Identification and Characterization of CYP6 Family Genes from the Oriental Fruit Moth (Grapholita molesta) and Their Responses to Insecticides

Simple Summary Eight CYP6 family genes were identified in Grapholita molesta (Busck). The expression of individual gene members differed between the developmental stages and insect tissues. High expression was found in third/fourth-instar larvae and in the midgut and Malpighian tubules. The response patterns of the genes exhibited diverse response patterns to the three representative insecticides were diverse. Abstract Cytochrome P450 (CYP) monooxygenases comprise a superfamily of proteins that detoxify xenobiotics and plant secondary metabolites in insects. The CYP6 family is unique to the class Insecta, and its members participate in the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of eight CYP6 family genes from Grapholita molesta (Busck), a global pest of pome fruits. P450 genes with the exception of CYP6AN35, which was most highly expressed in adults, consistently showed high expression in third- or fourth-instar larvae. The analysis of different tissues of adults showed that most of these genes were predominantly expressed in the midgut, Malpighian tubules, and/or fat body. The expression of these eight CYP6 genes was differentially affected by three representative insecticides: malathion (organophosphate), deltamethrin (pyrethroid), and chlorantraniliprole (carbamate). All eight CYP6 genes responded to malathion treatment. Only three CYP6 genes were highly expressed in deltamethrin-treated individuals. Chlorantraniliprole treatment exerted weak effects on gene expression. Interestingly, CYP6AN35 was a highly expression level in the adult head and its expression was induced by all three insecticides. CYP6AN35 may be a key gene in the metabolism of insecticides. This study provides a fundamental understanding of the functions of the CYP6 gene family in insecticide metabolism in G. molesta.


Introduction
The global fruit-borne pest Grapholita molesta (Busck) (Lepidoptera: Tortricidae) causes serious harm to horticultural crops such as pear and peach. Specifically, this pest affects the yield and quality of crops by drilling into fruits and sprouts [1][2][3]. Due to the increases in the orchard area and climate change, the population of G. molesta has increased sharply and the damage to fruit trees has become increasingly severe [3][4][5]. The chemical control of this pest remains the dominant strategy, but the repeated use of large amounts of chemical pesticides has led to the development of resistance in this insect [6]. In the United States, Canada and Insects 2022, 13, 300 3 of 15 tubules, spermary, and ovary were harvested from healthy adults of G. molesta within 24 h of emergence, with sixty adults comprising a replicate and three biological repetitions were established.
RNA was extracted from the obtained samples and used as a template for reverse transcription to cDNA. Quantitative PCR (qPCR) was used to quantify the initial expression levels of different genes at different developmental stages and in different tissues of G. molesta adults.

Bioinformatics Analysis
The nucleic acid sequences of eight CYP6 genes were obtained from the transcriptome of G. molesta [27]. ORFfinder (http://www.ncbi.nlm.nih.gov/orffinder, 25 January 2022) was employed to screen nucleic acid sequences with complete ORFs, and the sequences were submitted to the Cytochrome P450 International Nomenclature Committee for naming. NCBI BLASTp was used to align the protein sequences, and the ExPASy ProtParam tool (http://web.expasy.org/protparam/, 25 January 2022) was used for the analysis of parameters such as protein molecular weight, amino acid composition, acid-based amino acid number, and total average hydrophilicity. Signal peptides were examined with the SignalP4.1 server (http://www.cbs.dtu.-dk/services/SignalP/, 25 January 2022). ProtScale (http://web.expasy.org/protscale/, 25 January 2022) was applied for the prediction of protein hydrophobicity and AlphaFold2 (https://colab. research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb, 25 February 2022) was applied for the prediction and simulation of the protein tertiary structure. A MEGA 7.0 cluster analysis of amino acid sequences was performed, and forty-three P450 genes were chosen from B. mori. The neighbor-joining (NJ) method with 1000 bootstrap replicates was used to construct a phylogenetic tree in MEGA 7.0 [30].
We isolated total RNA from the adults using RNAiso Plus (Takara, Dalian, China). The RNA concentration and purity were estimated by using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) with 1 µL of a 50-fold dilution of the RNA. RNA integrity was detected by using 2-5 µL of each sample for agarose gel electrophoresis.
First-strand cDNA was synthesized by following the instructions for the Takara Reverse Transcriptase Kit (Takara, Dalian, China). The reaction product was stored at −20 • C. β-actin of G. molesta was used as an internal reference gene [32]. The qPCR primers were designed using Primer Premier 5.0 software (Supplementary Table S1) and synthesized by Sangon Biotech (Shanghai, China). The PCR primers used are detailed in the Table S1.

