南京农业大学学报  2015, Vol. 38 Issue (3): 409-416   PDF    
http://dx.doi.org/10.7685/j.issn.1000-2030.2015.03.009
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文章信息

仇汝龙, 刘众杰, 李岳虎, 张克云. 2015.
QIU Rulong, LIU Zhongjie, LI Yuehu, ZHANG Keyun. 2015.
昆虫病原线虫Heterorhabditidoides rugaoensis及其共生菌Serratia nematodiphila R187与黒腹果蝇免疫系统的互作研究
Interaction between immune system of adult Drosophila melanogaster with entomopathogenic nematode Heterorhabditidoides rugaoensis and its symbiotic bacterium Serratia nematodiphila R187
南京农业大学学报, 38(3): 409-416
Journal of Nanjing Agricultural University, 38(3): 409-416.
http://dx.doi.org/10.7685/j.issn.1000-2030.2015.03.009

文章历史

收稿日期:2014-08-01
昆虫病原线虫Heterorhabditidoides rugaoensis及其共生菌Serratia nematodiphila R187与黒腹果蝇免疫系统的互作研究
仇汝龙, 刘众杰, 李岳虎, 张克云     
南京农业大学生命科学学院, 江苏 南京 210095
摘要[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DiptericinDrosomycin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。
关键词昆虫病原线虫     黑腹果蝇     突变体     抗菌肽     体液免疫     Toll和Imd途径    
Interaction between immune system of adult Drosophila melanogaster with entomopathogenic nematode Heterorhabditidoides rugaoensis and its symbiotic bacterium Serratia nematodiphila R187
QIU Rulong, LIU Zhongjie, LI Yuehu, ZHANG Keyun     
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
Abstract: [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 than that of FADD mutants. Needle-pricking method had the highest infect efficency and caused 100% death of flies in 20 h. [Conclusions] The rhabditid entomopathogenic nematode H.rugaoensis and its symbiotic bacterial strain S.nematophila R187 infected hemocoel of adult D.melanogaster together or separately could activate humoral immune responses of both Toll and Imd pathway in flies. Imd was the main immune response pathway for fly when faced with the infection of S.nematophila R187. There might be some immune escape mechanism to help S.nematodiphila R187 to infect flies successfully in the process of intestinal infection.
Keywords: entomopathogenic nematode     Drosophila melanogaster     mutant     antimicrobial peptide     humoral immune     Toll and Imd pathway    

细菌、真菌和病毒与果蝇免疫途径的互作已有报道[19, 20],而昆虫病原线虫与果蝇的免疫途径互作的研究近年来刚刚启动,主要集中于斯氏线虫科共生菌Xenorhabdus nematophila和异小杆科的Heterorhabditis bacteriophora及其共生菌Photorhabdus luminescens。果蝇的体液免疫途径主要有Toll途径和Imd途径,Toll途径主要是抵抗真菌和革兰氏阳性菌[21],Imd途径主要是抵抗革兰氏阴性菌[22]。其中Diptericin是由Imd途径激活后产生的抗菌肽,Drosomycin是由Toll途径激活后产生的抗菌肽[23, 24, 25]Heterorhabditis bacteriophora及其共生菌P.luminescens侵染果蝇幼虫后会明显引发Diptericin和Drosomycin的表达[26],侵染果蝇成虫则引发果蝇Toll和Imd途径的免疫反应[27]X.nematophilaP.luminescens肠道侵染无法导致果蝇死亡[28]。在果蝇血腔的免疫反应研究中,在对发光杆菌(P.luminescens)和致病杆菌(X.nematophila)是否在血腔中引发免疫反应还是逃避免疫反应,研究结果存在分歧[29, 30]H.rugaoensis/Serratia类昆虫病原线虫及其共生菌与昆虫体液免疫系统的互作机制目前尚不清楚。

Heterorhabditidoides rugaoensis是小杆科中致病性最强的昆虫病原线虫品系[16],其携带的共生菌Serratia nematodiphila R187与斯氏科和异小杆科线虫的共生菌均不同,对其侵染果蝇的免疫反应研究还未见报道。因此本研究采用多种侵染方法(线虫携带共生菌自然侵染、人工饲喂线虫/共生菌、共生菌针刺侵染果蝇成虫血腔),系统模拟线虫及其共生菌对果蝇成虫侵染的主要过程,以期揭示该类昆虫病原线虫及其共生菌对果蝇的致病性及其侵染机制。

