Eucalyptol, the main active component of Artemisia vulgaris (A. vulgaris) essential oil, can reduce the activity of Tribolium castaneum (T. castaneum) metabolic detoxification enzymes. However, the molecular mechanism underlying the toxic effect of eucalyptol on T. castaneum is still unclear. Here, results showed that the eucalyptol caused time‐dependent increase in larval mortality. Further, 1669 differential expressed genes (DEGs) expressed between the control group and the experimental group were detected via RNA sequencing, of which 504 were upregulated and 1165 were downregulated. The validity of the RNA‐Seq results was verified by qRT‐PCR. Further, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to analyze the DEGs, focusing on immune system process, response to stimulus, and developmental process in the GO analysis and on the excretory system and the metabolism of terpenoids and polyketides in the KEGG analysis. The results showed that the upregulation of Apolipoprotein D and the downregulation of V‐type proton ATPase subunits, farnesol dehydrogenase, receptor tyrosine‐protein phosphatase ε and α may affect the growth and metabolism of T. castaneum larvae. In the KEGG pathway analysis, we focused on the role of certain metabolic detoxification enzymes in the regulation of eucalyptol‐stimulated T. castaneum. Among them, the expression of metabolic detoxification enzymes OBP10, CYP4C1, CYP6BS1, CYP4g15, CYP301A1, CYP49A1, UGTs2C1, UGTs2B2, UGTs2B16, and Est FE4 was downregulated. The expression of OBPC01, OBPC09, OBPC12, OBPC02, OBPC11, CYP345B1, CYP346B1, Est E4, Est 6, and GST1 increased significantly. These metabolic detoxification enzymes were involved in the detoxification of eucalyptol by T. castaneum, and their downregulation or upregulation may indirectly affect insect survival and death. Our results provide an overview of transcriptomic changes in eucalyptol‐stimulated T. castaneum and enriches the candidate transcripts that can be exploited for pest control initiatives.

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Choline acetyltransferase (Tcchat) is essential for metamorphosis, reproduction, and susceptibility to insecticide in Tribolium castaneum. However, the mechanisms through which Tcchat contributes to these biological processes remain poorly understood. Here we report the findings of RNA sequencing‐based transcriptome profiling of T. castaneum following RNA interference against Tcchat. In all, 173 differentially expressed genes were identified: 120 upregulated genes and 53 downregulated genes. Gene Ontology analysis classified the genes into 30 functional groups. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the genes were distributed over 22 different pathways. Silencing Tcchat reduced the activity of endopeptidase and inhibited regulation of protein decomposition in the beetles. Hence, knockdown of Tcchat inhibited the synthesis of chitinase and serine protease, which disturbed embryonic development in T. castaneum, thus affecting its development and reproduction. RNA interference of Tcchat also suppressed the synthesis of cytochrome P450 in the extracellular region and adversely affected normal functioning of the lysosomal pathway, which might have hindered the insects' ability to recognize, phagocytose, and degrade exogenous toxic substances, resulting in increased mortality. Furthermore, knockdown of Tcchat increased the susceptibility of larvae to carbamate and organophosphate insecticides, which supports the notion that Tcchat is indeed involved in susceptibility to insecticide in T. castaneum. Finally, Tcchat knockdown adults showed progressively reduced locomotor activity. These results provide novel insights into biological processes affected by Tcchat and provide a potential theoretical basis for the biological control of T. castaneum.

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