T. castaneum were fed in an artificial climate box with a temperature of 30°C and humidity of 40%. The food was made of flour and yeast (19:1) under standard conditions (Xiong et al., 2019).

A. vulgaris was collected in December 2017 at Mowan Village, Tangyin City, Henan Province, China (l35.922862°N, 114.480455°E). The collection site has a warm temperate continental monsoon climate, with adequate light, an average annual temperature of 14°C, average annual rainfall of 580 mm, and is located at an altitude of approximately 63 m above sea level. Fresh leaves and stems of A. vulgaris were washed with ddH₂O then placed inside a dark box at 37°C to dry for 1 week. Dried leaves were shattered using a Swing Medicinal material grinder (Baijie Industrial Co., Ltd., Shanghai, China). The powder was filtered using 30 mesh sieves. Filtered powders (200 g) were loaded into a 5-L extraction tank. When the vacuum pressure in the tank reached 100 kPa, a subcritical solvent (dimethylmethane, butane, dimethyl ether, and tetrafluoroethane mix) was injected into the tank. Extraction began at 35°C, with a liquid-solid ratio of 15: 1. Each extraction was 30 min, with three repeats during the whole extraction process. At the end of the process, the liquid solvent reached the separation tank and, after the solvent was removed by evaporation, the EO-AV was collected. The extract was diluted with acetone to obtain six concentrations: 0, 2.5, 5, 10, 15, and 20%.

The contact killing effect of EO-AV against late larvae (20 days old) of T. castaneum was measured according to the drip method of Lu et al. (2012). Briefly, 30 synchronous individuals in each group were loaded into 1.5-mL EP tubes and exposed to 100 μL EO-AV or acetone. After soaking for 1 min, the treated larvae were placed on filter paper and allowed to air dry for 2 min. Each group was then transferred to an 8-mL glass vial and maintained under standard conditions described in section “Insect Rearing.” Survival of individual larvae in the different treatment groups was recorded from 12 to 72 h after EO-AV exposure. Beetles were considered dead if they were unable to move and failed to respond when disturbed with a tweezer or brush. Each bioassay was replicated five times.

EO-AV had a strong contact killing effect on late larvae of T. castaneum. Therefore, 36 h after the treatment with 5% essential oil (TG) or 0% essential oil (CK), the late larvae were collected to extract RNA for RNA-Seq (Gao et al., 2020). Expression of the gene OBPC11 was significantly upregulated in the TG vs CK samples; consequent experiments in this study focused on the role and function of this gene. Primers were designed against OBPC11 from T. castaneum (TcOBPC11) to obtain the full-length cDNA sequence of the open reading frame (ORF) (Table 1). Total RNA was isolated from the late larvae using Trizol reagent (Vazyme Biotech, Nanjing, China), and 750 ng total RNA was used to synthesize cDNA templates using HiScript Reverse Transcriptase (Vazyme Biotech). These cDNA templates were used to clone the OBP gene by PCR in a 50 μL reaction comprising 2 μL cDNA, 25 μL 2 × Primer STAR Mix, 1 μL forward and reverse primers (10 μM), and 21 μL sterile water. The PCR amplification procedure was 95°C for 3 min, then 35 cycles of 95°C for 20 s, 55°C for 15 s, and 72°C for 30 s, followed by a final extension at 72°C for 10 min. The PCR product was detected using 1.2% agarose gel and the expected product was purified by FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech), cloned into Blunt-Zero Vector via Blunt-Zero Cloning Kit (Vazyme Biotech), and then transformed into competent cells of Escherichia coli. Positive clones were identified by blue-white screening and the resulting vector was extracted and sequenced (Springen). In addition, 30 late larvae were collected to extract RNA for qPCR after treatment with 5 or 0% essential oil for 12, 24, 36, 48, 60, and 72 h.

Pooled samples of male and female T. castaneum in different developmental stages, including early eggs (1 day old), late eggs (3 days old), early larvae (1 day old), late larvae (20 days old), early pupae (1 day old), late pupae (5 days old), early adults (1 day old), and late adults (10 days old), were collected, snap-frozen in liquid nitrogen, and immediately stored at −80°C to extract RNA for qPCR. Pooled samples of different tissues from the late larvae (whole larva, central nervous system, epidermis, fat body, Malpighian tubule, gut, and hemolymph) and from early adults (whole adult, central nervous system, epidermis, fat body, Malpighian tubule, gut, antennae, testis, and ovary) were dissected and collected in RNA-free centrifuge tubes to extract RNA for qPCR. Three biological replicates for each developmental stage and tissues were conducted.

