The citrus red mite, Panonychus citri (McGregor) (Trombidiformes: Tetranychidae), a highly destructive pest in citrus groves around the world, has developed resistance to many registered acaricides. Hexythiazox is a selective miticide that has been widely used to control citrus mites in a variety of crops. Forty-six cytochrome P450 mono-oxygenase genes related to general pesticide resistance in other insect species were obtained from the transcriptomes of the hexythiazox-resistant (RR) and hexythiazox-susceptible (SS) strains of P. citri and divided into 4 clans, 15 families and 24 subfamilies. Sequence analyses of each CYP resulted in detection of 3 mutationsin the CYP307A1 gene (841-A to C, 1395-T to C, 1491-T to C) that differed between the 2 strains. Only the change at an amino acid position (278-lysine to glutamine) resulted in a sense mutation. One SNP site was also detected in CYP381A2 (40-A to T) causing a sense amino acid mutation (14-threonine to serine). Seven of these P450s belonged to the CYP2 clan, CYP3 clan and CYP4 clan based on digital gene expression (DGE) library sequencing with a |log2 ratio| value greater than 2, but there were no significant differences revealed by qRT-PCR analysis. This study provides essential information for future research on the hexythiazox-resistance mechanism of P. citri. More methods are needed to further elucidate the molecular mechanisms of resistance to hexythiazox in P. citri.
The citrus red mite, Panonychus citri (McGregor) (Trombidiformes: Tetranychidae), is a worldwide pest of citrus causing significant yield losses annually. It has a short life cycle, a high reproductive rate and infests over 80 species of plants, e.g., citrus, rose, almond, pear, castor bean, and several broadleaf evergreen ornamentals (Lee et al. 2000; Zhang 2003). The citrus red mite is quite difficult to manage because of improper application of acaricides, and its capacity to rapidly develop resistance to many registered acaricides (Furuhashi 1994; Masui et al. 1995; Meng et al. 2000; Ran et al. 2008; Hu et al. 2010; Osakabe et al. 2010). Hexythiazox is a selective miticide that is active against various phytophagous mites. It has been widely used in integrated pest management programs on various crops, especially against the citrus red mite. However, phytophagous mites are capable of rapidly developing resistance to hexythiazox. Tetranychus urticae has developed a > 1,000-fold resistance in Australia (Gough 1990), and P. citri, developed > 23,000-fold resistance in Japan (Yamamoto et al. 1995) and > 3,500-fold resistance in Chongqing, China (Ran. et al. in press). In a previous study, we increased the resistance of P. citri to 3,532-fold by continuous selection with hexythiazox for 20 generations (Liu et al. 2011a). Nevertheless, the molecular mechanisms of resistance to hexythiazox in P. citri remain unknown.
Cytochrome P450s are a very large and diverse group of enzymes found in all domains of life. They are an extremely important system involved in the metabolism of endogenous compounds and xenobiotics such as drugs, pesticides, plant toxins, chemical carcinogens, mutagens, hormones, fatty acids and steroids (Guengerich et al. 1999; Waxman 1999; Eaton 2000; Ingelman-Sundberg 2001; Zhang et al. 2004; Kretschmer & Baldwin 2005; Li et al. 2007; Strode et al. 2008). Down- or up-regulation of P450 gene expression can significantly affect the disposition of xenobiotics or endogenous compounds in the tissues of organisms, and thus alter their pharmacological/toxicological effects (Liu et al. 2011b). In arthropods, an increase in P450 activity is associated with the enhanced metabolic detoxification of insecticides; and a constitutive over expression of P450 genes has been implicated in the evolution of resistance to insecticides (Cariño et al. 1994; Feyereisen 2005; Zhu et al. 2008a) and tolerance to plant toxins (Wen et al. 2003). The isolation, characterization, classification and nomenclature of specific insect P450s are critical first steps towards understanding their involvement in these important metabolic processes. Accumulating genomic and postgenomic technologies have made it easier to study large and complex gene families such as the cytochrome P450 superfamily. To date, more than 2000 insect P450s have been recorded in the National Center for Biotechnology Information (NCBI), most of which were described from Drosophila melanogaster Meigen (Amichot et al. 2004; Tijet et al. 2001), Culex quinquefasciatus (Liu et al. 2011b), Musca domestica (Markussen & Kristensen 2010), Helicoverpa armigera (Hübner) (Brun-Barale et al. 2010; Pittendrigh et al. 1997), Bombyx mori (L.) (Ai et al. 2011), Manduca sexta (L.) (Rewitz et al. 2006), Plutella xylostella (L.) (Bautsita et al. 2009) and Apis mellifera (Johnson et al. 2006).
