Identiﬁcation and Functional Characterization of the Transcription Factors AhR/ARNT in Dendroctonus armandi

: The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) belong to the bHLH-PAS (basic Helix–Loop–Helix–Period/ARNT/Single-minded) family of transcription factors, which participate in the sensing and transmitting stimuli of exogenous and endogenous chemical substances, and subsequently activates genes transcription involved in various detoxiﬁcation and physiological functions. However, they have not been identiﬁed in Dendroctonus armandi, and their roles in the detoxiﬁcation metabolism are unclear. In the present study, AhR and ARNT of D. armandi were characterized. Spatiotemporal expression proﬁling indicated that DaAhR and DaARNT were highly expressed in the adult and larval stages of D. armandi and mainly expressed in the midgut and Malpighian tubules of adults. Additionally, the expression of DaAhR and DaARNT signiﬁcantly increased after exposure to ( − )- β -pinene, (+)-3-carene, and ( ± )-limonene. Silencing DaAhR and DaARNT increased the susceptibility of D. armandi to ( − )- β -pinene, (+)-3-carene, and ( ± )-limonene, and the activities of detoxiﬁcation enzyme were also remarkably reduced. Moreover, DaCYP6DF1 and DaGSTs2 were signiﬁcantly down-regulated after injections of dsAhR and dsARNT in the male and female adults, with the expression of DaCYP6DF1 decreasing by higher than 70%. The present study revealed that the transcription factors AhR and ARNT of D. armandi were induced by terpenoids and participated in the regulation of DaCYP6DF 1 expression, which was associated with D. armandi ’s susceptibility to ( − )- β -pinene and ( ± )-limonene. These results may provide a theoretical basis for the integrated control of D. armandi and improve our comprehension of insect toxicology.


Introduction
The Chinese white pine beetle, Dendroctonus armandi Tsai and Li (Coleoptera: Curculionidae: Scolytinae), is an aggressive and destructive pest in coniferous forests in the middle Qinling Mountains of China. It invades not only healthy Pinus armandii but also attracts other pests to the host trees, which has resulted in the destruction of the forest ecological system and caused serious economic losses [1]. An important period of the bark beetle's life process is the host colonization stage, during which they must resist the host's defenses to reproduce successfully [2]. The resistance of P. armandii to bark beetles mainly depends on the composition and induced physical and chemical defense, and the induced oleoresin terpenes are the main defense components [3]. Oleoresin is a complex composed of dozens of monoterpenes, diterpenes, and a few sesquiterpenes [4]. According to a previous study, α-pinene, β-pinene, limonene, myrcene, and camphene are the main compounds in the volatile oleoresin terpenes, which were derived from the resin of P. armandi [3]. Moreover, they can harm or kill beetles due to their toxic effects [5,6].
In insects, the detoxification of toxic chemicals mainly contains the enhancement of cytochrome P450 monooxygenase, glutathione S-transferase, and esterase activity levels [7].

Developmental-and Tissue-Dependent Expression Profiles of DaAhR and DaARNT
D. armandi larvae were separated into the following stages: early larvae, late larvae, early pupae, late pupae, teneral adults, emerged adults, and feeding adults. The antennae, brain, hindgut, midgut, foregut, fat body, pheromone gland, hemolymph, and Malpighian tubules of emerged adults were collected by dissection and then stored at −80 • C. A total of three independent biological replicates were prepared for gene expression analysis.

Terpenoids Exposure
Fumigation treatment was performed as previously described [29]. The male and female of emerged adults were divided into six groups and treated with LC 50 concentrations of (−)-α-pinene, (−)-β-pinene, (+)-3-carene, and (±)-limonene for 2 h in 20 mL glass vial [33]. The group of DMSO exposure was used as a control. Each treatment contained 40 adults of essentially the same size. After the adults regained their vitality, they were transferred to an artificial climate cabinet. To explore the effect of terpenoids on the expression of DaAhR and DaANRT, the surviving adults were collected at 24 h postexposure to LC 50 of each terpenoid. Meanwhile, the DMSO-treated surviving adults were collected as controls at the same time point.