Firstly, 750 ng total RNA from each developmental stage and the different tissues were treated with 4 × gDNA wiper Mix to avoid the effects of genomic DNA on qPCR. Total RNA was then used as a template to synthesize single-stranded cDNAs using HiScript Q RT SuperMix for qPCR with Random primers and Oligo dT primer mix (Vazyme Biotech) in 25 μL reactions. The specific primers designed to quantify TcOBPC11 expression were OBP-2F and OBP-2R. The gene ribosomal protein S3 (rps3), which has a high degree of stability, was selected as the reference gene (Toutges et al., 2010; Horn and Panfilio, 2016; Xiong et al., 2019) and the primers were rps3-F and rps3-R. Primers were designed using Primer Premier 5.0 and sequences are shown in Table 1. Primers of the target gene and the reference gene have similar amplification efficiencies. A 10-μL reaction system containing 0.25 μL forward and reverse primers (10 μM), 5 μL 2 × AceQ Universal SYBR qPCR Master Mix, 3.5 μL ddH₂O water, and 1 μL cDNA was executed in an ABI Q6 (Applied Biosystems, Foster City, CA, United States) with the parameters of 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s, followed by 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. A melting curve of the amplification products was generated at the end of each reaction to confirm that only one PCR product was amplified. For each treatment, three technical repeats and three to seven biological replicates were executed.

To synthesize the dsRNAs, primers for TcOBPC11 and GFP were designed using Primer Premier 5.0, adding a T7 polymerase recognition promoter (Table 1). The target of the dsRNAs is located from 214 to 382 bp in the coding region of TcOBPC11. The PCR comprised 0.4 μL forward and reverse primers (10 μM), 10 μL 2 × Primer STAR Mix, 7.2 μL ddH₂O water, and 2 μL plasmid DNA with ORF cDNA of OBPC11. PCR procedures were carried out at 95°C for 5 min followed by 30 cycles of 95°C for 45 s, 60°C for 45 s, and 72°C for 15 s, with a final extension at 72°C for 10 min. PCR products were purified and served as templates to synthesize the dsRNAs using a TranscriptAi T7 High Yield Transcription Kit (Fermentas, Vilnius, Lithuania). The resulting dsRNA (200 ng) in a volume 150 nL solution was injected into the body cavity of each late larva of T. castaneum by InjectMan 4 (Eppendorf, Hamburg, Germany). Injection of equal volumes of 200 ng dsGFP and water served as the negative and blank controls, respectively. Three biological repetitions with independent injections were performed and each repetition injected 30 larvae. Late larvae injected with dsRNA were normally reared. Survival of the larvae was recorded after the fifth day. Ten random larvae were collected on the first, third, and fifth days to extract total RNA for qPCR to detect the silencing efficacy of the targeted gene. To verify the effect of non-target genes, expression of four non-target genes (TcOBPC01, TcOBPC02, TcOBPC04, and TcOBPC10) in T. castaneum, which share a high degree of similarity with TcOBPC11, were also examined; primer details for these genes are listed in Supplementary Table S1. Late larvae on the fifth day after water, dsGFP, dsOBPC11 injections, and non-injections were exposed to 5% EO-AV for 1 min. Insects were then transferred to the standard conditions, reared for 36 h, and their mortality was measured. Each treatment was repeated three to seven times.

Gene expression was calculated according to the 2^–△△Ct method (Livak and Schmittgen, 2001). Significant differences in gene expression levels between larvae of T. castaneum treated with 0.5% essential oil and 0% essential oil were compared using a Student’s t-test at the 0.05 level. Expression levels of TcOBPC11 among different developmental stages and RNAi treatments were analyzed by Fisher’s LSD test. After logarithmic transformation of levels of expression of TcOBPC11 in different tissues, significant differences were analyzed by Fisher’s LSD test. Statistical significance of the mortality or survival rate were analyzed by Kruskal-Wallis H test, except in the RNAi experiment, which was analyzed by Fisher’s LSD test. Interaction effects of the concentration of EO-AV and time after exposure to EO-AV on the mortality rate of T. castaneum larvae were investigated with a non-parametric two-way ANOVA (Scheirer–Ray–Hare test) to assess significant differences at the 0.05 level. All statistical analyses were performed with SPSS software, version 14.0.

There were significant differences in the mortality of larvae of T. castaneum exposed to different concentrations of EO-AV when reared for the same length of time (Figure 1 and Table 2). Larva mortality increased significantly as the concentration of EO-AV increased from 0 to 20% (12 h: x² = 27.217, df = 5, P < 0.001; 24 h: x² = 27.293, df = 5, P < 0.001; 36 h: x² = 27.872, df = 5, P < 0.001; 48 h: x² = 27.590, df = 5, P < 0.001; 60 h: x² = 27.620, df = 5, P < 0.001; 72 h: x² = 27.620, df = 5, P < 0.001). There were significant differences in the mortality of larvae at different time points after exposure to the same concentration of EO-AV (Figure 1 and Table 2). Mortality of T. castaneum larvae significantly increased following the extension of rearing time after exposure to EO-AV at concentrations of 10, 15, and 20% (10%: x² = 24.366, df = 5, P < 0.001; 15%: x² = 20.875, df = 5, P < 0.001; 20%: x² = 16.071, df = 5, P = 0.007), but there was no significant effect at concentrations of 0, 2.5, and 5% (0%: x² = 2.231, df = 5, P = 0.816; 2.5%: x² = 3.295, df = 5, P = 0.655; 5%: x² = 6.364, df = 5, P = 0.272). In a non-parametric two-way ANOVA, mortality rates of the red flour beetle were affected by concentration and rearing time after exposure to EO-AV, but the interaction was not significant (Table 2).

