【作者】 刘磊；

【导师】 刘怀；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2019， 博士

【摘要】 灰飞虱Laodelphax striatellus以水稻、小麦和玉米等作物的韧皮部汁液为食,同时,灰飞虱也是一种媒介昆虫,通过取食传播水稻条纹叶枯病(Rice stripe virus,RSV)和黑条矮缩病(Rice black-streak dwarf virus,RBSDV)等多种植物病毒病,已成为危害最严重的农业害虫之一。作为节肢动物中分布最广泛的细胞内共生细菌之一,Wolbachia可以在宿主中引起多种生殖异常现象,并调控宿主免疫和氧化还原等相关基因的表达,进而影响生物体对外源微生物入侵的抵御。自然界中的灰飞虱感染的Wolbachia株系是w Stri,该株系能诱导宿主产生强烈的胞质不亲和现象。然而,Wolbachia对灰飞虱免疫与氧化还原等方面的影响尚不清楚。本研究通过高通量测序技术、q RT-PCR技术和RNAi技术,明确了免疫与氧化还原等相关基因在感染和不感染Wolbachia两种灰飞虱品系之间的差异表达情况,以及免疫基因与Wolbachia密度之间的相关性;分析了两种灰飞虱品系在受到外源大肠杆菌感染后,免疫与氧化还原相关基因表达的变化情况;通过对两种灰飞虱品系的小RNA进行测序,分析响应Wolbachia感染的灰飞虱mi RNA潜在的作用。主要结果如下:1两种灰飞虱品系的转录组比较分析通过对感染和不感染Wolbachia的灰飞虱品系进行转录组测序分析,共获得了13.95 Gb clean data。对高质量的测序数据进行组装,共得到contig、transcript和unigene的总数分别为5,362,323、126,012和71,676,transcript与unigene的N50分别为2,287 bp和1,615 bp,其中长度在1kb以上的unigene有13,515条。与不感染Wolbachia的雄性灰飞虱相比,感染Wolbachia的雄性灰飞虱有560条基因上调,433条基因下调。对差异表达基因进行注释分析,结果表明,与免疫相关的基因在感染Wolbachia后发生了上调,包括防御素基因(defensin B,Ls Def-B)、黄嘌呤脱氢酶基因(xanthine dehydrogenase,Ls XDH)、漆酶2基因(laccase 2,Ls Lac-2)、转铁蛋白基因(transferrin,Ls Trf)、丝氨酸蛋白酶抑制剂B8基因(serpin-B8,Ls Spn-B8)、酚氧化酶原基因(prophenoloxidase,Lspro PO)、溶菌酶基因(i-type lysozyme6,Ls Lys i-6)、小分子热激蛋白基因(small heat shock protein,Ls Hsp20)、基质金属蛋白酶基因(Matrix metalloproteinase,Ls MMP)、丝氨酸蛋白酶基因(serine protease 4,Ls SP-4)。同时,一些与雄性生殖相关的基因在感染Wolbachia后发生了下调,包括精巢特异性丝氨酸/苏氨酸蛋白激酶3基因(testis-specific serine/threonine-protein kinase 3-like,Ls TSSK3)、精巢表达序列9蛋白基因(testis-expressed sequence 9 protein,Ls TEX9)、动力蛋白调节复合蛋白1基因(dynein regulatory complex protein 1,Ls DRC1)、外周致密纤维蛋白3基因(outer dense fiber protein 3-B-like,Ls ODF3B)。此外,Wolbachia引起7个涉及糖代谢基因的表达发生了变化。2 Wolbachia调节灰飞虱免疫与氧化还原相关基因的表达2.1 Wolbachia激活灰飞虱低日龄雄成虫、抑制低日龄雌成虫的免疫相关基因表达通过q RT-PCR检测,进一步分析了Wolbachia对不同日龄灰飞虱雌、雄成虫免疫相关基因表达的影响。结果表明,在雄成虫中,Wolbachia导致Lspro PO基因、Ls Spn-B8基因、Ls Lys i-6基因和Ls XDH基因的表达量在1-7日龄的雄成虫中以上调为主,而在9-15日龄期间以下调居多;Ls Lac-2基因在1-15日龄的雄成虫中均显著上调;Ls Def-B基因和Ls Trf基因的表达量在雄成虫3日龄和15日龄中上调,同时Ls Trf基因的表达量在7日龄和9日龄中上调,在11日龄和13日龄下调。在雌成虫中,Wolbachia导致Ls Lys i-6基因和Lspro PO基因的表达量既有上调又有下调,且1-7日龄主要表现出下调;Ls Spn-B8基因和Ls Trf基因的表达量在1-15日龄主要表现出显著下调;然而Ls Lac-2基因和Ls XDH基因的表达量在感染Wolbachia的灰飞虱雌成虫中整体表现出上调;此外,Ls Def-B基因的表达量变化较小。总体上来讲,Wolbachia激活灰飞虱雄成虫早期阶段体内免疫相关基因的表达,抑制灰飞虱雌成虫早期阶段体内免疫相关基因的表达。我们推测,灰飞虱雄成虫早期阶段免疫相关基因表达的升高可能与Wolbachia含量的增加有关,而灰飞虱雌成虫早期阶段免疫相关基因表达的降低可能与雌成虫维持其繁殖力有关。2.2 Wolbachia诱导灰飞虱免疫相关基因的上调与宿主NADPH氧化酶家族基因的上调有关通过检测Wolbachia诱导灰飞虱免疫基因上调的日龄中两种品系体内氧化还原相关基因表达水平的变化,分析了免疫基因上调与氧化还原相关基因变化之间的相关性。q RT-PCR结果显示,Wolbachia对3日龄雌成虫体内NADPH氧化酶基因的表达量没有影响,但Wolbachia导致3日龄雄成虫体内NADPH氧化酶基因Ls Nox4和Ls Nox5和抗氧化酶基因Ls SOD2和Ls POD2发生了显著上调。在11日龄中,Wolbachia引起雌成虫体内NADPH氧化酶基因Ls Nox5和Ls Duox和抗氧化酶基因Ls CAT和Ls SOD1显著上调。此外,Wolbachia导致11日龄雄成虫体内NADPH氧化酶基因Ls Nox4和Ls Duox和抗氧化酶基因Ls POD2基因显著上调。且感染Wolbachia的11日龄雌成虫体内NADPH氧化酶基因和抗氧化酶基因的上调程度高于相同日龄的雄成虫。暗示Wolbachia可能通过上调灰飞虱NADPH氧化酶家族基因的表达提升宿主体内的活性氧有水平,从而激活宿主免疫基因的表达。3沉默免疫相关基因导致灰飞虱雄成虫体内Wolbachia密度降低更显著利用RNA干扰技术,明确了灰飞虱雌成虫和雄成虫体内免疫相关基因下调对Wolbachia密度的影响。结果表明,干扰灰飞虱雌成虫体内免疫相关基因的表达后,仅在干扰Lspro PO基因后引起Wolbachia密度显著下降,而干扰Ls Def-B和Ls Lys i-6基因后Wolbachia密度也有一定程度的下降,但与对照相比差异不显著。干扰灰飞虱雄成虫体内免疫相关基因的表达后,Ls Def-B基因、Lspro PO基因、Ls Spn-B8基因和Ls Trf基因表达水平的下调均导致了Wolbachia密度的显著降低。