Statistical Analysis
After the reactions, the ABI 7500 software data were exported to Microsoft Excel (Microsoft, WA, USA) and analyzed using the 2 −∆∆CT method [33] to determine the expression levels of CYPs in G. molesta exposed to different concentrations.
One-way ANOVA followed by Tukey's honest significant difference (HSD) test at p < 0.05 were performed to determine whether the normalized expression level of the eight CYP6 genes differed significantly among developmental stages, tissues, and insecticide treatments. SPSS 22.0 software (IBM, Armonk, NY, USA) was used. All histograms were constructed using SigmaPlot 12.0 (Systat Software, Inc., San Jose, CA, USA).

Identification and Characteristics of CYP6 Gene Members in G. molesta
Eight CYP6 genes were identified from the transcriptomic data of G. molesta (Supplementary Table S2). These genes encode putative proteins consisting of 498-530 amino acid residues, with relative molecular weight of 58-61 kDa and isoelectric points (pIs) of 6.55-8.84. The instability coefficients were less than 40% and CYP6AB112-CYP6AB115 does not contain signal peptide (Supplementary Tables S3 and S4). Multiple-sequence alignment of the eight genes revealed common CYP signature sequences (Supplementary Figure S1). All genes contain the characteristic sequences of the heme-binding region, i.e., the 'FXXGXX XCXG/A' sequence in the meander, 'PXXFXPXXF', and the P450 oxygen binding sequence, i.e., 'A/GGXD/ETT/S'. The newly obtained protein sequences were aligned with protein sequences of known functions, and the structures and functions of the new protein sequences were predicted. The proteins encoded by the eight CYP6 genes had similar tertiary structures ( Figure 1). The purple region was the heme-binding region, which is the most conserved structural center of CYP6, with a noncovalently bound heme and the surrounding highly conserved cysteine sequence 'FXXGXXXCXG/A'. crosoft, Redmond, USA) and analyzed using the 2 −△△CT method [33] to determine the expression levels of CYPs in G. molesta exposed to different concentrations.
One-way ANOVA followed by Tukey's honest significant difference (HSD) test at p < 0.05 were performed to determine whether the normalized expression level of the eight CYP6 genes differed significantly among developmental stages, tissues, and insecticide treatments. SPSS 22.0 software (IBM, Armonk, NY, USA) was used. All histograms were constructed using SigmaPlot 12.0 (Systat Software, Inc., San Jose, CA, USA).

Identification and Characteristics of CYP6 Gene Members in G. molesta
Eight CYP6 genes were identified from the transcriptomic data of G. molesta (Supplementary Table S2). These genes encode putative proteins consisting of 498-530 amino acid residues, with relative molecular weight of 58-61 kDa and isoelectric points (pIs) of 6.55-8.84. The instability coefficients were less than 40% and CYP6AB112-CYP6AB115 does not contain signal peptide (Supplementary Table S3, Supplementary Table S4). Multiple-sequence alignment of the eight genes revealed common CYP signature sequences (Supplementary Figure S1). All genes contain the characteristic sequences of the heme-binding region, i.e., the 'FXXGXXXCXG/A' sequence in the meander, 'PXXFXPXXF', and the P450 oxygen binding sequence, i.e., 'A/GGXD/ETT/S'. The newly obtained protein sequences were aligned with protein sequences of known functions, and the structures and functions of the new protein sequences were predicted. The proteins encoded by the eight CYP6 genes had similar tertiary structures ( Figure 1). The purple region was the heme-binding region, which is the most conserved structural center of CYP6, with a noncovalently bound heme and the surrounding highly conserved cysteine sequence 'FXXGXXXCXG/A'.

Phylogenetic Analysis
A phylogenetic analysis of the G. molesta CYP6 proteins along with those known in B. mori was performed [34]. All eight genes belonged to the CYP3 clan and six of them and the B. mori CYP6 subfamily formed a cluster, whereas CYP6B74 and CYP6AB117, respectively, formed a cluster with other genes of the CYP6 subfamily in B. mori ( Figure 2).