The global transcriptome of resistant (RR) and susceptible (SS) strains of P. citri has been sequenced (Liu et al. 2011a). A total of 34,159, 30,466 and 32,217 transcripts were identified by assembling SS reads, RR reads and SS plus RR reads after filtration of the low quality reads, respectively, in which 121 unigenes were related to cytochrome P450 monoxygenases. Only 46 P450s were functionally annotated according to their degree of sequence matching, owing to the low sequence similarity of other P450s (Liu et al. 2011a). To clarify the relationship between resistance of P. citri and P450 genes, we compared the sequences of P450 genes between RR and SS strains. Furthermore, we compared the gene expression profiles of P. citri among different strains using a digital gene expression system and quantitative RT-PCR.
Supplemental File is displayed in supplementary material for this article online in Florida Entomologist 98(1) (March 2015) at http://purl.fcla.edu/fcla/entomologist/browse together with color versions of Figs. 4, 5, 6 and 7.
Materials and Methods
CITRUS RED MITE REARING AND RESISTANT MITE SELECTION
A colony of citrus red mite, P. citri, was collected from citrange (Citrus sinensis × Poncirus trifoliata ) next to citrus orchard fences (Citrus Research Institute, Chinese Academy of Agricultural Sciences), which had not been exposed to acaricides for more than 10 years. These mites were reared on citrange for 3 years under acaricide-free conditions (25 ± 1°, 80 ± 5% RH and 14:10 h L: D and were considered to be a susceptible strain (SS). The SS strain was treated with hexyzhiazox (about a 70% population mortality rate was observed at every spray application) and was continuously screened for 20 generations until it was regarded as a fully resistant (RR). The selection process and the resulting changes in resistance were shown in a previous study (Liu et al. 2011a). A mixture of eggs, larvae and adults was collected in PerkinElmer (PE) tubes with one sample for SS and RR respectively. The 2 samples were stored at -80° until use.
RNA ISOLATION AND INTEGRITY EXAMINATION
Total RNA was isolated from the following 2 samples: RR and SS of P. citri to hexythiazox. From each sample, approximately 8 mg of mites were homogenized with liquid nitrogen in a mortar. RNA was extracted with TRIzol® reagent (Invitrogen, USA) according to the manufacturer's instructions and was treated with RNase-free DNase I (Takara Biotechnology, China). RNA integrity was confirmed with a minimum RNA integrated number value of 8 by the 2100 bioanalyzer (Agilent).
DIGITAL GENE EXPRESSION (DGE) LIBRARY CONSTRUCTION AND SEQUENCING
Approximately 10 μg of total RNA from each specimen (a mixture of RNA from eggs, third-instar larvae, pupae, and adults at equal ratios) was used to construct the DGE libraries. Poly(A) mRNA was isolated with oligo-dT beads and then treated with the fragmentation buffer. The cleaved RNA fragments were then transcribed into first-strand cDNA using reverse transcriptase and random hexamer-primers. This was followed by second strand cDNA synthesis using DNA polymerase I and RNase-H. The double stranded cDNA was further subjected to end-repair using T4 DNA polymerase, Klenow fragment, and T4 polynucleotide kinase followed by a single <A> base addition using Klenow 3′ to 5′ exo-polymerase. It was then ligated with an adapter or index adapter using T4 quick DNA ligase. Adaptor ligated fragments were selected according to size and the desired range of cDNA fragments were excised from the gel. We performed a PCR reaction to selectively enrich and amplify the amount of the fragments. Finally, after validation on an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System, the DGE libraries was sequenced on a flow cell using an Illumina HiSeq2000.Bioinformatics analysis process of sequence data were presented in Supplemental File 1. In particular, the raw reads were cleaned up by removing adapter sequences, reads in which unknown bases are more than 10% and low quality reads of quality value ≤ 5 as described by Liu (Liu et al. 2011a).
