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Journal of Zhejiang University SCIENCE B

ISSN 1673-1581(Print), 1862-1783(Online), Monthly

Midgut immune responses induced by bacterial infection in the silkworm, Bombyx mori

Abstract: Insect gut epithelial cells produce reactive oxygen species (ROS) and antimicrobial peptides (AMPs) to protect hosts from pathogenic microorganisms. In this study, we evaluate the pathogenicity of Pseudomonas aeruginosa and Bacillus bombysepticus in the silkworm, Bombyx mori. Survival curves show that B. bombysepticus is deadly when larval silkworms are infected orally. Bacterial infection caused intestinal hydrogen peroxide (H2O2) and nitric oxide (NO) levels to increase significantly by 8 and 16 h post-infection (hpi), respectively. Real-time quantitative polymerase chain reaction (qPCR) analysis shows that the transcription levels of dual oxidase (Duox) and catalase (CAT) are highly up-regulated by P. aeruginosa infection at 8 hpi. P. aeruginosa infection induced nitric oxide synthase 2 (NOS2) expression at 16 hpi, which contributes to the generation of NO. mRNA levels of AMP genes, specifically Glovorin 2 and Glovorin 3, which obviously increase during the early infection stage. These results indicate that invading bacteria elevate intestinal ROS and NO levels and induce AMP gene transcription, which contributes to intestinal immune defense.

Key words: Bombyx mori, Midgut, Immune, Hydrogen peroxide, Nitric oxide, Antimicrobial peptide

Chinese Summary  <21> 细菌感染引起的家蚕中肠免疫反应研究

目的:探索经喂食细菌感染引起的家蚕肠道内免疫反应变化情况。
创新点:证明了家蚕肠道内的活性氧(ROS)、一氧化氮(NO)及抗菌肽在肠道免疫反应中的重要作用。
方法:通过绿脓杆菌(Pseudomonas aeruginosa)及黑胸败血菌(Bacillus bombysepticus)喂食感染家蚕以后,统计家蚕死亡率、检测感染后不同时间肠道内过氧化氢(H2O2)及NO的水平变化;同时利用实时荧光定量聚合酶链反应(qPCR)检测中肠组织中活性氧相关基因及抗菌肽基因的转录情况。
结论:死亡率结果显示,黑胸败血菌比绿脓杆菌具有更强的致病性。活性氧检测结果显示,喂食细菌感后8 h到16 h,家蚕肠道内H2O2及NO水平显著升高。通过qPCR研究ROS相关基因的表达变化的结果显示,P. aeruginosa感染后 8 h 可诱导肠道内双氧化酶(Duox)及过氧化氢酶(CAT)的转录上调,而感染后16 h,P. aeruginosa可诱导NO合成关键基因(一氧化氮核酶2,NOS2)的上调表达,喂食细菌感染同样可以诱导家蚕中肠抗菌肽基因的上调表达,而抗菌肽Glovorin 2及Glovorin 3在感染初期转录上调最为明显。实验结果进一步证明ROS、NO及AMP的产生在家蚕肠道免疫防御中的重要作用。

关键词组:家蚕;中肠;免疫;过氧化氢;一氧化氮;抗菌肽


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DOI:

10.1631/jzus.B1500060

CLC number:

S884.4

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On-line Access:

2015-10-03

Received:

2015-03-12

Revision Accepted:

2015-07-06

Crosschecked:

2015-09-17

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