海洋污染物对菲律宾蛤仔的免疫毒性

环境污染能够影响养殖贝类的免疫能力,是导致贝类大规模死亡的重要原因之一.探讨了大连周边4个海区污染物对采集的菲律宾蛤仔(Ruditapes philippinarum)免疫毒性影响.结果发现:污染物浓度和种类对蛤仔的免疫和生理指标具有重要影响,在重金属和石油污染物浓度较低的皮口海区,血细胞总数、亮氨酸氨基肽酶活性和血淋巴溶菌酶活性均显著高于其它3个海区(P＜0.05),而蛤仔的脂质氧化水平则较低;在重金属和石油污染物浓度较高的黑石礁海区,蛤仔血淋巴谷胱甘肽含量显著高于其它3个海区(P＜0.05);在重金属浓度较高的庄河海区,蛤仔表现出较高的超氧化物歧化酶活性(P＜0.05).     

Molecular cloning and characterization of Y-box gene (Rpybx) from Manila clam and its expression analysis in different strains under low-temperature stress

Manila clam, Ruditapes philippinarum, is an economically important marine bivalve species. Y-box proteins are members of the cold shock proteins family and highly conserved from bacteria to humans. In this study, a novel Y-box gene (Rpybx) was cloned from the Manila clam and gene expression profiling was performed on three shell color strains (white, zebra and white zebra) and two wild populations (Southern and Northern) of R. philippinarum. The complete ORF length of Rpybx is 1367 bp, encoding 253 amino acids residues. Based on the amino acid sequence analysis and phylogenetic analysis, the Rpybx gene was identified as a member of the invertebrate Y-box proteins family. Rpybx has a similar tertiary structure to human Y-box protein YB-1. The Rpybx mRNA levels were analyzed by qPCR under acute and gradually varied cold stress. Under acute low-temperature stress, the expression of Rpybx mRNA in gills and hepatopancreas was significantly increased in all selected strains and populations (P < 0.05). The Northern population showed the lowest relative expression level of Rpybx. The expressions of Rpybx were greatly upregulated in gills and hepatopancreas of different stains and populations at 5 or −2°C under gradually varied temperature stress (P < 0.05). The results shed light on the biological function of the Rpybx gene in defending against low-temperature challenge and further exploring the molecular mechanism of cold tolerance and resistance in R. philippinarum.     

菲律宾蛤仔EST-SSRs标记开发及不同地理群体遗传多样性

利用13对微卫星引物对大连、莆田、青岛3个地理群体蛤仔遗传多样性进行了检测。结果表明:13个基因座共检测到154个等位基因,每个座位检测到的等位基因数在2-7个之间,平均有效等位基因数为2.7657;3个群体平均观测杂合度分别为0.4387、0.4194、0.2383,平均期望杂合度分别为0.6488、0.6484、0.5526;群体间的遗传多样性差异不显著(P＞0.05)。NJ聚类结果显示大连和莆田群体的蛤仔亲缘关系较近,二者与青岛群体关系较远。3个群体均有不同程度的偏离Hardy-Weinberg平衡现象,表明各群体基因频率和基因型频率的稳定性相对较低。本研究所获得的微卫星标记的多态信息含量(PIC)>0.5,说明这些微卫星位点的多样性较高,可为下一步遗传图谱构建研究提供参考。     

Sediment Toxicity Bioassays Using Ruditapes decussatus Embryos and Larvae to Assess the Contamination in Four Sites on the Coast of Tunisia