Phylogenetic Analysis
A phylogenetic analysis of the G. molesta CYP6 proteins along with those known in B. mori was performed [34]. All eight genes belonged to the CYP3 clan and six of them and the B. mori CYP6 subfamily formed a cluster, whereas CYP6B74 and CYP6AB117, respectively, formed a cluster with other genes of the CYP6 subfamily in B. mori ( Figure 2).

Developmental Stage and Tissue Specificity of P450 Gene Expression
To gain insight into the functional differences, the relative mRNA expression levels of the eight genes in different developmental stages, including eggs, first-to fifth-instar larvae, pupae, and adults, were examined. As shown in Figure 3, CYP6AB112, CYP6AB114, and CYP6B74 expression increased as development progressed until it peaked in thirdinstar larvae, and CYP6B74 peaked in fourth-instar larvae. CYP6AB113 and CYP6AB117 exhibited the same expression pattern, which consisted of gradually increasing before the fourth-instar stage, peaking at the third-instar stage, and showing a small increase from the fourth-to fifth-instar larval stage. CYP6AN35 and CYP6AB115 were found at high expression levels in adults. CYP6AN35 expression increased gradually, and reached the highest level in adults, and CYP6AB115 expression peaked in the third-instar larval stage. CYP6AB116 showed little change in expression across the different stages and exhibited significantly higher expression in the egg stage than at the other stages.
The expression patterns of the eight CYP6 genes among different tissues, including head, foregut, midgut, hindgut, fat body, Malpighian tubules, spermary, and ovary, were also examined. The results showed that the highest expression of CYP6AN35 was found in the head and that the other seven CYP6 genes were highly expressed in the midgut. In addition, the expression of CYP6AB112, CYP6AB114, CYP6AB115, and CYP6AB117 was widely distributed in Malpighian tubule, and CYP6AB115 expression was significantly highest in Malpighian tubule than in other tissues. The expression of CYP6AB115 was higher in the ovary than in other tissues. CYP6AB113 and CYPAB116 exhibited high expression in fat bodies, and the expression of CYP6AB112, CYP6AB113, and CYP6AB116 was higher in the spermary ( Figure 4).

Developmental Stage and Tissue Specificity of P450 Gene Expression
To gain insight into the functional differences, the relative mRNA expression levels of the eight genes in different developmental stages, including eggs, first-to fifth-instar larvae, pupae, and adults, were examined. As shown in Figure 3, CYP6AB112, CYP6AB114, and CYP6B74 expression increased as development progressed until it Expression of eight CYP6 genes at different developmental stages of G. molesta Note: egg (Egg), first-instar larvae (1st), second-instar larvae (2nd), third-instar larvae (3rd), fourth-instar larvae (4th), fifth-instar larvae (5th), pupae (Pu), and adults (Ad). Each treatment included three biological replicates. The data in the bar charts are presented as the means ± SEs. Different letters indicate significant differences at the p < 0.05 level, as determined by ANOVA and Tukey's HSD test.
The expression patterns of the eight CYP6 genes among different tissues, including head, foregut, midgut, hindgut, fat body, Malpighian tubules, spermary, and ovary, were also examined. The results showed that the highest expression of CYP6AN35 was found

Response of CYP Genes Expression to Insecticide Exposure
Three common insecticides, namely, malathion, deltamethrin, and chlorantraniliprole were selected to evaluate their possible effects on the expression of eight CYP6 genes in this study. The transcriptional changes in the eight CYP6 genes in

