease 72h later, suggesting that excessive accumulation of ROS may have caused oxidative damage to the tissues. At a light intensity of 60μmol/m2/s, expression levels of TPX, GSTS, GSTm, and HSP70 genes, total antioxidant capacity, and GPX activity peaked at 48h, and the ROS content was significantly higher than in any other group 48h later (P<0.05). However, gene expression levels of TPX, GSTS, GSTm, HSP70, HSP90, and CAT, and reduced glutathione content began to decrease, indicating that the antioxidant system had been unable to perform its normal physiological functions to withstand the long-term stress of adverse environmental factors. These results suggest that the light intensity should be controlled at 5–15μmol/m2/s during H. discus hannai aquaculture, to maintain the organism's normal physiological metabolism. Statement of relevanceThe disc abalone Haliotis discus hannai is an economically important shellfish in China, with demand growing for this high-protein and low-fat seafood. In 2014, 11,5397 tons of H.discushannai were harvested from aquacultural sources, but supply cannot currently meet consumer demand. In recent years, the natural habitat of H. discus hannai has declined as a result of overfishing, marine reclamation, water pollution, among others, leading to a sharp decrease in the numbers of H. discus hannai in the wild. Thus, the development of an aquacultural system for farming H. discus hannai, as well as for its protection in the wild, are a focus of current research.Light, including photoperiod, quality and intensity, is one of the key environmental factors influencing the growth, culture and survival of aquatic organisms. Over evolutionary time, organisms have evolved both physiological and behavioral mechanisms that enable them to adapt to diurnal fluctuations in light. In Chinese traditional abalone aquaculture, a sunshade net is usually used because of the photophobic nature of abalone. In particular, the aquaculturist usually provides a darkened setting for adult abalones to increase their food intake rate, facilitate their growth and promote gonadal development. For abalones in their natural environment, the light intensity in the water area is relatively stable, suggesting that there is a key regulative effect of light intensity on their growth and development.Gao et al. (2016a,b) found that a dark environment not only adversely affected the daily aquaculture production, but was also associated with a lower growth rate compared with animals reared under higher light intensities, because of lower food conversion efficiency and greater energy losses through excretion and feces. Physiological metabolism and antioxidant defense systems may therefore be key indicators for measuring the suitability of light intensity for aquaculture production. We therefore examined the effects of light intensity on the metabolism and antioxidant defense system, and on the expression of related genes, in H. discus hannai using a light-emitting diode (LED) to replace the conventional fluorescent light source. The results of this study will improve our understanding of the physiological tolerance and antioxidant defense characteristics of abalone under different light intensities, and enable the optimization of environmental light conditions in abalone aquaculture.

Isolation and characterization of an antibacterium against Vibrio harveyi 11593 from a mixed pond with penaeus japonicus bate, portunus trituberculatus and ruditapes philippinarum in China

Aquaculture diseases, particularly Vibriosis, are becoming a pressing concern due to incurred aquacultural loss in China. Commercial antibiotics may be used to control it, but its increased antibiotic resistance to prominent pathogenic bacteria has become a prevalent problem nationwide, and a global threat to public health. Probiotics are recommended instead because they are healthy, environment-friendly, and capable of maintaining productivity. An antibacterium against Vibrio harveyi 11593 was isolated from a mariculture pond with shrimp, crabs, and shellfish in China. The bacterium, E14, has an inhibitory zone diameter (DIZ) of 24.5 ± 0.5 mm. The strain was identified as Bacillus pumilus based on morphological observation, conventional biochemical tests, and 16S rDNA sequence analysis. The gram-positiveand motile bacterium is around 1.10-1.32 pm × 0.67-0.83 μm in size. Optimized conditions for antimicrobial substance production of B. pumilus E14 require that it be cultured for 26 h at 28 °C, with an initial pH of 7.0 in 100 mL/500 mL LB with 3% NaCl. The B. pumilus E14 cultures were confirmed to be safe and efficacious and actually worked to protect the host animal in shrimp larvae (Penaeus chinensis) culture. The B. pumilus E14 obtained in this study strengthened the strain's defense against aquaculture disease and made a good candidate for an alternative probiotics and benefit to sustainability of aquaculture.