4外源大肠杆菌侵染对两种灰飞虱品系死亡率的影响及免疫与氧化还原相关基因的表达分析4.1 Wolbachia影响灰飞虱在大肠杆菌侵染时的死亡率通过给两种灰飞虱品系饲喂含有大肠杆菌的人工饲料,然后计算校正死亡率。结果表明,取食含有大肠杆菌的人工饲料后,感染Wolbachia的2日龄、6日龄和10日龄雄成虫的校正死亡率分别为23%、39%和66%,不感染Wolbachia的2日龄、6日龄和10日龄雄成虫的校正死亡率分别为11%、24%和35%。表明感染Wolbachia的雄成虫取食含有大肠杆菌的人工饲料后的死亡率高于不感染Wolbachia的雄成虫,且随着试虫日龄的增加,两种灰飞虱品系雄成虫取食含有大肠杆菌的人工饲料后的死亡率升高。4.2 Wolbachia操纵灰飞虱在应对外源大肠杆菌侵染时的免疫基因表达通过检测两种灰飞虱品系不同日龄的雌成虫和雄成虫取食含有大肠杆菌的人工饲料后免疫相关基因的表达水平,分析两种灰飞虱品系感染外源细菌后死亡率存在差异的内在机制。结果显示,雄成虫取食含有大肠杆菌的人工饲料后,Ls Def-B基因的表达趋势在两种品系之间差异较大,两种品系Ls Lys i-6基因的表达量都上调,Ls Trf基因只在不感染Wolbachia的品系中上调,Ls Spn-B8基因、Ls Lac-2基因和Lspro PO基因几乎不响应大肠杆菌的侵染。雌成虫取食含有大肠杆菌的人工饲料后,两种品系免疫基因的表达整体上被抑制。Ls Trf基因的表达趋势在两种品系之间有明显的区别,Ls Trf基因在不感染Wolbachia的雌成虫中显著上调,在感染Wolbachia的雌成虫中显著下调。4.3 Wolbachia改变灰飞虱氧化还原系统对外源大肠杆菌侵染的敏感性通过检测两种灰飞虱品系不同日龄的雌成虫和雄成虫取食含有大肠杆菌的人工饲料后氧化还原相关基因的表达水平,进一步分析两种灰飞虱品系在感染外源细菌后死亡率存在差异的内在机制。结果表明,大肠杆菌侵染雄成虫后,感染Wolbachia品系2日龄的抗氧化酶基因都显著上调,同时,Ls Nox4基因和Ls Duox基因的表达量显著上调;不感染Wolbachia品系2日龄的抗氧化酶基因既有上调,也有下调,Ls Nox5基因显著上调。6日龄灰飞虱雄成虫取食含有大肠杆菌的人工饲料后,感染Wolbachia品系的大多数抗氧化酶基因都显著上调,不感染Wolbachia品系只有Ls CAT显著上调。10日龄灰飞虱雄成虫取食含有大肠杆菌的人工饲料后,感染Wolbachia品系的抗氧化酶基因大多数都显著下调,不感染Wolbachia品系有3个抗氧化酶基因上调。大肠杆菌侵染后,两种品系6日龄和10日龄雄成虫的NADPH氧化酶家族基因的表达都被抑制。2日龄雌成虫取食含有大肠杆菌的人工饲料后,不感染Wolbachia的品系中有更多的抗氧化酶基因的表达量上调。6日龄雌成虫取食含菌的人工饲料后,不感染Wolbachia的品系中有2个抗氧化酶基因的表达量显著下调。10日龄雌成虫取食含菌的人工饲料后,两种品系之间抗氧化酶基因上调和下调的数量相近。感染大肠杆菌后,两种灰飞虱品系雌成虫体内的NADPH氧化酶家族基因被诱导,且不感染Wolbachia品系中的NADPH氧化酶家族基因上调数量更多。暗示Wolbachia改变灰飞虱氧化还原系统在应对外源细菌侵染的敏感程度。5两种灰飞虱品系mi RNA的表达分析及靶基因预测通过高通量测序平台构建了四个s RNA文库(FUI、FI、MUI和MI),并分别获得16,616,711、26020481、23,702,808和22,471,936个clean reads。四个文库共鉴定到152个mi RNA,其中包括49个已知的mi RNA和103个新预测的mi RNA,共有104个mi RNA在四个文库中共享。Wolbachia导致灰飞虱雌成虫和雄成虫中51个mi RNA的表达水平发生了变化。与不感染Wolbachia的品系相比,Wolbachia导致雄成虫中18个mi RNA上调和6种mi RNA下调。在雌性中,Wolbachia引起25个mi RNA上调和15个mi RNA下调。而且,在雌性和雄性的比较组中共享13个差异表达的mi RNA。对差异表达mi RNA靶基因进行GO富集分析,结果表明,在雌性和雄性比较组中,预测到了参与immune system process、reproductive process、reproduction、developmental process、response to stimulus、antioxidant activity和growth等过程的靶基因。差异表达mi RNA的靶基因的KEGG注释分类结果显示,在雌性和雄性比较组中,富集到了涉及免疫(m TOR信号通路、Hippo信号通路、Jak-STAT信号通路、lysosome和peroxisome)、氧化还原(Hedgehog信号通路)以及sphingolipid metabolism和steroid biosynthesis等通路的靶基因。而通路如endocytosis、phagosome、aminoacyl-t RNA biosynthesis、carbon metabolism、amino sugar and nucleotide sugar metabolism、glycerolipid metabolism和insect hormone biosynthesis仅在雌性比较组中得到富集。同时,通路ether lipid metabolism、neuroactive ligand-receptor interaction、N-glycan biosynthesis、notch signaling pathway和selenocompound仅在雄性比较组中得到富集。同时,与雌性繁殖力有关的lst-mi R-n36-5p(靶卵黄蛋白原-6(Lsvg6)基因)和lst-mi R-n10-3p(靶核激素受体Fushi tarazu-factor1β(LsβFTZ-F1)基因)在感染Wolbachia的雌成虫中下调,然而,与雄性生育能力有关的lst-mi R-n13-5p(靶保幼激素酯酶(Ls JHE)基因)和lst-mi R-n81-5p(靶精子相关抗原6(Ls Spag6)基因)在感染Wolbachia的雄成虫中上调。而参与氧化还原的lst-mi R-n23-3p(靶线粒体锰超氧化物歧化酶(Lsm Mn SOD)基因)在感染Wolbachia的雌成虫中下调,lst-mi R-n47-5p(靶硫氧还蛋白还原酶(Ls Trx R)基因)在感染Wolbachia的雄成虫中上调。此外,与自噬有关的lst-mi R-n5-5p(靶雷帕霉素靶蛋白(Ls TOR)基因)和与黑化反应有关的lst-mi R-n52-5p(靶储存蛋白hexamerin(Ls Hex)基因)在感染Wolbachia的雌性和雄性灰飞虱中都被下调。综合上述结果表明,Wolbachia激活了灰飞虱雄成虫早期阶段体内免疫相关基因的表达,抑制了灰飞虱雌成虫早期阶段体内免疫相关基因的表达。灰飞虱体内免疫相关基因表达的下调会导致Wolbachia密度降低,这种相关性在雄成虫中表现的更为密切。在外源大肠杆菌入侵时,Wolbachia阻制了灰飞虱体内Ls Trf基因的上调表达,并导致灰飞虱雄成虫体内氧化还原的平衡受损更严重,从而使得感染Wolbachia的雄成虫取食含有大肠杆菌的人工饲料后的死亡率高于不感染Wolbachia的雄成虫。同时,Wolbachia通过调控灰飞虱体内mi RNA的表达,进一步调节灰飞虱体内免疫与氧化还原基因的表达水平。研究结果不仅有助于揭示Wolbachia与灰飞虱互作的分子机制,也为进一步利用Wolbachia在昆虫中的生物防治研究提供了理论基础。更多还原

