Resistance irrelevant CYP417A2v2 was found degrading insecticide in Laodelphax striatellus

Abstract Cytochrome P450 monooxygenases (CYPs) usually overexpressed in resistant strain were found involved in oxidative detoxification of insecticides. In this study, an investigation was conducted to confirm if resistance irrelevant CYPs which were not overexpressed in resistant strain before, were capable of degrading insecticides. Three resistance irrelevant CYPs viz. CYP417A2v2, CYP425A1v2, and CYP4DJ1 from CYP4 family of Laodelphax striatellus were randomly selected for experiments. CYP417A2v2 and CYP425A1v2 were found expressed successfully in Sf9 cell line while CYP4DJ1 was not expressed successfully and out of two expressed CYPs, only CYP417A2v2 showed its efficient catalytic activity. For catalytic activity, three traditional model probe substrates and five insecticides were assayed. For the probe substrates screened, p‐nitroanisole and ethoxycoumarin were preferentially metabolized by CYP417A2v2 (specific activity 3.76 ± 1.22 and 1.63 ± 0.37 nmol min−1 mg protein−1, respectively) and they may be potential diagnostic probes for this enzyme. Among insecticides, only imidacloprid was efficiently degraded by CYP417A2v2. Incubation of imidacloprid with CYP417A2v2 of L. striatellus and subsequent HPLC, LC‐MS, and MS/MS analysis revealed the formation of imidacloprid metabolites, that is, 4′ or 5′hydroxy‐imidacloprid by hydroxylation. This result implies the exemption of CYPs character that it is not always, all the CYPs degrading insecticides being selected and overexpressed in resistant strains and the degrading CYPs without mutations to upregulate could be candidates during insecticide resistance evolution. This characterization of individual insect CYPs in insecticide degradation can provide insight for better understand of insecticide resistance development.


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MIAH et Ial. Brattsten, 1972). Some time the detoxification mechanism is so active that the insecticide fails to reach to effective level at its molecular target after being metabolized and degraded by these enzymes: such individuals become resistant to insecticides (Taylor & Feyereisen, 1996).
CYPs are mainly involved in Phase 1 (Primary) reaction of insecticides metabolism (Feyereisen, 2005;Liu, Li, Gong, Liu, & Li, 2015) and oxidation (hydroxylation) is considered as the most important among phase 1 reactions. In oxidation reaction, a portion of insecticide molecules that enter into insects is transformed to less toxic metabolites and finally excreted (Feyereisen, 2005). Hence, understanding of insecticides degradation through metabolism is crucial for the development of more selective insecticides (Hodgson & Levi, 2010).
The small brown planthopper, Laodelphax striatellus (Fallén) (Homoptera: Delphacidae), a notorious phytophagous, causes serious damage directly by feeding grain crops and indirectly by transmitting several plant viruses, that is, rice stripe virus and rice black-streaked dwarf virus (Kisimoto, 1967). It has a wide distribution range from Southeast Asia to Siberia and to Europe, attacking several important agricultural crops including rice, corn, wheat, oat, and barley (Liu, Zhai, & Liu, 2006). Furthermore, this pest is able to overwinter in the temperate zone of East Asia, like China, and Japan (Matsumura, Otuka, & Watanabe, 2006). In China, L. striatellus is considered as a serious pest since the late 1990s and found in all rice-growing areas. Its density has boosted up dramatically in the beginning of this century, especially in the middle and downstream Yangtze River (the coastal rice production regions of eastern China) and caused great economic damage.
Large outbreak of L. striatellus (Wei, 2007), serious damage (Liu et al., 2006), and yield loss (Gu, Xue, Shi, & Zhou, 2005) of rice and other crops were commonly occurred incidence in different rice growing areas in china. Usually farmers use insecticides as common practice to suppress L. striatellus populations, consequently, this pest develops resistance to various insecticides due to extensive use of chemical insecticides like organophosphate, carbamate, pyrethroid, and neonicotinoid (Wang et al., 2008) as well as cyclodiene organochlorines, phenylpyrazoles, and chitin biosynthesis inhibitors (http://www.pes ticideresistance.org). In recent years in China, L. striatellus resistance to insecticide has been a frequent incidence and it is field populations have developed variable resistance to different kinds of insecticides (Wang, Zhang, Han, Liu, & Fang, 2010). It is also well documented that field populations of L. striatellus developed different levels of (high to extremely high) resistance to imidacloprid, deltamethrin, buprofezin, fipronil, and chlorpyriphos in different areas in China (Gao, Wu, Huang, Mu, & Han, 2008;Ma, Gao, Wei, & Shen, 2007;Zhang, Chen, Chen, & Yu, 2007). Therefore, insecticide resistance management strategies must be developed to prevent further increase in resistance of L. striatellus.
Different insect CYPs overexpressed in resistant strains or involved in insecticide resistance have been studied and confirmed for their capability of catalyzing insecticide degradation. The CYPs overexpressed in L. striatellus resistant to deltamethrin (Xu, Wu, & Han, 2013), buprofezin (Zhang et al., 2012), and imidacloprid (Elzaki et al., 2015) and their capability to degrade insecticides were also studied (  (data published online)). However, little is known about those CYPs that are not overexpressed in any resistant strains of L. striatellus and even in any insects before. Therefore, this resistance irrelevant CYPs (not overexpressed, therefore not associated with insecticide resistance) need to be explored for their catalytic activity. Accordingly, in the present work, some gene members from CYP4 subfamily which are well documented for detoxification were randomly selected and functionally recombinant expressed in Sf9 cells and a biochemical investigation was conducted to establish whether resistance irrelevant CYPs in L. striatellus are capable of degrading insecticides.

