Insecticidal Activity Of Cinnamon Essential Oil And Functional Analysis Of GST In Response To Cinnamaldehyde In Tribolium Castaneum

The red flour beetle,Tribolium castaneum,is an important grain storage pest.The pest insect is widely distributed in China and causes serious economic losses every year.To date,the control of T.castaneum mainly relies on phosphine fumigation.However,the extensively use of phosphine will make the insect resistant to the chemical.Therefore,the developing of alternative control strategies of T.castaneum is urgently needed.Plant essential oil has fumigant activity against a variety of pest species and is environmentally friendly,making them a hot topic to replace chemical fumigants for controlling grain storage pests.In this study,the cinnamon essential oil,which has a good fumigant effect on T.castaneum,was selected from 10 plant essential oils,and its main active ingredient was identified as cinnamaldehyde.Furthermore,the effects of cinnamaldehyde on the activities of several detoxifying and protective enzymes were determined and the expression patterns of glutathione-S-transferase（GST）genes of T.castaneum were analyzed after cinnamaldehyde treatment.Moreover,recombinant TcGSTe2 protein was expressed in vitro to clarify its function in response to cinnamaldehyde stress.This study provides a theoretical basis for the development of cinnamaldehyde as a novel plant-derived insecticide for green control of grain storage pests,as well as a basis for understanding the molecular mechanisms of T.castaneum in response to cinnamaldehyde stress.The main findings of this study are as follows:1.Screening of plant essential oils against T.castaneum and insecticidal activity of cinnamon essential oilThe fumigant activity of 10 essential oils at a concentration of 20μL/L was measured against the T.castaneum using a flask fumigation method.The results showed that cinnamon essential oil had the best effect on T.castaneum,and the corrected mortality rate of the test insects was 100%after a 24 h treatment.Cinnamon essential oil was prepared at different concentrations（0.5,1,1.5,2 and 2.5μL/L）and the virulence against adult T.castaneum was determined at different treatment times（24,48 and 72 h）.The results showed that cinnamon essential oil exhibited significant temporal and dose effects with lethal medium concentrations(LC₅₀)of 5.97,4.73 and 2.89μL/L at 24,48 and 72 h,respectively.2.Identification of Main Insecticidal Active Components of cinnamon Essential OilThe chemical components of cinnamon essential oil were analyzed by gas chromatography-mass spectrometry（GC-MS）,and the relative contents of each component were calculated by area normalization method.The results showed that the main componentsofcinnamonessentialoilincludedcinnamaldehyde,o-methoxycinnamaldehyde,cinnamyl acetate,o-anisaldehyde,（+）-limonene and benzaldehyde.Among these,the relative content of cinnamaldehyde was the highest（75%）,followed with cinnamyl acetate（7.65%）.Next,cinnamaldehyde,and cinnamyl acetate were used to analyze the fumigation effects on T.castaneum.The results showed that only cinnamaldehyde had obvious fumigation activity,with a LC₅₀of 2.28,1.73 and 1.28μL/L,respectively,at 24,48 and 72 hours after the treatment.Therefore,cinnamaldehyde was considered as the main insecticidal active component of cinnamon essential oil.3.Effects of Cinnamaldehyde on the Activities of Detoxification and Protective Enzymes of T.castaneumThe T.castaneum adults were treated with 2.28μL/L(LC₅₀dose at 24 h)of cinnamaldehyde,and the activities of several detoxification enzymes and protective enzymes were analyzed.The results showed that the activities of GST,superoxide dismutase（SOD）and catalase（CAT）increased significantly.Although the activities of these enzymes decreased gradually with the increase of treatment time,the activities of these enzymes in the treatment group were always significantly higher than those in the control group.The activities of carboxylesterase（Car E）and peroxidase（POD）decreased with the increase of treatment time,and were significantly lower than those of the control group.Among the enzymes with increased activity,GST was most obviously.Its activity reached 5.59 times than that of the control group 36 h after treatment.This result shows that the GST plays an important role in responding to cinnamaldehyde stress.4.Expression analysis of GST genes in T.castaneumReal-time quantitative PCR was used to analyze the changes of GST expression level of T.castaneum after cinnamaldehyde treatment.The results showed that the expression levels of three delta-class GST genes were down-regulated,and six epsilon-class GST genes were up-regulated.Among them,TcGSTe2 was up-regulated most rapidly,with a2.95-fold up-regulation than that of the control group after 24 h of cinnamaldehyde treatment.It is possible that the protein encoded by this gene may be involved in the defense of cinnamaldehyde.5.Functional analysis of TcGSTe2Recombinant TcGSTe2 protein was expressed and purification from Escherichia coli.However,the results of HPLC analysis showed that the protein had no metabolic function on cinnamaldehyde.Alternatively,we found that cinnamaldehyde treatment increases the contents of malondialdehyde and hydrogen peroxide in T.castaneum,indicating the oxidative damage.TcGSTe2 has strong antioxidant function and can enhance the tolerance of cells to oxidative damage.The results showed that TcGSTe2 played an important role in the response to cinnamaldehyde in T.castaneum,which could improve the survival of T.castaneum under cinnamaldehyde stress.