Transcriptome analysis of male and female mature gonads of Japanese scallop Patinopecten yessonsis

The Japanese scallop Patinopecten yessoensis is an important commercial culture shellfish in China and Japan. In addition to its commercial interest, it has also attracted much attention because of its value in studying sex determination and differentiation mechanisms. Herein, two transcriptome libraries from male and female gonads at maturing stage were constructed. The two libraries were paired-end sequenced using Illumina sequencing techniques. A total of 81,986,898 cleaned reads were obtained, and assembled into 29,897 unigenes by using Trinity software and removing redundancy. Compared with the public Swiss-Prot and NR databases by BlastX, 9354 unigenes were significantly matched to known unique proteins. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes uncovered diverse biological functions and processes. After comparing the two transcriptome libraries, 8652 up-regulated and 2973 down-regulated genes were identified in the male gonads. According to annotation information, at least 30 genes related to sex determination and differentiation, such as Dmrt1, Sox9, fem1 and vasa, were identified and characterized. The expression patterns of the random eight genes were then validated by quantitative real-time PCR (qRT-PCR) suggesting the high reliability of RNA-Seq results. The study provides an archive for further studies of molecular mechanisms of bivalve sex determination and differentiation.

Transcriptomic Analysis of Differentially Expressed Genes During Larval Development of Rapana venosa by Digital Gene Expression Profiling.

During the life cycle of shellfish, larval development, especially metamorphosis, has a vital influence on the dynamics, distribution, and recruitment of natural populations, as well as seed breeding. Rapana venosa, a carnivorous gastropod, is an important commercial shellfish in China, and is an ecological invader in the United States, Argentina, and France. However, information about the mechanism of its early development is still limited, because research in this area has long suffered from a lack of genomic resources. In this study, 15 digital gene expression (DGE) libraries from five developmental stages of R. venosa were constructed and sequenced on the IIIumina Hi-Sequation 2500 platform. Bioinformaticsanalysis identified numerous differentially and specifically expressed genes, which revealed that genes associated with growth, nervous system, digestive system, immune system, and apoptosis participate in important developmental processes. The functional analysis of differentially expressed genes was further implemented by gene ontology, and Kyoto encyclopedia of genes and genomes enrichment. DGE profiling provided a general picture of the transcriptomic activities during the early development of R. venosa, which may provide interesting hints for further study. Our data represent the first comparative transcriptomic information available for the early development of R. venosa, which is a prerequisite for a better understanding of the physiological traits controlling development.