Double-Strand RNA (dsRNA) Synthesis and Injection
The synthesis of dsRNA was performed as previously described [26]. Briefly, the T7 Ribo-MAXTM Express RNAi System (Promega, Madison, MI, USA) was used for the synthesis of dsGFP (395 bp), dsAhR (412 bp), dsARNT (387 bp), and dsCYP6DF1 (455 bp). RNAi primers (Table S1) were designed based on the sequences obtained. Injection with dsGFP was used as a control. To prevent off-target effects, we chose specific target fragments to avoid any overlap with other genes, and the sequence specificity of target fragments was tested via NCBI BLAST. The final dsRNA products were diluted to 1000 ng/µL with diethylpyrocarbonate (DEPC)-treated water, then stored at -80 • C and used within 6 months. Before injection, D. armandi were placed in an ice bath for 10 min. The beetles were immobilized on an agarose plate using manual forceps. Afterward, each of the D. armandi emerged adults were microinjected with 0.2 µL dsRNA solution. Each treatment group contained 40 individuals, and 6 individuals from each treatment group were collected 24, 48, and 72 h after injection and then stored at -80 • C until qRT-PCR. Each treatment group contained three biological replicates. In addition, after injection at 48 h, beetles were also used for enzyme determination, and the P450, GST, and CarE activity levels were measured as previously described [7]. At the 48 h after dsRNA injection, the adults were treated with (−)-α-pinene, (−)-β-pinene, (+)-3-carene, and (±)-limonene as described previously, then they were reared in normal conditions for 48 h and the mortality rates were determined. We selected 12 genes from the three classes of D. armandi detoxifying enzymes: 6 CYP genes from the CYP3 clade, 4 GST genes from epsilon and sigma superfamilies, and 2 carboxylesterases, which are involved in the xenobiotic compounds' detoxification in D. armandi [34,35]. These detoxification gene expression levels (P450, CarE, and GST) after dsRNA injection at 48 h were measured by qRT-PCR. The primers are listed in Table S1. Each treatment group contained three biological replicates.

Statistical Analysis
SPSS Statistics 19.0 (IBM, Chicago, IL, USA) was performed in all the statistical data analyses. Post-hoc Tukey tests were used to check the difference through one-way ANOVA. The two-sample analyses were performed with Student's t-test. In addition, Prism 6.0 (GraphPad Software, San Diego, CA, USA) was used to plot graphs.

Sequencing and Bioinformatic Analysis
The full-length D. armandi AhR and ARNT cDNAs were cloned and characterized. The lengths of the coding regions of AhR and ARNT are 2412 bp and 2106 bp, which encode 803 and 701 amino acids, respectively. Moreover, the molecular weights (MW) of proteins of AhR and ARNT are 90.63 and 77.29 kDa, and the isoelectric points (PI) are 7.29 and 6.16, respectively (Table 1). A phylogenetic analysis showed that DaAhR ( Figure 1A) and DaARNT ( Figure 1B) have a high homology with their counterpart in Dendroctonus ponderosa, and they were also clustered with the Coleoptera group. An amino acid sequence alignment indicated that the bHLH and PAS domains of DaAhR and DaARNT are relatively conserved among different species ( Figure S1).

