| 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-factor1?(Ls?FTZ-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. |
PDF Full Text Request