MANUAL CURATION OF CYPS IN P. CITRI
We blasted P. citri transcriptomes with sequences from the NCBI nucleotide database and obtained genes related to general pesticide resistance in other insect species. In total, there were 121 related to cytochrome P450 monooxygenase (CYPs). We chose only 46 of these for functional annotation according to sequence matching (E-value< le-5) owing to low sequence similarity of other P450s. Nucleotide sequences were dynamically translated using the EXPASY Proteomics Server ( http://www.expasy.ch/tools/dna.html, Swiss Institute of Bioinformatics). All the identified sequences were searched with BLASTx against all the assembled contigs in the iceblast server using an E-value cut-off of 1e-5 and the results with more than 99% similarity with the query sequence were eliminated as allelic variants (note that from those sequences, only the longest contigs with the best coverage were manually curated). We also searched the P. citri P450s on the NCBI network and obtained 2 amino acid alignments of P. citri CYPs. Next, we manually curated each of the CYPs with the help of comparison with the CYP genome of the two-spotted spider mite ( http://drnelson.uthsc.edu/Two.spotted.spider.mite.htm). Then, we assigned gene names based on the homology of P. citri CYPs to T. urticae using defined nomenclature and naming rules (Nelson 2006).
PHYLOGENETIC ANALYSIS OF CYPS IN P. CITRI
MEGA 4.0 software (Tamura et al. 2007) was used to analyze the phylogenetic relationships between P450 of P. citri and several CYPs in T. urticae to predict their classification. The neighbor-joining method was used to create phylogenetic trees (Saitou & Nei 1987). Positions containing alignment gaps and missing data were eliminated with pairwise deletion. Bootstrap analysis of 1,000 replication trees was performed to evaluate the branch strength of each tree (Efron et al. 1996). The NJ tree included bootstrap confidence levels at nodes, which reflected the conﬁdence of trees sampled during the reconstruction that included each particular branch. Only clades with a bootstrap value higher than 50 were selected for the bootstrap consensus tree (Soltis & Soltis 2003).
SEQUENCE ALIGNMENT AND VERIFICATION OF MUTATIONS IN CYPS IN P. CITRI
Multiple nucleotide sequence alignment of each CYP between the resistant and susceptible haplotypes of P. citri was performed using ClustalX (1.83) software (Thompson et al. 1994) and followed methods of Liu et al. 2011a. This alignment process was also based on the Feng & Doolittle (1987) algorithm, but with an improved choice of alignment parameters using dynamic assignment of penalties. Two pairs of specific Primers (Table 2) for surveying the nucleotide differences between RR and SS were designed by using Primer Premier 5.0 software based on the transcriptome of P. citri. Analyses using PCR were carried out in a total volume of 25 μL and performed in a thermocycler(GeneAmp PCR system 9600; Perkin-Elmer Corporation, Norwalk, Connecticut). The PCR reaction contained 12.5 μL of 1×Taq MasterMix from (Tiangen Biotech), 10 pmol of each primer, 30 ng of cDNA. PCR was performed using a Mastercycler® Gradient using the following protocol: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, with a final extension step of 72 °C for 10 min. Samples (5 μL) of reaction mixtures were examined by electrophoresis through 2% agarose gels in TAE buffer. Bands were revealed by visualization with UV light after ethidium bromide staining. PCR products were purified and sequenced by Shanghai Sangong Biological Engineering Technology & Service CO., LID. All these primers mentioned above were also used as sequencing primers. Once a mutation was confirmed, both nucleotide sequences from the 2 strains were dynamically translated using the EXPASY Proteomics Server ( http://www.expasy.ch/tools/dna.html, Swiss Institute of Bioinformatics). Amino acid sequence alignment was performed by ClustalX (1.83) software to confirm whether the mutation was a sense mutation.
