Studys On The Mechanism Of The Contact Toxicity Of Carvone On The Tribolium Castaneum

Tribolium castaneum（Herbst）,a family of insects in the family Coleoptera,is a storage pest that has a huge impact on grain quality and safety and is widely distributed around the world.Although the strategy based on chemical synthesis has greatly controlled storage pests such as the T.castaneum,its use has been greatly limited by the disadvantages of pest resistance and environmental pollution.Plant-derived insecticides have become increasingly popular in recent years as environmentally friendly pesticides.Carvone is a terpenoid of plant secondary metabolites.In a previous study,we found that carvone has obvious contact and fumigant activities against the T.castaneum,with LD₅₀and LC₅₀reaching 5.08μg/adult and 4.34 mg/L air,respectively,but its toxic mechanism of action is still unclear.In this study,based on previous studies,we investigated the mechanism of carvone effect on topical application of T.castaneum by physiological changes,histopathology and key enzyme activities,and predicted the binding mode of carvone with potential target proteases by means of homology modeling and molecular simulation.The main findings were as follows:In terms of physiological changes,carvone showed neurotoxic symptoms from excitement to spasm and finally to coma and death for the T.castaneum,and the poisoned and dead test insects were obviously blackened;From the time of poisoning and death,the test insects died rapidly,and the survival rate decreased significantly from 8 h to 12 h after carvone treatment;In terms of physiological metabolism,both CO₂release and body weight loss of the T.castaneum reached significant levels under LC₅₀and LC₁₀₀treatments,with CO₂release showing an increasing trend followed by a decreasing trend,while body weight loss showed a continuous increasing trend.The above suggests that carvone may interfere with the normal physiological functions of the nervous system,respiratory system,and tissue structure of the T.castaneum.In terms of histopathology,the structure of the body wall of the T.castaneum was significantly changed after carvone topical application,mainly manifested by changes in the shape of the waxy particles on the surface of the test insects,loosening of the arrangement,widening of the gaps and inward depression of the hair bases with breakage.Paraffin sections of abdominal tissues showed that carvone had significant effects on the abdominal muscles and adipose tissues of the T.castaneum,in which the muscle tissues ruptured or even disintegrated,and the adipose tissues became blurred or even vacuolated.Damage to the body wall structure leads to massive water loss in the test worms,while muscle and adipose tissue can affect the normal physiological functions of the test insects,eventually leading to their poisoning and death.The results of the key enzyme activity assay showed that carvacrolone showed persistent and significant inhibition of T.castaneum acetylcholinesterase and succinate dehydrogenase,with the highest inhibition rate reaching 65.15%and 39.90%,respectively.Carvone showed activation followed by inhibition of T.castaneum Na⁺K⁺-ATPase,Ga²⁺Mg²⁺-ATPase and total ATPase.Carvone showed significant activation of glutathione-S-transferase,a detoxification metabolizing enzyme of the T.castaneum,and significant inhibition of carboxylesterase,indicating that glutathione-S-transferase plays a positive role in the detoxification metabolism of carvone.Overall,carvone had the strongest inhibitory effect on the activity of acetylcholinesterase of the T.castaneum,and the inhibition of this enzyme activity caused abnormal nerve conduction,which in turn led to the poisoning and death of the test insects.Molecular docking results showed that carvone interacted directly with the active site of T.castaneum acetylcholinesterase（PDB:6xyy）,in which carvone formed five hydrophobic interactions with four amino acids,6xyy glutamate（Glu 80）,tryptophan（Trp 83）,tyrosine（Tyr 370）,and leucine（Leu 479）,and two hydrogen bonding interactions with two amino acids,tryptophan（Trp 83 and Trp 472）.The ligand carvone interacts with the receptor acetylcholinesterase with a maximum binding energy of-6.89kcal/mol.In conclusion,the inhibition of acetylcholinesterase activity may be related to the direct interaction of carvone.The above results suggest that the inhibition of acetylcholinesterase activity may be related to the direct interaction of carvone.In summary,the present study initially revealed the mechanism of carvone topical application of T.castaneum from the effects of physiological changes,histopathology and key enzyme activities.In addition,the molecular docking technique was used to predict the binding pattern of carvone to potential targets,which provides a reference for the development and rational use of novel plant-derived insecticides.