1 材料与方法 1.1 试验材料

Heterorhabditidoides rugaoensis及其共生菌Serratia nematodiphila R187,由本课题组鉴定保存[16]。大肠杆菌DH5α购于南京基天生物公司。野生型黑腹果蝇w[1118]、FADD突变体黒腹果蝇w[1118]Fadd[f06954][31]Dif突变体黒腹果蝇w[1118]Dif[LA00958][32],购自Bloomington Drosophila Stock Center(USA)。试验中采用的果蝇均为3~5日龄的大小相同的成虫。培养温度为25 ℃,相对湿度75%,光照度2 000 lx,光照周期12 h。

1.2 线虫Heterorhabditidoides rugaoensis对野生型果蝇的侵染

将共生菌R187涂于肝-琼脂培养基平板[16]表面,37 ℃培养12 h后,共生菌R187将会在平板上形成一层菌苔,然后将表面消毒的EPN卵接入平板,将平板放置在温度25 ℃、相对湿度70%的温箱中培养至线虫长满整个平板,这样可以确保线虫都会携带共生菌。加入适量的无菌水将线虫从肝-琼脂培养基上冲洗下来,并将线虫悬浮液置入50 mL的离心管中,然后按照Castillo等[33]设计的线虫消毒及清洗过程对线虫进行处理,将处理好的线虫室温保存备用,可存活1周。

按照Castillo等[33]的方法用线虫侵染果蝇,选用口径、底径和高分别为10.0、5.0和5.5 cm的塑料盆。为了验证试验的可行性,设计了不同剂量的线虫侵染果蝇的试验。每个塑料盆放入15只果蝇,每只果蝇被线虫侵染的剂量设0(空白对照)、50、75、100和125个,共5个剂量,每个剂量3盆。根据果蝇死亡的速度,筛选出最佳侵染效率的剂量进行以后侵染试验。每组试验3次重复。

在试验中发现采用Castillo等[33]的方法有较多的果蝇意外死亡,所以对原方法进行了改进,在线虫侵染试验中加入1.5 mL的50 mmol · L-1蔗糖水保证果蝇的营养。选择最佳侵染剂量的线虫进行侵染试验,共侵染24 盆。于12、18、24和30 h分别提取果蝇RNA,同时分离果蝇体内的共生菌株R187,计算每毫升体液中的菌落数,然后统计每只果蝇体内的菌落数(CFU)。整个试验3次重复,每个时间点重复提取RNA 3次并进行反转录。

1.3 共生菌S.nematodiphila R187对果蝇的肠道侵染

参照Nehme等[30]的方法用共生菌饲喂果蝇,将共生菌R187培养24 h后4 000 r · min-1离心10 min,无菌水反复清洗菌体沉淀后,使用50 mmol · L-1的蔗糖水将菌体稀释成A600为0.1的菌悬液。将1 mL菌悬液滴入底部含有滤纸的果蝇培养管,管内分别接入野生型果蝇、FADD突变体果蝇和Dif突变体果蝇各15只,每种果蝇12个培养管。分别于12、18、24和30 h调查果蝇存活情况,同时在每个时间点提取果蝇RNA并重复提取3次,整个试验重复3次。对照试验采用大肠杆菌饲喂果蝇。试验中采用专门定制的果蝇管(直径2.5 cm,长度10 cm),每管可容纳15只果蝇。

1.4 共生菌S.nematodiphila R187对果蝇的血腔侵染

参照Apidianakis等[34]的试验方法,利用钨针沾菌悬液针刺果蝇背胸部,模拟自然界外部创伤感染细菌的方法接入共生菌R187,使共生菌侵入果蝇血腔。菌悬液的A600值为0.1,以大肠杆菌DH5α为对照,果蝇的品系为野生型、FADD突变体和Dif突变体。分别于0、6、12和18 h调查果蝇的存活情况。由于共生菌S.nematodiphila R187对氨苄青霉素有较强的抗性,并且菌落呈现红色,根据该特性可以测定共生菌 R187在果蝇血腔的生长动态。将单只果蝇在匀浆器加1 mL的无菌水粉碎后,梯度稀释,在含有20 μg · mL-1 氨苄青霉素的LB平板上涂板调查菌落数量(CFU)。每个时间点至少调查10只果蝇体内共生菌的生长情况。分别于0、6、12和18 h提取果蝇RNA,整个试验重复3次,每个时间点重复提取RNA 3次。

