舟形藻生物膜诱导下虾夷扇贝(Mizuhopecten yessoensis)幼虫的变态及其蛋白质组学响应

METAMORPHOSIS OF MIZUHOPECTEN YESSOENSIS LARVAE INDUCED BY NAVICULA sp.AND ITS PROTEOMIC RESPONSE

为深入了解舟形藻(Navicula sp.)生物膜诱导虾夷扇贝(Mizuhopecten yessoensis)幼虫附着变态的机制,选用舟形藻和虾夷扇贝幼虫作为研究对象,研究了4个浓度梯度舟形藻液(1A、10A、100A和1000A)下虾夷扇贝幼虫附着变态率以及幼虫响应舟形藻生物膜诱导的蛋白质组学差异。结果表明,稀释10倍后的藻液(1366 ind./mm^(2))所形成的生物膜对幼虫诱导率最高为45%,与空白组(13%)差异极显著(P<0.01),过高或过低浓度都导致诱导率降低。采用非标记定量技术(4D label free)蛋白质组学分析比较舟形藻生物膜和对照组幼虫发现,经舟形藻生物膜诱导后共316个蛋白质表达显著上调或下调。如谷胱甘肽S转移酶、谷胱甘肽过氧化物酶、组织蛋白酶K、组织蛋白酶L、NAD(P)H氧化酶(形成H2O2)、软骨基质蛋白、胶原蛋白和铁蛋白等。以上这些差异蛋白表达的上下调均与贝类幼虫在附着变态过程中形态学变化息息相关。因此,舟形藻生物膜诱导虾夷扇贝幼虫附着变态的有效成分可能是其产生的胞外多糖,幼虫体内的凝集素检测到舟形藻生物膜表面的多糖后,特异性与多糖结合,最终诱导幼虫完成附着和变态。研究结果可为虾夷扇贝人工培育提供理论依据。

To understand the mechanism of the effect of Navicula sp.film substract on the settlement and metamorphosis of Mizuhopecten yessoensis larvae,the settlement and metamorphosis rates of the larvae and the proteomic differences were investigated under four concentrations of Navicula sp.solution(1A,10A,100A,and 1000A;A is the multiple of dilution of the original concentration)and sterilized natural seawater as the control.Results show that the highest metamorphosis rate(45%)was found in Group 10A(1366/mm^(2)),which differed significantly(P<0.01)from that of the control(13%).Too high or too low concentration resulted in a lower induction rate.Using non-labeled quantitative techniques(4D label free)proteomic analysis to compare between Navicula sp.filmed substract and the control group larvae,we found that 316 proteins were significantly up-(198)or down-regulated(118)after induction by the Navicula sp.biofilm.The expressions of cathepsin K,cathepsin L,cartilage matrix proteins,collagen,ferritin,glutathione S-transferase,glutathione peroxidase,and lectin were elevated,while those of heat shock proteins 70 and NAD(P)H oxidase(forming H2O2)proteins were decreased.Proteins such as histone K,histone L,and heat shock proteins were associated with apoptosis,indicating that there is substantial apoptosis during larval settlement and metamorphosis.Proteins such as cartilage matrix proteins,collagen,and ferritin were associated with calcium carbonate shell formation,indicating that larvae induced by Navicula sp.film were in the process of completing the construction of adult shells.Proteins of glutathione S-transferase,glutathione peroxidase,and NAD(P)H oxidase(forming H2O2)were associated with immune function,indicating the maintenance of cellular homeostasis during larval settlement and metamorphosis.C-type lectins and galectin were bound to polysaccharides to trigger larval settlement and metamorphosis.The up-and down-regulation of these differential proteins were closely related to morphological changes in larvae during settlement and metamorphosis.The effective component of the biofilm inducing the settlement and metamorphosis of the scallop larvae might be the extracellular polysaccharides produced by Navicula sp.After detecting the polysaccharides on the surface of the Navicula sp.filmed substract,the lectins in the larvae would bind specifically to the polysaccharides,inducing the larvae to complete settlement and metamorphosis.This study provided a theoretical basis for artificial cultivation of bivalve seedlings.