高温胁迫下厚壳贻贝(Mytilus coruscus)消化腺代谢组学研究

在全球变暖的背景下,海水温度不断升高、海洋暖化逐渐加剧,高温严重影响着海洋生物的各种生命过程,但对于海洋双壳贝类如何应对热应激的研究仍然不足。为此,开展了以18℃(CT)为对照组在26℃(ST)和33℃(HT)下对厚壳贻贝消化腺组织进行了急性热胁迫下的代谢组学分析,以便于研究其代谢反应。采用LC-MS/MS技术,并结合生物信息分析手段对差异代谢物进行筛选,并分析确定相关的代谢通路的变化,共有2532种代谢物在厚壳贻贝消化腺中被鉴定。KEGG富集分析用于探索差异代谢物的潜在代谢途径,共有29条代谢通路被显著富集,与对照组相比,ST组显著富集于牛磺酸和次牛磺酸代谢、神经活性配体-受体相互作用、鞘脂类代谢和视黄醇代谢等代谢通路;HT组显著富集于酪氨酸代谢、亚油酸代谢、丙氨酸新陈代谢、酪氨酸代谢、色氨酸代谢、苯丙氨酸代谢等代谢通路。研究结果显示,厚壳贻贝消化腺主要通过调节色氨酸代谢、酪氨酸代谢、鞘脂代谢、苯丙氨酸代谢、氧化磷酸化,脂肪酸、赖氨酸降解等信号通路应对热应激,从而帮助维持身体内部环境的稳定状态。上述研究为多视角探究厚壳贻贝耐热机制与应对环境中温度变化的适应性进化提供理论基础。

海水酸化对厚壳贻贝(Mytilus coruscus)外套膜代谢组的急性影响

海洋酸化是当前全球面临的最为紧迫的环境问题之一,已显现出对海洋生物的严重影响。贻贝是我国重要的经济贝类之一,但目前尚缺乏海洋酸化背景下厚壳贻贝的分子响应相关研究。为此,对厚壳贻贝外套膜组织开展了急性酸化条件下的代谢组学分析,以期了解厚壳贻贝在酸化条件下,其外套膜组织的代谢物组成和含量变化。采用超高压液相色谱-串联质谱技术,结合代谢物数据库搜索和鉴定,从厚壳贻贝外套膜中鉴定代谢物总计7882种。与对照组相比,酸化处理后的厚壳贻贝外套膜组织中共有497种代谢物含量发生显著变化(P<0.05),其中,320种差异代谢物显著上调,177种显著下调。差异代谢物富集的代谢通路主要包括脂代谢、信号转导、维生素代谢、核酸代谢、氨基酸代谢以及ABC转运体。结合外套膜游离氨基酸组成分析以及抗氧化活性分析,推测厚壳贻贝通过激活尿素合成、增强细胞膜流动性以及加强渗透压调节和钙离子转运等方式来应对海洋酸化的威胁。上述研究为深入了解贻贝应对海洋酸化的分子策略,以及在海洋酸化大背景下的贻贝健康养殖奠定了基础。

Genetic Analysis of Cultured and Wild Populations of Mytilus coruscus Based on Mitochondrial DNA

DNA sequences from the mitochondrial gene cytochrome oxidase subunit I （mtDNA CO I） were used to estimate the genetic variability in two wild populations and two cultured populations of the hard shelled mussel, Mytilus coruscus. Thirty haplotypes were identified in the four populations. The cultured populations exhibited a lower number of haplotypes and genetic diversity than those of the wild populations, suggesting that a small number of effective founding breeders contributed to the genetic variation of the cultured populations. No significant differentiation was observed between the cultured population and local wild population, implying that persistent gene flow occurred in these populations. This genetic survey is intended as a baseline for future genetic monitoring of M. coruseus aquaculture stocks.

