Effects of three insecticides on the expression of cytochrome P450 CYP6B7 in Helicoverpa armigera

Cytochrome P450 genes can be induced by xenobiotics, which may contribute to insect's adaptability to the environments and resistance to insecticides. Previous studies indicated that cytochrome P450 CYP6B7 played a vital role in the resistance of Helicoverpa armigera to fenvalerate. However, effects of different insecticides on the expression of CYP6B7 in H. armigera are still unclear. In this study, resistance level of H. armigera to six insecticides was determined by topical application method, and effects of fenvalerate, phoxim and indoxacarb on the expression of CYP6B7 in susceptible (HDS) and fenvalerate‐resistant (BJR) strains of H. armigera were evaluated by RT‐qPCR. The results showed that BJR strain had an extremely high level of resistance to fenvalerate (1990.57‐fold), and the induction of CYP6B7 in different tissues of BJR strain was significantly higher than that of HDS strain after exposure to fenvalerate for 24 and 48 hr. The highest induction level by fenvalerate was observed in the midgut, which were 13.7‐fold in HDS strain and 127.9‐fold in BJR strain at 24 and 48 hr, respectively. After exposure to phoxim, the expression level of CYP6B7 in HDS and BJR strains was induced by 2.3‐ and 316.8‐fold at 24 hr, respectively. It is worth to note that CYP6B7 could be induced by phoxim at different time points in BJR strain, but only induced at 24 and 72 hr in HDS strain. After indoxacarb exposure, the expression of CYP6B7 was induced by 1.6‐fold at 72 hr in BJR strain, whereas it was induced at 24 and 48 hr in HDS strain. These results demonstrated that the expression level of CYP6B7 could be induced by fenvalerate, phoxim and indoxacarb, but the induction time and levels varied; moreover, the induction in BJR strain was markedly higher than that in HDS strain after exposure to fenvalerate and phoxim.

application of insecticides has led to the resistance of H. armigera to many insecticides. For example, different field strains of H. armigera have developed high level resistance to pyrethroids (Bajya et al., 2011;Sene et al., 2020;Xu et al., 2016); H. armigera in Pakistan showed low to moderate level of resistance to indoxacarb, with resistance ratios ranging from 5.36-to 20.40-fold (Qayyum et al., 2015).
Cytochrome P450s (P450s) is one of the major detoxification enzymes that can metabolize endogenous and exogenous compounds, including insecticides and other xenobiotics (Feyereisen, 2012;Scott, 2008). Inducibility is an important characteristic of P450s (Liu et al., 2015;Scott et al., 1996), which would help insects to better metabolize xenobiotics so that increase their adaptability to host plants and tolerance to insecticides (Mao et al., 2007;Tao et al., 2012). Previous studies have shown that insect cytochrome P450 genes can be induced by allelochemicals (Liu et al., 2006;Mao et al., 2007; and some insecticides (Giraudo et al., 2015;Zhu et al., 2016). For example, quercetin induced the expression of CYP6B6 and CYP6B8 (Chen et al., 2018), and xanthotoxin induced that of CYP6AE19 and CYP6AE20 in H. armigera ; fenvalerate induced the expression of CYP9A105 in Spodoptera exigua , DDT induced the expression of Cyp4p1 and Cyp4p2 in DDT resistant strain of Drosophila melanogaster (91-R) (Seong et al., 2019). The induction of P450s in response to phoxim and indoxacarb in Bombyx mori, Plutella xylostella and S. exigua has also been reported Hu et al., 2019;Wang et al., 2013). However, information on effects of insecticides on the expression of P450s in H. armigera is still limited.
Cytochrome P450 CYP6B7 was first isolated and identified in pyrethroid-resistant H. armigera by Ranasinghe et al. (1998), Ranasinghe et al. (1999) in Australia, and it was later reported being overexpressed in a fenvalerate-selected resistant strain of H. armigera (Zhang et al., 2010); the sensitivity of H. armigera to fenvalerate increased after knockdown of CYP6B7 by RNA interference (Tang et al., 2012). These results indicated that CYP6B7 played a vital role in the resistance of H. armigera to pyrethroids. Ranasinghe and Hobbs (1999) reported that CYP6B7 in fat body culture of H. armigera (in vitro) could be induced by α-pinene, phenobarbital and some pyrethroids; Tao et al. (2012) suggested that gossypol and xanthotoxin significantly induced the expression of CYP6B7, and the gossypol-induced multiple P450s contributed to the tolerance of H. armigera to deltamethrin. The expression of CYP6B7 in H. armigera could also be significantly induced by other allelochemicals, including 2-tridecanone (Xu et al., 2018), flavone, coumarin, DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and visnagin

| Insects
The susceptible HDS strain of H. armigera was collected from field of Handan, Hebei Province, China, in 1988, and since then has been reared in the laboratory without exposure to insecticides. The resistant BJR strain, which showed 114.7-fold resistance to fenvalerate when it was first collected from field of Beijing in 2014 (Xu et al., 2016), was further selected by fenvalerate for more than 15 generations. The two strains of H. armigera were reared on artificial diets as described by Xu et al. (2016), with temperature of 27 ± 1℃, relative humility of 70% ± 10% and light: dark photoperiod of 14:10 hr.