1.5 荧光定量PCR试验

RNA的提取方法为Trizol(购于宝生物工程有限公司)法。反转录试剂盒(BioTeke supermo Ⅲ RT Kit)购于百泰克生物技术有限公司。荧光定量试剂[Ultra SYBR Mixture(With ROX)]购于康为世纪生物科技有限公司,引物由英潍捷基贸易有限公司合成。荧光定量仪器ABI7500购于美国应用生物系统公 司。荧光定量反应体系(20 μL):10 μL Ultra SYBR Mixture(With ROX),0.4 μL上、下游引物(10 μmol · L-1),2 μL cDNA模板,7.2 μL ddH2O。荧光反应定量程序:95 ℃ 10 min;95 ℃ 15 s,60 ℃ 1 min,40个循环。结果分析采用2-ΔΔCT[35]

表 1 管家基因和抗菌肽基因引物序列 Table 1 Primer sequences of housekeeping gene and antimicrobial peptide gene
引物Primer 引物序列Primer sequence
RP49-f[34] 5′-GACGCTTCAAGGGACAGTATCTG-3′
RP49-r 5′-AAACGCGGTTCTGCATGAG-3′
Diptericin-f 5′-GCTGCGCAATCGCTTCTACT-3′
Diptericin-r 5′-TGGTGGAGTGGGCTTCATG-3′
Drosomycin-f 5′-CGTGAGAACCTTTTCCAATATGATG-3′
Drosomycin-r 5′-TCCCAGGACCACCAGCAT-3′

将反转录的cDNA稀释10倍后作为模板进行荧光定量。管家基因为RP 49 ,目的基因为抗菌肽基因DiptericinDrosomycin(表1)。对照组是试验组相同批次的未处理的正常果蝇,将对照组的正常果蝇的管家基因和目的基因作为对照,具体公式:ΔΔCT=(试验组目的基因-试验组管家基因)-(对照组目的基因-对照组管家基因)。

2 结果与分析 2.1 果蝇总RNA提取和cDNA合成

RNA的琼脂糖验证结果(图1-A)显示RNA完整性好,符合果蝇RNA条带18S亮度大于28S的条件,表明试验中提取的果蝇RNA质量较好。利用管家基因RP 49 验证反转录cDNA的质量,发现管家基因RP49条带清晰并且单一(部分结果见图1-B),符合相对荧光定量的要求,cDNA可用于后续试验。

图 1 果蝇总RNA提取(A)和管家基因RP 49 cDNA的验证(B) Fig. 1 The total RNA of fly(A)and cDNA amplification verification of houskeeping gene RP 49 (B) M:DNA marker DL2000;RNA:RNA 样品RNA sample;RP 49:RP49 cDNA
2.2 S.nematodiphila R187通过线虫携带侵染与果蝇血腔免疫系统的互作关系

在自制的塑料盆侵染装置内,用5种不同剂量的携带共生菌的侵染期线虫侵染果蝇(0个为空白对照),结果(图2-A)显示线虫最有效的侵染剂量为100个。线虫剂量过低(50和75个)时,线虫不能侵染全部果蝇并导致果蝇死亡;线虫剂量过高(125个)时,由于线虫凝集成团,和侵染位点竞争使很多线虫不能成功侵染。

图 2 线虫携带共生菌侵染野生型果蝇后与其免疫系统的互作关系 Fig. 2 Interaction relationships between symbiotic bacterium with wild-type flies after being infected by the bacterial strain carried by its nematode symbiont A.不同剂量线虫携带共生菌侵染野生型果蝇后果蝇的存活率Survival rates of wild-type flies infected by nematode with its bacterial symbiont at different doses;B.果蝇体内的菌落数The symbiotic bacterium colonies in hemocoel of infected wild-type flies;C.果蝇体内抗菌肽的相对表达水平The relative expression level of antibacterial peptide in infected wild-type flies. *P < 0.05.