Response of CYP Genes Expression to Insecticide Exposure
Three common insecticides, namely, malathion, deltamethrin, and chlorantraniliprole were selected to evaluate their possible effects on the expression of eight CYP6 genes in this study. The transcriptional changes in the eight CYP6 genes in adult moths exposed to the insecticides were determined by RT-qPCR. Malathion treatment increased the CYP6AB113, CYP6AB115, and CYP6AN35 expression levels in a concentration-dependent manners. In contrast, the expression level of the CYP6AB114 gene decreased and remained lower than the control level. With increases in the malathion concentration, the expression of CYP6AB116 and CYP6AB117 showed fluctuating trends, with peak expression at LC 30 , although the expression changes of these genes were small. No significant differences in CYP6AB112 expression were observed, and the expression of CYP6B74 was a high expression only with the LC 50 treatment ( Figure 5).
Insects 2022, 13, x FOR PEER REVIEW 8 of 15 adult moths exposed to the insecticides were determined by RT-qPCR. Malathion treatment increased the CYP6AB113, CYP6AB115, and CYP6AN35 expression levels in a concentration-dependent manners. In contrast, the expression level of the CYP6AB114 gene decreased and remained lower than the control level. With increases in the malathion concentration, the expression of CYP6AB116 and CYP6AB117 showed fluctuating trends, with peak expression at LC30, although the expression changes of these genes were small. No significant differences in CYP6AB112 expression were observed, and the expression of CYP6B74 was a high expression only with the LC50 treatment ( Figure 5). The expression of CYP6 genes in G. molesta treated with different concentrations of deltamethrin was analyzed ( Figure 6). Treatment with deltamethrin rapidly decreased the expression of CYP6AB112, CYP6AB114, and CYP6AB116 after 24 h exposure. With increases in the insecticide concentration, the expression of these genes did not increase significantly Insects 2022, 13, 300 9 of 15 and remained markedly lower than that of the control after 24 h exposure. These results demonstrated that the expression of these genes maybe be inhibited by deltamethrin within a period of time. CYP6AB115, CYP6AN35, and CYP6B74 showed the highest expression levels with the LC 30 concentration. Compared with the levels obtained with the control treatment, the CYP6AN35 and CYP6B74 expression levels only increased 2.5-3.0-fold after deltamethrin treatment. In addition, the expression of CYP6AB113 and CYP6AB117 did not significantly differ among the treatments with different deltamethrin concentrations.
The expression of CYP6 genes in G. molesta treated with different concentrations of deltamethrin was analyzed ( Figure 6). Treatment with deltamethrin rapidly decreased the expression of CYP6AB112, CYP6AB114, and CYP6AB116 after 24 h exposure. With increases in the insecticide concentration, the expression of these genes did not increase significantly and remained markedly lower than that of the control after 24 h exposure. These results demonstrated that the expression of these genes maybe be inhibited by deltamethrin within a period of time. CYP6AB115, CYP6AN35, and CYP6B74 showed the highest expression levels with the LC30 concentration. Compared with the levels obtained with the control treatment, the CYP6AN35 and CYP6B74 expression levels only increased 2.5-3.0-fold after deltamethrin treatment. In addition, the expression of CYP6AB113 and CYP6AB117 did not significantly differ among the treatments with different deltamethrin concentrations. Treatment with different concentrations of chlorantraniliprole (Figure 7) decreased the expression levels of CYP6AB114, CYP6AB116, CYP6AB117, and CYP6B74, and the fold changes in expression showed subtle differences among the treatments. Among these genes, CYP6AB116 and CYP6AB117 exhibited the same expression pattern. The expression of CYP6AB112 and CYP6AB113 peaked at LC 10 , reaching 1.8-4.0-fold that obtained with the control treatment, and then decreased with increases in the chlorantraniliprole concentration. Compared with the levels obtained with the control treatment, CYP6AB115 expression decreased with the lower chlorantraniliprole concentrations and increased with the highest concentration of chlorantraniliprole. The CYP6AN35 expression levels were highest with the LC 30 treatment, and these levels were higher than those obtained with the control treatment.
Treatment with different concentrations of chlorantraniliprole (Figure 7) decreased the expression levels of CYP6AB114, CYP6AB116, CYP6AB117, and CYP6B74, and the fold changes in expression showed subtle differences among the treatments. Among these genes, CYP6AB116 and CYP6AB117 exhibited the same expression pattern. The expression of CYP6AB112 and CYP6AB113 peaked at LC10, reaching 1.8-4.0-fold that obtained with the control treatment, and then decreased with increases in the chlorantraniliprole concentration. Compared with the levels obtained with the control treatment, CYP6AB115 expression decreased with the lower chlorantraniliprole concentrations and increased with the highest concentration of chlorantraniliprole. The CYP6AN35 expression levels were highest with the LC30 treatment, and these levels were higher than those obtained with the control treatment.  The data in the bar charts are presented as the means ± SEs. Different letters indicate significant differences at the p < 0.05 level, as determined by ANOVA and Tukey's HSD test.

