Molecular Mechanism Of Cantharidin On Plutella Xylostella

Cantharidin has been reported to be toxic to many pest insect species and is being developed as a bio-insecticide in China.Cantharidin also has synergistic interactions with some insecticides.The diamondback moth(DBM),Plutella xylostella,is a destructive pest attacking cruciferae crops worldwide and is able to develop insecticide resistance rapidly.It has been reported that cantharidin and its analogs exhibit good insecticidal activity against DBM larvae but the underlying mechanism of how cantharidin kills DBM and other pests remains has never been elucidated.In this work,the inhibitory effect of cantharidin on DBM serine/threonine protein phosphatases(PSPs)was investigated to reveal the action mode of cantharidin in insects,which might contribute to development of more efficient cantharidin-like insecticides.Responses of two kinds of detoxification enzymes,gluthione s-transferases(GSTs)and cytochrome P450 monooxygenases(P450s),as well as small heat shock proteins(sHSP),to cantharidin poisoning were monitored at their transcriptional levels to preliminarily explore the potential resistance mechanism of DBM to cantharidin.Significant were as follows:1.Cantharidin impedes the activity of DBM PSPsIn DBM 4th instar larvae fed with LC10,LC25 and LC50 doses of cantharidin,the in vivo PSP activity decreased in time-and dose-dependent manners.Residual activity of PSPs in LC50 treated larvae at 48 h was only 36.96%of the control.Cantharidin,at the final concentrations of 1 nM-100 ?M,inhibited the PSs activity in vitro The IC50 value was calculated as 5.39 ?M.2.Cantharidin affects DBM PSPs genes at the transcriptional levelsSix DBM PSP genes,PP1,PP2A,PP2B,PP4,PP5 and PP6,were cloned.Sequence alignment showed that PSP genes are highly conserved in eukaryote.The catalytic domain sequences of these six DBM PSP genes were highly similar.These six DBM PSP genes showed varied transcriptional profiles at different developmental stages and in various tissues.The highest expression levels of DBM PSP genes occurred in adults,except for PP6,which is more highly expressed in larvae.The mRNAs of all six genes were most abundant in the head.The LC10,LC50 and LC75 doses of cantharidin seemed to have no significant inhibitory effect on the transcription of DBM PSP genes.3.Cantharidin and its analogs inhibit the activity of recombinant PSP proteinsFor recombinant DBM PSPs expressed heterologously in Escherichia coli,only PP2B and PP5 were expressed in soluble form.The activity of purified recombinant PP2B and PP5 were tested using pNPP as the substrate.Recombinant PP2B showed no activity,while recombinant PP5 displayed good activity and its activity could be enhanced by arachidonic acid.The inhibitory activity of cantharidin and 11 analogs against recombinant PP5 protein were investigated.Cantharidin exerted the most potent inhibition at the IC50 value of 0.38 ?M.The 11 analogs impeded the activity of recombinant PP5 to varied degrees with the IC50 values ranging from 7.42 to 538.38 ?M.The rank of IC50 values of these 12 compounds was found to be consistent with their published toxicities(LC50 values)in DBM 3rd instar larvae with a correlation coefficient(R2)of 0.87.The increased Km value and unchanged Vmax value of recombinant PP5 in the presence of cantharidin indicated that cantharidin competitively inhibited recombinant PP5 by anchoring into its active site.4.Cantharidin suppresses the transcription of some DBM GSTs genes and inhibits theactivity of some recombinant GST proteinsNineteen GST genes were identified from the DBM transcriptomic database(KONAGAbase).Seventeen GST genes were grouped into six known insect GST families while two were unclassified.The LC50 dose of cantharidin treatment decreased expression of most GST genes.The transcription levels of GSTe4 and GSTu2 were down-regulated by 12.78-and 11.52-fold at 48 h post treatment,respectively.Nine out of twelve recombinant GST proteins produced in Escherichia coli possessed CDNB conjunction activity.Cantharidin could impair the activity of three recombinant GST proteins,GSTe3,GSTo2 and GSTu2.5.Cantharidin induces the overexpression of some P450 genes in the CYP6 familyFourteen P450s genes belonging to CYP6 family were identified from the KONAGAbase.At 6-48 h post treatment with an LC50 dose of cantharidin,one gene was consistently overexpressed;the mRNA levels of nine genes were decreased,then were increased;the transcription of one gene was up-regulated then was down-regulated;expression levels of three genes remained unchanged.6.Cantharidin up-regulates the expression of some sHSP genesFourteen sHSP genes were identified from the KONAGAbase.Only sHSP22.1 was overexpressed at all time points post treatment with an LC50 dose of cantharidin,the mRNA levels of sHSP22.1 were increased by 3.7-,9.73-,11.21-,and 16.31-folds at 6 h,12 h,24 h,and 48 h,respectively.Whereas,the expression levels of sHSP 19.5 and sHSP20.1 were significantly reduced in all cases.No significant expression changes were observed in other sHSP genes.In summary,given that cantharidin inhibited DBM PSP activity both in vivo and in vitro,and competitively inhibited recombinant PSP activity,and given the high correlation between inhibitory activity of cantharidin and 11 analogs against recombinant PSPs and their insecticidal activity toward DBM larvae,the results suggest that cantharidin,by anchoring into the active sites of PSP proteins,inhibits the activity of PSPs in DBM larvae,disrupting the physiological activities they control,and thus resulting in death.This is a new mode of action differing from other commercially available insecticides.In addition,the down-regulated expression of some GST genes and/or activity losses of some GST proteins caused by cantharidin might be related to the synergistic effect of cantharidin with some insecticides.The overexpression of some P450 genes and sHSP genes in response to cantharidin poisoning might also provide the basis for further investigation of the cantharidin resistance mechanism in DBM.