利用线粒体COI和微卫星标记分析文蛤7个地理群体的遗传变异

PDF HTML阅读 XML下载 导出引用 引用提醒 利用线粒体COI和微卫星标记分析文蛤7个地理群体的遗传变异 DOI: 10.5846/stxb201409151822 作者: 作者单位: 国家海洋环境监测中心,大连海洋大学,国家海洋环境监测中心,辽宁医学院,国家海洋环境监测中心,国家海洋环境监测中心 作者简介: 通讯作者: 中图分类号: 基金项目: 国家海洋公益项目(201305043,201305027);国家自然科学基金项目(31101899) Genetic diversity and differentiation of seven geographical populations of hard clam(Meretrix meretrix) assessed by COI and microsatellite markers Author: Affiliation: National Marine Environmental Monitoring Center,Dalian Ocean University,National Marine Environmental Monitoring Center,Liaoning Medical University,National Marine Environmental Monitoring Center,National Marine Environmental Monitoring Center Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:利用线粒体细胞色素氧化酶亚基I(COI)和微卫星标记分析了文蛤7个地理群体(朝鲜新义州,辽宁丹东、蛤蜊岗和盘山,山东东营,江苏如东和启东)的遗传多样性和群体分化。PCR扩增获得142条602 bp的COI核苷酸片段,比对到13个变异位点,包括11个转换和2个颠换,定义了22个单倍型,共享单倍型12个,新义州、丹东和启东群体分别拥有特有单倍型。单倍型多样性最高的是如东群体(h=0.900),最低的是东营群体(h=0.600);核苷酸多样性最高的是丹东群体(π=0.00350),最低的是蛤蜊岗群体(π=0.00115)。基于COI数据的Fu's Fs中性检验和核苷酸不配对分析揭示文蛤种群历史上曾经历过群体扩张事件。分子变异分析(AMOVA)表明,群体内遗传变异占71.64%,群体间遗传变异占28.36%,群体间发生显著的遗传分化(P < 0.05)。7个微卫星标记扩增280个个体共获得54个等位基因,平均等位基因数为7.7个,平均观测杂合度和期望杂合度分别为0.3878和0.7996。江苏群体具有较高的遗传多样性,但7个群体间遗传多样性不存在显著差异(Kruskal-Wallis检验, P > 0.05)。Hardy-Weinberg平衡检验结果显示49个群体-位点组合中有18个偏离平衡(P < 0.05),表现为杂合子缺失。单倍型邻接(NJ)树显示聚类未展示地域性特色,但某几个同一或者相近地理群体的单倍型具有聚类现象(如东和启东部分单倍型出现地理聚类)。依据群体间遗传距离以Kimura 2-parameter为模型建立UPGMA系统发育树,显示丹东群体和江苏的如东和启东群体聚为一支,暗示江苏苗种的异地养殖已经污染丹东文蛤的遗传背景。 Abstract:The hard clam(Meretrix meretrix) is a commercially important shellfish in China. With the rapid development of the aquaculture industry of M. meretrix, the demand for seed cultures has led to the over-exploitation of natural populations, which makes stock enhancement a high priority. Stock enhancement programs that use a small number of breeders for the production of hatchery-reared juveniles to be released to the environment may have negative effects on the genetic diversity of wild populations due to a reduction in the genetic variability of the released stock. In this study, the genetic diversity of seven geographical populations(Sinuiju in North Korea; Dandong, Geligang, and Panshan in Liaoning Province; Dongying in Shandong Province; and Rudong and Qidong in Jiangsu Province) of M. meretrix was assessed using the mitochondrial cytochrome c oxidase subunit I(COI) gene and microsatellite markers. We obtained a total of 142 COI sequences. Each COI sequence was 602 bp in length. These sequences contained 14 variant sites, including 12 transitions and 2 transversions. Twenty-two haplotypes were identified, with 12 haplotypes shared among populations. Population-specific haplotypes were identified in the Sinuiju, Dandong, and Qidong population, respectively. The haplotype diversity was highest in Rudong(h=0.900) and lowest in Dongying(h=0.600). The nucleotide diversity was highest in Dandong(π=0.00350) and lowest in Geligang(π=0.00115). Neutral test(Fu's Fs) and mismatch distribution analysis revealed that the hard clam experienced a population expansion event. Analysis of molecular variance(AMOVA) indicated that 71.64% of the genetic variance was within populations and 28.36% of the variance was among populations, demonstrating significant genetic differentiation among populations(P < 0.05). A total of 54 alleles were amplified from 280 individuals by using 7 microsatellite markers, with an average of 7.7 alleles per locus. The mean observed heterozygosity(Ho) and expected heterozygosity(He) was 0.387 and 0.7996, respectively. Compared with other populations, genetic diversity in the Jiangsu population was highest, but the difference was not significant(Kruskal-Wallis test, P > 0.05). Among the 49 population-locus combinations(7 populations×7 loci), 18 cases deviated from the Hardy-Weinberg equilibrium(P < 0.05), indicating heterozygote deficiencies. The neighbor-joining tree showed that the haplotypes were not clustered according to geographical location, but some haplotypes from the same or neighboring locations grouped together(e.g. Rudong and Qidong showing geographical clustering). The unweighted pair group with arithmetic mean(UPGMA) phylogenetic tree showed that the Dandong population was grouped with the Jiangsu population, suggesting that the introduced Jiangsu clam seed has contaminated the genetic background of the Dandong population. These results highlight the need to monitor the genetic effects of releasing large numbers of juveniles. 参考文献 相似文献 引证文献

Effect of LED light quality on respiratory metabolism and activities of related enzymes of Haliotis discus hannai

