Study On The Mechanism Of The Enantioselective Toxicity Of Isocarbophos To Locusta Migratoria Manilensis(Meyen)

Abstract:Chirality refers to the non-overlapping features of chemicals and their mirror image,which is widely cognitive in the biological field.Enantiomers of chiral materials tend to significantly vary in terms of biological activity.More than 25%of organphosphorus pesticides(Ops)have enantioselectivity.Known as a type of worldwide pest,Locusta migratoria(Meyen)exert dramatic harm on gramineae and cyperaceous.Current prevention and control works are primarily traditional chemical control methods that spray organophosphorus pesticides.However,insecticide resistance has emerged due to the long-time utilization of similar pesticides,which further results in the increase of the dosage and thus the insect-resistant cost as well as the destruction of the soil water and environment.In order to reveal the generation mechanism of the enantioselective toxicity of isocarbophos(ICP)to L.migratory manilensis,the transcription of genes of carboxylesterases(CarEs),Glutathione S-transferases(GSTs)and acetylcholinesterase(AChE),activities of these above enzymes in vivo and the inhibition of Ache in vitro were investigated.Choral separation of ICP race mates was achieved on a Chorale(?)OD column with the mobile phase of n-hexane and isopropanol at the ratio of 90:10(v/v)at the flow rate of 0.8 mL·min-1.The individual enantiomers with different concentration were topically applied to 3th-instar nymphs and the median lethal dose(LD50)of each ICP enantiomer was calculated by SPSS 19.0 software 24 h later.Genes expression level of CarEs,GSTs and AChE was assayed by RT-PCR and the enzymatic activity was detected in control group and experimental groups.The enzymes inhibition test was measured using crude enzyme extracted from control insects which were treated only with acetone.The median lethal dose(LD50)of(+)-enantiomer and(-)-enantiomer of ICP toward Locusta migratoria manilensis were 0.609 and 79.412 mg·kg-1 respectively,after 24 h of exposure,displaying a 130-fold diference between the two enantiomers.LmCesA4 and LmCesA5 were restrained by adding(+)-ICP and the inhibition activity of(+)-ICP to CarEs increased with its increasing concentration.In the locusts treated with(-)-ICP,the expression level of LmCesA4 mRNA reached 4.65 fold of the control level wich was far higher than the expression level of LmCesA5 mRNA.Expression of the general esterase and CarEs activity existed enantioselectivity.There is strong inhibition activity of(+)-ICP to CarEs while usingβ-NA as the substrate.In vivo,activity of GSTs increased with the increasing concentration of(-)-ICP.In vitro,the inhibition activity of(+)/(-)-ICP to GSTs increased with the increase of the individual enantiomer concentration,the half maximal inhibitory concentration(IC50)of(-)-ICP was 10.5 fold than that of(+)-ICP.The over-expression of LmGSTs4,LmGSTs7 and LmGSTtl can cause the Resistance of(+)-ICP,and the overexpression of LmGSTdl,LmGSTs1,LmGSTs3,LmGSTs5,LmGSTs6 and LmGSTs7 can cause the Resistance of(-)-ICP.The expression level of Lm-acelmRNA first increased and then decreased,and reached 4.38～14.9 fold of the control level,with the increasing concentration of(+)/(-)-ICP concentration.The maximum value 14.9 fold was appeared at 12 mg·L-1 for(+)-ICP and 12.0 fold at 3200 mg·L-1 for(-)-ICP.The AchE activities in locust decreased with the increase of(+)-ICP concentration,but increased with the increase of(-)-ICP concentration.The results showed that the activities of AChE in(+)-ICP treatment groups declined by 35.55%at 12mg·L-1 and 32.54%at 10mg·L-1,and the difference was extremely significant(P<0.01).In vitro,the inhibition activity of(+)/(-)-ICP to AChE increased with the increase of the individual enantiomer concentration.The half maximal inhibitory concentration(IC50)of(-)-ICP was 10-fold higher than that of(+)-ICP.Our results indicated that the enantioselective inhibition to CarE,different combining capacity of ICP enantiomers and GSTs,different inducing activity to the expression level of Lm-acelmRNA and the different inhabitation activity to AChE in Locust might be the reasons of the enantioselective activity of ICP.