Discussion
CYP monooxygenases constitute a common superfamily in insects. Studies have shown that these enzymes can participate in the detoxification of xenobiotics and plant secondary metabolites in insects [35,36]. The CYP6 family is unique to the class Insecta, and the biochemical functions of its members are predominantly associated with the metabolism of xenobiotics [37]. Many studies have contributed to our understanding of the relationships between the molecular characteristics and metabolic detoxification functions of CYP genes [38]. We previously performed transcriptome sequencing of G. molesta and obtained 77 P450 unigenes [27]. In the present study, eight CYP6 family genes containing conserved sequences of CYPs were identified by transcriptomic and bioinformatics analyses. These genes were named by the Cytochrome P450 International Nomenclature Committee as follows: CYP6AB112, CYP6AB113, CYP6AB114, CYP6AB115, CYP6AB116, CYP6AB117, CYP6AN35, and CYP6B74. According to their molecular characteristics, these eight genes had typical CYP structures and shared the closest relationships with genes from the Lepidoptera species C. pomonella, Manduca sexta, B. mori, Amyelois transitella, Heortia vitessoides, Ostrinia furnacalis, and Cnaphalocrocis medinalis [39][40][41]. These results provide an important basis for the construction of three-dimensional structures that are crucial for understanding protein function at the molecular level. Starting from the amino acid sequences of the interacting proteins, both the stoichiometry and the overall structure of the complex were predicted by homology modeling which could help to facilitate further study of the functions of the genes at the protein level [42,43].
In G. molesta, the expression of CYP6 gene members differed among the different developmental stages, which suggested that these genes may be involved in different metabolic processes. Seven of these eight CYP6 genes exhibited low expression in eggs, only CYP6AB116 which may participate in worm survival and egg development presented slightly high expression. For example, in Schistosoma mansoni, a high expression level of CYP3050A1 is found in eggs and double-stranded RNA (dsRNA) silencing results in worm death [44]. The expression of the CYP6AB genes was significantly higher during the third-instar larval stage of G. molesta and the highest expression of CYP6B74 was found at the fourth-instar larvae. These genes may be involved in the metabolism of foreign substances because, third and fourth instars are the stages involving the large-scale feeding of G. molesta, which possibly require more enzymes to metabolize exogenous substances. In Bradysia odoriphaga, CYP3356A1 presented the highest expression level in fourth-instar larvae and is related to the metabolism of exogenous substances [45]. CYP6AN35 and CYP6AB115, which are highly expressed in adults, may participate in the detection and metabolism of foreign substances. In L. migratoria, high expression levels of LmCYP6HQ1 were found in adults, through RNAi knockdown of LmCYP6HQ1, which suggested that the expression of genes enhances the locust's ability to detoxify insecticides [22]. CYP6BQ23 in Meligethes aeneus is also significantly and highly overexpressed (∼900-fold) in adults of pyrethroid-resistant strains compared with susceptible strains, and is the primary mechanism conferring pyrethroid resistance [46].
In G. molesta, the expression intensity of CYP6 members was tissue specific. CYP6AN35 was most highly expressed in the head and may be related to the recognition and metabolism of toxics substances. In Plutella xylostella (Linnaeus), high expression of CYP4BN6 in the head of Tribolium castaneum has been suggested to be associated with insecticide susceptibility [47]. Studies have shown that certain CYPs with a high expression level in the insect head participate in olfaction or detoxification [48]. Among the eight CYP6 genes analyzed in this study, all of the genes with the exception of CYP6AN35 exhibited higher levels to varying degrees in the Malpighian tubules and fat body. Similar results have been obtained in many other studies on P450s [49]. Most of the P450 enzymes functioning as metabolic detoxification are highly expressed in the midgut, fat body, and Malpighian tubules. Midgut and fat body are major tissues involved in the detoxification of allelochemicals [50,51]. In addition to maintaining the salt and water balance, the Malpighian tubules play key roles in the detoxification and excretion of xenobiotics [52]. CYP6AB56 is highly expressed in the fat body of Mamestra brassicae Linnaeus and RNA interference followed by a deltamethrin bioassay showed that it plays an important role in deltamethrin detoxification [53]. These findings indicate that in G. molesta, CYP6 genes may be involved in metabolic detoxification. The diversity of the gene distribution in the insect body indicates that the genes may be involved in different metabolic activities [20,54].