Oxygen consumption and ammonia excretion rates of abalone (Haliotis discus hannai) of differing sizes (wet weight 63.74±8.49g, 17.49±3.20g, and 3.52±0.94g) were evaluated under five different light emitting diode (LED) conditions (red, orange, purple, green, and blue light) and in dark and natural light environments. The weight-specific oxygen consumption/ammonia excretion rates decreased significantly with increasing body weight, and these relationships could be expressed by the exponential equations R=aWb and E=aWb (P<0.05), respectively. The oxygen consumption/ammonia excretion rates of abalone in the red light, orange light, and dark environment treatments were lower than those in the green, blue, natural, and purple light treatments, and the highest rates (3.27mgg−1 h−1/187μgg −1 h −1) occurred in the purple light treatment (P<0.05). The O/N ratio exceeded 10 in all light quality groups, indicating that the energy requirements were met by carbohydrate and fat. In another experiment, the effects of different light qualities on the activities of hexokinase (HK), pyruvate kinase (PK), superoxide dismutase (SOD), and catalase (CAT) in juvenile abalone (wet weight 17.49±3.20g) were investigated at 0.5, 1, 3, 6, 12, 24, and 72h of light exposure. HK and PK activities in the green, purple, blue, and natural light groups were significantly higher than those in the red light, orange light, and dark environment (P<0.05). SOD activity increased immediately when the abalones were placed in green, purple, blue, and natural light, then decreased gradually and returned to its original level. CAT activity did not increase until 12h and was higher in the green, purple, blue, and natural light groups than in the red light, orange light, and dark environment (P<0.05). These results suggest that more carbohydrates were provided and utilized by the glycolytic cycle when the abalones were exposed to adverse light conditions. Therefore, the selection of appropriate light conditions (red and orange light) and the control of metabolic waste ammonia will have a significant impact on high-intensity aquaculture in recirculating cultivation systems. Statement of relevanceThe disk abalone Haliotis discus hannai is an economically important shellfish in China, with demand growing for this high-protein and low-fat seafood. In 2012, 90 694t of H. d. hannai were harvested from aquacultural sources, but supply cannot currently meet consumer demand. In recent years, the natural habitat of H. d. hannai has declined as a result of overfishing, marine reclamation, water pollution, among others, leading to a sharp decrease in the numbers of H. d. hannai in the wild. Thus, the development of an aquacultural system for farming H. d. hannai, as well as for its protection in the wild, are a focus of current research.Light, including photoperiod, quality and intensity, is one of the key environmental factors influencing the growth, culture and survival of aquatic organisms. Over evolutionary time, organisms have evolved both physiological and behavioral mechanisms that enable them to adapt to diurnal fluctuations in light. This is particularly relevant to coastal aquatic environments, where various phytoplanktonic species and dissolved organic matter in the water can absorb or scatter blue light, converting the light quality of transmitted light from blue-green to green-orange. Blue light (470nm) has the strongest penetrability in clean seawater, whereas red light and ultraviolet (UV) are quickly absorbed in clean seawater. In its natural environment, H. d. hannai is mostly distributed in shallow inshore waters at a depth of less than 10m. Such waters are also inhabited by dense populations of phytoplankton and contain high levels of dissolved organic matter, which both diminish the transmission capacity of light, particularly of blue light, and this can result in an extended range of red and orange light, rather than blue light, in the water.In Chinese traditional abalone aquaculture, a sunshade net is usually used because of the photophobic nature of abalone. In particular, the aquaculturist usually provides a darkened setting for adult abalones to increase their food intake rate, facilitate their growth and promote gonadal development. For abalones in their natural environment, the light quality in the water area is relatively stable, suggesting that there is a key regulative effect of light quality on their growth and development.The aim of the present study was to evaluate the effects of different LED light qualities on the respiratory metabolism and physiological response of abalones by measuring oxygen consumption rate, ammonia excretion rate, glycolytic enzyme activity, and antioxidant defense. Our results may be used to optimize the light conditions for optimal growth and survival of abalones and to provide basic theory for regulation and control of light conditions in closed recirculating aquaculture systems in general.

Temporal and Spatial Variation in the Abundance of Total and Pathogenic Vibrio parahaemolyticus in Shellfish in China.

We investigated the abundance of total and pathogenic Vibrio parahaemolyticus in shellfish sampled from four provinces in China during May 2013 and March 2014 using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 67.7% of 496 samples. A total of 38.1% and 10.1% of samples exceeded 1,000 MPN g-1 and 10,000 MPN g-1, respectively. V. parahaemolyticus densities followed a seasonal and geographical trend, with Guangxi and Sichuan shellfish possessing total V. parahaemolyticus levels that were 100-fold higher than those of the Liaoning and Shandong regions. Moreover, the levels of V. parahaemolyticus were at least 10-fold higher in the summer and autumn than in the cooler seasons. Pathogenic V. parahaemolyticus levels were generally lower than total V. parahaemolyticus levels by several log units and tended to be high in samples contaminated with high total V. parahaemolyticus levels. The aqua farms had a lower prevalence but higher abundance of total V. parahaemolyticus compared to retail markets. The catering markets showed the lowest levels of total V. parahaemolyticus, but 20.0% of samples exceeded 1,000 MPN g-1. The levels of both total and pathogenic V. parahaemolyticus in oysters were higher than in clams. The log-transformed abundance of V. parahaemolyticus was significantly correlated with both water temperature and air temperature but not water salinity. These results provide baseline contamination data of V. parahaemolyticus in shellfish in China, which can be applied to local risk assessments to prioritize risk control to key sectors and evaluate the effectiveness of future control measures.