线虫侵染果蝇后,对果蝇体内共生菌菌株R187的菌落统计结果显示:在12 h时共生菌菌落数为每只果蝇700 CFU左右,而在48 h菌落数则达到约6 000 CFU,这表明果蝇的免疫系统无法有效地抑制共生菌的繁殖(图2-B)。抗菌肽的荧光定量PCR结果显示,携带共生菌的线虫侵染果蝇后,会引发DiptericinDrosomycin的同时表达;随着线虫的持续侵染,Drosomycin的表达持续加强;Diptericin的表达一直较弱,在24 h的表达量最高(约为Drosomycin表达量的50%),30 h时基本检测不到(图2-C)。这表明携带共生菌的线虫在侵染的同时激活了Toll途径和Imd途径,但随着线虫和共生菌的繁殖,Toll途径介导的Drosomycin呈先上升再下降的表达趋势,Imd途径介导的Diptericin的表达呈先上升然后消失的趋势。

2.3 S.nematodiphila R187针刺侵染与果蝇血腔免疫系统的互作关系

共生菌R187对氨苄青霉素产生抗性并且呈红色菌落(图3-A)。统计被共生菌R187侵染的野生型果蝇血腔内的菌落数,绘制共生菌的生长动力学曲线(图3-B)。结果显示对照组大肠杆菌在果蝇血腔内无法繁殖,菌落数基本维持不变,并且12 h后菌落数有下降的趋势;共生菌菌株R187却不受抑制而且快速繁殖,菌落数从104 CFU增长到106 CFU。

图 3 共生菌R187在含氨苄青霉素的LB平板上的菌落形态(A)与在野生型果蝇血腔内的繁殖情况(B) Fig. 3 The colony morphology of symbiotic bacterium R187 in LB ampicillin plate(A)and its reproducing

colonies in fly haemocoel(B)
*P < 0.05;* *P<0.01.The same as follows.

感染R187的果蝇在20 h基本全部死亡,2种突变体果蝇的死亡情况不同(图4-A)。0~18 h内,Toll信号途径的免疫突变体果蝇(Dif)与野生型果蝇存活率相差不大,而Imd信号途径的免疫突变体果蝇(FADD)存活率则远远小于野生型果蝇和Dif突变体果蝇。对照组中,通过针刺将大肠杆菌转移到果蝇血腔,可以发现野生型果蝇和Dif突变体果蝇基本不死亡,而FADD突变体果蝇则逐渐死亡。这说明共生菌在果蝇血腔内导致FADD突变体果蝇快速死亡,并且Imd途径突变体对血腔中的共生菌更加敏感。

图 4 野生型及突变体果蝇血腔感染共生菌R187后的存活率及其体内抗菌肽的相对表达水平 Fig. 4 The survival rate of wild-type and mutant flies infected by symbiotic bacterium R187 to hemocoel

and relative expression level of antibacterial antipeptide in these flies
A.野生型果蝇、FADD突变体果蝇、Dif突变体果蝇分别被线虫共生菌R187和大肠杆菌侵染后的存活率The rate of survival of flies infected by symbiotic bacterium R187 and E.coli in hemocoel,the fly stocks were wild-type,FADD mutant(Imd)and Dif mutant(Toll)(WT:野生型果蝇wild-type fly;FADD:FADD突变体果蝇FADD mutant fly;Dif:Dif突变体果蝇Dif mutant fly);B.野生型果蝇血腔感染共生菌后体内抗菌肽的相对表达水平The relative expression level of antibacterial peptide in wild-type infected by symbiotic bacterium R187;C.FADD突变体果蝇血腔感染共生菌后体内抗菌肽的相对表达水平The relative expression level of antibacterial peptide in FADD mutant flies infected by symbiotic bacterium R187;D.Dif突变体果蝇血腔感染共生菌后体内抗菌肽的相对表达水平The relative expression level of antibacterial peptide in Dif mutant flies infected by symbiotic bacterium R187.

荧光定量结果显示,共生菌侵入果蝇血腔后引发了DiptericinDrosomycin的相对表达量上调,说明共生菌在果蝇血腔内同时激活了Toll途径和Imd途径(图4-B~D)。在野生型果蝇体内,2种抗菌肽的表达量都很高;在FADD突变体果蝇中,由于Imd途径中的FADD突变,Diptericin的表达量要远远低于Drosomycin;在Dif突变体中,情况则相反。结合突变体果蝇存活率,说明在抵抗共生菌R187中起主要作用的是Imd途径产生的Diptericin。荧光定量结果显示,Diptericin和Drosomycin在野生型、FADD突变体和Dif突变体果蝇血腔内的表达在18 h内都会经历先上调后下降的过程,18 h果蝇抗菌肽下调与果蝇濒临死亡有关。

