| Cotton bollworm(Helicoverpa armigera)is an important agricultural pest in the world.Cotton and corn are the main hosts of cotton bollworm in China.The larvae have powerful ability of feeding,digestion and defense,which can cause destructive damage to a variety of crops and bring serious losses to agricultural production.Its feeding,digestion and defense ability is closely related to the well-developed midgut tissue.The midgut efficiently digests and absorbs nutrients to provide nutritional raw materials for the normal operation of the physiological activities of the larvae.At the same time,the midgut acts as the first physical and immune barrier to effectively defend the pathogenic microorganisms entering the intestinal cavity during feeding in larvae.The larval midgut has also become a target for a variety of microbial insecticides and transgenic Bt crops,and serious damage to the midgut cells can lead to larval death.However,the larval midgut damage can induce the regeneration mechanism.When the intestinal cells regeneration rate exceeds the injury rate,the larval survival will increase.This could reduce the effect of insecticides.It is known that the injury and repair process of the midgut in Drosophila is regulated by a complex signal pathway network,but the mechanism of the Lepidoptera insects has not been clear.1.Transcriptome analysis of midgut tissues of Helicoverpa armigera after feeding onEnterobacter cancerogenus strain Ha Ec1Previous studies showed that midgut cell apoptosis and stem cell proliferation induced by fedding on Ha Ec1 were time-dependent in cotton bollworm larvae.In order to clarify the gene regulation pathway,transcriptome sequencing was used to analyze the gene m RNA expression in midgut cells of the fifth instar larvae of Helicoverpa armigera fed or not fed on Ha Ec1.Results showed that there were significant differences in the expression of 551 genes,of which 275 genes were significantly up-regulated and 276 genes were significantly down-regulated.Compared with the control group,117 genes were significantly up-regulated and 75 genes were down-regulated in the larvae fedding on Ha Ec1 for 2 hours.The KEGG pathway enrichment analysis of these genes showed that the signal pathways related to the regulation of apoptosis and proliferation included MAPK,Apoptosis,Fox O,PI3K-Akt,Wnt,Hedgehog,Hippo,Insulin,Jak-STAT and p53.The up-regulated expression of Apoptosis pathway and down-regulated expression of p53 pathway may be involved in the regulation of midgut apoptosis.The down-regulated or up-regulated genes of the Fox O,PI3K-Akt,Wnt,Hedgehog,Hippo,Insulin and Jak-STAT pathways may be involved in the proliferation of midgut cells.After feeding on Ha Ec1 for 6 hours,32 genes were up-regulated and 60 genes were down-regulated compared with the control,and no signal pathways related to the regulation of cell proliferation were found by KEGG pathway enrichment analysis.In addition to the enrichment of pathways related to the regulation of apoptosis and proliferation,the pathways significantly enriched by feeding on Ha Ec1 for 2h include ribosome pathway,oxidative phosphorylation pathway and thermogenesis pathway,which are closely related to protein translation and heat production,which may reflect the physiological feedback of larvae to bacterial infection,such as raising body temperature.The significantly enriched pathways of larvae fedding on Ha Ec1 at 6 h also included pancreatic secretion pathway,protein digestion and absorption pathway,galactose metabolism pathway,carbohydrate digestion and absorption pathway,which were significantly down-regulated,which may be related to excessive injury of midgut cells.Neither of the two treatments was enriched to the immune pathway against Gram-negative bacteria.The above results showed that there were related signal pathways to regulate the damage and repair of midgut cells in Helicoverpa armigera.Enterobacter cancerogenus escaped the activation of midgut immune response and seriously damaged the midgut cells of Helicoverpa armigera.2.Detection of midgut cell response of Helicoverpa armigera after feeding with Ha Ec1for different timeAccording to the results of transcriptome analysis,the