Differntial Responses Of Midgut Cells To Bt Toxin Cry1Ac In Bt-Resistant And Suseptible Strains Of Helicoverpa Armigera

Cotton is one of the most important economic crops in China,it plays a vital role in agriculture.As a major pest in the cotton,cotton bollworm(Helicoverpa armigera Hubner),has posed a serious threat to its production.Bacillus thuringiensis crystal protein is a crucial biological insecticide.Since 1997,transgenic cotton expressing Bt toxin Cry1Ac has widely used for controlling cotton bollworm in China,thus leading to reduced the population of them and increased significant benefits of economy and environment.However,Due to the expanding area of transgenic cotton,it is easy to evolve resistance to the Bt cotton when cotton bollworm under long-term selection pressure.The monitoring data in field illuminated Cry1Ac resistance allele frequencies in population from northern China were sustained growth in recent years,especially for dominant resistance alleles.The insect intestinal epithelium is an important organ which isolates insect from the outside world,it is essential to growing development and physiological processes.Many researches have showed that the target of Bt toxin is the insect gut.Cry toxins released as protoxin when invade the gut and activated by gut proteases.After enzymatic activation,the active toxin binds to specific receptor located in the midgut microvilli,then it leads to membrane penetration,followed by forming transmembrane pores or channels that cause osmotic lysis and death of larval midgut epithelial cells.Studies on the histopathologic changes of lepidopterous larvae which fed on Bt endotoxin received the attention of many workers.These studies showed that crystal endotoxin causes the significant destruction of columnar cells such as notable changes in the microvilli and mitochondria,moreover,Cry toxin also included the midgut regeneration response.However,almost all of previously research materials were susceptible strains.This study focus on the differential response of midgut cells to Bt toxin Cry1Ac in Bt-resistance and susceptible strains of cotton bollworm,including histopathology,apoptosis and proliferation.The aim of this study is to complement the resistance mechanism in cellular level.1.Histologic response of midgut cells to CrylAc toxin of H.armigeraThis study did some comparison of the differential responses of midgut cells to CrylAc in resistance and susceptible strains.The result showed that the susceptible strain(SCD)presented histopathological changes after an hour to two hours,such as columnar cells with protuberances and vesicles were identified,then they sloughed into the midgut lumen,extensive vacuolization in cytoplasm and degeneration in columnar cells,and finally they can’t maintain normal condition.Due to lacking the significant receptor(cadherin),the midgut cells of recessive resistance strain(SCD-r1)only showed protuberances,a few cells sloughed to the lumen and recover to normal at 12 hours.To our surprised,the dominant resistance strain(AY2)also showed histopathological changes which were similar to the susceptible strain(SCD),but they recover to normal at 12 hours.Obviously,the midgut cells of dominant resistance strain(AY2)repaired after fed on toxin for 12 hours.This study found that the midgut cells of SCD and AY2 both can proliferate and differentiation after exposed toxin for 6 hours.The difference was SCD could not defeat the toxin and died,while AY2 recorded to normal.This may be related to the capacity of stem cells regeneration.I speculate this phenomenon implied the vital function of healing in resistance mechanism,but how to impact will to be explored.2.Apoptosis and proliferation of midgut cells to CrylAc toxin of H.armigeraThe pathological result showed that CrylAc toxin caused a series of histopathologic changes,and may induce apoptosis and regeneration response.In addition,the response of midgut cells to CrylAc of the dominant resistance strain(AY2)illustrated cells healing plays a key role in promoting resistance to Bt.Thus,to further explore the function of cells apoptosis and regeneration,this study used TUNEL and BrdU labeling to detect.The result of TUNEL labeling showed that midgut cells of the control 5th larvae of H.armigera were not detected apoptosis,while CrylAc could induced apoptosis more or less,especially the susceptible strain(SCD)and the dominant resistance strain(AY2).After fed on toxin for 2 hours,some cells near to the lumen started to apoptosis,after 6 hours,these cells sloughed into lumen and the nuclei became larger.Finally the cells gap of SCD grew bigger and some matured cells with big nuclei showed TUNEL positive result after 12 hours,while midgut cells of AY2 back to normal and did not detected apoptosis.The result of BrdU labeling showed,midgut cells of the control 5th larvae of H.armigera were undergo regeneration slightly,which may be normal physiological activity.However,After fed on Cry1Ac toxin,the stem cells of the susceptible strain(SCD)and the dominant resistance strain(AY2),not the recessive resistance strain(SCD-rl),started to proliferate.The result of TUNEL labeling was consistent with BrdU,Thus I speculated the apoptosis may promote proliferate.Quantitative of regerative cells showed that the number of regenerative cells of AY2 more than those cells of SCD,it illustrated that the capacity of stem cells may contribute to generate resistance.