Identification and expression profiles of putative cytochrome P450 monooxygenase genes from Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Insects

Fourth-instar larvae of C. medinalis were collected from an experimental field of Anhui Agricultural University, Hefei, Anhui, China. The field studies did not involve endangered or protected species. No specific permits were required for the described field collections. Cnaphalocrocis medinalis is a common agricultural pest and is not included in the “List of Protected Animals in China”. Individuals were anesthetized on ice and dissected into different tissues (integument, midgut, Malpighian tubules and fat body). Each sample was immediately frozen in liquid nitrogen and stored at −80°C until use.

Insecticide treatment

Abamectin (purity >90%) was purchased from Veyong Bio-Chemical Co., Ltd, Shijiazhuang, Hebei, China, and diluted with analytical-grade acetone. The fourth-instar larvae of C. medinalis were exposed to the chemical by topical application, as described previously (Huang et al. 2011). Briefly, a sub-lethal concentration (LD₂₀, 0.12 ng per larva) of abamectin was applied topically on the dorsal part of the larval middle abdomen by using a capillary microapplicator. Control insects were treated with acetone only. Abamectin-treated and control insects were collected after 24 h, frozen in liquid nitrogen immediately, and stored at −80°C before RNA extraction. Each treatment was biologically replicated three times and in each replicate fifteen larvae were collected.

RNA isolation and cDNA synthesis

Total RNA was isolated from different tissues of the C. medinalis larvae, as well as the abamectin-treated and control larvae, using Trizol reagent (Life Technologies, Carlsbad, CA, USA) following the manufacturer's protocol. The concentration of each RNA sample was determined using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Total RNA (1 μg) from each sample was reverse-transcribed using the PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian, China), and then diluted to 10 ng/μL with nuclease-free water.

Transcriptome searching

Previously, a transcriptome dataset of C. medinalis was released (NCBI SRA Acc. no. SRA058899, Li et al. 2012). We searched the dataset using the Basic Local Alignment Search Tool (BLAST, Altschul et al. 1997) to obtain putative P450 genes. The process could be divided into three stages. First, we performed a TBLASTN search to identify all P450 sequences that have full-length or partial open reading frames (ORFs), by using the annotated P450s from other insect species as references. Second, these partial P450 sequences were used as queries to re-screen the dataset; some of them could be assembled and extended to full-length or near full-length genes. Last, the BLASTX best matches of these partial P450s also were used as templates to predict potential targets, and the outputs were examined manually to determine the closest relative to the partial gene. As a result, 36 putative P450 sequences were identified. These putative P450 genes were amplified from whole larva cDNA and were confirmed to be the target genes by direct DNA sequencing (primers were listed in File S1). Furthermore, these sequences were compared by searching against the NCBI non-redundant protein database using BLASTX (cut-off e-value of 10^–5).

Bioinformatic analyses

The functional domains were predicted by searching the NCBI's conserved domain database (http://www.ncbi.nlm.nih.gov/structure/cdd/cdd.shtml). The transmembrane anchors were determined with TMHMM (http://www.cbs.dtu.dk/services/tmhmm). The amino acid sequences of C. medinalis P450s together with their orthologs were aligned using Clustal Omega (http://www.ebi.ac.uk/tools/msa/clustalo). A phylogenetic tree was constructed in MEGA5.05 software using the neighbor-joining method with 1000-fold bootstrap resampling (Tamura et al. 2011). P450 sequences used are listed in File S2.

Real-time quantitative PCR

Real-time quantitative polymerase chain reaction (qPCR) was run on a StepOne Plus Real-time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Premix ExTaq II (Tli RNaseH Plus) (Takara, Dalian, China). Each reaction (20 μL) contained a SYBR Premix ExTaq (10 μL), a cDNA template (1 μL), a sense primer and an anti-sense primer (0.2 μM each) and nuclease-free water. The thermal conditions were one cycle of 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 25 s. A heat-dissociation protocol was added at the end of the thermal cycle to verify that only one PCR product was detected by the fluorescence dye. A pre-run test confirmed the constant expression of the house-keeping gene (β-actin; GenBank Acc. no. JN029806) in each sample, and a no template control (NTC) was also included in the experiment to detect potential contamination. Each sample was run in triplicate from three biological replicates. Quantification of gene expression levels was carried out according to the 2^–ΔΔCt method (Livak & Schmittgen 2001). Primer pairs for qPCR were list in File S1.

Data analyses

The qPCR results were analyzed using the Data Processing System (DPS) software v9.5 (Tang & Zhang 2013). The paired Student's t-test and one-way analysis of variance (anova) with the least significant difference (LSD) test were applied, respectively, for comparing the difference between two samples and the differences among multiple samples. The level of significance was set at P < 0.05.

