Integrated analysis of cytochrome P450 gene superfamily in the red flour beetle, Tribolium castaneum
© Zhu et al.; licensee BioMed Central Ltd. 2013
Received: 17 December 2012
Accepted: 27 February 2013
Published: 14 March 2013
The functional and evolutionary diversification of insect cytochrome P450s (CYPs) shaped the success of insects. CYPs constitute one of the largest and oldest gene superfamilies that are found in virtually all aerobic organisms. Because of the availability of whole genome sequence and well functioning RNA interference (RNAi), the red flour beetle, Tribolium castaneum serves as an ideal insect model for conducting functional genomics studies. Although several T. castaneum CYPs had been functionally investigated in our previous studies, the roles of the majority of CYPs remain largely unknown. Here, we comprehensively analyzed the phylogenetic relationship of all T. castaneum CYPs with genes in other insect species, investigated the CYP6BQ gene cluster organization, function and evolution, as well as examined the mitochondrial CYPs gene expression patterns and intron-exon organization.
A total 143 CYPs were identified and classified into 26 families and 59 subfamilies. The phylogenetic trees of CYPs among insects across taxa provided evolutionary insight for the genetic distance and function. The percentage of singleton (33.3%) in T. castaneum CYPs is much less than those in Drosophila melanogaster (52.5%) and Bombyx mori (51.2%). Most members in the largest CYP6BQ gene cluster may make contribution to deltamethrin resistance in QTC279 strain. T. castaneum genome encodes nine mitochondrial CYPs, among them CYP12H1 is only expressed in the final instar larval stage. The intron-exon organizations of these mitochondrial CYPs are highly diverse.
Our studies provide a platform to understand the evolution and functions of T. castaneum CYP gene superfamily which will help reveal the strategies employed by insects to cope with their environment.
KeywordsInsecticide resistance Gene cluster Molecular docking Mitochondrial CYPs
Insects appeared more than 450 million years ago [1, 2] and have been known to be the unprecedented evolutionally successful metazoans on the earth. One of the factors that may contribute to this success is the ability of insects to adapt to almost every ecological niche by virtue of traits such as metamorphosis and flight [3, 4]. In the meantime, the radiation of insects into diverse habitats and food sources largely enhanced the risk for them to be exposed to toxic or otherwise life-threatening conditions. Insect CYPs impact on the ability of insect adaptation to diverse habitats. On one hand, CYPs have very important physiological functions during all life stages of insects. They might be involved in the biosynthesis pathway of endogenous compounds, such as molting hormone (20-hydroxyecdysone, 20E is the most active form)  and juvenile hormone (JH)  that are the key factors in regulating metamorphosis, development, and reproduction. Some insect CYPs are also involved in the degradation of pheromones [6, 7] as well as catalysis and hydroxylation of fatty acids , which are critical for chemical communication, behavior [7, 9] and metabolism. On the other hand, as a group of environmental response genes , some CYPs protect insects by detoxifying xenobiotics including synthetic insecticides [11–13] and plant allelochemicals [14, 15], resulting in the adaption of insects to the chemical stresses. In a way, the functional and evolutionary diversification (“bloom”) of insect CYPs has shaped the success of insects.
As microsomal pigments, CYPs have an absorption peak at 450 nm when reduced and saturated with carbon monoxide . The bacterial CYPs are water soluble while eukaryotic CYPs are membrane-bound proteins located either on the endoplasmic reticulum or the inner mitochondrial membrane. P450s are hemoproteins and act as the terminal oxidases in the monooxygenase system . The three components of the P450 monooxygenase system are P450, which acts as the substrate binding protein, NADPH-cytochrome P450 reductase (CPR), which transfers electrons from NADPH to CYPs, and cytochrome b5, which transfers electrons from NADH to CYPs in some P450 monooxygenase systems as an additional potential electron donor .
CYPs constitute one of the largest and oldest gene superfamilies that are found in virtually all aerobic organisms . Insects typically contain tens to more than one hundred individual P450 genes in their genomes (http://drnelson.uthsc.edu/CytochromeP450.html). Genome annotation efforts identified 90 CYPs in D. melanogaster, 111 CYPs in Anopheles gambiae, 84 CYPs in B. mori, 48 CYPs in Apis mellifera, 164 CYPs in Aedes aegypti, 204 CYPs in Culex quinquefasciatus, 38 CYPs in Pediculus humanus humanus, and 143 CYPs in T. castaneum. T. castaneum, commonly known as the red flour beetle, is the first beetle having its genome sequenced. T. castaneum is a notorious worldwide pest of stored grains and farinaceous materials [26, 27]. It has developed resistance to all five classes of insecticides and fumigants used against it . Moreover, the functional genomics method, RNAi, works systemically in almost every tissue and developmental stage of T. castaneum[28, 29]. These characters make T. castaneum an ideal insect model for conducting functional genomics, investigating the mechanisms of insecticide resistance, and exploiting potential new insecticide targets for pest control. Although several T. castaneum CYPs, CYP6BQ9, CYP306A1[31–33], CYP314A1[31–33] had been functionally investigated in our previous studies, the role of the majority of CYPs remains largely unknown. Here, we analyzed the phylogenetic relationship of all T. castaneum CYPs with genes in other insect species, examined the CYP6BQ gene cluster organization, function and evolution, as well as investigated the mitochondrial CYPs intron-exon and gene expression patterns which provide platform to understand the evolution and predict the functions of T. castaneum P450 genes.