2.4 S.nematodiphila R187肠道侵染与果蝇免疫系统的互作关系

野生型和Toll信号途径的免疫突变体果蝇(Dif)在72 h的死亡率相同,约为75%,死亡率变化也基本相同;Imd信号途径的免疫突变体果蝇(FADD)的死亡率约为85%(图5-A)。该结果显示Imd途径的突变体比Toll途径突变体和野生型果蝇对于侵染肠道的共生菌更加敏感,这表明果蝇的Imd途径在对抗共生菌的肠道侵染中相对于Toll途径起着更重要的作用。荧光定量PCR结果(图5-B)显示:饲喂共生菌后,野生型果蝇的2种抗菌肽在12 h基本无表达,18 h时有微弱表达,24 h后果蝇的2种抗菌肽的相对表达水平呈快速下降的趋势。根据18 h果蝇死亡率的快速上升和2种抗菌肽的微弱上调的结果,可以得出结论:共生菌在侵染果蝇肠道时,繁殖到一定数量时会被宿主免疫系统识别,并激活Toll途径和Imd途径,使2种体液免疫反应的抗菌肽产物在18 h有微弱表达。在24和30 h两种抗菌肽相对表达水平趋零,但是果蝇的死亡率仍然上升,这说明共生菌逃避了宿主的免疫系统并继续导致果蝇死亡。这与Nehme等[30]关于细菌侵染果蝇肠道的结果相同,共生菌在被上皮细胞识别并吞噬后是无法引起体液免疫反应的。

图 5 野生型果蝇及突变体果蝇肠道感染细菌后的存活率(A)和野生型果蝇肠道感染R187后体内抗菌肽的相对表达水平(B) Fig. 5 The survival rate of flies infected by symbiotic bacterium R187 in their intestine(A)and the relative expression level of antibacterial peptide in wild-type flies infected by symbiotic bacterium R187 in their intestine(B)
3 讨论

H.rugaoensis线虫和共生菌S.nematodiphila R187在侵染果蝇时,由于线虫对果蝇组织的破坏,DiptericinDrosomycin的表达都会有一定的上调,但是这种上调是微弱的,所以抗菌肽的持续上调还与线虫释放共生菌有关。在线虫侵染过程、共生菌肠道侵染过程和共生菌侵染血腔的过程中,Diptericin表达都会经历一个快速下降的过程,但是Diptericin的相对表达下调的原因不同。在共生菌血腔侵染18 h时,Diptericin的下调是与果蝇的组织死亡有关,而在线虫侵染果蝇和肠道共生菌侵染中,Diptericin的下调与Nehme等[30]的研究结果相同,与共生菌在肠道上皮细胞中被吞噬有关,被吞噬细胞吞噬的共生菌将不会引发Imd途径的抗菌肽Diptericin的表达,这种吞噬作用使共生菌R187逃避了宿主免疫系统并在果蝇体内继续繁殖。H.rugaoensis线虫携带共生菌侵染黑腹果蝇成虫的研究结果与Castillo等[28]对异小杆线虫及其共生菌发光杆菌侵染黑腹果蝇成虫的研究结果相一致,携带共生菌的线虫将会激活黑腹果蝇成虫的Toll和Imd途径,并导致DiptericinDrosomycin的上调和侵染后期主要是Diptericin的相对表达水平的下调。然而,共生菌直接侵染黑腹果蝇血腔的研究结果与Castillo等[28]的不同,Castillo等[28]研究发现发光杆菌(P.luminescens)在血腔中是无法引起免疫反应的,而本研究的结果与Aymeric等[29]的结果相一致,3种共生菌S.nematodiphila R187、发光杆菌(P.luminescens)和致病杆菌(X.nematophila)在血腔都会激活Toll途径和Imd途径。尽管共生菌不同,但是都会在果蝇血腔中激活Toll途径和Imd途径,引发DiptericinDrosomycin的表达量上调。另外在肠道侵染试验中,共生菌R187在饲喂果蝇后快速导致果蝇死亡,而Aymeric等[29]描述的发光杆菌(P.luminescens)和致病杆菌(X.nematophila)在果蝇肠道和大肠杆菌一样无法引起果蝇的死亡。这说明共生菌R187在肠道的毒性比发光杆菌(P.luminescens)和致病杆菌(X.nematophila)强。