A Preliminary Analysis on the Construction and Expression of Eukaryotic Expression Vectors of Mytilin and Myticin from Mytilus coruscus

[Objective] The aim was to study the construction and expression of eukaryotic expression vectors of antibacterial peptides （mytilin and myticin） from Mytilus coruscus.[Method] By the screening of antibacterial peptide genes of mytilin and myticin of Mytilus coruscus,five antibacterial peptide genes were selected.Then,the relative eukaryotic expressing vectors were constructed by the use of PCR technique and DNA recombinant technology.Subsequently,they were transferred in to S78 Saccharomyces cerevisia by using LiAC transformation method,and then preliminary expressing analysis was carried out.[Result] Five eukaryotic expressing vectors of antibacterial peptides from Mytilus coruscus were successfully constructed,and the results of mRNA detection revealed that the five antibacterial peptides from Mytilus coruscus were successfully transcribed.[Conclusion] The results provide a basis for using genetic engineering to express antibacterial peptides of mytilin and myticin from Mytilus coruscus,and for developing the further study of antibacterial peptides from Mytilus coruscus based on this.

厚壳贻贝Mytilus coruscus足丝盘及足丝的多巴分析(英文)

多巴(3,4-l-dihydroxyphenylalanine,DOPA)是贻贝足丝粘附蛋白中的一种特殊的氨基酸,由酪氨酸经羟化后生成,与贻贝足丝粘附蛋白的强粘附性能具有直接联系.目前,已鉴定的多种贻贝足丝蛋白序列中均发现有不同含量的DOPA存在.蛋白中DOPA的定量检测对于了解DOPA在蛋白粘附中的作用以及粘附蛋白的粘附机理具有重要意义.为了解厚壳贻贝足丝盘和足丝纤维中DOPA的含量以及DOPA与厚壳贻贝足丝蛋白的粘附性能之间的关系,利用氮蓝四唑(nitrobluetetrazolium,NBT)对DOPA的特异染色原理,结合蛋白质电泳、组织化学染色以及玻璃包被实验,分析和比较了厚壳贻贝足丝不同部位的DOPA含量、蛋白组成特点以及粘附能力.NBT可以在不水解蛋白质的情况下进行DOPA的定量分析,且与DOPA的氧化状态无关.研究结果表明,厚壳贻贝足丝盘中蛋白的DOPA含量明显高于足丝,两者DOPA含量相差2.33倍;蛋白质电泳结果表明,厚壳贻贝足丝盘和足丝纤维的蛋白质组成差异较大,足丝盘中的蛋白质分子量范围明显低于足丝纤维;玻璃包被实验结合POQuest软件半定量分析表明,厚壳贻贝足丝盘蛋白的粘附能力明显高于足丝纤维,且蛋白在玻璃表面的吸附能力与DOPA含量具有相关性.通过以上研究,建立了一种蛋白中DOPA的定量分析方法并初步了解了厚壳贻贝足丝盘和足丝纤维的蛋白组成、DOPA含量以及粘附性能之间的关系,为深入了解贻贝足丝的粘附性能、粘附机制以及开发新型生物粘合剂奠定了基础.

Effect of Salinity-Changing Rates on Filtration Activity of Mussels from Two Sites within the Baltic Mytilus Hybrid Zone:The Brackish Great Belt(Denmark)and the Low Saline Central Baltic Sea

Mussels from two sites within the Baltic mussel (Mytilus edulis × M. trossulus) hybrid zone were used in a comparative study on the effects of salinity-changing rates on filtration activity. The acute effect of varying salinity-changing rates was found to be similar in M. edulis from the brackish Great Belt and in M. trossulus from the low saline Central Baltic Sea, and the relationships could be described by linear regression lines through 0.0 indicating that the acute effect of deteriorating conditions at decreasing salinities is the opposite as for improving conditions when the salinity is subsequently increased. Further, both M. edulis and M. trossulus acclimatized to 20 psu reacted to an acute salinity change to 6.5 psu by immediately closing their valves whereupon the filtration rate gradually increased during the following days, but only M. trossulus had completely acclimatized to 6.5 psu within 5 days which may be explained by different genotypes of M. edulis and M. trossulus which probably reflected an evolutionary adaptation of the latter to survive in the stable low-salinity Baltic Sea.