Surveillance of hepatitis E virus contamination in shellfish in China.

Background: Hepatitis E virus (HEV) has been confirmed to be a zoonotic virus of worldwide distribution. HEV contamination in the water environment has not been well examined in China. The objective of this study was to evaluate HEV contamination in shellfish in a coastal area of China. Such contamination would be significant for evaluating public health risks. Methods: samples of three species shellfish were collected from thirteen points of estuarine tidal flats around the Bohai Gulf and screened for HEV RNA using an in-house nested RT-PCR assay. The detected HEV-positive samples were further verified by gene cloning and sequencing analysis. Results: the overall HEV-positive detection rate is approximately 17.5% per kilogram of shellfish. HEV was more common among S. subcrenata (28.2%), followed by A. granosa (14.3%) and R. philippinarum (11.5%). The phylogenetic analysis of the 13 HEV strains detected revealed that gene fragments fell into two known 4 sub-genotypes (4b/4d) groups and another unknown group. Conclusions: 13 different sub-genotype 4 HEVs were found in contaminated shellfish in the Bohai Gulf rim. The findings suggest that a health risk may exist for users of waters in the Bonhai area and to consumers of shellfish. Further research is needed to assess the sources and infectivity of HEV in these settings, and to evaluate additional shellfish harvesting areas.

Cadmium content in shellfish from the coast of the South China Sea: the risk to human health

In many regions,the marine environment is contaminated with cadmium(Cd),a toxic but widely used industry material.Because shellfish live in the benthos and have a relatively fixed location,their health is largely dependant on the quality of the surrounding environment.A number of researchers have reported finding heavy metals in shellfish samples.Given the importance of shellfish in China,we measured levels of Cd contamination in 5 shellfish species: Crassostrea rivularis,C.gigas,Ruditapes Philippinarum,Perna viridis,and Meretrix meretrix collected from the coast of the South China Sea in 2007 and 2009.We measured the Cd content and evaluated the dietary exposure to Cd from shellfish using the point estimate method.We found significant differ-ences in Cd content among shellfish species.Mean concentrations were 1.80,0.38,0.26,0.15,and 0.12 mg.kg?1 in C.rivularis,C.gigas,P.viridis,M.meretrix,and R.Philippinarum,respectively(range 0.92?2.39 mg.kg?1,0.31?0.46 mg.kg?1,0.21?0.30 mg.kg?1,0.08?0.22 mg.kg?1,and 0.10?0.13 mg.kg?1,respectively).The distribution and seasonal variation in Cd content was correlated with marine environmental factors such as dissolved oxygen,salinity,and temperature.The Cd concentration was higher than the limit for deleterious material by non-environmental pollution of aquatic products in C.rivularis.Conversely,Cd levels were below this limit in the remaining species.In comparison,the levels of Cd were lower than foreign limits for all species.A comparison with survey data collected from other coastal areas in past years suggests the current levels of Cd are within a normal range.Based on our risk assessment,dietary exposure to Cd from consumption of C.rivularis exceeds levels recommended by the Joint Expert Committee on Food Additives(JECFA),suggesting there is a risk to hu-man health.Conversely,the other 4 shellfish species likely pose no risk as the Cd levels were lower than the val-ues recommended by JECFA.The ratio of Cd∶Zn was much lower in C.rivularis than the suggested critical value of 0.015 proposed by some authors as effectively providing protection from Cd induced health impacts.Increased intake of bioavailable zinc from C.rivularis reduces excessive cadmium absorption.Therefore,con-sumption of C.rivularis likely does not pose a risk to human health.

Development of 53 novel polymorphic EST-SSR markers for the hard clam Meretrix meretrix and cross-species amplification

The hard clam (Meretrix meretrix) is a commercially important maricultured shellfish in China. The transcriptome data of M. meretrix provided a valuable resource to mining large amount of EST-SSR markers quickly and cost-effectively. Here we isolated and characterized 53 novel polymorphic EST-SSR markers in 30 individuals. The number of alleles varied from 2 to 8, with an average of 4.19 alleles per locus. The observed and expected heterozygosities ranged from 0.040 to 1.000 and from 0.040 to 0.799, respectively. Thirty-one loci significantly deviated from HWE (P < 0.01) after Bonferroni correction. Interspecific transferability revealed that 19 polymorphic loci in Meretrixlamarckii and 10 in Meretrix lyrata were firstly characterized. These novel polymorphic SSR markers will be particularly useful for further diversity analysis, evolutionary studies, genetic trait mapping and marker assisted selection.