This paper aims to assess the marine sediment quality along the Tunisian coast using clam Ruditapes decussatus embryos and larval bioassays tests. Elutriate samples prepared with sediment from four sites (Monastir Lagoon, Chebba, Mahres, and Zarat) were used for chemical analysis and clam bioassays tests (embryogenesis, larval growth. and metamorphosis). For embryogenesis test, five elutriates concentrations (12%, 25%, 50%, 75%, and 100%) were prepared. Metal concentrations were highest at the Mahres and Monastir Lagoon sites. Clam embryogenesis success was significantly (p < 0.05) reduced in all elutriates preparations at concentrations up to 50% except Chebba, where inhibition was evident at only the highest concentration (100%). Compared to the control (9.4 μm day^?1), larval growth rate was significantly (p < 0.05) reduced only in two sites, Monastir Lagoon (6.8 μm day^?1) and Zarat (5.6 μm day^?1). However, larval survival was lower (42.9%) in elutriates prepared from Mahres sediment compared to other studied sites and control treatment. We did not note any significant (p > 0.05) effect of elutriate preparations on clam metamorphosis; conversely, survival in this stage was significantly affected in three sites (Monastir: 59.0 ± 6.7%; Zarat: 52.1 ± 6.3%, and Mahres: 64.1 ± 5.4%) compared to the control (83.2 ± 4.7%). This work shows that both clam embryos and larvae are sensitive to contaminants and can be used to evaluate sediment contamination and monitor pollution.     

Invasion in tidal zones on complex coastlines: modelling larvae of the non‐native Manila clam,Ruditapes philippinarum,in the UK

Aim To evaluate whether natural larval transport and behaviour alone can explain the pattern of invasion and establishment of the non‐indigenous Manila clam, Ruditapes philippinarum ( Adams & Reeve, 1850 ), and its spread beyond the point of introduction in the UK. Location The study is focused on Poole Harbour, south England, the point of introduction of the Manila clam in the UK. Methods We use fine‐resolution hydrodynamic models coupled with a water salinity model and an individual behaviour model of Manila clam larvae. The model was informed by experimental studies on the vertical response of larvae to salinity and field studies of the species in its natural and new environments. Results Variations in the behavioural response of larvae to salinity in the model considerably affected the retention of clam larvae within the harbour. High levels of predicted larval retention occurred in two of five zones in the harbour when the salinity target was set at 17 practical salinity units. Persistently high densities of adult clams and recruits are accurately predicted in these regions. Main conclusions Even within a relatively small region such as Poole Harbour, there is both localized retention of larvae or ‘closed’ areas and areas that are considerably more ‘open’ and potentially connected. The behavioural response of larvae to salinity significantly affected the degree of retention and ‘openness’ of the harbour to this species. Although, through natural transport, larvae could theoretically reach the next available habitat within the duration of their pelagic stage our study indicates that areas of sufficiently reduced salinity may be necessary for sufficient retention, recruitment and establishment of new adult populations in estuaries. High resolution hydrodynamic models, coupled with larval behaviour, can accurately simulate and predict biological invasion along complex coastlines and contribute to risk assessment of the introduction of non‐indigenous species for aquaculture and spatial management of marine protection.     

基于大叶藻成苗率的新型海草播种技术评价

蛤蜊播种技术是一种新型海草播种技术,该技术中,种子通过糯米糊粘在蛤蜊贝壳上,随蛤蜊穴居被埋入底质。为评价蛤蜊和糯米糊对种子成苗率的影响,以菲律宾蛤仔(Ruditapes philippinarum)和大叶藻(Zostera marina)种子为试验对象,设置了直接播撒种子(A1组)、直接埋种(A2组)、包埋糯米后播撒(B1组)、包埋糯米后埋入底质(B2组)、蛤蜊播种(C)5种处理,每种处理设置3个重复,在实验室的水槽中对其进行试验。结果表明:糯米糊对种子萌发有负面影响,但由于蛤蜊的行为使底质具有透气性,减轻了糯米糊对种子萌发的影响,种子成苗率可达到23.2%;海区试验中利用蛤蜊播种的两个样方中的成苗率分别为19.1%和9.9%。试验表明,蛤蜊播种技术适合作为一种经济、有效的播种技术用于海区海草床的建立和修复。     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

Molecular phylogeography of Ruditapes philippinarum in the Northwestern Pacific Ocean based on COI gene