Biochemical response of the mussel Mytilus coruscus(Mytiloida:Mytilidae) exposed to in vivo sub-lethal copper concentrations

Many aquatic organisms are negatively affected by exposure to high copper concentrations. We investigated the biochemical response of the mussel Mytilus coruscus （Mytiloida： Mytilidae） to copper exposure. In vivo bioassays using M. coruscus and different copper concentrations were conducted. The activity of six biomarkers, namely superoxide dismutase （SOD）, catalase （CAT）, acid phosphatase （ACP）, alkaline phosphatase （AKP）, glutamic-oxaloacetic transaminase （GOT） and glutamic-pyruvic transaminase （GPT） were measured. Survival rates decreased with increased copper concentrations and exposure times. The LCs0 values at 48, 72, and 96 h exposure were 0.48, 0.37, and 0.32 rag/L, respectively. Within digestive glands, CAT activity increased with increasing Cu concentrations. The activity of AKP showed no significant change, while the remaining four enzymes showed decreasing activity with increasing Cu concentrations. Within the gills, AKP activity increased when the Cu concentration was 0.05 mg/L, but showed no significant changes at higher concentrations. Activity of CAT and ACP within gills tended to decrease with increasing Cu concentration. The activity of SOD and GPT decreased at an exposure concentration of 0.2 mg/L. GOT activity within gills decreased at 0.1 mg/L and increased at an exposure concentration of 0.2 mg/L. Within the adductor muscle, AKP activity increased at 0.05 mg/L but did not change at higher exposure concentrations. ACP activity within adductor muscle tissue showed no change, while activities of CAT, GOT and GPT decreased with increasing Cu concentrations. SOD activity within the adductor muscle tissue significantly decreased at the 0.02, 0.05 and 0.2 mg/L exposure concentrations. Our results show tissue specific differences for the six biomarkers in for M. coruscus. Our findings provide the basis for the establishment of reference activity levels against which biomarker changes can be estimated, and are essential preliminary steps in development of in vivo bioassays.

Expression profiles of two small heat shock proteins and antioxidant enzyme activity in Mytilus galloprovincialis exposed to cadmium at environmentally relevant concentrations

Small heat shock proteins encompass a widespread but diverse class of proteins, which play key roles in protecting organisms from various stressors. In the present study, the full-length cDNAs of two small heat shock proteins （MgsHSP22 and MgsHSP24.1） were cloned from Mytilus galloprovincialis, which encoded peptides of 181 and 247 amino acids, respectively. Both MgsHSP22 and MgsHSP24.1 were detected in all tissues examined by real-time PCR, with the highest expression being observed in muscle and gonad tissues. The real-time PCR results revealed that Cd significantly inhibited MgsHSP22 expression at 24 h and MgsHSP24.1 at 24 and 48 h under 5 ug/L Cd2＋ exposure. MgsHSP24.1 expression was also significantly inhibited after 50 ug/L Cd2＋ exposure for 48 h. With regard to antioxidant enzymes, increased GPx and CAT activity were detected under Cd2＋ stress （5 and 50 ug/L）, while no significant difference in SOD activity was observed throughout the experiment. Overall, both MgsHsps and antioxidant enzymes revealed their potential as Cd stress biomarkers in M. galloprovincialis.

Beneficial effects of high-pressure homogenization on the dispersion stability of aqueous hydrolysate from Mytilus edulis

Owing to the formation of aggregation and gelation during storage,certain proteins and peptides exhibit limited applications in aqueous protein food products.The purpose of this study was to investigate the influence of homogenization and xanthan gum addition on the dispersion stability of Mytilus edulis hydrolysate(PHM).High-pressure homogenization(HPH)at 360 and 40 bar in the first and second values,respectively,and adding xanthan gum at a concentration of 1 mg/mL showed significantly improvement on the stability of the PHM solution.PHM-xanthan gum solutions(PHMX)showed the highest polypeptide precipitation rate and turbidity retention rate compared with those of PHM.Moreover,the centrifugal precipitation rate of PHMX without HPH was higher than that of homogeneous PHMX.After HPH treatment at 400 bar,the percentage of smaller particles in PHM and PHMX was increased,the aqueous system became more uniform,and the fluorescence intensity reached its maximum.HPH pretreatment improved the polypeptide dispersion stability and turbidity retention rate of PHM and PHMX and reduced the fluorescence intensity.The interactions of xanthan gum and polypeptide render the network microstructure more uniform under the conditions of homogenization,thus improving the dispersion stability of PHMX solutions.Therefore,under the premise of adding xanthan gum,HPH can better enhance the dispersion stability of the polypeptide in PHM.