Results and discussion
Annotation of T. castaneumCYPs
Number of T. castaneum CYP families, subfamilies, pseudogenes, and genes in each insect P450 clan
7 (CYP15, 18, 303-307)
8 (CYP12, 49, 301, 302, 314, 315, 334, 353)
6 (CYP6, 9, 345-348)
5 (CYP4, 349-352)
Phylogenetic analysis of T. castaneumCYPs
Within mitochondrial clan, four out of nine T. castaneum CYPs show distinct 1:1:1:1 orthologies with genes from other insect species (Figure 1B). The three CYPs CYP302A1, CYP314A1, and CYP315A1 encoded by D. melanogaster Halloween genes disembodied (dib), shade (shd), and shadow (sad) respectively are involved in ecdystroid biosynthesis . Since the T. castaneum CYP302A1, CYP314A1, and CYP315A1 share high sequence similarity with those of D. melanogaster, they very likely have similar functions in T. castaneum. The T. castaneum genome contains a single CYP12H1 gene in CYP12 family that includes genes associated with insecticide resistance in the house fly Musca domestica and D. melanogaster.
T. castaneum genome encodes largely expanded CYP3 (27 subfamilies, 79 individual genes) and CYP4 (15 subfamilies, 47 individual genes) clans, especially the families 4 (27 genes), 6 (40 genes), and 9 (23 genes) (Figure 1C and D, see Additional file 1). Genes in these two clans appear to undergo exceedingly species-specific radiations. The CYP6 family is evolutionary related to vertebrate CYP3 and CYP5 families [1, 43]. T. castaneum CYP6 family merely has one CYP6B subfamily. All CYP6 genes in T. castaneum and A. gambiae are clustered in one clade within species, whereas CYP6 genes in D. melanogaster clustered into several clades in the phylogenetic tree (Figure 1C). In dipteran and lepidopteran insects, a number of CYP6 genes were shown to be involved in resistance to a wide range of insecticides and detoxification of plant allochemicals through either constitutive overexpression and/or inducible expression in resistant strains [11, 15, 44, 45]. In T. casta-neum deltamethrin-resistant QTC279 stain, CYP6BQ9, a brain-specific P450, is constitutively overexpressed in resistant strain and is responsible for the majority of delta-methrin resistance . D. melanogaster CYP6 gene, Cyp6a20, is expressed in the non-neuronal support cells of olfactory sensilla associated with pheromone-sensing, and its expression level is correlated with the influence of social experience on aggressiveness [7, 9].
T. castaneum CYP9 family is the second biggest family in the Clan 3 (Figure 1C). Several members in this family are known to be associated with insecticide resistance and metabolism of odorant compounds [46–50]. Genes in CYP4 clan show high diversity in their sequences and functions. In Clan 4, CYP4 family is the largest gene family that has members from the vertebrates and insects as well as Caenorhabditis elegans. It is the only family in Clan 4 that has been studied in other insect species (Figure 1D, see Additional file 1). Except CYP4AA1, CYP4G7, CYP4G14 have 1:1:1:1 orthologs in three other species, all other CYP4 genes in T. castaneum are clustered in several clades within species (Figure 1D). Members of family CYP4 in other insects are known to be associated with biosynthesis of endogenous compounds [51, 52], pheromone metabolism [46, 53], and pyrethroid insecticide resistance [49, 54–56]. It is interesting that Antheraea yamamai CYP4G25 is associated with diapauses in the pharate first instar larvae , indicating that the large complement of CYP4 CYPs might have much more diverse functions beyond what we appreciated, perhaps even more diversified than the CYP3 clan .