| Determine resistance level of H. armigera to insecticides
Topical application method was used to determine the resistance levels of BJR strain of H. armigera to six commonly used insecticides via comparing with the susceptible HDS strain. Stock solution of six insecticides was prepared by acetone and diluted to 5-7 serial solutions of various concentration. One microlitre of test solution was applied on the thoracic dorsum of the third instar larvae (weight between 15 and 20 mg) of HDS and BJR strains by Hamilton syringe.
Mortality was recorded at 48 hr after treatment with insecticide and acetone (as control). All treatments were conducted with at least three replicates, and twelve larvae for each replicate. The resistance ratios (RR) were calculated by dividing the LD 50 of the resistant BJR strain by the LD 50 of the susceptible HDS strain.

| RNA isolation and cDNA synthesis
After the fifth instar larvae were treated by fenvalerate, phoxim and indoxacarb for 24, 48 and 72 hr, respectively, total RNA was extracted from the midgut, fat body and cuticle of larvae using TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa) according to the manufacturer's recommendations. The quality and integrity of RNA were measured using 1% gel electrophoresis and a Nanodrop microspectrophotometer (Denovix company). One microgram total RNA was reverse-transcribed into cDNA using FastQuant RT Kit (With gDNase) (TIANGEN) according to the manufacturer's protocol. The quality and quantity of cDNA were checked using a Nanodrop microspectrophotometer before quantitative real-time PCR.

| Quantitative real-time PCR
Quantitative RT-PCR was performed on an ABI Prism7500 Real-

Time PCR System (Applied Biosystems by Life Technologies)
with SuperReal PreMix Plus (SYBR Green, TIANGEN), following a two-step protocol: 95°C for 15 min, 40 cycles of denaturing at 95°C for 10 s, annealing at 60°C for 20 s and extension at 72°C for 32 s. A melting curve was added as a final step to make sure the PCR product was unique and specific. PCR primers were designed using the Primer Premier 6.0 software based on the sequences published in NCBI for CYP6B7 (Accession No: DQ497428.2, CYP6B7-F: AATGACCACACCCTGCCTAC, CYP6B7-R: RPS15-R: TGTTGGTCAGCGCACTACTT), was used to normalize the gene expression level (Shakeel et al., 2015). At least three sample repetitions and three technical repetitions were required for each test.

| Statistical analysis
The insecticide LD 50 values to H. armigera and the differences between control and treatment were analysed with SPSS (version 16.0). The gene expression level was calculated according to the 2 −△△Ct method (Livak & Schmittgen, 2001). The values were shown as means ± standard deviation derived from three biological repeats.
A p-value <.05 indicates statistically significant differences according to Duncan test.

| Resistance level of H. armigera to six insecticides
The toxicity of six commonly used insecticides to H. armigera was determined by topical application method. When compared with the susceptible HDS strain, BJR strain showed extremely high level of resistance to fenvalerate, with LD 50 and resistance ratio (RR) values of 242.850 μg/larva and 1990.57-fold, respectively; and it showed middle level of resistance to deltamethrin, with LD 50 and RR values of 2.607 μg/larva and 5.48-fold, respectively; meanwhile, BJR strain was susceptible to the other four insecticides, including phoxim, chlorpyrifos, methomyl and indoxacarb, with LD 50 values ranging from 0.010 to 1.569 μg/larva, and RR values ranging from 1.48-to 2.44-fold (Table 1). These results indicated that there was crossresistance between pyrethroid insecticides fenvalerate and deltamethrin, but no cross-resistance between pyrethroids and other insecticides tested.