Spatiotemporal Expression Pattern of DaAhR and DaARNT
AhR and ARNT were expressed in all developmental stages of D. armandi. They were expressed the highest in the adult stage, followed by the larval stage, and the lowest in pupae ( Figure 2). Moreover, as for the adult stage, the expression level of AhR in females was significantly higher than that in males in the feeding adults ( Figure 2A). While ARNT showed the opposite result in the feeding adults, there was no statistical significance (Fig-

Spatiotemporal Expression Pattern of DaAhR and DaARNT
AhR and ARNT were expressed in all developmental stages of D. armandi. They were expressed the highest in the adult stage, followed by the larval stage, and the lowest in pupae ( Figure 2). Moreover, as for the adult stage, the expression level of AhR in females was significantly higher than that in males in the feeding adults ( Figure 2A). While ARNT showed the opposite result in the feeding adults, there was no statistical significance ( Figure 2B). In addition, the expression of AhR and ARNT was found at different levels in tissues, and there were gender differences in some tissues. Specifically, AhR and ARNT were expressed predominantly in the midgut and Malpighian tubules ( Figure 3). Moreover, the expression of AhR in female adults was higher than in male adults in the midgut ( Figure 3).

Spatiotemporal Expression Pattern of DaAhR and DaARNT
AhR and ARNT were expressed in all developmental stages of D. armandi. They were expressed the highest in the adult stage, followed by the larval stage, and the lowest in pupae ( Figure 2). Moreover, as for the adult stage, the expression level of AhR in females was significantly higher than that in males in the feeding adults ( Figure 2A). While ARNT showed the opposite result in the feeding adults, there was no statistical significance (Figure 2B). In addition, the expression of AhR and ARNT was found at different levels in tissues, and there were gender differences in some tissues. Specifically, AhR and ARNT were expressed predominantly in the midgut and Malpighian tubules ( Figure 3). Moreover, the expression of AhR in female adults was higher than in male adults in the midgut ( Figure 3).

Exposure to Terpenoids
As shown in Figure 4

Functional Analysis of DaAhR and DaARNT by RNAi Silencing
To further investigate the role of DaAhR and DaARNT in host chemical defe expression of two genes was repressed by RNAi in male and female adults.

Functional Analysis of DaAhR and DaARNT by RNAi Silencing
To further investigate the role of DaAhR and DaARNT in host chemical defense, the expression of two genes was repressed by RNAi in male and female adults. Compared with the control group, after dsRNA injection, the expression levels of DaAhR and DaARNT of adults were significantly downregulated at 24, 48, and 72 h, except for in male adults at 24 h ( Figure 5A,D). In addition, the expression level of DaAhR and DaARNT decreased most at 72 h in male and female adults, reaching 80.3% and 57.0%, 86.0%, and 55.7%, respectively ( Figure 5A,D). Moreover, the relative activities of P450, GST, and CarE remarkably reduced compared to the control after the injection of dsRNA ( Figure 5B,E). Additionally, knocking down DaAhR and DaARNT increased the beetle's susceptibility to terpenoids. The mortality of dsAhR and dsARNT-injected beetles elevated by 37.1% and 21.2%, 22.4% and 23.3, and 43.4% and 38.1% in male adults ( Figure 5C), respectively, after the (−)-β-pinene (+)-3-carene and (±)-limonene treatment when compared with the control. The female adults were enhanced by 34.3% and 18.7%, 20.3% and 20.1, and 36.4% and 19.5%, respectively ( Figure 5F). Nevertheless, there was no significant change in (−)-α-pinene susceptibility ( Figure 5C,F 19.5%, respectively ( Figure 5F). Nevertheless, there was no significant change in (−)pinene susceptibility ( Figure 5C,F). The mortality of adults exposed to four terpenoids was assessed at 48 h after dsRNA injection. The relative expression levels were normalized with β-actin and CYP4G55. The asterisk indicates a sig nificant difference between treatment groups (* p < 0.05, ** p < 0.01, *** p < 0.001, independent Stu dent's Test). All values are mean ± SE, n = 3.