Using Microsatellites to Assess Genetic Variation in a Selective Breeding Program of Chinese Bay Scallop (Argopecten irradians irradians)

This study aimed to improve our understanding of the genetics of the Chinese bay scallop (Argopecten irradians irradians), one of the most important maricultured shellfish in China. Ten polymorphic microsatellite loci were examined to assess the allelic diversity, heterozygosity, and genetic variation between two domesticated populations selected for fast growth in breeding programs, and their base population. Forty-one alleles were found throughout the loci and the mean number of alleles per locus ranged 3.30-3.50. The average heterozygosity ranged 0.38-0.45, whereas the polyamorphic information content ranged 0.1504-0.7518. Genetic differences between the three populations were detected based on the number of alleles per locus, effective number of alleles, Shannon index, inbreeding coefficient (Fis), p values, genetic distance, and pairwise Fst values. There was no significant loss of genetic variability in the breeding program but changes in gene frequencies were detectable over the populations, implying that thea loci were saffected by the pressures of selective culture.

Using Microsatellites to Assess Genetic Variation in a Selective Breeding Program of Chinese Bay Scallop (Argopecten irradians irradians)

This study aimed to improve our understanding of the genetics of the Chinese bay scallop (Argopecten irradians irradians), one of the most important maricultured shellfish in China. Ten polymorphic microsatellite loci were examined to assess the allelic diversity, heterozygosity, and genetic variation between two domesticated populations selected for fast growth in breeding programs, and their base population. Forty-one alleles were found throughout the loci and the mean number of alleles per locus ranged 3.30-3.50. The average heterozygosity ranged 0.38-0.45, whereas the polyamorphic information content ranged 0.1504-0.7518. Genetic differences between the three populations were detected based on the number of alleles per locus, effective number of alleles, Shannon index, inbreeding coefficient (Fis), p values, genetic distance, and pairwise Fst values. There was no significant loss of genetic variability in the breeding program but changes in gene frequencies were detectable over the populations, implying that thea loci were saffected by the pressures of selective culture.

Identification of host-defense genes and development of microsatellite markers from ESTs of hard clam Meretrix meretrix

The hard clam Meretrix meretrix is an economically important shellfish in China. However, genomic research on this species is still at early stage, and few genomic resources are available. The objective of the present study was to generate expressed sequence tags (ESTs), and identify host-defense genes and microsatellite markers for M. meretrix. Three cDNA libraries for intestine, mantle and hepatopancreas were constructed using highly efficient SMART (Switching Mechanism At 5' end of the RNA Transcript) method. A total of 3224 random clones were single-pass sequenced from 5'-ends, resulting in 3129 high-quality (>100 bp) ESTs averaging 734 bp. All the ESTs were assembled by software Cap 3, producing 1796 unigenes-1490 singletons and 306 contigs. All the unigenes were compared to the public protein database using tblastx, and 696 (38.8%) were homologues to known genes while the remaining 1100 (61.2%) appeared to be novel sequences. A total of 31 EST clusters were related to immune and defense functions. They included immune recognition receptors, proteases and protease inhibitors, and other immune-related genes. The screening of 1796 unigenes identified 55 (3.1%) microsatellite-containing sequences, with 20 having sufficient flanking sequences for primer design. Polymerase chain reaction amplification was successful for 12 primer pairs and 7 of them showed polymorphic. The EST collection and microsatellite markers obtained in this study provide a useful resource for further gene discovery and population genetic analysis in M. meretrix.

Technical Note Isolation and characterization of polymorphic microsatellite loci in the hard-shelled mussel, Mytilus coruscus (Mytilidae)

Mytilus coruscus is one of the most important cultured species of marine shellfish in China. Using an expressed sequence tag-library and two microsatellite-enriched genomic libraries of M. coruscus, we isolated and characterized 12 polymorphic microsatellites in a test population; the number of alleles ranged from three to seven, and the observed and expected heterozygosities ranged from 0.0333 to 0.8571 and from 0.3452 to 0.8267, respectively. Four loci deviated from Hardy-Weinberg equilibrium. These polymorphic microsatellite loci can be employed in genetic diversity analysis and molecular marker-assisted breeding of M. coruscus.