| Tribolium castaneum is one of the important stored grain pests.At present,it mainly relies on chemical agents for control.However,the long-term use of high-dose chemical agents has resulted in different degrees of resistance to the insecticides.Therefore,it is urgent to develop new green environmental protection insecticide.Plant essential oils have been widely used to control stored grain pests due to their high insecticidal activity,diverse modes of action,low toxicity,and environmental friendliness.Plant essential oils not only have fumigating,repelling,contact killing,and antifeedant effects on stored grain pests,but also inhibit their growth and reproduction.The previous research found that the main component of tea tree essential oil,terpinene-4-ol,has a significant fumigation effect.On this basis,this thesis explored the effect of terpinene-4-ol on the larval pupation and pupal eclosion of T.castaneum,screened out differential metabolites and their enrichment pathways based on metabolomics,and further analyzed the effects of larval pupation and pupal eclosion.The activity of key enzymes and the expression levels of related genes in the metabolic pathway of pupal eclosion,and the interaction sites and binding activities of terpinene-4-ol and related proteins were simulated by molecular docking technology.The research results provided a theoretical basis for the development of green control technology for T.castaneum.The results were as follows.1、Terpinene-4-ol treatment affects the larval pupation and pupal eclosion of T.castaneum.Using the larvae and pupae of T.castaneum as test insects,the effects of terpinene-4-ol on the larval pupation and pupal eclosion of T.castaneum were studied under the effect of time and dose.Under the same treatment time,with the increase of concentration,the pupation rate of larvae decreased,and the pupation rate decreased by more than 20% when fumigated for different days at a concentration of 14.4 μL/L;Under the condition of treatment time,the pupal eclosion rate decreased with the increase of concentration;in addition,the abnormal eclosion rate of pupae increased significantly,and the abnormal eclosion rate reached more than 80% when fumigated for different days at a concentration of 8 μL/L,even up to 100% in 2d,and the abnormally eclosion adults of T.castaneum died rapidly in a short period of time.The results showed that the treatment with terpinene-4-ol could reduce the pupation rate and eclosion rate of T.castaneum,increase the abnormal eclosion rate.2、Terpinene-4-ol treatment affects the contents and metabolic pathways of multiple metabolites in T.castaneumAnalyzing and processing the metabolome data of the larvae and pupae,it was found that terpinene-4-ol fumigation treatment of the larvae had significant effects on fatty acid degradation,biosynthesis of amino acids,ABC transporters,carbon metabolism,propanoate metabolism and lysine degradation had obvious effects,among them,the content of trehalase in the treatment group decreased by 40% compared with the control group of T.castaneum larvae.Biosynthesis of amino acids,alanine,aspartate and glutamate metabolism,beta-alanine metabolism,ABC transporters,glycine,serine and threonine metabolism,aminoacyl-t RNA biosynthesis in terpinene-4-ol fumigation treatment of T.castaneum pupae have obvious roles.Among them,the contents of Laspartic acid and β-alanine in the treatment group were 1.61 times and 1.84 times of those in the control group,respectively.L-Aspartic acid and β-alanine are involved in alanine,aspartate and glutamate metabolism pathways.3、Terpinene-4-ol treatment has a significant effect on the enzyme activities of Cht,Tre and HKT.castaneum were treated with triangular flask fumigation to determine the activities of chitinase(Cht),trehalase(Tre)and hexokinase(HK).The results showed that after fumigation at a concentration of 14.4 μL/L for different periods of time,the activity of Cht enzyme decreased by more than 28% compared with the control group,the activity of Tre enzyme decreased by more than 21% compared with the control group,and the activity of HK enzyme decreased by more than 34% compared with the control group.After fumigation with 6.0 μL/L concentration for different time to treat the pupae,the Cht activity decreased by 30% after 2 days,With the prolongation of time,the enzyme activity of Cht fluctuated somewhat,but the overall enzyme activity was significantly lower than that of the control group.The enzyme activity of HK decreased by more than 31% at 4 d and 5 d,which had obvious inhibitory effect,while the activity of Tre enzyme increased by40% overall.The above has a significant promotion effect.4、Terpinene-4-ol treatment affects the m RNA expression levels of chitin and melanin synthesis genesUsing RT-q PCR technology to detect the m RNA levels of related genes in chitin and melanin synthesis pathways.The results showed that the gene expression levels of CHS2,HK,Tre2 and Cht showed a trend of down-regulation after fumigation of terpinene-4-ol to treat the larvae,among which the expression level of Cht decreased significantly.The gene expression levels decreased by more than 80% at 3 d;the gene expression levels of CHS2 and HK also decreased by more than 80% at 4 d and 5 d;the expression level of Tre2 decreased by 50% at different time treatments.After fumigation with terpinene-4-ol to treat the pupae,the expression level of CHS2 gene decreased by more than 35% at 2 d and 4 d;and the expression level of the TRE5 treatment group increased by more than 2.8 times compared with the control group,with a significant difference.The expression levels of melanin synthesis genes showed an overall up-regulation trend,among which the Lac2,Nat and CPRA2B2 gene expressions after treatment increased by more than 50%,40% and30% compared with the control group,and the CPRA2B3,CPR7,CPR17 and CPR18.6genes compared with the control group,the expression levels increased by 1.16 times,2.4times,1.07 times and 1.4 times respectively;while the expression levels of ADC and Ebony were significantly increased by more than 11.1 times and more than 2.8 times at 2days,3 days and 5 days;indicating that terpinene-4-ol may induce the expression of genes related to melanin synthesis.5、Molecular docking simulation of binding sites and interaction modes of terpinene-4-ol with several proteins.Using Alpha Fold2 software to predict the protein structure of ADC,Cht,Ebony,Lac2 and Tre.The binding sites and binding modes of ADC,Cht,Ebony,Lac2,CPR and Tre to terpinene-4-ol were analyzed by computer.The results showed that the binding of ADC,Cht,Ebony,Lac2 and Tre to terpinene-4-ol mainly depended on hydrogen bonds and hydrophobic interactions.The analysis of the binding energy found that the binding energy of terpinene-4-ol to Tre was the lowest-52.8328 kcal/mol,The binding energies of terpinene-4-ol to ADC,Cht,Ebony,and Lac2 were all lower than the threshold-7.0kcal/mol,showing strong binding activity,while CPR lacked protein cavities and could not carry out molecular docking with terpinene-4-ol. |
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