Analysis of DaAhR and DaARNT Regulation of Detoxification Genes
To further explore the role of DaAhR and DaARNT in the detoxification metabolism of D. armandi, the expression levels of detoxification-related genes were determined using qRT-PCR when DaAhR and DaARNT were silenced. Among the P450, GST, and CarE genes, CYP6CR2, CYP6DF1, GSTs1, GSTs2, and CarE4 expression levels were significantly downregulated when DaAhR and DaARNT were reduced in male adults ( Figure 6A). The expression levels of CYP6BX1, CYP6DE5, CYP6DF1, GSTe1, GSTe4, GSTs2, and CarE3 were significantly inhibited when silencing DaAhR and DaARNT in female adults ( Figure  6B). These results indicated that DaAhR and DaARNT regulate the expression levels o detoxification genes and the metabolic detoxification enzyme activity of the correspond ing genes participating in D. armandi's susceptibility to host allelochemicals. (C,F) The mortality of adults exposed to four terpenoids was assessed at 48 h after dsRNA injection. The relative expression levels were normalized with β-actin and CYP4G55. The asterisk indicates a significant difference between treatment groups (* p < 0.05, ** p < 0.01, *** p < 0.001, independent Student's Test). All values are mean ± SE, n = 3.

Analysis of DaAhR and DaARNT Regulation of Detoxification Genes
To further explore the role of DaAhR and DaARNT in the detoxification metabolism of D. armandi, the expression levels of detoxification-related genes were determined using qRT-PCR when DaAhR and DaARNT were silenced. Among the P450, GST, and CarE genes, CYP6CR2, CYP6DF1, GSTs1, GSTs2, and CarE4 expression levels were significantly downregulated when DaAhR and DaARNT were reduced in male adults ( Figure 6A). The expression levels of CYP6BX1, CYP6DE5, CYP6DF1, GSTe1, GSTe4, GSTs2, and CarE3 were significantly inhibited when silencing DaAhR and DaARNT in female adults ( Figure 6B). These results indicated that DaAhR and DaARNT regulate the expression levels of detoxification genes and the metabolic detoxification enzyme activity of the corresponding genes participating in D. armandi's susceptibility to host allelochemicals. Moreover, the expression of CYP6DF1 and GSTs2 significantly decreased after DaAhR and DaARNT injection, with CYP6DF1 being reduced by higher than 70% ( Figure  6). To further reveal the role of CYP6DF1 in the detoxification metabolism, we determined the susceptibility of D. armandi to four terpenoids after silencing CYP6DF1. The expression of DaCYP6DF1 in adults was down-regulated significantly at 24, 48, and 72 h when compared to the control after dsRNA injection. In addition, the expression level of DaCYP6DF1 decreased the most at 72 h in male and female adults, reaching 81.7% and 62.4%, respectively ( Figure 7A,B). The mortality significantly elevated in the dsCYP6DF1injected group after treatment with (−)--pinene and (±)-limonene compared with the control, with 38.7% and 37.3% in male adults, 21.4% and 31.3% in female adults, respectively ( Figure 7C,D). Nevertheless, there was no significant change in (−)--pinene and (+)-3carene susceptibility. Moreover, the expression of CYP6DF1 and GSTs2 significantly decreased after DaAhR and DaARNT injection, with CYP6DF1 being reduced by higher than 70% ( Figure 6). To further reveal the role of CYP6DF1 in the detoxification metabolism, we determined the susceptibility of D. armandi to four terpenoids after silencing CYP6DF1. The expression of DaCYP6DF1 in adults was down-regulated significantly at 24, 48, and 72 h when compared to the control after dsRNA injection. In addition, the expression level of DaCYP6DF1 decreased the most at 72 h in male and female adults, reaching 81.7% and 62.4%, respectively ( Figure 7A,B). The mortality significantly elevated in the dsCYP6DF1-injected group after treatment with (−)-β-pinene and (±)-limonene compared with the control, with 38.7% and 37.3% in male adults, 21.4% and 31.3% in female adults, respectively ( Figure 7C,D). Nevertheless, there was no significant change in (−)-α-pinene and (+)-3-carene susceptibility. The mortality of adults exposed to four terpenoids was asses h after dsRNA injection. The relative expression levels were normalized with β-actin and C The asterisk indicates a significant difference between treatment groups (* p < 0.05, ** p < 0 < 0.001, independent Student's Test). All values are mean ± SE, n = 3.

