Tolerance Effect To Imidacloprid By Quercetin In Apis Cerana Cerana Fabricius

Honey bees are the most important pollinators which play an important role in maintaining plant diversity and increasing crops' yield.However,since 2006,Colony Collapse Disorder has occurred in America,Europe,and Asia,resulting in a sharp decline in honey bee populations.Imidacloprid,a neonicotinoid insecticide,has been recognized contributing to the decrease of honey bee populations.Quercetin is an important phytochemical widely presented in pollen and nectar,which has been proved to enhance the pyrethroid pesticides tolerance of Apis mellifera Linnaeus(Hymenoptera:Apidae).But,until now,whether phytochemicals can affect the lethal and sublethal effects of imidacloprid on honey bees is little known.Apis cerana cerana Fabricius is an important native honey bee in China.However,no information is available on the role of phytochemicals in regulating the tolerance of the native honey bee to pesticides.Therefore,to protect A.cerana cerana for safe pollination and reduce the effects of neonicotinoid insecticides on native pollinators,this research explored the effects of quercetin on the lethal and sublethal effects of imidacloprid on A.cerana cerana workers,and determined the difference of three detoxification enzymes activities in workers after exposed to quercetin and imidacloprid.To clarify the regulation mechanism of quercetin on the imidacloprid tolerance of A.cerana cerana,transcriptome sequencing and real-time quantitative PCR(qRT-PCR)were performed to evaluate the expression profiles of the detoxification enzyme genes induced by imidacloprid and quercetin.We screened the critical genes up-regulated by quercetin and involved in the detoxification of imidacloprid.Finally,the effect of the key P450 s genes induced by quercetin on the imidacloprid tolerance of A.cerana cerana was evaluated by RNA interference(RNAi).The main results are as follows:1 Quercetin reduced the lethal and sublethal effects of imidacloprid on A.cerana ceranaThe survival and Proboscis Extension Response(PER)experiments were conducted to explore the effects of quercetin on the imidacloprid tolerance of A.cerana cerana.The results showed that chronic exposure to imidacloprid of 100 ?g/L significantly reduced the survival time of workers by 10.81 d.Acute exposure impaired sucrose responsiveness and olfactory learning of workers.The A.cerana cerana workers acutely exposed to 20 ?g/L imidacloprid showed significantly lower sucrose responsiveness.The workers chronically exposed to 100 ?g/L imidacloprid survived much longer when treated with quercetin at 37.8 mg/L for 24 h.The sucrose responsiveness of workers acutely exposed to imidacloprid was improved at 75.6 mg/L quercetin.These results indicated that quercetin reduced the lethal and sublethal effects of imidacloprid on A.cerana cerana,and enhanced the imidacloprid tolerance.However,the effect of quercetin on the lethal effect of imidacloprid was dosage-dependent.Quercetin at 75.6 mg/L and 151.2 mg/L reduced the longevity of workers chronically exposed to 100 ?g/L imidacloprid.2 Responses of detoxification enzymes of A.cerana cerana to quercetin and imidaclopridThe effects of quercetin and imidacloprid on the activities of P450 s,carboxylesterases(Car Es),and glutathione S-transferases(GSTs)of A.cerana cerana were evaluated in our study.The results showed that the quercetin and imidacloprid treatment significantly increased the activities of three detoxification enzymes,indicated that P450 s,Car Es,and GSTs participated in the detoxification of A.cerana cerana to two xenobiotics.However,the P450 s might be the key detoxification enzyme for the detoxification of quercetin and imidacloprid,because these two xenobiotics increased the activity of P450 s more than Car Es and GSTs.Quercetin increased the P450 s activity of A.cerana cerana dramatically compared with imidacloprid.The workers treated with quercetin for 12 h-1 h showed significantly higher P450 s activity.The workers exposed to imidacloprid for 12 h-2 d did not affect the P450 s activity relative to control.However,with the increasing of time,the P450 s activity of workers in the imidacloprid group increased significantly at 5 d and 10 d.Our results suggested that the response of P450 s to imidacloprid exposure was relatively delayed,while the rapid response to quercetin might contribute to the regulation of imidacloprid tolerance in A.cerana cerana.The Car Es and GSTs activities of A.cerana cerana exposed to 20?g/L imidacloprid for 12 h,1 d,5 d,and 10 d were significantly higher than control,however,the 100 ?g/L only significantly increased the enzyme activities at 1 d and 5 d,which suggested that Car Es and GSTs played critical roles in detoxifying imidacloprid of low concentration in A.cerana cerana.3 Screening and expression of key detoxification enzyme genes of A.cerana cerana under quercetin