Immune Response Of Locusta Migratoria To Metarhizhium Anisopliae Strain(IMI330189)and MAD1 Protein

Metarhizium anisopliae?IMI330189?is a potent and entomopathogenic fungal strain could be effectively used against insect pests.Similarly,MAD1 protein adheres to insect cuticle,causing virulence to insects.Efficacy of M.anisopliae strain?IMI330189?and MAD1 protein alone or in combination by feeding method to overcome immune-related enzymes and Toll-like pathway genes were investigated in the Locusta migratoria.I confirmed a maximum 55%of mortality when M.anisopliae?IMI330189?and MAD1 was applied in combination.Similarly,increased PO activity was observed in locust with a combined dose of MAD1+IMI330189 whereas PO,POD,and SOD activities reduced using MAD1 independently.Four Toll-like signaling pathway genes?MyD88,Cactus,Pelle,and CaN?were investigated from the midgut and the body of the L.migratoria after 72 h of treatments.Subsequently,the expression of MyD88 in the midgut and body significantly decreased with the application of MD1 and MAD1+IMI330189.Performance of these treatments was absolutely non-consistent in both parts of insects.Meanwhile,IMI330189 significantly raised the expression of Cactus in both midgut and body.However,the combined treatment?MAD1+IMI330189?significantly reduced the Cactus expression in both body parts.Pelle expression was significantly increased in the midgut with the application of independent treatment of MAD1 and IMI330189 whereas the combined treatment?MAD1+IMI330189?suppressed the Pelle expression in midgut.Its expression level was absolutely higher in the body with the application of IMI330189 and MAD1+IMI330189 only.On the other hand,MAD1significantly increased the expression of CaN in the midgut.However,all three treatments significantly affected and suppressed the expression of CaN gene in the body of locust.This shows that the applications of M.anisopliae and MAD1 protein significantly affected Toll signaling pathway genes,which ultimately increased the level of susceptibility of locust.However,their effect was significantly different in both parts of locust which recommends that the Toll-related genes are conserved in midgut instead of locust body.I also checked that the activities of detoxifying enzymes through dietary method enzymes such as Multi-Function Oxidase?MFO?,chitinase?CHI?and AMPs significantly increased in the body of the host after 72 hr of the feeding of these proteins.The increased activities of MFO,CHI and AMPs subsequently activated the locust immune system in the response to feeding with M.anisopliae strain IMI330189 and MAD1.I also detected six antioxidant enzymes through spray method,enzymes as Chitinase?CHI?,Phenol oxidase?PO?,Super oxidase?SOD?,Peroxidase?POD?,Reactive oxygen species ROS),and Antimicrobial peptide?AMP?on 3^rd,5^th and 7^th day after treatments application using ELISA method.I also checked the expression of three genes Chitinase,Diptericin,and GNBP3 in L.migratoria body on3^rd,5^th and 7^th day after treatments application using qRTPCR.Our results showed that the activity of antioxidant enzymes Chitinase,Phenol oxidase,Super oxidase,Peroxidase,Reactive oxygen species,and Antimicrobial peptide significantly increased on 3^rd,5^th and 7^th day after MAD1,MAD+IMI330189and IMI33019 treatments application.Our qRT PCR results showed that IMI330189 significantly inhibit the Chitinase on 3^rd and 7^th day of treatment application however MAD1,MAD1+IMI330189,and IMI330189 significantly inhibit the Chitinase on 5^th day after treatments application.Application of MAD1+IMI330189 significantly inhibits the Diptericin on 3^rd,5^th and 7^thh day after treatment application.The MAD1+IMI330189 and IMI330189 significantly inhibit diptericin expression on 7^th day.Application of MAD1+IMI330189 significantly inhibits the GNBP3 on 3^rd,5^th and 7^th day after treatment application.As compared to control MAD1 significantly inhibit the GNBP3 expression on 3^rdd day after the application of the treatments.From the present results,it was concluded that all the enzymes Chitinase,Phenol oxidase,Super oxidase,Peroxidase,Reactive oxygen species,and Antimicrobial peptide increased the activity and activated the immunity of L.migratoria in response of MAD1 and M.anisopliae strain IMI330189.However inhibition in expression of genes namely Chitinase,Diptericin and GNBP3 caused susceptibility in L.migratoria and caused mortality in locust.On behalf of treatments application,it can be suggested that a combined dose of MAD1+IMI330189can give good results in mortality of L.migratoria.Conclusion:1.MAD1 increase the PO,SOD,MFO,CHI and AMPs activity,and inhibited the expression of MyD88,Pelle,Cactus,and CaN through feeding method,which reduced the immunity of locust and caused mortality in L.migratoria.2.MAD1 enhance the receptor recognition of locust through spray method,trigger the immune response of L.migratoria,while the expression of AMP showed a trend of increasing first and then decreasing,which resulted increased in the mortality.