To examine the pattern of phylogeography of Ruditapes philippinarum, a length of 644-bp gene fragment of the mtDNA COI was sequenced. A total of 170 individuals from 19 locations were analyzed yielding 74 haplotypes, most of which were unique. The levels of haplotype diversity and nucleotide diversity for the species ranged from 0.80 ± 0.16 (Notsuke Bay) to 1.00 ± 0.13 (Nanao Bay, Miyazu Bay, Dalian 1 and Ariake), and from 0.002 ± 0.001 (Notsuke Bay) to 0.011 ± 0.006 (Qingdao), respectively. Both the phylogenetic (NJ tree) and minimum spanning trees (MST) showed three significant genealogical clusters corresponding to sampling localities, a genetic differentiation speculated to be caused by the isolation of the marginal seas of the Northwestern Pacific during Pleistocene low sea-level stands. Both AMOVA and pairwise Fst analyses revealed significant genetic differentiation between the populations from Japan and China. The pattern of isolation by distance was also detected in this species (r = 0.50, P < 0.001). Both mismatch distribution analysis and the neutrality tests showed that R. philippinarum had undergone a recent population expansion. The estimate of population expansion time was about 425 kya-1580 kya.     

可食用文蛤中氧化性胁迫的生物标记物与甲基毒死蜱和草甘膦的生物富集

近几十年期间,在种群和个体受干扰后,对作为早期指示剂的生物标记物的研究受到越来越多的关注。我们用对水生生态系统污染敏感的生物标记物双壳类软体动物(文蛤)来评估两种有机磷杀虫剂(甲基毒死蜱、草甘膦)的影响。文蛤是水生生态系统污染的一种敏感的指示物种。在不同时间段测定文蛤中不同组织的非酶的(谷胱苷肽)和酶的(过氧化氢酶)抗氧化剂,作为文蛤中生物标记物的反应。在实验室条件下,测定了脂质过氧化作用、蛋白羰基含量、总蛋白含量、总脂质含量以及胆碱酯酶的活性。对不同的生物标记与杀虫剂的生物体内积累的相互关系进行了研究。甲基毒死蜱在文蛤组织中具有最大的诱导氧化胁迫的潜能,导致脂质过氧化反应增加并抑制抗氧化剂。而且,鳃是对该反应最敏感的器官。文蛤是一种极好的甲基毒死蜱的积聚者,因为暴露60天后,可以测定到其组织中浓度为824.0 mg/kg w.w的甲基毒死蜱。随着在草甘膦中暴露时间的增加,与背景水平相比,组织中草甘膦的浓度增加大约8×103mg/kg w.w。可以得出这样的结论:在一种生物中测定几种生物标记物是有用的。在双壳类中,蛋白质的羰基诱导可用于双壳类中化学污染物诱导的氧化胁迫的生物指示剂。抗氧化剂的防御成分是敏感的参数,是评估污染的水生生态系统的有用的生物标记物。辅以蛤组织的化学分析,生物标记参数能够提供一种有力的监测工具。     

大蒜对菲律宾蛤仔早期生长发育的影响

实验了不同大蒜汁浓度(0、2、4、8、16、32 mg/L)对菲律宾蛤仔受精卵孵化率,幼虫生长、存活、变态及稚贝生长与存活的影响,总结了室内大规模人工育苗过程中大蒜防病效果。结果表明:随着大蒜汁浓度的增加,孵化率降低;大蒜汁浓度达到16mg/L,胚胎发育延迟;达到32mg/L,受精卵不能孵化为正常幼虫。浮游期间,幼虫的生长受大蒜汁抑制,幼虫的存活率则随着大蒜汁浓度增加先升高后降低;幼虫的变态率随着大蒜汁浓度的增加先升高后降低,以16mg/L为最适浓度;变态规格随着大蒜汁浓度增加而减小。室内培育期间,稚贝生长与存活随着大蒜汁浓度的增加先升高后降低,以8mg/L为最适浓度;室内大规模人工育苗过程中,使用浓度为8-10mg/L大蒜汁可以起到较好的防病效果。