综上所述,H.rugaoensis线虫及其共生菌S.nematodiphila R187在侵染果蝇成虫时,尽管都可以激活Toll途径和Imd途径,但是起主要抵抗作用的还是针对革兰氏阴性菌的Imd途径。H.rugaoensis与斯氏线虫(Steinernema)和异小杆线虫(Heterorhabditis)侵染果蝇后果蝇抗菌肽DrosomycinDiptericin的相对表达水平相似,能同时激活Toll和Imd免疫途径。共生菌S.nematodiphila R187在肠道和血腔都可以激活Toll和Imd途径,而发光杆菌(P.luminescens)和致病杆菌(X.nematophila)没有相关报道,并且S.nematodiphila R187在线虫侵染和肠道侵染中都表现出了Diptericin的快速下调,暗示其逃避宿主免疫系统是与果蝇上皮细胞等的吞噬功能相关。因此,H.rugaoensis线虫和共生菌S.nematodiphila R187与斯氏线虫(Steinernema)和异小杆线虫(Heterorhabditis)及它们的共生菌相比具有强致病性,而这种强致病性主要体现在共生菌逃避免疫系统并且快速繁殖杀死宿主的能力和侵染过程中释放毒素因子导致宿主死亡的能力。

参考文献(References)
[1] Dillman A R, Chaston J M, Adams B J, et al. An entomopathogenic nematode by any other name[J]. PLoS Pathog, 2012, 8(3):e1002527
[2] Poinar J G, Hess R, Thomas G. Isolation of defective bacteriophages from Xenorhabdus spp.(Enterobacteriaceae)[J]. IRCS Medical Science:Microbiology, Parasitology and Infectious Diseases, 1980, 8(3/4):141
[3] Götz P, Boman A, Boman H G. Interactions between insect immunity and an insect-pathogenic nematode with symbiotic bacteria[J]. Proc R SOC Lond B, 1981, 212(1188):333-350
[4] Milstead J E. Heterorhabditis bacteriophora as a vector for introducing its associated bacterium into the hemocoel of Galleria mellonella larvae[J]. Journal of Invertebrate Pathology, 1979, 33(3):324-327
[5] Akhurst R J. Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae[J]. Journal of General Microbiology, 1982, 128(12):3061-3065
[6] Jarosz J, Balcerzak M, Skrzypek H. Involvement of larvicidal toxins in pathogenesis of insect parasitism with the rhabditoid nematodes, Steinernema feltiae and Heterorhabditis bacteriophora[J]. Entomophaga, 1991, 36(3):361-368
[7] Poinar J O, Jr. Taxonomy and biology of Steinernematidae and Heterorhabditidae[M]//Gaugler R, Kaya H K. Entomopathogenic Nematodes in Biological Control.London:CRC Press Inc., 1990:23-61
[8] Bedding R A, Molyneux A S. Penetration of insect cuticle by infective juveniles of Heterorhabditis spp.(Heterorhabditidae:Nematoda)[J]. Nematologica, 1982, 28:354-359
[9] Akhurst R J. Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae[J]. Journal of General Microbiology, 1982, 128:3061-3065
[10] Akhurst R J, Boemare N E. Biology and taxonomy of Xenorhabdus[M]//Gaugler R, Kaya H K. Entomopathogenic Nematodes in Biological Control.London:CRC Press Inc., 1990:75-90
[11] Morgan J A W, Kuntzelmann V, Tavernor S, et al. Survival of Xenorhabdus nematophilus and Photorhabdus luminescens in water and soil[J]. Journal of Applied Microbiology, 1997, 83:665-670
[12] Fischer-Le S M, Viallard V, Brunel B, et al. Polyphasic classification of the genus Photorhabdus and proposal of new taxa:P.luminescens subsp.luminescens subsp.nov, P.luminescens subsp.akhurstii subsp.nov., P.luminescens subsp.laumondii subsp.nov., P.temperata sp.nov., P.temperata subsp.temperata subsp.nov.and P.asymbiotica sp.nov.[J]. International Journal of Systematic and Evolutionary Microbiology, 1999, 49:1645-1656
[13] Bonifassi E, Fischer-Le S M, Boemare N, et al. Gnotobiological study of infective juveniles and symbionts of Steinernema scapterisci:a model to clarify the concept of the natural occurrence of monoxenic associations in entomopathogenic nematodes[J]. Journal of Invertebrate Pathology, 1999, 74:164-172
[14] Zhang C X, Liu J R, Xu M X, et al. Heterorhabditidoides chongmingensis gen.nov., sp.nov.(Rhabditida:Rhabditidae), a novel member of the entomopathogenic nematodes[J]. J Invertebr Pathol, 2008, 98:153-168