| Insects
The field population of L. striatellus was collected from the paddy field in Jianhu, Jiangsu Province, China, in June 2015 and has been reared since then without any contact with insecticides. All insects were reared on rice seedlings planted in tissue laid (soil less) plastic boxes at 26 (±2)°C under a 12:12-h light: dark regime at 70%-80% relative humidity.

| Functional expression of CYP genes in Sf9 cell and microsomal protein isolation
The entire coding region of three CYP genes of L. striatellus which were not reported overexpressed in any resistant strain before was obtained from NCBI (http://www.ncbi.nlm.nih.gov), including CYP417A2v2, CYP425A1v2, and CYP4DJ1. The ORF of each gene, in accordance with the cDNA sequences was amplified by polymerase chain reaction (PCR) using the specific primers designed. For convenient cloning, the restriction sites (underlined) were introduced into the forward and the reverse primers. The forward primer contained a Kozak translation sequence (bolded) and an ATG start codon for proper initiation of translation. Besides, the cytochrome P450 Reductase (CPR) was also constructed for the enzyme system to degrade insecticides (Gene accession number of each gene and gene-specific primers used in this study are listed in Table 1 Sf9 Cells were maintained in suspension culture serum-free (SF-900 II SFM, Gibco) medium supplemented with 10% (V/V) fetal bovine serum (FBS, Sigma) in the T-25 flask. The cells were grown in a 5% CO 2 humidified incubator at 27°C. The cells were used only from a 3 to 4 days old suspension culture in mid-log phase with a viability >97%.

The recombinant baculovirus DNA (Recombinant Bacmid) was transferred into Sf9 insect cells (Gibco) through Bac-to-Bac Baculovirus
Expression System (Invitrogen) according to the manufacturer's instructions. The titer of the recombinant viruses was determined following the standard protocols of the supplier. Prior to transformation, the cells were plated in a six-well culture plate and when the cells were at 50%-60% confluence, they were transformed using the Cellfectin reagent (Invitrogen). Sf9 cells transformed by EGFP (Enhanced Green Fluorescent Protein) were used for positive control and untranformed cells for negative control.
Insect cells grown to a density of 2 × 10 6 cells/ml were coinfected with recombinant baculoviruses containing different CYP genes and CPR with various MOI (multiplicity of infection) ratios to identify the optimal conditions. After 48 hr cells were harvested and washed with PBS, and the microsomes of the membrane fraction were prepared according to standard procedures and stored at 80°C (Phillips and Shephard, 2005). Briefly, the cells were washed twice with 0.1 mol/L, pH 7.8, sodium phosphate buffer and resuspended in precooled lysis buffer (0.1 mol/L, pH 7.8, sodium phosphate buffer, containing 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PTU, and 1 mmol/L proteinase inhibitor PMSF). The suspension was sonicated in an ice bath and again centrifuged at 10,000 rpm for 10 min. The supernatant was used immediately or kept in −70°C as the enzyme source for checking CYP proteins, catalytic activity, and insecticide metabolism. For detecting the target recombinant CYP protein, 12% SDS-PAGE gels was run on the Bio-Rad Mini-Protein II apparatus according to Laemmli (1970) and proteins were visualized by staining with Coomassie Blue.
The expression of functional P450 protein was first estimated by resuspending the microsomes of the membrane in Spectrum Buffer (100 mmol/L Tris-HCl, pH 7.4, 10 mmol/L CHAPS, 20% (v/v) glycerol, 1 mmol/L EDTA) (Pritchard et al., 1998), adding about 1 mg/ml of sodium dithionite (Na 2 S 2 O 4 ) as a reducing agent and recording the absorption spectra (500-400 nm) change after exposing to CO for 1 min.
The peak height at 450 nm was used to calculate the P450 concentration (Omura & Sato, 1964). Total protein content was determined by the Bradford method (Bradford, 1976)

| UPLC-MS analysis and identification of metabolites
The main metabolites of insecticides were identified by UPLC-MS analysis according to the method described by Karunker et al. (2009).
All samples obtained from insecticide metabolism assays were sub-