Transcriptome analysis revealed that 39 chemosensory-related genes had significant differences in expression between the TG group and the CK group (Gao et al., 2020). These genes included six OBPs, five CSPs, six odorant receptors, and three gene families encoding odorant-degrading enzymes: 14 cytochrome P450s, three esterases, and five glutathione S-transferases (Supplementary Table S2). Among these genes, expression of TcOBPC11 was highest in the TG group compared with the CK group (Gao et al., 2020). To appraise the underlying function of TcOBPC11 in the tolerance of T. castaneum to EO-AV, cDNA of TcOBPC11 with an ORF of 402 bp, encoding a protein of 134 amino acids, was cloned (GenBank accession no. XM_962706). A phylogenetic tree of the TcOBPC11 protein with homologous proteins of other insects was reconstructed (Supplementary Figure S1). The similarity of TcOBPC11 with homologous genes from other insects was very low. TcOBPC11 was most related to an OBP in Asbolus verrucosus and Tenebrio molitor (Supplementary Figure S1). The amino acid identities of TcOBPC11 with OBP of A. verrucosus and T. molitor were 40.04 and 35.50%, respectively.

Expression of the gene TcOBPC11 was significantly higher in TG than CK samples using qPCR methods (36 h: t = 4.917, df = 3, P = 0.016) (Figure 2); this was consistent with the transcriptome analysis results of Gao et al. (2020). TcOBPC11 had significantly higher expression at each time point after exposure to 5% EO-AV compared with 0% EO-AV (Figure 2). There were significant differences in expression among 12, 24, 36, 48, 60, and 72 h at a concentration of 5% EO-AV (F_{5, 23} = 29.300, P < 0.001). Meanwhile, after exposure to 5% EO-AV, expression of TcOBPC11 rapidly increased from 24 h, peaked at 36 h, and then decreased gradually (Figure 2). This indicated that EO-AV induced expression of the gene TcOBPC11.

qRT-PCR revealed that TcOBPC11 was expressed throughout all stages of development of T. castaneum and there were significant differences in expression of TcOBPC11 among developmental stages (F_{7, 24} = 1,660.983, P < 0.001) (Figure 3). TcOBPc11 was highly expressed at the late larvae and adult stage, and expression at these stages was significantly greater than at other developmental stages (Figure 3).

Expression of TcOBPC11 was further surveyed in various tissues from late larvae and early adults. Relative transcript levels of TcOBPC11 in different tissues from the larvae of T. castaneum showed that TcOBPC11 was mainly expressed in the fat body, Malpighian tubule, and hemolymph, and at much lower levels in other tissues (F_{6, 14} = 318.323, P < 0.001) (Figure 4A). Expression of TcOBPC11 in larvae was highest in the fat body, closely followed by the Malpighian tubule and hemolymph, and then other apparatus (Figure 4A). Relative transcript levels of TcOBPC11 in different tissues from adult T. castaneum showed that TcOBPC11 was mainly expressed in the epidermis, fat body, and antennae, and at much lower levels in other tissues (F_{6, 14} = 318.323, P < 0.001) (Figure 4B). Expression of TcOBPC11 in adults was highest in the epidermis, closely followed by the fat body and antennae, and then other apparatus (Figure 4B). TcOBPC11 was highly expressed in olfactory and non-olfactory tissues, suggesting that TcOBPC11 has multiple physiological functions in T. castaneum.

To further determine the effects of TcOBPC11 on insect resistance to EO-AV, RNAi was used to silence the gene TcOBPC11. RNAi targeting of TcOBPC11 in late larvae distinctly lowered its expression level but did not change the transcript level of non-target genes (Supplementary Figure S2). This indicates that RNAi of TcOBPC11 was absent of off-target effects. Relative expression levels of TcOBPC11 mRNA significantly decreased after injection of dsOBPC11 (day 1: F_{2, 6} = 36.116, P < 0.001; day 3: F_{2, 6} = 46.395, P < 0.001; day 5: F_{2, 6} = 88.310, P < 0.001) (Figure 5). At day 5 after injection of water, dsOBPC11, or dsGFP, survival rates of T. castaneum larvae were 96.33 ± 3.67%, 98.38 ± 0.83%, and 93.92 ± 3.25%, respectively, and there were no significant differences between the treatments (F_{2, 6} = 0.605, P = 0.576).

Dip bioassays of EO-AV were performed on the fifth day after late larvae were injected or not with dsRNA. The cumulative mortality rate of T. castaneum larvae increased in the non-injection, water, dsGFP, and dsOBPC11 groups because of the exposure to essential oil. However, the mortality rate of the larvae injected with dsOBPC11 significantly increased compared with the water and dsGFP groups (F_{3, 12} = 6.707, P = 0.007) (Figure 6). This supported the conclusion that the enhanced mortality rate of T. castaneum larvae detected after exposure to essential oil was primarily because TcOBPC11 genes were silenced. Obvious correlation effects between the reduction in TcOBPC11 transcript level and the enhanced mortality rate after exposure to essential oil suggest that TcOBPC11 plays a significant role in the defense of T. castaneum larvae against EO-AV.