DIFFERENTIAL EXPRESSION OF CYPS IN P. CITRI
The uniquely mapped reads for a specific transcript were counted by mapping reads to assembled sequences using SOAP (Li et al. 2009). Then the RPKM value for each transcript was measured in reads per kilobase of transcript sequence per million mapped reads (Mortazavi et al. 2008). The transcript fold change was calculated by the formula of log2 (RR_ RPKM / SS_RPKM). If the value of either RR_ RPKM or SS_RPKM was zero, we used 0.01 instead of 0 to calculate the fold change. We modified Audic's (Audic & Claverie 1997) method to analyze differential expression. The probability of a specific gene being expressed equally between the 2 samples was defined by the following formula:
In this equation, N1 and N2 indicate the total number of clean reads in the SS and RR haplotypes and x and y represent the mapped clean read counts of one transcript in the 2 samples. The false discovery rate (FDR) method was used to determine the threshold of the P value in multiple tests. In this study, we used ‘FDR ≤ 0.001 and the absolute value of the log2 ratio ≥ 1’ as the threshold to judge the significance of differential gene expression.
QUANTITATIVE RT-PCR ANALYSIS
Quantitative Reverse transcription polymerase chain reaction (RTPCR) detection was used to verify 5 differentially expressed genes (2 up-regulated and 3down-regulated genes), e.g., CYP389A6, CYP385A2, CYP307A1, CYP307A2, CYP389A1. Multiple specific Primers (Table 2) were designed by using Primer Premier 5.0 software (PREMIER Biosoft International, California). The expression study was performed on an Mx3000P™ Multiple Quantitative PCR System (Stratagene, La Jolla, California, USA) with ELF1A, elongation factor-1 alpha, used as reference gene (Niu et al. 2012). The reaction system and reaction parameters were same as Niu et al. (2012) described earlier. After collecting the Ct values, the relative quantitative expression of these 5 genes were calculated by the 2-δδCT method (Livak & Schmittgen 2001).
DIGITAL GENE EXPRESSION (DGE) LIBRARY SEQUENCING
Based on the transcriptome sequence data, 2 DGE libraries were constructed to identify the expression profiles of the various strains. After removing the low-quality reads, each library generated more than 7 million clean reads. Among these clean reads, approximately 2.8 million and 2.9 million (36.92% and 40.36%, respectively) were mapped to unigenes in each library (Table 3). The percentage of clean reads ranged from 98.68% to 99.70%, reflecting a high quality of sequencing.
MANUAL ANNOTATION IDENTIFIED 48 CYPS IN P. CITRI
Manual annotation and curation of the CYPs in the P. citri transcriptome sequence assembly ( http://www.ebi.ac.uk/ena/data/view/ERP000885) produced 121 sequences related to cytochrome P450 monooxygenase (CYPs). Of these, 46 were manually curated, as the remainder was found to have either low sequence similarities to other P450s according to sequence matching (E-value< le-5), or they contained too many sequencing errors. In addition, 2 full length P450s were detected by Jiang et al. (Jiang et al. 2010), which were the first detected P450 genes of P. citri. The latter were added to our data, and these 48 P450 sequences were named by D. R. Nelson in accordance with the P450 nomenclature committee conventions ( http://drnelson.uthsc.edu/cytochromeP450.html). Based on the closest BLASTX matches in the NCBI nr database first, and when possible, by phylogenetic analyses with CYPs in T. urticae ( http://drnelson.uthsc.edu/Two.spotted.spider.mite.htm), the P450s of P. citri were assigned to all 4 major insect CYP clans (CYP2, CYP3, CYP4 and mitochondrial). The 4 clans can be further subdivided into 15 families, 24 subfamilies, and 48 putatively functional isoforms (Fig. 1). Comparison of the number of functional CYP genes in different transcriptomes and genomes (Table 1) shows that the CYP number is lower than in the typical invertebrate or insect with the exception of the honeybee (Claudianos et al. 2006) and the New World screwworm (Carvalho et al. 2010).
Comparison of the number of functional CYP genes in different genomes or transcriptomes of various arthropods.
PHYLOGENETIC ANALYSIS OF CYPS IN P. CITRI.
