| Chilo suppressalis(Lepidoptera:Pyralidae)is one of the most harmful and frequent insect pest on rice and other gramineous plants.In recent years,the habitat environment of C.suppressalis has been greatly improved and the population increased significantly owing to the changes in our farming systems,rice varieties,and global warming.For a long time,control of C.suppressalis is primarily dependent on the application of insecticides which are known to cause resistance problems,pesticide residues,and environmental pollution because of long-term improper use.Hence,it is necessary to seek safer biological control measures.Our previous study showed that vetiver grass could effectively attract female adults of rice stripped stem borer(SSB)Chilo suppressalis to laid eggs,and it has been considered as a trap plant of C.suppressalis in the paddy field and as an important component of ecological engineering to suppress SSB population density.C.suppressalis did not complete their life cycle in vetiver grass.Vetiver grass as a dead-end trap plant of C.suppressalis have been conducted in the paddy field,however,the lethal mechanism of vetiver to C.suppressalis has not been reported up to now.Midgut is the second largest organ of insects,and it is a major organ to secrete digestive enzymes,detoxification enzymes and other important enzymes.The midgut is an important tissue to digest food,absorb nutrients and detoxify exotic toxin.In this study,we make a primary research on lethal mechanism of vetiver grass to C.suppressalis by using Illumina sequencing.The main results were summarized as follows:1.Transcriptome analysis of C.suppressalis midgutWe analyzed midgut transcriptome of C.suppressalis larvae 2 days after feeding on rice and vetiver grass,respectively.The midgut transcriptome generated 48,944 unigene with a total length of 48,666,252nt,and the longest and shortest unigene lengths are 35,885nt and 205nt.The average length of assembly is 994,325nt and GC content is 39.6%.The N50 and N90 are]874nt and 375nt respectively.According to the blast results,the E-value distribution showed that 53.69%of transcripts have significant homology(smaller than 1.0E10-50)matching in the NCBI database,while 46.31%of transcripts ranged between 1.0E10-5 and 1.0E10-50.GO functional classification showed 6738,9993 and 9052unigenes belong to categories of cells components,biological processes,and molecular function,respectively.A total of 716 transcripts grouped into KEGG pathways,which contains 14 pathways.The top three best match transcripts pathway is:Metabolic pathways and Ribosome,accounting for 9.02%and 3.67%respectively.2.Verification of the transcriptome resultsWe used the midgut of C.suppressalis as samples,which were 2 days after feeding on rice and vetiver respectively,to verify some important genes,including digestion-related genes,immune responsive genes,detoxification-related genes and protection-related genes form the transcriptome by using real-time PCR.The results showed 19 genes expressed up-regulated,including chymotrypsin,trypsin,aminopeptidase N2,carboxypeptidase A1,carboxypeptidase,dipeptidyl-peptidase,serine protease 20,pancreatic lipase,neutral lipase,glucosidase,alpha-amylase 2,alpha-amylase 1,peptidoglycan recognition protein,beta-glucan recognition protein,Hdd1,Hdd13,CYP306A1,GSTs-like protein,and catalase,while 4 genes expressed down-regulated,including cathepsin L2,carboxypeptidase inhibitor,sugar transporter 4,CYP6A2.Our results showed that avbove 2/3 of genes expressionwere consistant with the results of transcriptome,which means that the transcriptome results are credible.3.The change of gene expression in midgut of Chilo suppressalis in different time after feeding vetiver grassWe determined gene expression levels of C.suppressalis 2,4,6,8,10 days after feeding on rice and vetiver grass by using real-time PCR.The results showed that the relative expression level(80%)of pancreatic lipase and serine protease 20 in vetiver grass feeding larvae midgut were much higher than those expression levels in rice feeding larvae midgut.The relative expression level of serine protease inhibitor has reached 80%8 days after larvae feeding vetiver grass.The other genes expressed higher in the control of larvae midgut feeding on rice.Immune responsive-related gene Hddl had a higher expression level in vetiver group and its relative expression level was between 60%-70%.Hdd13 had a higher expression level in feeding rice group and its relative expression level was around 85%.In addition,peptidoglycan recognition protein and beta-glucan recognition protein gene have a similar expression level in two groups.When C.suppressalis was fed on vetiver for several days,all the determined 5 detoxification-related genes showed high expression levels in rice group.CYP306A1 and carboxylesterase expression levels in vetiver group were lower than those in rice group.Their relative expression levels were between 75%-100%.Thioredoxin peroxidase expression levels in the two groups showed no obvious change with the feeding time going on.The other protection-related genes showed a lower expression level in vetiver group and their relative expression levels were below 50%.In general,these genes may play some different but important roles in the lethal process of vetiver to C.suppressalis.4.Important function-related genes expression in the different tissues of C.suppressalis larvaeWe determined the 35 genes expression level in the head,midgut,fatbody and cuticle of C.suppressalis by using real-time PCR.The results indicated that digestive-related gene cathepsin L2 showed head-specific expression and its relative expression level in the other three tissues was below 15%.Cathepsin B and carboxypeptidase inhibitor showed expression level of 60%in cuticle and expression levels of less than 20%in the other three tissues.The remaining genes showed specific expression in the fatbody and midgut.Most of the digestion-related genes,immune responsive-related genes(except Hddl),detoxification-related genes and protection-related genes had relative high expression level in fatbody.Overall,most of the digestion-related genes,detoxification-related genes and protection-related genes showed much lower expression level in midgut of C.suppressalis feeding on vetiver.The reducing synthesis of digestive enzyme may affect the absorption and metabolism of protein,fat,carbohydrate and other nutriments.Hence,C.suppressalis may die because of malnutrition.The reducing synthesis of detoxification metabolic enzymes and protective enzymes affect the detoxification of toxic substance and defence mechanism.These factors maybe the reasons of the lethal action of vetiver grass to C.suppressalis. |
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