| Fenvalerate
To analyse the effects of fenvalerate on the expression of CYP6B7 in different tissues of H. armigera, the fifth instar larvae of HDS and BJR strains were fed on diets containing 0.025 and 0.1 mg/g (W/W) fenvalerate for 24, 48 and 72 hr, respectively, and the relative transcript levels of CYP6B7 in midgut, fat body and cuticle of HDS and BJR strains were determined. The results were as following: In HDS strain, the expression level of CYP6B7 in midgut was increased by 4.6-and 13.7-fold, respectively, while that in fat body and cuticle was not changed significantly, after exposure to 0.025 and 0.1 mg/g fenvalerate for 24 hr when compared with that of the control ( Figure 1a); the expression level of CYP6B7 in midgut and fat body was induced by 1.8-and 3.2-fold, respectively, after exposure to 0.1 mg/g fenvalerate for 48 hr; but no significant induction was observed after exposure to 0.025 mg/g fenvalerate, as well as in cuticle after exposure to 0.025 and 0.1 mg/g fenvalerate ( Figure 1b); the expression level in fat body was increased by 14.4-and 27.9-fold after exposure to 0.025 and 0.1 mg/g fenvalerate for 72 hr, respectively, while that in midgut and cuticle were not induced (Figure 1c).
In BJR strain, the expression level of CYP6B7 in midgut was significantly induced by 86.6-fold after 24 hr exposure to 0.025 mg/g fenvalerate; meanwhile that in fat body and cuticle was induced by 14.4-and 27.9-fold, respectively, after exposure to 0.1 mg/g fenvalerate in diets when compared with the control (Figure 1d). The expression level of CYP6B7 was induced by 15.6-and 4.6-fold in midgut and fat body, respectively, after 48 hr of 0.025 mg/g fenvalerate exposure; meanwhile the expression level was induced by 127.9-, 2.2-and 5.7-fold in midgut, fat body and cuticle, respectively, after 48 hr-exposure to 0.1 mg/g fenvalerate (Figure 1e). The expression level was induced by 3.2-fold in fat body after 72 hr exposure to 0.025 mg/g fenvalerate; and that was induced by 3.9-and 10.5-fold in fat body and cuticle, respectively, after 72 hr exposure to 0.1 mg/g fenvalerate (Figure 1f). These results suggested that the induction of CYP6B7 in the midgut enhanced with the increase of fenvalerate concentration at 48 hr in both HDS and BJR strains; moreover, the induction level in BJR strain was significantly higher than that in HDS strain. Due to the highest expression level of CYP6B7 was observed both in the midgut of HDS and BJR strains of H. armigera, we further investigated the effects of phoxim and indoxacarb on relative transcript levels of CYP6B7 in midgut of the two strains.

| Phoxim
The results indicated that the expression level of CYP6B7 in midgut of HDS strain of H. armigera was induced by 2.3-and 1.6-fold, respectively, after 24 hr of 0.00625 and 0.025 mg/g phoxim exposure and induced by 1.3-fold after 72 hr of 0.00625 mg/g phoxim exposure; however, it was significantly inhibited after 48 and 72 hr of 0.025 mg/g phoxim exposure when compared with that of the control (Figure 2a).
The expression level of CYP6B7 in midgut of BJR strain of H. armigera was induced by 183.4-, 2.2-and 1.6-fold at 24, 48 and 72 hr, respectively, after exposure to 0.00625 mg/g phoxim and was induced by 316.8-, 10.3-and 1.6-fold at 24, 48 and 72 hr, respectively, after exposure to 0.025 mg/g phoxim when compared with that of the control (Figure 2b).

| Indoxacarb
The results demonstrated that the expression level of CYP6B7 in midgut of HDS strain of H. armigera was induced by 4.4-fold at 24 hr after 0.00625 mg/g indoxacarb exposure and was induced by 1.3-fold at 48 hr after 0.025 mg/g indoxacarb exposure; but no significant induction was observed at 48 hr after 0.00625 mg/g indoxacarb exposure; moreover, the expression was significantly inhibited at 72 hr after 0.00625 and 0.025 mg/g indoxacarb exposure when compared with the control (Figure 3a).
The expression level of CYP6B7 in midgut of BJR strain of H. armigera was induced by 1.6-and 1.3-fold, respectively, at 72 hr after exposure to 0.00625 and 0.025 mg/g indoxacarb, but no induction was observed at 24 and 48 hr (Figure 3b).  insects to the environment, as well as the development of insecticide resistance (Clements et al., 2017;Mao et al., 2007;Tao et al., 2012).
Previous study has proved that CYP6B7 could be induced by fenvalerate in fat body culture of H. armigera (in vitro) (Ranasinghe et al., 1999), in addition, six P450 genes (CYP6AE14, CYP6B2, CYP6B6, CYP6B7, CYP9A12, and CYP9A17) and four P450 genes (CYP6AE14, CYP6B2, CYP6B6, CYP9A14) were significantly induced by deltamethrin in the midgut and fat body of H. armigera, respectively . In the present study, the expression of CYP6B7 could be induced in three major tissues (midgut, fat body and cuticle) of H. armigera after exposure to fenvalerate in diets; however, the induction level varied in different tissues; the maximum induction of CYP6B7 was observed in midgut of both susceptible and resistant strains, followed by fat body and cuticle. These results suggested that the induction of P450s is tissue-dependent, and P450s in midguts is more easily responded to xenobiotics when they enter the insect by feeding, which may make it easier for the insects adapt to the environment. reported that the induction of P450s in response to cypermethrin was more significant by leaf dip method than by topical application in Plutella xylostella, and Zhang et al. (2016) suggested that the induction of P450 genes was related to the exposure dose and time of fipronil in Solenopsis invicta. In the present study, we investigated and compared the inducing effect of CYP6B7 in response to three insecticides (fenvalerate, phoxim and indoxacarb) in HDS and BJR strains of H. armigera. The induction of CYP6B7 in midgut enhanced with the increase of fenvalerate concentration at 48 hr in both HDS and BJR strains; simultaneously, the expression level of CYP6B7 was increased with the extension of exposure time; after exposure to 0.1 mg/g fenvalerate, the expression was not changed significantly at 24 hr, but induced by 127.9-fold at 48 hr in BJR strain. In addition, the expression of CYP6B7 was induced the most strongly by fenvalerate, followed by phoxim and indoxacarb. Taken together, our results suggested that the inducibility of CYP6B7 in H. armigera was affected by the exposure dose, time and type of insecticide, which further confirmed the results of previous studies mentioned above.
When compared the effect of three insecticides on the expression of CYP6B7 between the susceptible HDS and resistant BJR strains of H. armigera, we found that the induction of CYP6B7

