›› 2012, Vol. 55 ›› Issue (5): 527-534.doi:

• 研究论文 • 上一篇    下一篇

拟双角斯氏线虫D43品系鞘蛋白对大蜡螟幼虫的免疫抑制作用

杨君, 曾洪梅, 邱德文, 林华峰, 杨秀芬, 郭立华, 袁京京   

  1. 安徽农业大学植物保护学院, 合肥 230036
  • 收稿日期:2012-01-08 修回日期:2012-03-28 出版日期:2012-05-20 发布日期:2012-05-20
  • 通讯作者: 林华峰 E-mail:hf.lin@163.com
  • 作者简介:杨君, 男, 1981年生, 河北抚宁人, 博士研究生, 研究方向为害虫生物防治, E-mail: yang22181@163.com
  • 基金资助:

    国家自然科学基金项目(31071741); 农业部“948”项目(2011-G4)

Suppression of Galleria mellonella (Lepidoptera: Pyralidae) larval immune responses induced by sheath proteins of the entomopathogenic nematode Steinernema ceratophorum strain D43

YANG Jun, ZENG Hong-Mei, QIU De-Wen, LIN Hua-Feng, YANG Xiu-Fen, GUO Li-Hua, YUAN Jing-Jing   

  1. College of Plant Protection, Anhui Agricultural University, Hefei 230036, China
  • Received:2012-01-08 Revised:2012-03-28 Online:2012-05-20 Published:2012-05-20
  • Contact: LIN Hua-Feng E-mail:hf.lin@163.com
  • About author:yang22181@163.com

摘要: 侵染期的拟双角斯氏线虫Steinernema ceratophorum D43品系体外都包裹着一个透明的体鞘。为探明体鞘对线虫侵染力的影响, 了解鞘蛋白(sheath proteins, SPs)对大蜡螟Galleria mellonella 幼虫的免疫抑制作用, 本研究通过化学方法使拟双角斯氏线虫D43脱鞘, 以对寄主的致死率和侵入点数量为指标, 与包鞘线虫比较对大蜡螟幼虫的侵染力; 采用乙醇提取的方法获得线虫鞘蛋白, 利用双向电泳和质谱技术对鞘蛋白进行鉴定分析; 从血细胞数量和酚氧化酶活力两个方面评价鞘蛋白对大蜡螟幼虫免疫反应的抑制作用。结果表明: 0.5%次氯酸钠处理20 min可以保证95%以上的线虫存活和脱鞘。与包鞘线虫相比, 脱鞘线虫对大蜡螟幼虫的致死率显著降低, 致死时间延后, 节间膜侵入点数量显著减少。以35%乙醇提取的鞘蛋白提取物可鉴定出6种鞘蛋白, 其中一个被鉴定为丝氨酸蛋白酶。此外, 血腔注射鞘蛋白提取物可导致试虫血细胞数量明显降低, 酚氧化酶活力受到显著抑制。由此说明, 体鞘对拟双角斯氏线虫D43的侵染力具有显著影响, 鞘蛋白在抑制寄主昆虫免疫反应中发挥重要作用。

关键词: 昆虫病原线虫, 拟双角斯氏线虫, 大蜡螟, 鞘蛋白, 免疫反应

Abstract: Infective juveniles (IJs) of Steinernema ceratophorum strain D43 are all ensheathed in a transparent sheath. In order to investigate the effect of sheaths on infectivity of S. ceratophorum D43 and determine the roles of sheath proteins (SPs) in immune suppression of Galleria mellonella larvae, we first obtained the desheathed IJs with chemical exsheathment method. And then, the infection rate of desheathed and ensheathed IJs to G. mellonella larvae was compared according to the host mortality and number of penetration sites on the host. Moreover, the SPs from IJs were extracted with ethanol and characterized by two dimensional electrophoresis (2-DE) and MALDI-TOF-MS. Finally, the influence of SPs on hemocyte number and phenoloxidase (PO) activity of G. mellonella larvae was evaluated. The results indicated that over 95% ensheathed nematodes lost their sheaths after being exposed to 0.5% sodium hypochlorite at room temperature for 20 min and kept alive. Desheathed nematodes caused lower mortality of G. mellonella larvae, longer lethal time and less penetration sites compared to ensheathed nematodes. The SPs extracted in cold 35% ethanol showed six protein spots on 2-DE, and only one was successfully identified as a serine protease by peptide mass fingerprinting. The hemocyte number and PO activity in G. mellonella larvae injected with the extracted SPs were significantly reduced and suppressed, respectively. These results suggest that sheaths have an important role in pathogenicity of S. ceratophorum D43, and the SPs are implicated in the suppression of host immune responses.

Key words: Entomopathogenic nematode, Steinernema ceratophorum, Galleria mellonella, sheath proteins (SPs), immune responses