【Abstract】 The small brown planthopper(SBPH),Laodelphax striatellus(Fallén),feeds on the phloem sap of crops such as rice,wheat and corn,and it is also a kind of vector insect that spreads a variety of plant viral diseases such as rice stripe virus(RSV)and Rice black-streak dwarf virus(RBSDV)by feeding,it has become one of the most serious agricultural pests.As one of the most widely distributed intracellular symbiotic bacteria in arthropods,Wolbachia can cause a variety of reproductive abnormalities in its host,and can regulate the expression of genes related to immunity and redox of host,thereby affecting the response of the organism against various exogenous microorganisms.SBPH is naturally infected with the Wolbachia strain w Stri,which induces strong cytoplasmic incompatibility of its host.However,the impact of Wolbachia on the immune and redox effects of SBPH is unclear.In this study,we detected the differential expression of immune and redox-related genes between Wolbachia-infected and Wolbachia-uninfected SBPH using high-throughput RNA sequencing and q RT-PCR technology,and the correlation between immune genes and Wolbachia densities were clarified by RNAi technology;the changes of the expression of immune and redox-related genes between two strains of SBPH after infection by exogenous E.coli were analyzed;we also analyze the potential role of mi RNA in SBPH in response Wolbachia infection by sequencing of small RNA in two strains of SBPH.The main results and conclusions of this study are as follows:1 Comparative transcriptomes of two SBPH strainsIn this study,the transcriptome of male SBPH secquencing produced 13.95 Gb clean data in Wolbachia-infected and Wolbachia-uninfected strains.The high-quality sequencing data was assembled,and a total number of contig,transcript,and unigene was 5,362,323,126,012,and 71,676,respectively.The N50 of transcript and unigene was 2,287 and 1,615,respectively,there were 13,515 unigenes with a length of longer than 1 kb.In comparsion with male SBPH of Wolbachia-uninfected strains,560 genes were up-regulated and 433 genes were down-regulated in Wolbachia-infected male SBPH.By analyzing the results of differentially expressed genes,we found that some immune-related genes were up-regulated after Wolbachia infection,including defensin B(Ls Def-B)and xanthine dehydrogenase(Ls XDH),laccase 2(Ls Lac-2),transferrin(Ls Trf),serpin-B8(Ls Spn-B8),prophenoloxidase(Lspro PO),i-type lysozyme 6(Ls Lys i-6),small heat shock protein(Ls Hsp20),two matrix metalloproteinase(Ls MMP),serine protease 4(Ls SP-4).At the same time,some genes related to male reproduction were down-regulated after Wolbachia infection,including testis-specific serine/threonine-protein kinase 3-like(Ls TSSK3)and testis-expressed sequence 9 protein(Ls TEX9),dynein regulatory complex protein 1(Ls DRC1),and outer dense fiber protein 3-B-like(Ls ODF3B).In addition,Wolbachia infection has also caused some changes in the expression of 7 genes involved in sugar metabolism.2 