Insecticidal Activity of Artemisia vulgaris Essential Oil and Transcriptome Analysis of Tribolium castaneum in Response to Oil Exposure

Abstract

Red flour beetle (Tribolium castaneum) is one of the most destructive pests of stored cereals worldwide. The essential oil (EO) of Artemisia vulgaris (mugwort) is known to be a strong toxicant that inhibits the growth, development, and reproduction of T. castaneum. However, the molecular mechanisms underlying the toxic effects of A. vulgaris EO on T. castaneum remain unclear. Here, two detoxifying enzymes, carboxylesterase (CarEs) and cytochrome oxidase P450 (CYPs), were dramatically increased in red flour beetle larvae when they were exposed to A. vulgaris EO. Further, 758 genes were differentially expressed between EO treated and control samples. Based on Gene Ontology (GO) analysis, numerous differentially expressed genes (DEGs) were enriched for terms related to the regulation of biological processes, response to stimulus, and antigen processing and presentation. Our results indicated that A. vulgaris EO disturbed the antioxidant activity in larvae and partially inhibited serine protease (SP), cathepsin (CAT), and lipase signaling pathways, thus disrupting larval development and reproduction as well as down-regulating the stress response. Moreover, these DEGs showed that A. vulgaris indirectly affected the development and reproduction of beetles by inducing the expression of genes encoding copper-zinc-superoxide dismutase (CuZnSOD), heme peroxidase (HPX), antioxidant enzymes, and transcription factors. Moreover, the majority of DEGs were mapped to the drug metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Notably, the following genes were detected: 6 odorant binding proteins (OBPs), 5 chemosensory proteins (CSPs), 14 CYPs, 3 esterases (ESTs), 5 glutathione S-transferases (GSTs), 6 UDP-glucuronosyltransferases (UGTs), and 2 multidrug resistance proteins (MRPs), of which 8 CYPs, 2 ESTs, 2 GSTs, and 3 UGTs were up-regulated dramatically after exposure to A. vulgaris EO. The residual DEGs were significantly down-regulated in EO exposed larvae, implying that partial compensation of metabolism detoxification existed in treated beetles. Furthermore, A. vulgaris EO induced overexpression of OBP/CYP, and RNAi against these genes significantly increased mortality of larvae exposed to EO, providing further evidence for the involvement of OBP/CYP in EO metabolic detoxification in T. castaneum. Our results provide an overview of the transcriptomic changes in T. castaneum in response to A. vulgaris EO.

Keywords: Artemisia vulgaris; Tribolium castaneum; development; insecticidal activity; metabolism system; reproduction.

FIGURE 1

(A) The effect of Artemisia vulgaris essential oil (EO) on Tribolium castaneum larvae. Larvae were treated with eight different concentrations (0.1, 1, 1.25, 1.7, 2.5, 5, 10, and 100%) of A. vulgaris EO or with acetone (control). Larval mortality was observed at 24, 48, and 72 h post-treatment. (B) Contact toxicity of A. vulgaris EO against T. castaneum larvae. At different times with 5% A. vulgaris EO on carboxylesterase (CarEs) (C), cytochrome P450 (CYPs) (D), and glutathione S-transferase (GSTs) (E) in larval T. castaneum in vivo. Fold change of the total enzyme activity was calculated by dividing the enzyme activity of each treatment by that of the acetone only control, which had been ascribed an arbitrary value of one. The times of 12, 24, 36, 48, 60, and 72 h were six different test points after insecticide treatment. Data represent mean ± SE of three independent experiments. Asterisks indicate significant differences between the control and EO treatment groups (^∗P < 0.05, ^∗∗∗P < 0.001; Student’s t-test).

FIGURE 2

(A) Gene Ontology (GO) enriched terms of differentially expressed genes (DEGs) of T. castaneum after exposure of its larvae to 5% A. vulgaris essential oil (EO) versus the control group. The x-axis is the number of DEGs involved in each term. The y-axis lists the sub-GO terms under categories of biological processes, cellular components, and molecular functions. (B) The most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of T. castaneum after exposure of its larvae to 5% A. vulgaris EO. Pathway significance is shown together with Q-value (color), rich factor (vertical ordinate), and a number of involved genes (size of circles).

FIGURE 3

(A) Heat map of 758 genes differentially expressed in larvae exposed to 5% A. vulgaris essential oil (EO) and control. Rows represent a single gene and columns are comparisons between genes in the exposure treatment and the control. The left-hand column clusters samples (N = 6) based on similarity of log10 transformed gene expression. Lighter colors indicate lower levels of differential expression. (B) Changes in the gene expression profile between the control and 5% A. vulgaris EO exposure groups. (C) A comparison of the expression profiles of the selected genes as determined by RNA-sequencing and qRT-PCR. OBP, Odorant binding protein; CYP, Cytochrome P450; Ugt, UDP-glucuronosyltransferase; EST, Esterase; GST, Glutathione S-transferase; MRP, Multidrug resistance protein; BG, Beta-glucuronidase; XDH, Xanthine dehydrogenase; FMO, Flavin-containing monooxygenase. Effects of 5% A. vulgaris EO exposure on the accumulation of TcCYP4BN6 (D) and TcOBPC11 (E) in 20-day-old larvae of T. castaneum. Acetone treatment represents the negative control group, which has been ascribed an arbitrary value of 1. The 12, 24, 36, 48, 60, and 72 h are six different test time points following EO treatment. (F) RNAi-mediated gene silencing. Twenty-day-old larvae were used for dsRNA injection. RNA was extracted and quantified by qRT-PCR at fifth day. Control beetles were injected with the same amount of green fluorescent protein (GFP) dsRNA. (G) Effect of TcCYP4BN6/TcOBPC11 silencing by injection of ds-TcCYP4BN6/ds-TcOBPC11 on toxicity of EO to 20-day-old larvae. Larvae injected with ds-TcCYP4BN6/ds-TcOBPC11/ds-GFP only served as untreated controls. Following injection with ds-TcCYP4BN6/ds-TcOBPC11/ds-GFP, larvae were treated with EO for 72 h. Data shown are mean ± SE (n = 3). An asterisk above bars indicated significant differences in the mRNA expression among the control and treatments (**P < 0.01, ***P < 0.001; Student’s t-test). Different letters above bars indicate significant differences (P < 0.05) according to LSD multiple comparison tests.

FIGURE 4

Schematic of the mechanism of insect response to stimulation by A. vulgaris essential oil. OBPs, Odorant-binding proteins; CSPs, chemosensory proteins; CYPs, cytochrome P450 monooxygenases; CarE, carboxylesterase; Ugt, UDP-glucuronosyltransferase; GST, Glutathione S-transferase; MRP, multidrug resistance protein; (HPX), heme peroxidase; HSP, heat shock protein; CuZnSOD, copper-and zinc-containing superoxide dismutase.