Discussion
During D. armandi attack host trees, they must overcome the induced defense of the pine trees, which contains a large number of terpenoids [3]. To avoid host t insects have evolved a complicated regulatory mechanism to solve xenobiotics ex including enhanced detoxification metabolisms [36]. Although the relationship b the induction of relevant detoxification genes and insect tolerance to exogenous ch has been widely reported, the regulatory cascades of these xenobiotic response g D. armandi are still basically unknown. In the present study, we clarified the ef transcription factors DaAhR and DaARNT on D. armandi downstream-related det tion genes and relevant regulation metabolism in plant toxin resistance.
Stage-dependent expression patterns of DaAhR and DaARNT were detected ferent developmental stages with RT-qPCR, and the results indicated that DaA DaARNT were expressed in all developmental stages of D. armandi. The highest sion level was detected in the larval and adult stage, and the lowest was in the which may be involved in detoxifying xenobiotics in food. Moreover, the expre DaAhR and DaARNT was further investigated in various tissues of adults, suggest it was expressed predominantly in the midgut and Malpighian tubules compared parts. The midgut is not only the place where insects digest and absorb but also key role in resisting exogenous substances [37]. Previous studies have reported Malpighian tube also has a function in xenobiotics detoxification [38,39]. It wa noting that the expression of DaAhR in female adults was higher than in male a The mortality of adults exposed to four terpenoids was assessed at 48 h after dsRNA injection. The relative expression levels were normalized with β-actin and CYP4G55. The asterisk indicates a significant difference between treatment groups (* p < 0.05, ** p < 0.01, *** p < 0.001, independent Student's Test). All values are mean ± SE, n = 3.