Cryopreservation of strip spawned sperm using programmable freezing technique in the blue mussel Mytilus galloprovincialis

In this study, a programmable freezing technique has been developed for strip spawned sperm in the blue mussel, M ytilus galloprovincialis. The optimized key parameters include cooling rate, endpoint temperature, thawing temperature, sugar addition and sperm to oocyte ratio. The sperm quality was assessed by the fertilization rate or the integrity of sperm component and organelle. The highest post-thaw sperm fertilization rate was 91%, which was produced with sperm cryopreserved in 8% dimethyl sulfoxide at the cooling rate of-4°C/min from 2°C to-30°C before being plunged into liquid nitrogen for at least 12 h, thawed in a 20°C seawater bath and fertilized at sperm to egg ratio of 50 000:1. The addition of glucose, sucrose or trehalose to 8% dimethyl sulfoxide could not further improve fertilization rates. The fluorescent assessments showed that the post-thaw sperm plasma membrane integrity and acrosome integrity were significantly damaged in comparison with fresh sperm.

An integrated biomarker response index for the mussel Mytilus edulis based on laboratory exposure to anthracene and field transplantation experiments

Organic pollution is a serious environmental problem in coastal areas and it is important to establish quantitative methods for monitoring this pollution. This study screened a series of sensitive biomarkers to construct an integrated biomarker response （IBR） index using Mytilus edulis. Mussels were exposed to the polycyclic aromatic hydrocarbon anthracene trader controlled laboratory conditions and the activities of components of the glutathione antioxidant system, and the concentrations of oxidative-damage markers, were measured in the gills and digestive glands. Anthracene exposure resulted in increased levels of malondialdehyde （MDA） and superoxide radicals （O2-·）, indicating that oxidative damage had occurred. Correspondingly, anthracene exposure induced increased activities of glutathione S-transferase （GST）, glutathione peroxidase （GPx） and reduced glutathione （GSH） in digestive glands, and GPx and glutathione reductase （GR） in gills, consistent with stimulation of the antioxidant system. A field experiment was set up, in which mussels from a relatively clean area were transplanted to a contaminated site. One month later, the activities of GST, GPx and GR had increased in several tissues, particularly in the digestive glands. Based on the laboratory experiment, an IBR, which showed a positive relationship with anthracene exposure, was constructed. The IBR is suggested to be a potentially useful tool for assessing anthracene pollution.

Carnosine concentration and expression profiles of carnosine related genes in Mytilus after beta-alanine injection

Carnosine and its analogues are histidine-containing dipeptides(HCDs)playing diverse functions in vertebrates.However,the distribution and the metabolism of carnosine in invertebrates are still unknown.In this study,Mytilus coruscus,a shellfish with important economic value in China,was selected for the investigation of HCD content and the expression profiling of carnosine-related genes in various mussel tissues.Quantification of HCD by amino acids analyzer revealed a low concentration of anserine in muscular tissues in Mytilus,indicating the presence of HCD even in an invertebrate.mRNA expression of five carnosine metabolic-related genes was profiled in various tissues,and the results highlighted the relative higher expression level of these genes in muscular tissues.Considering the fact that beta-alanine supplementation can increase the HCD content in vertebrates,a beta-alanine injection was performed and the changes of HCD concentration and the mRNA expression of carnosine related genes were investigated in five mussel tissues.The results revealed the increase of HCD concentration,as well as the up-regulated expression level of related genes,in tested tissues of beta-alanine injected mussels.Transcriptomic analysis for the whole soft tissue of mussel before and after beta-alanine injection were performed,and 3569 differential expression genes(DEGs)were identified in the beta-alanine injected group when compared to their expression levels in the control.These data indicated the complex eff ects of betaalanine on M.coruscus metabolism,and those DEGs enriched in pathways of cancers,muscle contraction,and tyrosine metabolism highlighted the possible functions of beta-alanine in cell proliferation,sports,and melanogenesis,respectively.