Wolbachia regulates the expression of immune-and redox-related genes in SBPH2.1 Wolbachia activates early age male adults and inhibits expression of immune-related genes in early age female adultsThe effect of Wolbachia infection on the expression of immune-related genes in different days old female and male adults of SBPH were further verified by q RT-PCR.The results showed that,in male adults,Wolbachia infection caused the expression levels of Lspro PO,Ls Spn-B8,Ls Lys i-6 and Ls XDH to be mainly up-regulated during 1-7 day old adult,and were mainly down-regulated during 9-15 day old adult;the expression of Ls Lac-2 gene was significantly up-regulated from 1 to 15 day old,and the expression levels of Ls Def-B and Ls Trf were up-regulated in 3 and 15 day-old male adult,at the same time,the expression level of the Ls Trf gene was up-regulated at 7 and 9 day-old male adult,and was down-regulated at 11 and 13 day old male adult.In female adults,Wolbachia infection caused the expression levels of Ls Lys i-6 and Lspro PO were both up-regulated and down-regulated,and were mainly down-regulated from 1 day old to 7 day old.The expression level of Ls Spn-B8 and Ls Trf mainly showed significant down-regulation in Wolbachia-infected females,while the expression levels of Ls Lac-2 and Ls XDH were up-regulated in Wolbachia-infected adult females.In addition,the expression level of Ls Def-B was less affected after Wolbachia infection.In general,Wolbachia activates the expression of immune-related genes in the early stages of male adults and inhibits the expression of immune-related genes in the early stages of female adults.We speculate that the increased expression of immune-related genes in the early stages of male adult may be related to the increase in Wolbachia load,while the decreased expression of immune-related genes in the early stages of female adults may be related to the maintenance of female fecundity.2.2 Wolbachia induces up-regulation of the immune-related genes related to up-regulation of the NADPH oxidase family genes in SBPHThe expression levels of redox-related genes in two strains of the age-inducing immune gene up-regulated by Wolbachia were detected.The correlation between the up-regulation of immune genes and the changes of redox-related genes was analyzed.The results of q RT-PCR showed that Wolbachia had no effect on the expression of NADPH oxidase genes in 3 day old female adults.However,Wolbachia resulted in the NADPH oxidase genes Ls Nox4 and Ls Nox5,and antioxidant enzyme genes Ls SOD2 and Ls POD2 were significantly up-regulated in 3 day old male adults.At 11 day old,Wolbachia caused a significant up-regulation of the NADPH oxidase genes Ls Nox5 and Ls Duox,and antioxidant enzyme genes Ls CAT and Ls SOD1 in female adults.In addition,Wolbachia resulted in a significant up-regulation of the NADPH oxidase genes Ls Nox4 and Ls Duox,and antioxidant enzyme gene Ls POD2 in 11 day old male adults.The NADPH oxidase genes and antioxidant enzyme genes were up-regulated to a greater extent in Wolbachia-infected 11 day old female adults than male adults.It was suggested