Repeated and exhaustive searching for P. citri transcriptome assemblies and nucleotides in the NCBI uncovered a total of 48 CYP genes. Based on inferred amino acid sequences, these genes fell into 14 CYP gene families. Molecular phylogenetic analysis (Fig. 2) by the neighborjoining method (Saitou & Nei 1987) shows the relationships among CYP genes and gene families in P. citri and several T. urticae P450s. Positions containing alignment gaps and missing data were eliminated with pairwise deletion. Bootstrap analysis of 1,000 replication trees was performed to evaluate the branch strength of each tree (Efron et al. 1996). The NJ tree included a bootstrap confidence level at nodes that reflects the conﬁdence of trees sampled during the reconstruction that included each particular branch. In general, the tree demonstrates that the 4 major clans found in insects (Feyereisen 2006), which include CYP2, CYP3, CYP4 and mitochondrial clans, encompass all of the CYPs in P. citri. All nucleotide and amino acid sequences of cytochrome P450 monooxygenase genes in P. citri are available in the database ( http://www.ebi.ac.uk/ena/data/search?query=Panonychus citri).
Specific RNA primers used in sequencing and quantitative RT-PCR of Panonychus citri.
The CYP2 clan of P. citri contains 12 members, which can be separated into 2 distinct families (CYP307, CYP392). The spook gene CYP307A1, which is expressed in the prothoracic gland (Niwa et al. 2005), is a conserved Halloween gene involved in the early stages of ecdysone synthesis (Rewitz et al. 2006; Rewitz & Gilbert 2008), and can convert 7-dehydrocholesterol to δ4-diketol with the gene product of spookier (CYP307A2) (Gilbert 2008). These 2 genes are close paralogs that are believed to mediate the same enzymatic reaction at different stages of development (Rewitz & Gilbert 2008). The other CYP2 clan family in P. citri (CYP392) is divided into 3 subfamilies.
The CYP3 clan of P. citri contains 12 CYPs. Genes in the CYP3 clan are the most numerous among insect P450 genes, and are often found in large clusters associated with oxidative detoxiﬁcation of xenobiotics (Kretschmer & Baldwin 2005; Li et al. 2009; Waxman 1999) and endobiotics (Strode et al. 2008; Zhang et al. 2004). The first insect P450 gene (CYP6A1) was isolated from an insecticide-resistant strain of the housefly, M. domestica L. (Feyereisen et al. 1989). The first P450 gene (CYP6A2) was isolated from D. melanogaster, and is also associated with resistance to insecticides (Waters et al. 1992). In P. citri, the CYP3 clan contains 12 genes and 5 subfamilies. Eight genes belong to the CYP385 family and just 1 gene (CYP382A1) is part of the CYP382 family.
Alignment statistics of the DGE-SEQ analysis of Panonychus citri RNA extracted from the hexythiazox-resistant (RR) and susceptible (SS) strains.
The CYP4 clan of P. citri contains 19 genes and can be divided into 6 families and 10 subfamilies. Interestingly, the number of CYP4 clan genes varies in different species. For example, there are 32 in D. melanogaster (Tijet et al. 2001), 45 in Anopheles gambiae Giles (Holt et al. 2002), 44 in Tribolium castaneum (Herbst) (Richards et al. 2008), 32 in B. mori (L.) (Bin et al. 2005), 59 in Aedes aegypti L. (Strode et al. 2008), and only 4 in Apis mellifera L. (Claudianos et al. 2006). The great diversity of genes in the CYP4 clan was also reflected in a great diversity of functions. CYP4s in other insects have been implicated in functions as diverse as 20-hydroxyecdysone biosynthesis (Maibeche-Coisne et al. 2000), pheromone metabolism (Maibeche-Coisne et al. 2000) and pyrethroid insecticide resistance (Pridgeon et al. 2003).
The mitochondrial clan of P. citri probably originated in the CYP2 clan, which includes P450s with essential physiological functions, and contains 5 members in 3 families and 3 subfamilies. Two of the members, CYP302A1 and CYP314A1, are highly conserved halloween genes, involved in ecdysone synthesis (Rewitz et al. 2006, 2008). The disembodied (dib) gene (CYP302A1) codes a cytochrome P450 enzyme that adds a hydroxyl group to the carbon-22 position of 2, 22, dE- Ketotriol to make 2- Deoxyecdysone (Gilbert 2004). The dib mutants are defective in producing cuticle and have severe defects in morphological processes such as head involution, dorsal closure and gut development (Chávez et al. 2000). The shade gene (CYP314A1) codes for a P450 enzyme which reportedly adds hydroxyl group to the 2C- position of ecdysone to make 20-hydroxyecdysone, as the final step in the biosynthetic pathway (Petryk et al. 2003). CYP381 was a single family in P. citri.