Many results have demonstrated that CYPs participate in the insecticides' metabolism. To clarify which CYP genes are involved in the metabolism of insecticide, we investigated the effect of the three insecticides on CYP gene expression. Gene exposure to endogenous and exogenous substances was induced differently at different times. Zhang et al. found that the expression of many P450 genes reached the highest level at 24 h after induction [55]. Our study considered gene expression changes after 24 h exposure, and the result indicated a complex relationship between CYP genes and insecticide treatments. Among these eight CYP6 genes, CYP6AB112 was only highly expressed after chlorantraniliprole treatment, was uninfluenced by malathion, and was downregulated by deltamethrin. This result preliminary certifies that this gene may play roles in the chlorantraniliprole metabolism. Similar results have been reported for CYP6FD1 in L. migratoria, which was inhibited by deltamethrin treatment and no significant increase in mortality was found after RNAi knockdown [37]. We also found that CYP6AB115 and CYP6AN35 expression increased after treatment with the three insecticides, and these two genes may play an important role in metabolic detoxification in G. molesta. The same result for B. odoriphaga CYP3356A1 revealed its association with the metabolism of three xenobiotics, imidacloprid, thiamethoxam, and beta-cypermethrin through RNAi [45]. The expression of CYP6AB114 showed the same decreasing pattern in response to different insecticides after 24 h exposure. Treatment with different concentrations resulted in lower expression levels of this gene than those obtained with the control treatment, and this gene may thus play a role in the adaptive response to external stimuli. Decreased expression of P450 is a homeostatic or adaptive response, and suppressed in response to pathophysiological signals and various exogenous or endogenous compounds [56,57]. CYP6AB116 and CYP6AB117 expression showed little change across the different concentrations of these three insecticides, which suggested little involvement of these genes in resistance to these chemicals. CYP6B74 expression was induced by malathion and deltamethrin, and may participate in the metabolism of these two insecticides. A study on the CYP6B family showed that CYP6B1 metabolizes organophosphorus diazinon, and CYP6B8 metabolizes both diazinon and the pyrethroid cypermethrin [58]. CYP6BB2 upregulation can be observed in pyrethroid-resistant populations and is associated with pyrethroid resistance [59].
CYP6AN35 exhibited a special expression pattern in the head of G. molesta adults, and our results showed that the expression of this gene tended to be induced by three insecticides, which indicates that this gene might be a key gene for further exploration. For example, the upregulation of CYP6G1 confers resistance to DDT [60] and high expression of CYP9AQ2 plays an important role in deltamethrin detoxification in L. migratoria [61]. An analysis of insect detoxification enzymes has been proposed as a means for identifying the main enzymes involved in insecticide detoxification [62]. Determining the genes that participated in the insecticide metabolism remains difficult in our study due to the diversity, rapid evolution, and lack of information about the substrate specificity of cytochrome P450 enzymes [63]. Our study clearly demonstrates that the expression of multiple P450 genes was significantly enhanced after treatment with different insecticides. The diversity of cytochrome P450 genes in organisms constitutes an adaptation of insects to their plant hosts and environmental change. This result provided possible target genes for the exploration of the metabolic detoxification function in G. molesta and corroborated the finding that G. molesta cytochrome P450 responds by changing its expression in the face of pesticide stress. Our findings revealed the functional importance of cytochrome P450 genes in response to insecticide exposures, the detoxification of insecticides, the selection of insecticides for G. molesta control, and the evolution of insecticide resistance in G. molesta.

Conclusions
In this study, eight cytochrome P450 genes were identified and found to exhibit various expression levels at different developmental stages and in different tissues. Most of these eight genes were highly expressed in third-or fourth-instar larvae and were distributed in detoxification-related tissues, such as the midgut, Malpighian tubules, and/or fat body in adults. Interestingly, CYP6AN35 was found at a high expression level in the adult head and its expression was induced by all three insecticides. CYP6AN35 may be a key gene in the metabolism of insecticides.