小卷蛾斯氏线虫侵染对草地贪夜蛾幼虫天然免疫反应的影响

Effects of infection of the entomopathogenic nematode Steinernema carpocapsae All on the innate immune response in Spodoptera frugiperda(Lepidoptera:Noctuidae)larvae

【目的】探讨昆虫病原线虫小卷蛾斯氏线虫Steinernema carpocapsae All侵染对草地贪夜蛾Spodoptera frugiperda幼虫天然免疫反应的影响。【方法】借助倒置显微镜观察和鉴定草地贪夜蛾幼虫的血细胞类型,并对小卷蛾斯氏线虫侵染后不同时间的草地贪夜蛾幼虫血细胞总数目进行统计;通过倒置显微镜观察草地贪夜蛾幼虫对侵入的小卷蛾斯氏线虫的包囊反应;利用倒置荧光显微镜观察小卷蛾斯氏线虫侵染后的草地贪夜蛾幼虫血细胞对金黄色葡萄球菌Staphylococcus aureus的吞噬活性;检测小卷蛾斯氏线虫侵染后的草地贪夜蛾幼虫血淋巴中酚氧化酶(phenoloxidase,PO)活性、体内抗菌肽基因相对表达水平以及血浆的抗菌活性。【结果】从草地贪夜蛾幼虫体内共发现5种不同类型的血细胞,分别为原血细胞、粒细胞、类绛色细胞、珠血细胞和浆血细胞。注射1μL侵染期(infective juveniles,IJs)小卷蛾斯氏线虫(3 IJs/μL)后9和12 h,草地贪夜蛾幼虫的血细胞总数目显著增多。草地贪夜蛾幼虫的血细胞不能包囊活的以及冷处死的小卷蛾斯氏线虫,但可以包囊热处死的线虫。活的小卷蛾斯氏线虫会显著抑制草地贪夜蛾幼虫血细胞对金黄色葡萄球菌的吞噬活性,但冷处死和热处死的线虫不能。注射1μL(3 IJs/μL)小卷蛾斯氏线虫后,草地贪夜蛾幼虫血淋巴PO活性总体呈“下降-升高-下降”变化趋势;体内抗菌肽基因Attacin-A2,Attacin-B1,Cecropin-B3,Cecropin-D,Gallerimycin,Gloverin-3以及Lebocin-2的表达水平在线虫侵染后12 h时显著上调,24 h时恢复到对照水平或低于对照水平;血淋巴抗菌活性水平在小卷蛾斯氏线虫侵染后12 h时显著升高,24 h时与对照无显著差异。【结论】小卷蛾斯氏线虫在侵入早期会抑制草地贪夜蛾幼虫的天然免疫反应来建立感染;随后草地贪夜蛾的免疫系统会被激活试图抵御小卷蛾斯氏线虫的侵染;后期随着线虫的成功定殖,草地贪夜蛾的免疫系统最终被抑制或破坏。本研究所得结果为进一步揭示线虫-草地贪夜蛾的免疫互作机理奠定了基础,也为改善昆虫病原线虫对草地贪夜蛾的防治效果提供了理论依据。

【Aim】To investigate the effects of Steinernema carpocapsae All infection on the innate immune response in larvae of the fall armyworm,Spodoptera frugiperda.【Methods】The hemocyte types of S.frugiperda larvae were observed and identified under an inverted microscope,and the total numbers of hemocytes in S.frugiperda larvae at different time after infection by S.carpocapsae All were counted.The encapsulation of invading S.carpocapsae All nematodes by S.frugiperda hemocytes was observed under an inverted microscope.The phagocytoic activity of fluorescent Staphylococcus aureus by hemocytes of S.frugiperda larvae was observed under an inverted fluorescence microscope.The phenoloxidase(PO)activity in the hemolymph,the relative expression levels of antibacterial peptide genes,and the antibacterial activity of plasma in S.frugiperda larvae infected with S.carpocapsae All were detected.【Results】Five types of hemocytes,prohemocyte,granulocyte,oenocytoide,spherulocyte and plasmatocyte,were found in S.frugiperda larvae.The total numbers of hemocytes in S.frugiperda larvae increased significantly at 9 and 12 h after injection of 1μL S.carpocapsae All infective juveniles(IJs)at the dose of 3 IJs/μL.The hemocytes from S.frugiperda larvae failed to encapsulate the live and cold-killed S.carpocapsae All nematodes but could encapsulate heat-killed nematodes.The phagocytic activity of fluorescent S.aureus by S.frugiperda hemocytes was significantly inhibited after incubation with live S.carpocapsae All nematodes,but not with cold-and heat-killed S.carpocapsae All nematodes.The PO activity in the hemolymph of S.frugiperda larvae decreased first,then increased,and finally decreased after injection of 1μL S.carpocapsae All at the dose of 3 IJs/μL.The relative expression levels of antimicrobial peptide genes Attacin-A2,Attacin-B1,Cecropin-B3,Cecropin-D,Gallerimycin,Gloverin-3 and Lebocin-2 in S.frugiperda larvae were significantly induced at 12 h after S.carpocapsae All infection,and then recovered to the control level or lower than the control level at 24 h after infection.The antibacterial activity of S.frugiperda plasma increased significantly at 12 h after S.carpocapsae All infection,but was not significantly different between the treatment group and the control group at 24 h after infection.【Conclusion】In the early stage of infection,S.carpocapsae All would inhibit the innate immune response in S.frugiperda larvae,then the immune system in S.frugiperda would be initiated for trying to defend against S.carpocapsae All,and in the late stage the immune system in S.frugiperda would be inhibited or destroyed with the successful colonization of nematodes.The results obtained in this study provide a basis for further understanding the immune mechanisms involved in the interaction between nematodes and S.frugiperda,and lay a theoretical foundation for further improving the control efficacy of entomopathogenic nematodes against S.frugiperda larvae.