The Mechanism Of Insecticide Resistance Influenced By Heavy Metal Copper In Spodoptera Litura

The indiscriminate application of pesticides can lead to pest resistance and ultimately develop drug resistance.Heavy metal pollution is also an important aspect of the current deterioration of the ecological environment.In the natural environment,pests are vulnerable to the dual stress of pesticides and heavy metals,and pests have evolved and developed corresponding resistance strategies.However,whether pests have similar resistance mechanisms in response to pesticides and heavy metal stress,it is unclear whether there is cross-resistance between the pests.In the paper,Spodoptera litura,which is an important herbivorous pest,was used to study the effect of heavy metal copper on its drug resistance.The inducing effect of heavy metal copper stress on resistance to Spodoptera litura was explored through enzyme activity assay and analysis of the function of the detoxifying enzyme gene and its mechanism of action.Results listed as following:1.The third-instar larvae of Spodoptera litura larvae were treated with heavy metal copper at a concentration of 200 mg/kg in the artificial diet for 72 h.Compared with the non-copper-extracted control group,the body weight gain was reduced by 9.34%and the pupae weight was reduced by 18.92%.When the copper concentration in the artificial diet was below 100 mg/kg,there was no significant difference between body weight gain and pupae weight of Spodoptera litura and the control group.The copper treatment within 100 mg/kg does not affect the normal growth and development of Spodoptera litura.2.The copper concentration gradients were set 0,25,50,75,and 100 mg/kg,and the resistance to lambda-cyhalothrin and imidacloprid was determined after 72 h stress on Spodoptera litura.It was found that lowdose copper stress can significantly improve the larvae tolerance to lambda-cyhalothrin and imidacloprid.The results of the pesticide feeding method showed that the tolerance to lambda-cyhalothrin increased for Spodoptera litura treated with concentrations of 25,50,75,and 100 mg/kg copper,respectively,compared with the control group without copper consumption,increased by 26.98%,32.02%,43.88%and 30.74%.At the copper ion concentrations of 50,75,and 100 mg/kg,the tolerance to imidacloprid increased by 24.92%,35.75%,and 24.09%,respectively.The pesticide drop test results showed that the resistance of Spodoptera litura to lambda-cyhalothrin was gradually improved with the increase of copper concentration in the feed.When the concentration of copper in the feed was 100 mg/kg,Spodoptera litura finally mortality rate was 51.91%of the control group.And the copper-stressed offspring still had resistance to lambda-cyhalothrin.Compared with the control group,the final mortality of the larvae was reduced by 34.7%.It shows that low-dose heavy metal copper can effectively induce the emergence of Spodoptera litura resistance,and the induction can be inherited.3.The activities of P450 enzymes in the midgut of Spodoptera litura significantly increased,which had no significant effect on the activity of GST and CarE,suggesting that copper might affect drug resistance through the P450 enzyme system.The effect of copper stress on the expression of P450 gene was detected by Real-time quantitative PCR.It was found that the expression of the two P450 genes,CYP6AB12 and CYP6B50,was induced by copper stress.Under the stress of 75 mg/kg copper,CYP6AB12 and CYP6B50 were found in the larval midgut,the gene expression levels were 10.21 and 4.60 times that of the control group,respectively.The fulllength sequences of these two genes were cloned and their conserved domains were analyzed.It was found that both have a conserved P450 binding and catalytic sites.The results of quantitative PCR showed that CYP6AB12 was mainly expressed in the larval stage.The expression level of CYP6AB12 was the highest at 6th instar.It was basically not expressed in the pupae stage.Tissue distribution was found specific high expression in the midgut.CYP6B50 reached the highest peak at the third instar and sixth instar stage,It did not express at the pupae stage and adult stage.Similar to CYP6AB12,CYP6B50 is also highly specific expressed in the midgut.The developmental expression patterns and tissue distribution characteristics of the two genes indicate that they may be involved in the metabolic detoxification process of Spodoptera litura.Lambdacyhalothrin had no significant effect on the expression of CYP6AB12 and CYP6B50 in the midgut of Spodoptera litura larvae,but imidacloprid effectively induced the expression of these two genes in the midgut.In addition,imidacloprid can effectively induce the upregulation of midgut P450 activity of Spodoptera litura,and lambda-cyhalothrin has no significant effect on P450 activity.Both imidacloprid and heavy metal copper can effectively induce the expression of these two P450 genes and stimulate the up-regulation of P450 activity,suggesting that there may be the same induction mechanism,lambda-cyhalothrin may not be involved in copper induced the process of emergence of drug resistance.4.RNAi technology was used to down-regulate the expression of CYP6AB12 and CYP6B50 in Spodoptera litura.The expression of the two genes was only 35.49%and 18.30%of the control group injected with the green fluorescent protein gene(GFP)double-stranded RNA.The death rate of Spodoptera litura increased significantly after the suppression of CYP6B50 expression,and its tolerance to imidacloprid decreased significantly,the extent of resistance to imidacloprid in Spodoptera litura induced by low-dose copper(75 mg/kg)also decreased significantly.But dsCYP6AB12 dose not have this effect.Those indicate that the CYP6B50 was involved in the process of copper-induced resistance of Spodoptera litura to imidacloprid.The results demonstrate that heavy metal copper accumulates in the midgut and fat body of Spodoptera litura and effectively stimulates the expression of P450 genes(CYP6AB12 and CYP6B50),CYP6B50 enhances the resistance of nicotinic pesticide imidacloprid to Spodoptera litura.Copper contamination may be an important potential factor for resistance of Spodoptera litura to the nicotinic pesticide imidacloprid or even development of resistance to insecticides.This study will help to in-depth understand the relationship between heavy metal pollution and the outbreak of insect populations.Meanwhile,it provides guidance for guiding the rational use of pesticides and controls for heavy metal pollution.