Effects Of Host Plants On Immune Response Of Spodoptera Exigua To Multicapsid Nucleopolyhedrovirus

Studies have shown that host plants can regulate insect susceptibility to viruses,but the mechanisms that reveal the differences in such susceptibility are not clear.In this paper,we adopted the plants(soybean,cabbage and water convolvulus),insect herbivore(Spodoptera exigua)and Spodoptera exigua multicapsid nucleopolyhedrovirus(SeMNPV)as research objects,used ecological,physiological,biochemical and molecular biological methods to explore the mechanism that plants mediate the susceptibility of insect herbivores to virus.The following were our main study results:1.The virus production and menanization rate of NPV-infected larvae feeding on different diets significantly varied on hours 12 h,24h,36 h,48h and 72 h after treatment.The order for virus production is soybean > artificial diet > cabbage > water convolvulus.The virus production increased exponentially with the hours after NPV infection.The melanization rate of the SeMNPV-infected larvae was significantly higher on cabbage and water convolvulus than on soybean.The exponential fitting for the relationship between melanization rate and virus production manifested that there was a negative correlation,i.e.,the melanization rate of hemolymph decreases with the increase of virus proliferation.These results manifested that the differences in melanization response affected by host plants might lead to the difference in virus production in larval hemolymph.2.In order to explore the mechanism of the difference in melanization,we determined the activity of the key enzymes(phenoloxidase dopamine decarboxylase and tyrosine hydroxylase)in melanization of larvae.Experiment indicated that diets affected the activities of the key enzymes(dopa decarpboxylase,phenoloxidase and tyrosine hydroxylase)in melanization of infected larvae.When the larvae were not infected,four diets significantly affected the activities of the three enzymes respectively,and the three enzymes of larvae fed on four diets all indicated the rising trend with the prolongation of time after treatment respectively.In the virus treatment group,the highest activity of phenol oxidase in hemolymph of larvae was on water convolvulus,followed by artificial diet and cabbage and the lowest on soybean,the activities of dopamine decarboxylase and tyrosine hydroxylase also showed the similar phenomena.Correlation analysis suggested that there was a positive correlation between the activities of the key enzymes and the melanization rate,and a negative correlation between the activities and the amount of virus proliferation.In general,the results showed that dopa decarpboxylase,phenoloxidase and tyrosine hydroxylase were involved in larval melanization.Meanwhile,it suggests that water convolvulus and collard give rise to a higher ability for larvae to resist against the virus through humoral immunity,while soybean leads to a lower resistance against virus.3.Based on the determination of enzyme activity,the transcription levels of three key enzymes(dopa decarpboxylase,phenoloxidase and tyrosine hydroxylase)of melanization in hemolymph of larvae were further determined.The changes of gene expression of the three key enzymes were consistent with those of the enzyme activities,the trend is increased and then decreased.The highest activities of dopa decarpboxylase?phenoloxidase and tyrosine hydroxylase for infected larvae were all on water convolvulus,followed by artificial diet and cabbage,and the lowest on soybean.The highest phenoloxidase gene expression level was on hours 24 after treatment in each virus infection group,while the highest dopa decarpboxylase and tyrosine hydroxylase gene levels were both on hours 36 after treatment but there are significant differences among different diets.The dopa decarpboxylase,phenoloxidase and tyrosine hydroxylase gene levels were higher on water convolvulus than on soybean from hours 12 to 72 respectively.These results suggest that host plantsaffect the activities of the enzyme by regulating the gene expression of the melanization in SeMNPV-infected larvae.4.Designed to study the impacts of host plants on the cellular immunity and the physiology of Spodoptera exigua larvae infected with SeMNPV.The results from flow cytometer indicated that the apoptosis rate of hemocytes in non-infected larvae was low during the 72 hours after treatment.However,the apoptosis rate for SeMNPV-infected larvae increased rapidly after SeMNPV treatment,and the hours after treatment significantly affected apoptosis rate of infected larvae feeding on each diet.Also,four diets significantly affected apoptosis rate of infected larvae on each time point.Generally,the apoptosis rate was vastly higher on water convolvulus and cabbage than on soybean and artificial diet.It was proved that the cellular immune response of the larvae to SeMNPV was higher on water convolvulus and cabbage than on soybean.The apoptosis rate of hemocyte was positively correlated with the relative proliferation of virus in hemolymph,which can be fitted with exponential functions.5.To further investigate the influences of host plants on the molecular effects of Spodoptera exigua larvae treated by SeMNPV.The expression levels of caspases and IAP,two key proteases that regulate apoptosis,were detected by RT-PCR.The relative expression levels of caspase,which promoted hemocyte apoptosis,vastly increased and the relative expression levels of inhibitor of apoptosis proteins Se-IAP decreased from hours 12 to 72 after SeMNPV treatment.The relative expression levels of caspase were higher on water convolvulus and cabbage than on soybean and artificial diet,while the relative expression level of Se-IAP gene presented an opposite.This reveals that the host plant played an important role in regulating the expression of caspase and IAP genes in the apoptosis of beet armyworm.These results provide references to explore the molecular mechanism that host plants affected hemocyte apoptosis of SeMNPVinfected larvae.To sum up,this paper concluded that the interaction between virus and host plant significantly affected the virus production,melanization and hemocyte apoptosis of Spodoptera exigua larvae.Form the aspect of insect immunity,this paper reveals plantmediated effect on insect susceptibilities to entomovirus,and provides a new way and method to explore the relationships among plant,insect and entomovirus.