SEQUENCE ALIGNMENT AND VERIFICATION OF MUTATIONS IN CYPS OF P. CITRI
ClustalX (1.83) software (Thompson et al. 1994) was used to analyze alignment differences in each CYP between the RR and SS of P. citri. After sequence verification, 3 SNP sites were detected inCYP307A1 (1688 bp, Fig. 4). The first nucleotide change located at 841(A to C), the second change located at 1395 (T to C), and the final change located at 1491 (T to C). With alignment of the 2 predicted amino acid sequences of the CYP307A1 (Fig. 5), a mutation was detected for one amino acid (278-lysine to glutamine). Therefore, the mutation was a sense mutation and the other 2 were nonsense mutations. A single SNP site, 40 (A to T), was detected in CYP381A2 (619 bp, Fig. 6). With alignment of the 2 predicted amino acid sequences of the CYP381A2 (Fig. 7), there was a sense amino acid mutation detected (14-threonine to serine). In insects, steroidogenic CYPs are products of the Halloween genes phantom (CYP306A1), disembodied (CYP302A1), shadow (CYP315A1) and shade (CYP314A1) and are responsible for the last 4hydroxylations in the pathway leading to 20E (Niwa et al. 2004; Niwa et al. 2005; Warren et al. 2002),which are biochemically similar to one that yields 20E in crustaceans (Lachaise et al. 1993). In D. melanogaster, mutations in these genes disrupt 20E production and cause the arrest of embryonic development and death. Spook (CYP307A1) is another member of this CYP group and, when mutated, results in low 20E mutants (Niwa et al. 2005). In B. mori, the nucleotide transition of C to T at position 1049 was found in phantom (CYP306A1), resulting in a nonsense mutation at amino acid position 286. The morphology of the phantom mutant is normal until stage 14 when there is a failure of dorsal closure and head involution and embryos become compacted. The phantom mutant exhibited severe reductions in the epidermal expression of these 20- hydroxyecdysone-inducible genes (Niwa et al. 2004).
DIFFERENTIAL EXPRESSION OF CYPS IN RR AND SS OF P. CITRI
A rigorous algorithm (see Methods) was developed to identify genes differentially expressed between resistant (RR) and susceptible (SS) mite populations treated with hexyzhiazox by referring to “The significance of digital gene expression profiles” (Audic & Claverie 1997). To explore the gene expression levels further, the reads per kb per million reads (RPKM) method (Mortazavi et al. 2008) was adapted to eliminate the influence of variation in gene length and the total reads number. Results showed 22 P450s were up-regulated and 24 were down-regulated (Table 4). Furthermore, the significance of gene expression differences were determined using the false discovery rate (FDR≤ 0.001) and the absolute value of the log2 ratio (≥ 1). Nine genes were differentially expressed between the 2 samples at significant levels, including 5 down-regulated and 4 up-regulated genes (Table 4). Seven members of P450s had a greater value than 2 based on a log2 ratio formula, and these members were distributed as 2, 2 and 3 in the CYP 2 clan, CYP 3 clan and CYP 4 clan, respectively. It was straightforward to identify the highest up-regulated expression gene (log2 ratio = 12.14) as CYP389A6, which belonged to the CYP4 clan. The gene of highest expression might be analogous with those CYP4s in other insects that have been implicated in pesticide metabolism. For example, CYP4G8 is over expressed in pyrethroid-resistant strains of H. armigera (Pittendrigh et al. 1997). CYP4C27 in A. gambiae is over expressed in a DDT-resistant strain (David et al. 2005) and CYP4G19 expression in Blattella germanica is correlated with pyrethroid resistance (Pridgeon et al. 2003). Several CYP4 genes are over expressed in pesticide resistant C. pipiens and Diabrotica virgifera (Scharf et al. 2001; Shen et al. 2003). The expression level of CYP385A2 (log2 ratio = 9.46), belonging to the CYP3 