Genomic distribution of T. castaneumCYPs
CYP6BQ cluster in T. castaneum
The tissue specific expression pattern of genes within the same Clan is largely conserved in QTC279 strain (Figure 4B). The only exception is CYP6BQ11; this may imply potentially novel physiological functions for this gene. Genomic sequence analysis showed that the 12 members of CYP6BQ cluster share highly similar intron-exon structures (see Additional file 3). Each gene consists of three exons, with exon one having 1123-1147 bp, exon two containing about 253-256 bp, and exon three consisting of approximately 163-169 bp. There are only two exceptions, CYP6BQ3P containing a single exon and CYP6BQ5 containing two exons. The length of intron ranges from about 44 bp to 54 bp. The first introns in all genes follow the canonical GT/AG rule. The differential expression profiles of the cluster genes showed that with the exception of CYP6BQ3P and CYP6BQ5, all other 10 genes have significantly higher mRNA levels in QTC279 resistant strain than in the Lab-S susceptible strain (see Additional file 4). Moreover, six out of these 10 genes were induced by deltamethrin (115 mg/48 cm2 that caused 50-60% mortality of beetles in QTC279 strain) after 12 h exposure to the chemical (see Additional file 5) suggesting a potential contribution of these cluster members to the deltamethrin resistance in QTC279 strain.
Mitochondrial CYPs in T. castaneum
The mitochondrial CYPs form a unique branch in the phylogenic tree of animal CYPs . To date, mitochondrial CYPs are only found in animals, but not in fungi and plants . There is a minor group in the total P450 family members of animals compared with the microsomal CYPs. In T. castaneum, only nine out of 143 CYPs are found in mitochondria. In vertebrates, mitochondrial CYPs are generally specialized in the metabolism of steroid or vitamin D, in contrast with microsomal CYPs that show considerably extensive substrate specificities [1, 62]. Whereas, insect mitochondrial CYPs show somewhat structural and functional diversity, which suggests that they have undergone several blooms [1, 2]. There are at least two groups of mitochondrial CYPs in insects. One is CYP12 family including variable number of genes across different taxa that are rapidly evolving . Three CYP12A genes were cloned from the house fly . Among them, CYP12A1 is constitutively overexpressed in diazinon resistant strain and metabolizes insecticides and other xenobiotics but not ecdysteroids. D. melanogaster has seven CYP12 members in its CYPome (Figure 1B). The overexpression of CYP12A4 in a natural population confers the lufenuron resistance . CYP12D1 was observed to be overexpressed in a DDT-resistant strain and induced by xenobiotics [42, 65]. There are four CYP12F genes in A. gambiae CYPome (Figure 1B). It was reported that CYP12F1 is constitutively overexpressed in both DDT-resistant strain (ZAN/U) and permethrin-resistant strain (RSP) . The close association with xenobiotic resistance in the group of insect CYP12 genes demonstrates the evolutionary differentiation between insects and vertebrates. The other group of insect mitochondrial CYPs show sequence conservation (Figure 1B) and include three Halloween genes that are the orthologs of the C22, C2, and C20 hydroxylases that function in the biosynthesis of ecdysteroids  as well as genes with unknown functions. These genes are considered to perform essential physiological functions during insect development and reproduction .
In summary, integrated studies including annotation, phylogenetic analysis, gene expression as well as molecular modeling and docking were conducted for T. castaneum CYPs. 143 CYPs were identified and classified into 4 clans, 26 families and 59 subfamilies suggesting the CYP number in T. castaneum is three fold higher than honeybee and body louse, substantially higher than D. melanogaster and A. gambiae but significantly lower than A. aegypti and C. quinquefasciatus. The relatively large CYP gene superfamily in T. castaneum may contribute to the remarkable ability of insecticide resistance in this beetle. Current studies provided insights into the evolution of T. castaneum CYP gene superfamily and developed a valuable resource for the functional genomics research which will help to understand the strategies employed by insects to cope with their environment and to exploit potential new insecticide targets for pest control.
Red flour beetle strains
Three red flour beetle strains were used in this study. QTC279, originally collected from wheat storage in Malu, Queensland, Australia in 1984, was selected with pyrethroids for 10 generations until it was homozygous for the major pyrethroid resistance factor (2). LBS is an insecticide-susceptible strain. GA strain was used in whole genome sequencing project. These three strains were obtained from Dr. R.W. Beeman (U.S. Grain Marketing Research Laboratory of USDA, KS). Beetles were reared in whole wheat flour with brewers’ yeast (10% by weight) and maintained in darkness at 32°C and 55±2% relative humidity.