Relationship between oxygen concentration, respiration and filtration rate in blue mussel Mytilus edulis

The large water-pumping and particle-capturing gills of the filter-feeding blue mussel Mytilus edulis are oversized for respiratory purposes. Consequently, the oxygen uptake rate of the mussel has been suggested to be rather insensitive to decreasing oxygen concentrations in the ambient water, since the diffusion rate of oxygen from water flowing through the mussel determines oxygen uptake. We tested this hypothesis by measuring the oxygen uptake in mussels exposed to various oxygen concentrations. These concentrations were established via N2-bubbling of the water in a respiration chamber with mussels fed algal cells to stimulate fully opening of the valves. It was found that mussels exposecl to oxygen concentrations decreasing from 9 to 2 mg O2/L resulted in a slow but significant reduction in the respiration rate, while the filtration rate remained high and constant. Thus, a decrease of oxygen concentration by 78% only resulted in a 25% decrease in respiration rate. However, at oxygen concentrations below 2 mg O2/L M. edulis responded by gradually closing its valves, resulting in a rapid decrease of filtration rate, concurrent with a rapid reduction of respiration rate. These observations indicated that M. edulis is no longer able to maintain its normal aerobic metabolism at oxygen concentration below 2 mg O2/L, and there seems to be an energy-saving mechanism in bivalve molluscs to strongly reduce their activity when exposed to low oxygen conditions.

紫贻贝(Mytilus edulis Linnaeu)大量死亡原因的初步研究

从发病紫贻贝(Mytilus deulis)的内脏团内分离出一株菌SS1,通过人工感染试验,证实其为致病菌。经形态、生理生化等几项指标鉴定,该菌株性状与溶藻弧菌基本相符。药物敏感试验结果表明羧苄青霉、万古霉素、克林霉素、氨苄青霉素、阿莫西林等9种药物对该菌株均有明显的抑制作用。

紫贻贝(Mytilus edulis Linnaeus)大量死亡原因的初步研究

从发病紫贻贝(Mytilusedulis)的内脏团内分离出一株菌SS1,通过人工感染试验,证实其为致病菌。经形态、生理生化等几项指标鉴定,该菌株性状与溶藻弧菌基本相符。药物敏感试验结果表明羧苄青霉、万古霉素、克林霉素、氨苄青霉素、阿莫西林等9种药物对该菌株均有明显的抑制作用。

Trace Metals in Mussels <i>Mytilus galloprovincialis</i>from Dakar Coast (Senegal)

Mussel samples from the Dakar coast (Senegal) were analysed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Microwave acid digestion was also employed for metals (Mn, Pb, Cd, As) determination. Dakar coast usually receives numerous domestic and industrial discharges without prior treatment. The contents of Arsenic were, in all cases, higher than other metals. However, the bivalve molluscs present themselves as effective bio-monitors when assessing marine aquatic pollution by contaminants in the Dakar coast. The ANOVA analysis allows concluding that significant differences were found between mussels from different sampling points. In all cases, the February samples have in all cases a higher content than those collected in October.

Effect of Salinity on Growth of Mussels,Mytilus edulis,with Special Reference to Great Belt(Denmark)

