Toxicological Mechanism Of Carvacrol On Lymantria Dispar Larvae

With the long-term and large-scale use of chemical pesticides,the 3R problems(resistance,rampant regeneration and residue)have attracted widespread attention.For this reason,it is inevitable to develop new pesticides with high efficiency,low residue and safety to non-target organisms.In the process of growth and development,a variety of secondary metabolites are produced in plants to resist harmful organisms.These products have the advantages of high efficiency,easy degradation,and complex mechanism of action against non-target organisms.They are used as substitutes for chemical pesticides.As one of the secondary metabolites of plants,the monoterpenoid carvacrol has biological activities such as antibacterial,anti-tumor and anti-inflammatory,but its insecticidal activity and mechanism to the forest pest Lymantria dispar is still unclear.In this study,Lymantria dispar is used as the research object.The feed mixture method is used to determine the effect of carvacrol on the toxicity,food utilization,detoxification enzyme and acetylcholinesterase(AchE)activity of the Lymantria dispar larvae;the transcriptomics sequencing analysis is used to explore the effect of carvacrol on the mRNA transcription level of Lymantria dispar larvae;on the basis of screening the differential expression genes(DEGs)related to the redox system,the redox system indicators,antioxidant enzyme activity and mRNA expression are determined to analyze the effect of carvacrol on the redox system of the Lymantria dispar larvae;according the determination to the contents of soluble protein,carbohydrate and lipid,as well as metabonomic sequencing analysis,the relationship of carvacrol on the nutritional metabolism of the Lymantria dispar larvae is explained;by the determination of carvacrol on the effect of Lymantria dispar larvae digestive enzyme activityand their mRNA relative expression and gut microbial community,the effect of carvacrol on the digestive function of Lymantria dispar larvae was clarified.This study systematically explores the toxicological mechanism of carvacrol on Lymantria dispar larvae by measuring the food utilization,detoxification mechanism,mRNA transcription level,redox system,nutritional metabolism and digestive function of carvacrol on Lymantria dispar larvae,which can provide theoretical basis and new ways for the development and utilization of carvacrol and the control of Lymantria dispar larvae in the future.The main results of this study are as follows:(1)Carvacrol has insecticidal activity on Lymantria dispar larvae,whose sublethal concentration(LC20)and median lethal concentration(LC50)are 0.297 mg/mL and 1.120 mg/mL at 72 h,respectively.Carvacrol has antifeeding and growth inhibitory effects on Lymantria dispar larvae.After eating carvacrol-containing artificial feed,the weight gain,relative food intake and defecation of Lymantria dispar larvae all decreased significantly(P<0.05).Under carvacrol stress,the relative growth rate,food utilization rate and food conversion rate of Lymantria dispar larvae decreased significantly.Lymantria dispar larvae can reduce the adverse effects of carvacrol by regulating their own food digestibility;carvacrol can significantly inhibit Carboxylesterase(CarE)and Glutathione S-transferase(GsT)in Lymantria dispar larvae,has an activating effect on Acid phosphatase(ACP)activity,but no significant effect on Alkaline phosphatase(ALP)activity,and has an inhibitory effect on AchE activity.(2)At the mRNA level,carvacrol can cause significant changes in the expression levels of 296 genes(142 up-regulated and 154 down-regulated)in Lymantria dispar larvae.Most of the DEGs are annotated in heterologous substances,carbohydrates,lipid metabolism,redox process and oxidoreductase activity;qRT-PCR results is consistent with that of transcriptome,which verifies the accuracy of transcriptome analysis;a variety of genes related to the metabolism of heterologous toxic substances were identified from the differential genes,including 6 CYP450s,2 ESTs,1 UTGs,and 2 HSPs.(3)Through the comparative analysis of DEGs,a total of 22 DEGs related to the redox system of the Lymantria dispar larvae are found;the content of hydrogen peroxide and MDA in the Lymantria dispar larvae increase significantly under carvacrol stress(P<0.05)At the same time,the total antioxidant capacity is significantly reduced(P<0.05);carvacrol can significantly activate the four antioxidant enzyme activities of Superoxide dismutase(SOD),Catalase(CAT),Peroxidase(POD)and Polyphenol oxidase(PPO)in Lymantria dispar larvae,and significantly up-regulated the gene expression level of SOD enzymes at 12 h and 24 h(P<0.01),and the gene expression level of CAT enzyme is significantly up-regulated at 24 h(P<0.01),indicate that carvacrol can damage the redox system of Lymantria dispar larvae.(4)Through the comparison and analysis of DEGs,a total of 27 DEGs related to the nutritional metabolism of Lymantria dispar larvae are found;carvacrol can significantly decrease the soluble protein and carbohydrate content in Lymantria dispar larvae while increasing its lipid content;The results of the metabolomics analysis show that 161 differential metabolites in the Lymantria dispar larvae are found under carvacrol stress,of which 89 metabolites are significantly up-regulated and 72 metabolites are significantly down-regulated;the differential metabolites are mainly concentrated in lipids and lipid molecules,organic acids and their derivatives,organic oxygen compounds and organic heterocyclic compounds;there are 15 different metabolites from the metabolic process,and up to 7 different metabolites in the lipid metabolism process;in addition,the differential metabolites are also enriched in the necrotic process,sphingolipid signaling,sphingolipid metabolism and protein digestion and absorption,it is shown that carvacrol can affect the nutritional metabolism of Lymantria dispar larvae.(5)Under carvacrol stress,the two digestive enzyme activities of Lipase(LIP)and Amylase(AMS)in the Lymantria dispar larvae show a trend of inhibition,and the activity of Protease show a trend of first inhibition and then activation;LIP gene expression is significantly down-regulated at 12 h and 72 h(P<0.05),the expression of AMS and Protease genes is significantly down-regulated at 12 h(P<0.05),and the expression of AMS and Protease genes are significantly up-regulated at 24 h;the structure,abundance,and function of Lymantria dispar larvae gut microbial communitys has changed.There are 4 different OTUs in gut microbial communitys.The dominant phyla of Lymantria dispar larvae intestinal bacteria changes from Proteobacteria to Firmicutes.The dominant genus changed from Lauerella to Lactobacillus.Lymantria dispar larvae gut microbial communitys G(carbohydrate transport and metabolism)and V(defense mechanism)two functional abundances are up-regulated.carvacrol can inhibit the abundance of H(coenzyme transport and metabolism)and Q(biosynthesis,transport,and catabolism of secondary metabolites),thereby reducing the detoxification ability of Lymantria dispar larvae to carvacrol.The decline of the functional abundance of C(energy production and conversion)and I(lipid transport and metabolism)of two kinds of bacteria further shows that carvacrol can affect the nutritional utilization of Lymantria dispar.In summary,carvacrol can inhibit the detoxification and digestion functions of the Lymantria dispar larvae,damage the redox system in the Lymantria dispar larvae,and change the nutritional metabolism and food utilization capacity of the Lymantria dispar larvae,thereby produce a toxic effect on the Lymantria dispar larvae.