CYP6B7
in midgut of BJR strain was significantly higher than that of HDS strain after exposure to fenvalerate for 24 and 48 hr, and the induction of CYP6B7 in fat body and cuticle of BJR strain was significantly higher than that of HDS strain after exposure to fenvalerate for 72 hr. The induction of CYP6B7 in BJR strain was also stronger than that in the HDS strain after exposure to phoxim for different time points, but similar results were observed only after 72 hr of exposure to indoxacarb. These results suggested that the responses of CYP6B7 to insecticides between susceptible and resistant strains of H. armigera were different. Such difference has also been reported in other insects. In D. melanogaster, CYP6A2 was over-transcribed in a resistant strain, but not significantly changed in a susceptible strain after DDT treatment (Sun et al., 2011); in Culex quinquefasciatus, CYP6AA7, CYP9J34 and CYP9M10 were induced by permethrin in a resistant strain; however, no significant induction was observed in a susceptible strain (Liu et al., 2011); in P. xylostella, the induction of Cyp4M19, Cyp4M21, Cyp4M22, Cyp4M23, Cyp6CN1, Cyp6BF1, Cyp6BG1 and Cyp9G4 were more obvious in a resistant strain than that in a susceptible strain after exposure to 1 ppm cypermethrin (Baek et al., 2010); in Chilo suppressalis, the induction of CYP6CV5, CYP9A68, CYP321F3 and CYP324A12 was stronger in a chlorantraniliprole resistant strain than that in a susceptible strain (Xu et al., 2019).

Many previous studies suggested that the induction of insects
P450s by xenobiotics was complicated (Stevens et al., 2000;Tian et al., 2019;, this was further confirmed in our study. In addition to the results mentioned above, the response of   (Poupardin et al., 2008).
Many studies have shown that cytochrome P450s were involved in the metabolism and detoxification of insecticides War et al., 2011). In H. armigera, CYP6B6 was significantly induced by pyrethroid insecticides and involved in the detoxification of esfenvalerate (Feyereisen, 2006;Tian et al., 2017;; in Spodoptera litura, CYP9A40 was induced significantly by deltamethrin and methoxyfenozide and participated in the detoxification of the two insecticides . In our study, the expression of CYP6B7 was significantly induced by fenvalerate, phoxim and indoxacarb, since previous studies have confirmed that CYP6B7 was involved in fenvalerate detoxification in H. armigera (Tang et al., 2012;Zhao et al., 2017), herein, we speculated that CYP6B7 may also be involved in the metabolism of phoxim and indoxacarb in H. armigera. However, further study is needed for this conclusion.
In summary, the results of the present study indicated that CYP6B7 could be induced by fenvalerate, phoxim and indoxacarb in both resistant and susceptible strains of H. armigera, but the induction levels were different among the three insecticides; The induction of CYP6B7 is tissue-dependent, with the highest induction level observed in midguts; Moreover, the induction of CYP6B7 in the resistant BJR strain was more significant than that in the susceptible HDS strain after exposure to fenvalerate and phoxim, while that was opposite after indoxacarb treatment. Taken together, our results suggested the induction of CYP6B7 in H. armigera varied with the types of insecticides, exposure dose and time, insect tissues and strains.

ACK N OWLED G EM ENTS
This work is supported by the National Natural Science Foundation of China (Grant/Award Number: 31772195).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are publicly available