Physiological And Biochemical Responses Of Insect Midgut Cells To Rice Resistance

The brown planthopper（BPH）Nilaparvata lugens sucks rice sap through the piercing-sucking mouthparts to obtain nutrients,and at the same time it can spread rice diseases,which causes rice yield loss.Currently,spraying chemical pesticides and breeding resistant rice varieties are the main steps to control this pest.But BPH can develop resistance to pesticides and coevolve with rice to produce new virulence.The midgut is the main interaction interface between rice and BPH.Investigation of the interaction will help in understanding the interaciton mechanism of BPH and rice,and gaining theoretical evidence for management of this pest.In this study,biotypeⅠof brown planthopper was used as the experimental material.BPH insects were fed with different resistant rice TN1,MH63,B5 and Mudgo for 24 h.Then TUNEL Reagent,Reactive Oxygen Species kit,Phospho-Histone H3 antibody,and PROX1 antibody were used to detect the apoptosis,the reactive oxygen species,the number of enteroendocrine cells,and the number of mitotic cells in the treated midgut tissue.The results showed that with the increase of rice resistance,more apoptotic cells were found in the midgut tissues;but there was no significant difference in the content of reactive oxygen species（ROS）;The signals representing the enteroendocrine cells in the intestines of brown planthopper treated with Mudgo and B5 for 24 h,were more than that in the midguts treated with TN1 and MH63.The signals representing the mitotic cells showed an upward trend in the midguts treated with TN1,MH63,Mudgo and B5,respectively.Immunohistochemical staining of paraffin sections of midgut tissue showed that the enteroendocrine cells quantity was increasing in the midguts exposed to TN1,MH63,Mudgo and B5 rice.The mitotic cells in the midguts treated with TN1,MH63,Mudgo and B5 rice increased gradually.These results are consistent with the data obtained in the whole mount in situ immunol hybridization of midguts.This work demonstrates that host rice resistance imposed a serious injury on BPH midgut cells and tissues,even causing apoptosis.On the other hand,to restore the structure and function of the injured midgut,stem cells accelerated their mitosis,and then differentiated into more enteroendocrine cells.The latter differentiated into enterocytes.This physiological reaction would maintain the midgut homeostasis.To reveal the mechanism of the midgut apoptosis,degradation of DNA with acetylcholinesterase（ACh E）was carried out,which confirmed that ACh E can directly degrade the plasmid DNA and the genomic DNA of BPH at the presence of Mg²⁺.It is supposed that the ACh E acted as an endonuclease to digest the genomic DNA,which resulted in the midgut apoptosis.In order to further explore the ROS content and mitosis in midgut cells in response to resistant rice,Helicoverpa armigera midgut cells were treated with rice juices of TN1,MH63,Mudgo and B5 at 24 h,48 h and 72 h.The results showed that the mitotic cells in the sample treated with TN1 increased over time,those treated with MH63 ROS first then fell down,and the cells treated with Mudgo and B5 increased.In addition,the ROS content increased in the sample treated with TN1.While the content showed an increasing pattern then falling down in the cells treated with MH63.Comparatively,the ROS content decreased in the cells exposed to Mudgo and B5.The data demonstrated that the higher the rice resistance level is,the more or stronger the toxin is in the rice allelochemicals,which creates more severe damage to the midgut tissue and cells.The physiological and biochemical reaction of BPH midgut to host rice resistance were studied in this work.The results laid a foundation for understanding the interaction mechanism of BPH and rice,and developing novel management of this pest.