that Wolbachia may increase the level of reactive oxygen species in the SBPH by up-regulating the expression of the NADPH oxidase family genes,which activates the expression of immune genes in host.3 Silencing of immune-related genes leads to more significant reduction in Wolbachia density in male adult of SBPHThe effects of down-regulation of immune-related genes on the density of Wolbachia in female adults and male adults were determined by RNA interference technology.The results showed that,after interfering with the expression of immune-related genes in female adult,only the down-regulation of Lspro PO result in the density of Wolbachia to decrease significantly,the density of Wolbachia was also decreased after interference with Ls Def-B and Ls Lys i-6,but the difference was not significant.After interfering with the expression of immune-related genes in the male adults,the down-regulation of Ls Def-B,Lspro PO,Ls Spn-B8 and Ls Trf expression resulted in a significant decrease in Wolbachia density.4 Effect of exogenous E.coli on mortality of two SBPH strains and analysis of expression of immune and redox related genes4.1 Wolbachia affects mortality of SBPH after infected with exogenous bacteriaThe artificial diet containing E.coli was fed to two SBPH strains and the corrected mortality was calculated.The results showed that,after feeding on artificial diet containing E.coli,the corrected mortality rates of 2,6 and 10 day-old Wolbachia-infected male adults was 23%,39% and 66%,respectively;the corrected mortality rates of 2,6 and 10 day-old Wolbachia-uninfected male adults was 11%,24%,and 35%,respectively.It indicated that the Wolbachia-infected male adults had higher mortality than Wolbachia-uninfected male adults after feeding on artificial diet containing E.coli,and with the increase of the day old of SBPH,the mortality of male adults of two SBPH strain increased after feeding on the artificial diet containing E.coli.4.2 Wolbachia manipulates immune gene expression of SBPH in response to infection by exogenous bacteriaBy detecting the expression levels of immune-related genes in different day old female and male adults of two SBPH strains after feeding on the artificial diet containing E.coli,and the mechanism of differences in the mortality of two SBPH strains infected with exogenous bacteria were analyzed.The results showed that,after male adults fed on artificial diet containing E.coli,the expression trend of Ls Def-B was significantly different between the two strains,the expression levels of Ls Lys i-6 gene were up-regulated in both strains,and the Ls Trf gene was only up-regulated in Wolbachia-uninfected strains,Ls Spn-B8,Ls Lac-2 and Lspro PO hardly respond to E.coli infection.When the female adult feeds on the artificial diet containing E.coli,the expression of immune genes in two SBPH strains was suppressed as a whole.The expression trend of the Ls Trf was significantly different between two SBPH strains.The Ls Trf was significantly up-regulated in Wolbachia-uninfected female adults and significantly down-regulated in Wolbachia-infected female adults.4.3 