and imidacloprid treatmentApproximately 69.45 Gb clean data were obtained from nine samples of A.cerana cerana,with the clean reads for the control,quercetin,and imidacloprid group was79.54,76.13,and 77.28 Mb,respectively.Compared with the reference genome sequence,a total of 429.41 Mb mapped reads were obtained.Treated with 37.8 mg/L quercetin,122 genes were up-regulated and 200 genes were down-regulated in A.cerana cerana.By analyzing the function of differentially expressed genes(DEGs),these genes were enriched in the oxidation-reduction process,such as detoxification mediated by P450 s and the antioxidation process.In addition,the DEGs were also involved in the metabolism,neural signal transmission,and behaviour.Some DEGs showed similar expression as that in the imidacloprid group,suggesting that quercetin as an important phytochemical might produce detrimental effects on honey bees.Exposed to 100 ?g/L imidacloprid,669 genes were up-regulated while 194 genes were down-regulated in A.cerana cerana.The up-regulated DEGs were enriched in the neuroactive ligand-receptor interaction,G-protein coupled receptor signaling pathway,G-protein coupled receptor activity,and phototransduction-fly;the down-regulated DEGs were enriched in the carbon metabolism,biosynthesis of amino acids,carbohydrate metabolic process,and innate immune response.It indicated that the imidacloprid exposure significantly affected the neural signal transmission,metabolism,immunity,vision,and olfaction and brought in multiple lethal and sublethal effects on A.cerana cerana.According to the comment information of the GO,KEGG,and NR database,the DEGs involved in the xenobiotic detoxification were screened in the quercetin and imidacloprid groups.A total of 8 DEGs was obtained.The four key up-regulated DEGs were AccCYP6a14,AccCYP305a1,AccCYP303a1,and AccEsterase FE4,indicating that these DEGs participated in the detoxification of quercetin and imidacloprid.The q RT-PCR experiment confirmed that the expression of AccCYP303a1 was significantly up-regulated after being treated with quercetin for 1 d.AccCYP6a14 and AccCYP305a1 were also up-regulated.These results suggested that quercetin might affect the imidacloprid tolerance of A.cerana cerana by up-regulating the expression of AccCYP303a1,AccCYP6a14,and AccCYP305a1.4 AccCYP6a14 and AccCYP305a1 participated in the regulation of imidacloprid tolerance of A.cerana ceranaRNAi experiment was performed to evaluate the effect of key P450 s induced by quercetin on the imidacloprid tolerance of A.cerana cerana.Injection and feeding of ds RNA to workers significantly down-regulated the expression of AccCYP6a14,AccCYP305a1,and AccCYP303a1 by 60.86%-80.85%.Silencing AccCYP6a14 and AccCYP303a1 dramatically increased the mortality of workers exposed to 100 ?g/L imidacloprid,indicating that these key P450 s genes were related to the imidacloprid tolerance of A.cerana cerana.In addition,the mortality of workers in the ds AccCYP6a14 group was higher than that in the ds AccCYP303a1 group,which indicated that AccCYP6a14 played a greater role in the detoxification of A.cerana cerana to imidacloprid compared with AccCYP303a1.However,down-regulating the AccCYP6a14,AccCYP305a1,and AccCYP303a1 did not affect the survival of workers exposed to 20 ?g/L imidacloprid,suggesting that the P450 s induced by quercetin had little effect on the detoxification of the low concentration of imidacloprid.Injection of the ds RNA of AccCYP305a1 significantly affected the mortality of workers exposed to100 ?g/L imidacloprid,but feeding ds RNA had no effect.We speculated that the different interference efficiency in the injection and feeding experiments accounted for this result.In conclusion,37.8 mg/L quercetin protected the honey bees from imidacloprid exposure by up-regulating the expression of AccCYP6a14 and AccCYP303a1,and enhanced the imidacloprid tolerance of A.cerana cerana.However,the P450 s induced by quercetin did not affect the tolerance of honey bees to imidacloprid of low concentration,as indicated by the fact that treated with quercetin did not influence the sublethal effects of 20 ?g/L imidacloprid.In addition,AccCYP6a14 and AccCYP303a1 not only participated in the imidacloprid detoxification but were also involved in the quercetin metabolism.Consequently,with increasing the concentration of quercetin and the exposure time of imidacloprid,excessive quercetin metabolized by P450 s might reduce the detoxifying efficiency of imidacloprid and enhance the lethal effects of the pesticide.Our results confirmed the vital role of quercetin and the key P450 s genes induced by quercetin in regulating the imidacloprid tolerance of A.cerana cerana.It provided a new strategy to protect A.cerana cerana and reduce the risk of pesticides on honey bees,but also inspired further study exploring the effect of phytochemicals on the other pesticides tolerance of A.cerana cerana.