[15] Zhang C X, Yang S Y, Xu M X, et al. A novel species of Serratia, family Enterobacteriaceae:Serratia nematodiphila sp.nov., symbiotically associated with entomopathogenic nematode Heterorhabditidoides chongmingensis(Rhabditida:Rhabditidae)[J]. Int J Syst Evol Microbiol, 2009, 59:1603-1608
[16] Zhang K Y, Liu X H, Tan J, et al. Heterorhabditidoides rugaoensis n.sp.(Rhabditida:Rhabditidae), a novel highly pathogenic entomopathogenic nematode member of Rhabditidae[J]. Journal of Nematology, 2012, 44(4):348-360
[17] Shapiro-Ilan D I, Han R, Dolinksi C. Entomopathogenic nematode production and application technology[J]. Journal of Nematology, 2012, 44(2):206-217
[18] Shapiro-Ilan D I, Gouge D H, Piggott S J, et al. Application technology and environmental considerations for use of entomopathogenic nematodes in biological control[J]. Biological Control, 2006, 38:124-133
[19] Ramet M. The fruit fly Drosophila melanogaster unfolds the secrets of innate immunity[J]. Acta Paediatrica, 2012, 101:900-905
[20] Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster[J]. Annu Rev Immunol, 2007, 25:697-743
[21] Kounatidis I, Ligoxygakis P. Drosophila as a model system to unravel the layers of innate immunity to infection[J]. Open Biol, 2012, 2(5):120075
[22] Rutschmann S, Kilinc A, Ferrandon D. Cutting edge:the Toll pathway is required for resistance to Gram-positive bacterial infections in Drosophila[J]. J Immunol, 2002, 168:1542-1546
[23] Lemaitre B, Kromer-M E, Michaut L, et al. A recessive mutation, immune deficiency(imd), defines two distinct control pathways in the Drosophila host defense[J]. Proc Natl Acad Sci USA, 1995, 92:9465-9469
[24] Bulet P, Hetru C, Dimarcq J L, et al. Antimicrobial peptides in insects:structure and function[J]. Dev Comp Immunol, 1999, 23:329-344
[25] Hetru C, Hoffmann J. NF-κB in the immune response of Drosophila[J]. Cold Spring Harb Perspect Biol, 2009, 1(6):a000232
[26] Uvell H, Engstrom Y. A multilayered defense against infection:combinatorial control of insect immune genes[J]. Trends Genet, 2007, 23(7):342-349
[27] Hallem E A, Rengarajan M, Ciche T A, et al. Nematodes, bacteria, and flies:a tripartite model for nematode parasitism[J]. Curr Biol, 2007, 17:898-904
[28] Castillo J C, Shokal U, Eleftherianos I. Immune gene transcription in Drosophila adult flies infected by entomopathogenic nematodes and their mutualistic bacteria[J]. J Insect Physiol, 2013, 59:179-185
[29] Aymeric J L, Givaudan A, Duvic B. Imd pathway is involved in the interaction of Drosophila melanogaster with the entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus luminescens[J]. Mol Immunol, 2010, 47:2342-2348
[30] Nehme N T, Liegeois S, Kele B, et al. A model of bacterial intestinal infections in Drosophila melanogaster[J]. PLoS Pathog, 2007, 3(11):e173
[31] Apidianakis Y, Mindrinos M N, Xiao W, et al. Profiling early infection responses:Pseudomonas aeruginosa eludes host defenses by suppressing antimicrobial peptide gene expression[J]. Proc Natl Acad Sci USA, 1995, 102(7):2573-2578
[32] Costa A, Jan E, Sarnow P, et al. The Imd pathway is involved in antiviral immune responses in Drosophila[J]. PLoS ONE, 2009, 4(10):e7436
[33] Castillo J C, Shokal U, Eleftherianos I. A novel method for infecting Drosophila adult flies with insect pathogenic nematodes[J]. Virulence, 2012, 3(3):339-347
[34] Apidianakis Y, Rahme L G. Drosophila melanogaster as a model host for studying Pseudomonas aeruginosa infection[J]. Nat Protoc, 2009, 4(9):1285-1294
[35] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25(4):402-408