Wolbachia alters the sensitivity of redox system to exogenous bacterial infection in SBPHBy detecting the expression levels of redox-related genes in different day old female and male adults of two SBPH strains after feeding on the artificial diet containing E.coli,and the mechanism of differences in the mortality of two SBPH strains infected with exogenous bacteria were further analyzed.The results showed that,after male adult infected with E.coli,the antioxidant enzyme genes of Wolbachia-infected strain were significantly up-regulated,and the expression levels of Ls Nox4 and Ls Duox were significantly up-regulated.The antioxidant enzyme genes were both up-regulated and down-regulated in Wolbachia-uninfected 2 days old males,and the Ls Nox5 was significantly up-regulated.After the 6-day-old adult males fed on the artificial diet containing E.coli,most of the antioxidant enzyme genes strain were significantly up-regulated in Wolbachia-infected SBPH,and only Ls CAT was significantly up-regulated in Wolbachia-uninfected strain.After the 10 day old male fed on the artificial diet containing E.coli,most of the antioxidant enzyme genes were significantly down-regulated in Wolbachia-infected strain,and the three antioxidant enzyme genes were up-regulated in Wolbachia-uninfected strain.After infection by E.coli,the expression of NADPH oxidase family genes was inhibited in both 6 and 10 day old male adults of two strains.After 2 day old female adults fed on the artificial diet containing E.coli,more antioxidant enzyme genes was up-regulated in Wolbachia-uninfected strain.After 6 day old female adults fed on the artificial diet containing E.coli,two antioxidant enzyme genes was down-regulated in Wolbachia-uninfected strain.After 10 days old female adults fed on the artificial diet containing E.coli,the number of up-regulation and down-regulation of antioxidant enzyme genes between the two strains was similar.After infection with E.coli,the NADPH oxidase family genes were induced in the female adults of two strains,and the NADPH oxidase family gene in Wolbachia-uninfected strain was up-regulated in greater numbers.It was suggested that Wolbachia changes the sensitivity of redox system of SBPH in response to exogenous bacterial infection.5 Expression analysis,and prediction of target genes of mi RNAs in two SBPH strainsFour s RNA libraries(FUI,FI,MUI,and MI)were constructed using a high-throughput sequencing platform,and 16,616,711,26020481,23,702,808 and 22,471,936 clean reads were obtained,respectively.In total,152 mi RNAs were obtained from the four libraries,including 49 known mi RNAs and 103 novel mi RNAs,and a total of 104 mi RNAs were shared among the four libraries.Wolbachia infection caused the expression levels of 51 mi RNAs were changed in females and males,including 4 known mi RNAs and 47 novel mi RNAs.Compared with uninfected strains,Wolbachia infection resulted in up-regulation of 18 mi RNAs and down-regulation of 6 mi RNAs in male,while 25 mi RNAs were up-regulated and 15 mi RNAs were down-regulated in female.Moreover,13 differentially expressed mi RNAs were shared in the