Phylogenetic tree construction
All CYP sequences in insects which have the full open reading frames (ORFs) were extracted from the National Center for Biotechnology Information (NCBI) (Bethesda, MD) (http://www.ncbi.nlm.nih.gov/). The insect CYP amino acid sequences were analyzed using ClustalW alignment through Molecular Evolutionary Genetic Analysis software version 5 (MEGA 5) (http://www.megasoftware.net/) . To significantly improve the alignments, the pair-wise alignment was performed with the gap opening penalty at 10 and the gap extension penalty left at default 0.1. The multiple alignment was conducted with the gap opening penalty at 3 and the gap extension penalty at 1.8 . The sites containing obviously missing data or alignment gaps were eliminated in a pair-wise manner. A p-distance < 0.8 when carrying out the compute overall mean distance suggested the alignment was acceptable . Subsequently, the alignment result was converted to a MEGA file (.meg) and submitted to construct the phylogenetic tree with neighbor-joining algorithm. A total of 2,000 bootstrap replications were used to test of phylogeny. Ultimately, the condensed tree was created with cut-off value of 50%.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from adult beetles using TRI reagent (Molecular Research Center Inc., Cincinnati, OH). The qRT-PCR was performed in Applied Biosystems StepOnePlus™ Real-Time PCR System (Life technologies™, http://Carlsbad, CA). Total RNA was isolated from 3 adult beetles or 3-30 tissues for each sample and the RNA was treated with DNase I (Ambion Inc., Austin, TX). cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). DNase I treated total RNA was used as a template. Each qRT-PCR reaction (10 μl final volume) contained 5 μl FastStart SYBR Green Master (Roche Diagnostics, Indianapolis, IN), 1.0 μl of cDNA, 3.6 μl ddH2O, and 0.4 μl each of forward and reverse gene specific primers (stock 10 μM). An initial incubation of 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, 55°C for 60 s settings were used. A fluorescence reading determined the extension of amplification at the end of each cycle. The most stable reference gene, rp49, was used for the housekeeping gene . Both the PCR efficiency and R2 (correlation coefficient) value were taken into consideration in estimating the relative quantities. Each experiment was repeated at least three times using independent biological samples.
Gene fragment isolation
Total RNAs were isolated from beetles in QTC279, GA, and LBS and cDNA was synthesized using iScript cDNA synthesis kit as described as above. The PCR products for CYP6BQ8, CYP6BQ9, CYP6BQ10 and CYP6BQ11 were amplified using primer pairs that were designed based on the sequences in NCBI database. The PCR products were cloned into pGEM®-T Easy Vector (Promega) and sequenced. Cloning and sequence analyses of P450 gene fragments were repeated at least three times with different preparations of RNAs. Three clones from each replication were sequenced.
Deltamethrin induction experiments
One to two weeks old resistant QTC279 beetles were exposed to filter paper surface treated with deltamethrin [26, 27]. According to preliminary study, 115 mg/48 cm2 deltamethrin that resulted in 50-60% mortality for QTC279 beetles was chosen for the experiment. The surviving beetles were collected for RNA extraction after 0, 6, 12, 24 h exposure to deltamethrin. The experiments were repeated three times. The statistical significance of the gene expression was calculated using a one-way analysis of variance (ANOVA) for multiple sample comparisons (SAS v9.4 software). A value of P ≤ 0.05 was considered statistically significant.
Homology modeling and ligand docking
Initial protein models were constructed by submitting the translated amino acid sequences to the I-TASSER server . The I-TASSER output includes up to 5 predicted models as well as predicted ligand binding sites. In the case of the P450 proteins modeled for this study the top scoring ligand binding site predictions all included heme bound to the conserved iron binding cysteine. For further model refinement the top scoring model was submitted to the FG-MD server for fragment guided molecular dynamics structure refinement . The coordinates for heme were manually transferred to the refined model PDB file and a covalent bond was created between the heme iron and the conserved cysteine residue for each P450 modeled in this study. Model quality was examined by Ramachandran plots generated with Procheck . Ramachandran plots of the P450 models gave a range of 97.8% to 98.9% of residues within the generously allowed regions and 2.2% or less in disallowed regions. Molecular docking was performed with Autodock Vina v1.1.2. . Ligand (deltamethrin, permethrin, DDT, and imidacloprid) structures were retrieved from the Zinc database . Proteins and ligands were prepared for docking with Autodock Tools v1.5.4  For all dockings a search space with a grid box of 20 x 20 x 20 Å, centered at the heme bound Fe for each P450.
NADPH-cytochrome P450 reductase
Open reading frame
quantitative real-time PCR
We thank Dr. R.W. Beeman (U.S. Grain Marketing Research Laboratory of USDA) for providing Tribolium castaneum strains and Dr. Byron Reid (Bayer Environmental Sciences) for the gift of technical grade deltamethrin. This work was supported by the US Department of Agriculture National Research Initiative Cooperative State Research, Education, and Extension Service Grant 2007-04636 and National Research Initiative of the USDA-NIFA (2011-67013-30143) to S.R.P. This is contribution number 13-08-41 from the Kentucky Agricultural Experimental Station.
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