昆虫病原线虫Heterorhabditidoides rugaoensis及其共生菌Serratia nematodiphila R187与？\腹果蝇免疫系统的互作研究

[目的] 本研究通过Heterorhabditidoides rugaoensis/Serratia nematodiphila R187侵染黑腹果蝇的免疫突变体,来研究该类昆虫病原线虫/共生菌成功侵染果蝇成虫的相关机制。[方法] 分别利用携带共生菌的线虫侵染、钨针沾共生菌菌悬液针刺和人工饲喂共生菌3种方法将S.nematodiphila R187植入果蝇血腔或肠道;通过果蝇死亡率、细菌菌落计数,结合果蝇体液免疫信号系统调控的2种抗菌肽――Diptericin和Drosomycin表达的Q-PCR检测,研究H.rugaoensis-S.nematodiphila R187与果蝇体液免疫系统的互作。[结果] H.rugaoensis携带共生菌和共生菌针刺侵染后同时激活果蝇的Toll和Imd途径;荧光定量PCR结果显示侵染过程中Diptericin和Drosomycin的相对表达均呈先上升再逐渐减弱的趋势;共生菌侵入果蝇血腔后,FADD突变体的Drosomycin相对表达量在6 h达到最高(100%),Dif突变体和野生型的Diptericin的相对表达量在12 h达到最高(90%)。共生菌侵染果蝇肠道时,仅在18 h时检测到抗菌肽的微弱表达。3种侵染方法均可导致果蝇死亡;Dif突变体果蝇和野生型果蝇的死亡进程相似,略慢于FADD突变体;以共生菌针刺侵染的致病力最强,20 h 果蝇全部死亡。[结论] S.nematodiphila R187侵染果蝇时,Toll途径和Imd途径都被激活;果蝇抵抗S.nematodiphila R187入侵的体液免疫信号通路主要是其Imd途径;S.nematodiphila R187可能采用免疫逃避机制从其肠道成功侵染果蝇。
[Objectives] This study focused on interaction mechanism between H.rugaoensis/S.nematophila R187 complex and humoral immune system in Drosophila melanogaster infected by the nematode/bacteria complex. [Methods] Three methods, nematode with symbiotic bacteria strain R187 infecting flies, needle-pricking the fly with the symbiotic bacterial strain R187 and feeding flies with symbiotic bacterium, were conducted to tranfer the bacterial strain R187 into hemocoel or intestine of flies. Lethal rate of flies and colonies statistic of symbiotic bacterium combined with Q-PCR detection of expression of the two antimicrobial peptides Diptericin and Drosomycin regulated respectively by Imd and Toll pathway were analyzed to recover the interaction between H.rugaoensis-S.nematodiphila R187 and the humoral immune system in D.melanogaster. The fly stocks used in this experiment were the wild type, FADD mutant(Imd)and Dif mutant(Toll). [Results] Infecting of H.rugaoensis with its symbiotic bacterial strain R187 or needle-pricking with the bacterial strain on flies could activate both the two humoral immune responses, Toll and Imd pathway. Results of fluorescence quantitative PCR detection showed that expression of Diptericin and Drosomycin in the two infecting methods had the same trend that up-regulated firstly and then gradually down-regulated. In the needle-pricking infection with symbiotic bacterium R187, the relative expression level of Drosomycin in FADD mutants was in the highest(100%)in 6 h, and those of Diptericin in Dif mutant and wild type were in the highest(90%)in 12 h. Only very weak expression of Diptericin and Drosomycin were detected in intestine infection of S.nematodiphila R187 on flies. All the three infecting methods could result in 100% lethal of flies. Lethal process of Dif mutant and wild type was similar, slowly