female and male comparison groups.GO enrichment analysis target genes of differentially expressed mi RNA showed that,target genes are involved in the process of immune system process,reproductive process,reproduction,developmental process,response to stimulus,antioxidant activity,and growth were predicted in the female and male comparison groups.KEGG annotation classification of target genes of differentially expressed mi RNAs,the results show that,target genes involved in immunity(m TOR signaling pathway,Hippo signaling pathway,Jak-STAT signaling pathway,lysosome and peroxisome),redox(Hedgehog signaling pathway),sphingolipid metabolism and steroid biosynthesis were enriched in female and male comparisons.Pathways such as endocytosis,phagosome,aminoacyl-t RNA biosynthesis,carbon metabolism,amino sugar and nucleotide sugar metabolism,glycerolipid metabolism and insect hormone biosynthesis were only enriched in the female comparison group.At the same time,pathways such as ether lipid metabolism,neuroactive ligand-receptor interaction,N-glycan biosynthesis,notch signaling pathway and selenocompound were only enriched in the male comparison group.At the same time,we found that lst-mi R-n36-5p(target vitellogenin-6(Lsvg6))and lst-mi R-n10-3p(target nuclear hormone receptor Fushi tarazu-factor1β(LsβFTZ-F1))related to female fecundity were down-regulated in Wolbachia-infected females,however,lst-mi R-n13-5p(target juvenile hormone esterase(Ls JHE))and mi RNA lst-mi R-n81-5p(target sperm associated antigen 6(Ls Spag6))related to male fertility was up-regulated in Wolbachia-infected males.The lst-mi R-n23-3p(target mitochondrial manganese superoxide dismutase(Lsm Mn SOD))involved in redox showed down-regulation in Wolbachia-infected females,lst-mi R-n47-5p(target thioredoxin reductase(Ls Trx R))was up-regulated in Wolbachia-infected males.In addition,lst-mi R-n5-5p(target rapamycin target protein(Ls TOR))associated with autophagy and lst-mi R-n52-5p(target hexamerin(Ls Hex)associated with melanization were down-regulated in both Wolbachia-infected females and males.In conclusion,Wolbachia activated the expression of immune-related genes in the early stage of adult male,and inhibited the expression of immune-related genes in the early stage of female adult.Down-regulation the expression of immune-related genes in the SBPH results in a decrease in Wolbachia density,this correlation is more closely in male adults.In the invasion of exogenous E.coli,Wolbachia blocked the up-regulated expression of Ls Trf in SBPH,and caused the redox balance of the adult male to be more severely damaged,so that the mortality of Wolbachia-infected male adults was higher than Wolbachia-uninfected male adults after feeding on artificial diet containing E.coli.At the same time,Wolbachia further regulates the expression levels of immuneand redox-related genes by regulating the expression of mi RNA of SBPH.The results not only reveal the molecular mechanism of Wolbachia interaction with SBPH,but also provide a theoretical basis for further utilization of Wolbachia in biological control research in insects.更多还原