| Functional expression of L. striatellus CYPs
Three resistance irrelevant CYPs like CYP417A2v2, CYP425A1v2, and CYP4DJ1 were checked for their functional expression in Sf9 cell lines.
The cell microsomes were prepared and subjected to SDS-PAGE analysis. The results showed that CYP417A2v2 and CYP425A1v2 were successfully expressed and the distinct band of recombinant proteins with expected molecular weight was identified, whereas there was no characteristic protein band exhibited in the microsomes prepared from Baculovirus infected cells treated with CYP4DJ1 and uninfected Sf9 insect cells (Figure 1).
The reduced CO-difference spectrum was tested in order to confirm the successful expression of intact recombinant CYP proteins in Sf9 cell. The result showed that only the expressed P450 protein of CYP417A2v2 had a characteristic absorption peak at 450 nm, which is the character of the functional P450 proteins (Omura & Sato, 1964) whereas CYP425A1v2 had no characteristic absorption peak. Thus, CYP417A2v2 protein has been expressed in its P450 form indicating a good-quality functional enzyme (Figure 2).

| CYP417A2v2 catalytic activity against standard P450 model substrates
For checking recombinant CYP417A2v2 for its catalytic activity, three standard P450 model substrates (Fluorescent and chemiluminescent) were first tested, which are routinely used in the pharmaceutical industry (Cohen, Remley, Raunig, & Vaz, 2003) and for diagnostic monitoring of P450 levels for insecticide resistance (Inceoglu et al., 2009

| CYP417A2v2 capability to metabolize insecticides
The metabolism of five insecticides including imidacloprid, deltamethrin, buprofezin, chlorpyrifos, and fipronil were assayed in vitro with  Results are shown as means ± SE. Significant differences were determined by one tailed T-tests. b nd, not detectable.

| DISCUSSION
Cytochrome P450 genes (CYPs) are usually overexpressed in resistant strains and involved in insecticide detoxification that ensuing insecticide resistance. Therefore, these CYPs are termed as resistance relevant genes. In this study, three CYPs which were not reported overexpressed before in any resistant strain of L. striatellus and not associated with insecticide resistance (resistance irrelevant CYPs) were selected to confirm the catalytic ability for insecticide degradation. As results showed in the SDS-PAGE analysis, among the three CYPs, CYP417A2v2 and CYP425A1v2 were successfully expressed in Sf9 cells while CYP4DJ1 was not found to be expressed. Not surprisingly, finally only CYP417A2v2 was confirmed for its catalytic activity through a series of biochemical investigation whereas the expressed CYP425A1v2 was not confirmed for its catalytic activity. In the past, resistance associating CYPs were usually thought and proved as the detoxification enzymes, such as Tetranychus urticae CYP392A16 could detoxify abamectin (Riga et al., 2014), B. tabaci CYP6CM1 hydroxylated pymetrozine (Nauen et al., 2013), CYP9A12 and CYP9A14 of Helicoverpa armigera metabolized esfenvalerate (Yang et al., 2008), and Meligethes aeneus CYP6BQ23 could hydroxylate deltamethrin and tau-fluvalinate (Zimmer et al., 2014).  (Stegeman & Livingstone, 1998). Insect CYPs are the most important groups of environmental response genes that play a vital role in the interactions of insects with insecticides and host plants (Wen, Zhang, & Zhang, 2011) and focused primarily on the metabolism of xenobiotics (Scott, 2008). A remarkable feature of CYPs is the large variation in substrate specificity of different CYPs (Scott, 1999). Therefore, some CYPs are capable of metabolizing a very wide range of compounds whereas some are limited to a highly restricted set of reactions (Kulkarni & Hodgson, 1980 On the other hand, CYPs from the same family shear higher similarity not only in gene sequence but also in function. In insects, members of the CYP family 4, 6, 9, and 12 have all been involved in detoxifying functions and among these families, the members of the CYP4 and CYP6 groups are most commonly implicated in metabolism and resistance to xenobiotics (Feyereisen, 2005;Li, Schuler, & Berenbaum, 2007). Previous research has documented that some members of the CYP4 family were highly expressed in insects and demonstrated their ability to metabolize a diverse synthetic insecticide. For example, CYP49A1 expressed in Bombyx mori to metabolize phoxim , Cyp4BN13v1, and Cyp4BN15 were highly expressed in Leptinotarsa decemlineata larvae and involved in cyhalothrin detoxification (Wan et al., 2013) and CYP4 genes in Diaphorina citri are associated with the development of insecticide resistance (Killiny, Hajeri, Tiwari, Gowda, & Stelinski, 2014). For imidacloprid metabolism, previous work has demonstrated that several CYPs in different insect species, such as CYP6A1 in N. lugens (Ding et al., 2013), CYP6CM1vQ in B. tabaci (Karunker et al., 2009), and CYP6G1 in D. melanogaster (Joußen et al., 2008) have degrading capability.
Most of them are resistance associating and from family 6. In our laboratory, the resistance associating CYPs from family 6 has also been confirmed for degrading imidacloprid in L. striatellus. Here we pres-