Discussion
During D. armandi attack host trees, they must overcome the induced defense system of the pine trees, which contains a large number of terpenoids [3]. To avoid host toxicity, insects have evolved a complicated regulatory mechanism to solve xenobiotics exposure, including enhanced detoxification metabolisms [36]. Although the relationship between the induction of relevant detoxification genes and insect tolerance to exogenous chemicals has been widely reported, the regulatory cascades of these xenobiotic response genes in D. armandi are still basically unknown. In the present study, we clarified the effects of transcription factors DaAhR and DaARNT on D. armandi downstream-related detoxification genes and relevant regulation metabolism in plant toxin resistance.
Stage-dependent expression patterns of DaAhR and DaARNT were detected in different developmental stages with RT-qPCR, and the results indicated that DaAhR and DaARNT were expressed in all developmental stages of D. armandi. The highest expression level was detected in the larval and adult stage, and the lowest was in the pupae, which may be involved in detoxifying xenobiotics in food. Moreover, the expression of DaAhR and DaARNT was further investigated in various tissues of adults, suggesting that it was expressed predominantly in the midgut and Malpighian tubules compared to other parts. The midgut is not only the place where insects digest and absorb but also plays a key role in resisting exogenous substances [37]. Previous studies have reported that the Malpighian tube also has a function in xenobiotics detoxification [38,39]. It was worth noting that the expression of DaAhR in female adults was higher than in male adults in the feeding adults and midgut, while there was no gender difference in DaARNT. This indicates that DaAhR plays an important role in the location and detoxification of host volatiles in female adults. Therefore, the overexpression of DaAhR and DaARNT in the detoxification organ of D. armandi may play a key role in the detoxification of exogenous substances. Further studies can be performed to show the function of DaAhR and DaARNT in other different developmental stages and tissues of insects.
Under normal circumstances, the increased metabolism in insects arises from the increased detoxification effects attributed to amplification, constitutive overexpression, and mutation of genes [40]. Elements in promoters and transcription factors mediate transcriptional regulation and participate in the expression of detoxification genes. The suppression of hepatocyte nuclear factor 4 by transcription factor results in the resistance to imidacloprid in N. lugens through regulating UDP-glycosyltransferase (UGT) and P450 genes [41]. The promoter region motif may be a candidate region for the transcription of tolerance-related genes [42,43]. A cis-acting element was called a xenobiotic response element (XRE) of flavonoids in Helicoverpa zea, which mediated the expression of CYP321A1 induced by flavonoids [44]. In Spodoptera litura, Nrf2 activated the GSTe1 expression by binding to the cis-acting element in its promoter in response to insecticides and phytochemicals [45]. As vital transcription factors for xenobiotic sensors, AhR and ARNT regulate a large number of metabolic genes in various insect species [10]; however, their roles in D. armandi detoxification remain unclear.
In the present study, the expression of DaAhR and DaARNT significantly increased after treatment with (−)-β-pinene, (+)-3-carene, and (±)-limonene, and silencing DaAhR and DaARNT enhanced the susceptibility of D. armandi, with the decrease of enzyme activity level corresponding to greater sensitivity. This revealed that the AhR/ ARNT signal pathway in D. armandi may be involved in metabolic responses to environmental stresses associated with host allelochemical exposure. In addition, DaAhR and DaARNT are homologous to other species. For instance, the AhR signal pathway is induced when mediating chemical-induced developmental toxicity in zebrafish [46]. Silencing LmAhR enhanced the susceptibility to chlorpyrifos in L. migratoria [25]. Moreover, the expression of the transcription factor SlituCncC is induced after indoxacarb exposure in S. litura [47]. Additionally, silencing NlAhR and NlARNT affects isoprocarb, imidacloprid, and etofenprox susceptibility in N. lugens [48].
The change in detoxification enzyme activity is due to the change in the expression of detoxification genes. In this study, silencing DaAhR and DaARNT resulted in the differential downregulation of some detoxification enzyme genes, with DaCYP6DF1 being downregulated by higher than 70%. These results indicated that DaCYP6DF1 was supposed to be a candidate gene that mainly participated in host chemical susceptibility. Similar results have also been reported in other insects. For example, AhR/ARNT of M. persicae regulated the expression of CYP6CY3 and CYP6CY4 cooperatively, leading to the nicotine adaption to tobacco [23]. The cis-regulatory elements AhR/ARNT and CncC/Maf participated in the expression regulation of some GST detoxification enzyme genes in Spodoptera exigua. Moreover, the constitutive overexpression of CncC, Maf, and AhR promoted the increased expression of several detoxification-related genes and led to chlorpyrifos and cypermethrin resistance [49]. In addition, NlAhR and NlARNT were induced by pesticides and participated in the NlCarE7 regulation, which was involved in the susceptibility to etofenprox and isoprocarb in N. lugens [48]. Previous studies have reported that CYP genes in D. armandi were remarkably increased in response to (−)-α-pinene, (−)-β-pinene, (+)-3-carene, and (±)-limonene treatments [29,34,35]. Because knockdowns of DaCYP6DF1 significantly increased D. armandi susceptibility to (−)-β-pinene and (±)-limonene, we supposed that DaAhR and DaARNT regulate the susceptibility by targeting DaCYP6DF1.

Conclusions
These results indicated that the transcription factors DaAhR and DaARNT of D. armandi were induced by terpenoids and involved in mediating the expression level of DaCYP6DF1, which was associated with D. armandi susceptibility to (−)-β-pinene and (±)-limonene. Our study showed the potential roles of DaAhR and DaARNT in the detoxification metabolism of D. armandi. These findings uncovered the molecular mechanisms of host allelochemical detoxification in insects and may provide a theoretical basis for controlling this pest.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11233856/s1, Figure S1: Multiple amino acid sequence alignments of DaAhR (A) and DaARNT (B) with six other insect species. Three characteristic domains (bHLH, PAS-A and PAS-B) of DaAhR and DaARNT are boxed in red. Table S1: Primer sequences used in the research.