The effects of salinities between 10 and 30 psu on the growth of blue mussels, Mytilus edulis, were studied in laboratory feeding experiments and compared to the growth of mussels suspended in net-bags in the brackish water Great Belt, Denmark. In the laboratory, 3 series of growth experiments were conducted: in Series #1, groups of mussels were exposed to 10, 15, 25 and 30 psu, in Series #2, two groups of mussels were exposed to 10 and 30 psu, respectively, for 15 days (first period) where upon the mussels were exposed to the reversed salinities for another 15 days (second period). In Series #3, two groups of mussels were initially exposed to 15 and 25 psu for 22 days whereupon the mussel groups were exposed to the reversed salinities for another 17 days. In the laboratory experiments there was a tendency towards reduced growth with decreasing salinity, reflected as reduced shell growth rate and decreasing weight specific growth rate with falling salinity. The shell growth rate was relatively low in the first feeding period compared to the second period, and mussels that were initially exposed to 10 psu, where the growth was low, exhibited fast growth when subsequently exposed to 30 psu, and reversed when 30 psu mussels were exposed to 10 psu. The study showed that mussels are able to adjust growth at changing salinities, and the observed effect of salinity could partly be explained by a temporary shell valve closure after a sudden change in salinity. The specific growth rate of mussels measured in laboratory experiments at salinities between 15 to 25 psu (4.2% to 4.8% d–1) were comparable to the growth of mussels in the field experiment (3.2% to 4.0% d–1) where the salinity varied between 24 and 13 psu during the growth period.

Genotoxic Potential of Copper Oxide Nanoparticles in the Bivalve Mollusk Mytilus trossulus

Copper oxide nanoparticles(CuO-NPs) are among the most widely used metal oxide nanoparticles,which increases the chance of their being released into the marine environment.As the applications of these particles have increased in recent years,their potential impact on the health of marine biota has also increased.However,the toxicological effects of these NPs in the marine environment are poorly known.In the present study,the DNA damaging potential of CuO-NPs in the marine eastern mussel Mytilus trossulus was evaluated and compared to that of dissolved copper exposures.Genotoxicity was assessed by the single cell gel electrophoresis(comet) assay in mussel gill and digestive gland cells.The results showed that copper in both forms(CuO-NPs and dissolved copper) was accumulated to different extents in mussel tissues.The mussel exposed to the dissolved copper attained higher concentrations of copper in the gills than in the digestive gland.In contrast to these results,it was found that CuO-NPs could induce much higher copper accumulation in the digestive gland than in the gills.A clear and statistically significant increase in DNA damage was found in both tissues of the Cu-exposed group compared to the control mussels.Our results indicated that the CuO-NP exposure produced remarkable effects and increased DNA damage significantly in mussel gill cells only.It should be noted that the digestive gland cells were prone to accumulation following CuO-NPs when compared to the gill cells,while the gill cells were more sensitive to the genotoxic effects of CuO-NPs.These results also suggested the need for a complete risk assessment of engineered particles before its arrival in the consumer market.

Metabolism of pectenotoxins in brown crabs Cancer pagurus fed with blue mussels Mytilus edulis

We investigated the metabolism of pectenotoxins in brown crabs(Cancer pagurus).The crabs were fed with blue mussels(Mytilus edulis) for 21 d then depurated for 42 d.We extracted the toxins from the digestive glands of contaminated crabs,uncontaminated crabs(control group),and the meat of blue mussels using methanol.Extracts of the crab digestive glands were fractionated by liquid-liquid partitioning and solid phase extraction.The fractions were analyzed by liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS) and liquid chromatography coupled with ion-trap mass spectrometry(LC-MSn).We detected a new PTX-like compound,designated as metabolite-1.The MS2 mass spectrum of the metabolite-1 [M+Na]+ ion at m/z 897.5 revealed fragment ions at m/z 853.5 and 555.5,typical of those exhibited by other pectenotoxins.

An improved glucose-DAB-nickel immunohistochemistry method and its application on protozoan Stylonychia mytilus

Using a modification of the very sensitive glucose-DAB-nickel(GDN)immunohistochemistry method,FOS(the expression protein of the oncogene c-fos)-,neuropeptide Y(NPY)-,somatostatin(SOM)-,leu-enkephalin(L-Enk),cholecystokinin(CCK)-,neurotensin(NT)-,and tyrosine hydroxylase(TH)-immunoreactivities were firstdemonstrated in the protozoan,Stylonychia mytilus.The GDN method and expression ofFOS,neuropeptides and TH in the Stylonychia mytilus were discussed.