fifth instar larvae of Helicoverpa armigera fed on Ha Ec1 strain for 2 hours and 6 hours,there were more signal pathways corresponding to strong cellular response,but the number of differentially expressed genes detected in each pathway was less,which may be due to the dynamic transmission of signal pathways,and the main functional signal pathways could not be accurately detected in the two treatment times.In this paper,TUNEL and Ed U immunofluorescence staining techniques were used to detect the response of midgut cells of Helicoverpa armigera larvae after feeding on Ha Ec1 for different time,so as to provide a reference for accurate analysis of the corresponding signal pathway.The midgut tissues of the fifth instar larvae of Helicoverpa armigera fed with Ha Ec1 for 1 h,3 h,6 h and 12 h were collected and sliced,and stained with TUNEL and Ed U immunofluorescence.The results showed that the peak of apoptosis and proliferation occurred at 3 h and 6 h,only a little apoptosis and proliferation signals were detected in midgut cells at 1 h,and the apoptosis and proliferation reaction were over at 12 h.After feeding Ha Ec1,TUNEL positive apoptotic cells gathered and distributed along the midgut,while the morphology,size and intestinal distribution of Ed U positive proliferative cells were specific,most of the proliferative cells were rectangular embedded between intestinal epithelial cells,and a small number of small round cells were distributed near the basement membrane,which may be stem cells produced by asymmetric proliferation and differentiated newly mature cells.The above results suggest that the larvae of Helicoverpa armigera fed on Ha Ec1 can quickly cause midgut cell damage,mature cell damage and stem cell proliferation almost occur at the same time.3.Expression Analysis of key genes in each signal Pathway of Helicoverpa armigeraafter feeding on Ha Ec1 for different timeIn order to further analyze the key signal pathways activated by Ha Ec1 in the process of midgut injury and repair in H.armigera larvae,the relative expressions of key genes in Apoptosis,Hippo,MAPK,Insulin,Hedgehod,Notch,JAK-STAT and IMD pathway were detected and analyzed by RT-q PCR after feeding on Ha Ec1 for 1 h,3 h and 6 h.The results showed that the relative expression of key genes in the signal pathways changed significantly at different times except Hedgehod and JAK-STAT pathways.After feeding on Ha Ec1 for 1 hour,the expression of hippo-B,yki-x1-x3 and yki-x2 in Hippo signal pathway of the larvae increased by 2.6,2.2 and 2.3 times,respectively;the expression of raf-x2 in MAPK signal pathway increased 2.7 times;the expression of akt,cyc G2 and cyc B in Insulin signal pathway increased 2.9 times,4.7 times and 2.9 times respectively;and the expression of csl in Notch signal pathway increased 1.8 times.The expression of relish in IMD signal pathway increased 3.3 times,and the expression of lamin,diap-x2-x3-x6 and diap-x4 in Apoptosis signal pathway increased 2.9 times,5.9 times and 2.9 times,respectively.After feeding on Ha Ec1 for 3 hours,the expression of pi3 k in Insulin signal pathway and caspase 1 in Apoptosis signal pathway of the larvae increased by 4.6 times and 4.6 times,respectively.Six hours after feeding on Ha Ec1,the expression of caspase 1,htra and diap-x2-x3-x6 in Apoptosis signaling pathway increased by 2.9,13.7 and 4.2 times,while the expression of cyct609 decreased by 91.5%.The expression of p38 and its two transcripts p38-x1,p38-x2 and mkk4 decreased by 71.1%,72.9%,90.6% and 56.2%,respectively.In Insulin signal pathway,the expression of Pi3 k increased by 2.9 times,while cyc B decreased by 77.1%.These results suggest that Apoptosis and IMD signal pathways may regulate the apoptosis of midgut cells after Ha Ec1 injury,while Hippo,MAPK,Insulin and Notch signal pathways may be involved in regulating the proliferation of midgut stem cells.The above results can provide a theoretical basis for clarifying the molecular regulatory network of midgut repair induced by midgut damage in H.armigera and fill the research gap in the field of Lepidoptera,and provide molecular targets for the development of new pesticides and other control measures targeting midgut cells. |