Identification of P450 genes in C. medinalis

Thirty-six putative P450 genes were identified from the C. medinalis transcriptome dataset. Of these, 29 genes have completed ORFs while 7 genes are incomplete cDNAs, lacking 5′- and/or 3′-termini (Table 1). These sequences were verified by cDNA amplification and direct sequencing. The specific names for these P450 genes were assigned by the Cytochrome P450 Nomenclature Committee (D Nelson, University of Tennessee, Memphis, TN, USA) and were classified into 19 families and 30 subfamilies (Table 1). A new P450 subfamily, CYP321F, which was recently discovered in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) (Wang et al. 2014), was also found in C. medinalis (Table 1).

TMHMM prediction showed that transmembrane regions composed of hydrophobic residues were found in the N-termini of the 20 P450s (Table 1). The region was known to anchor P450s in the endoplasmic reticulum membrane (Feyereisen 2012).

Almost all the P450s showed highly identities to those of other lepidopteran species, with more than 52% identity at the amino acid level (Table 1). The exceptions were CYP314A1 and CYP4G111, which showed 88% and 95% identities, respectively, to their orthologs from the parasitoid wasp Microplitis demolitor (Hymenoptera: Braconidae). Additionally, CYP4L18 identified in this study is a partial fragment of the previously isolated C. medinalis CYP4L18 (GenBank Acc. no. CAX94851). Also, CYP6AE28, CYP6CV1 and CYP4M25 have extremely high identities (>98%) with the previously isolated C. medinalis CYP6AE28 (CAX94849), CYP6CV1 (CAZ65618) and CYP4M25 (CAX94850), respectively, with some small changes in their amino acid residues (Table 1).

A multiple alignment analysis was performed for the deduced protein sequences of these P450s, and the structural characteristics were highlighted (Fig. 1). Among these sequences, the helix-C (WxxxR), helix-K (ExxR) and haem-binding domain (PFxxGxRxCxG) were well conserved, while the PERF motif (PxxFxPE/DRF) and helix-I (GxE/DTT/S) were more variable. One microsomal P450, CYP307A2, adds three residues to the helix-C motif (Fig. 1).

Phylogenetic analysis

In order to better understand the relationships of insect P450s, the C. medinalis P450s together with previously annotated P450s from Drosophila melanogaster (Tijet et al. 2001), Apis mellifera (Claudianos et al. 2006), Bombyx mori (Ai et al. 2011) and Ch. suppressalis (Wang et al. 2014) were included in a phylogenetic analysis. In the neighbor-joining tree (Fig. 2), the 36 C. medinalis P450s fell into four major clades: (i) the mitochondrial clan; (ii) CYP2 clan; (iii) CYP3 clan; and (iv) CYP4 clan (Feyereisen 2012). Most of the C. medinalis P450s (16 genes) were clustered within the CYP3 clan, followed by the CYP4 clan (9 genes). The C. medinalis P450s from the CYP3 clan were clustered into divergent families, such as the CYP6, CYP9, CYP321, CYP324, CYP337, CYP338, CYP345 and other families, whereas P450s from the CYP4 clan were mainly clustered into the CYP4 family (Fig. 2). The mitochondrial and CYP2 clans included six and five C. medinalis P450 genes, respectively. In addition, all of the C. medinalis P450s have close relationships with orthologs from lepidopteran species B. mori and Ch. suppressalis (Fig. 2).

Transcriptional profiles of P450 genes in the CYP6 family

Because the CYP6 P450 family in insects is the major family related to the detoxification of xenobiotics, the expression profiles of five C. medinalis P450 genes (CYP6AW1, CYP6AB62, CYP6CV1, CYP6AE28 and CYP6AE76) in the CYP6 family were investigated by qPCR assays. The results showed that these genes were differentially expressed in various larval tissues, including integument, Malpighian tubules, midgut and fat body (Fig. 3). Midgut expressed significantly higher levels of CYP6AW1 and CYP6CV1 than other tissues. CYP6AE76 was predominantly expressed in the fat body (Fig. 3). The remaining two genes, CYP6AB62 and CYP6AE28, showed no significant difference between Malpighian tubules, midgut and fat body (Fig. 3).

Further, the transcriptional changes of these five genes in larvae exposed to abamectin were determined by qPCR. The transcriptional levels of three genes, CYP6AB62, CYP6CV1 and CYP6AE28, were increased after exposure, but only CYP6AE28 showed significant difference between treated and control individuals (Fig. 4). On the contrary, CYP6AW1 and CYP6AE76 were slightly down-regulated following insecticide exposure, but their differences between abamectin-treated insects and controls were not significant (Fig. 4).