clan, was a little lower than that of CYP389A6. The CYP3 clan is related to metabolism of pyrethroids, chlorinated hydrocarbons (e.g., DDT) and neonicotinoids (e.g., imidacloprid) (Daborn et al. 2002; Jiang et al. 2010; Karunker et al. 2008; Komagata et al. 2010; Nikou et al. 2003). The most down-regulated gene (log2 ratio = -11.816) was CYP389A1, which belonged to the CYP4 clan. The second lowest (log2 ratio = -11.079) was CYP307A2, which belonged to the CYP2 clan. The genes with the absolute value of the log2 ratio (≥ 2) might be related to the lower activity of hexythiazox-poisoned P. citri individuals (Liu et al. 2011a). However, quantitative RT-PCR results showed that the upregulated value of log2 ratio (RR/SS) in CYP385A2 and CYP307A1 were only 0.28 and 0.17, the down-regulated value of log2 ratio (RR/SS) in CYP389A6, CYP307A2 and CYP389A1 were just -0.69, -1.46 and -1.08, respectively (Fig. 3). All sequences at http://www.ebi.ac.uk/ena/data/search?query=Panonychus citri
Insecticide resistance of the citrus red mite will seriously disrupt chemical control efficiency, and cross-resistance to various acaricides could further increase the difficulty of resistance management (Hu et al. 2010). Previously, only 2 P450 sequences of P. citri were available in GenBank. In this study, 46 additional unique sequences encoding P450 genes were selected. All 48 P450 (CYP) genes of the citrus red mite (Jiang et al. 2010; Liu et al. 2011a) were placed in the P450 repertoire for the insect genome or transcriptome that has been reported so far. The number of P450 sequences of the citrus red mite is fewer than the number found in other species (Table 1), which is more proof of the diversity of CYP.
Despite decades of study on P450 enzymes, the molecular mechanism mediated by this super-family of enzymes during metabolism is yet to be fully elucidated. Gene mutations and over-expression are considered to be responsible for resistance. Amichot et al. (2004) first demonstrated that point mutations were associated with insecticide resistance in the Drosophila cytochrome P450 and CYP6A2 enabled DDT metabolism by the insect's cytochrome P450. Previously, sequence polymorphism of CYP6A1 and CYP6D1 had been documented in the house ﬂy, but there was no link established between these polymorphisms and insecticide resistance (Kasai & Scott 2000; Scott et al. 1998). These results are in contrast with cytochrome P450 polymorphisms in humans, which are known to affect the metabolism of drugs (Guengerich et al. 1999; Ingelman-Sundberg 2001) and even pesticides (Eaton 2000). In fact, only 2 examples of pesticide resistance linked to mutations in a cytochrome P450 have been described. Thus, a single substitution in CYP51 of the saccharomycete Candida albicans (C.P.Robin) Berkhout (T315A) (Lamb et al. 1997) and of the pathogenic fungus Uncinula necator (Schwein.) Burrill (F136Y) (Delye et al. 1998) confer resistance to the fungicides ﬂuconazole and triadimenol respectively. Recently, 2 CYP9M10 haplotypes were isolated from susceptible (JHB) and resistant (JPal-per) strains of C. quinquefasciatus Say to insecticides. The results showed that cis-acting mutations and duplications in the CYP9M10 haplotype might be responsible for the insecticide resistance (Itokawa et al. 2011). In this study, sequence variation was detected in 2 CYPs. The A841C mutation on CYP307A1 and A40Tmutation on CYP381A2 caused the amino acid mutations CYP307A1 (K278Q) and CYP381A2 (T14S) respectively. These variations may be linked with metabolic resistance of P. citri to hexythiazox.
In many cases, the overexpression of P450 genes results in increased levels of total P450s and their activities are responsible for insecticide resistance (Cariño et al. 1994; Zhu et al. 2008b). Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides (Pavek & Dvorak 2008). Expression of the P450 genes CYP6AA7, CYP9J40, CYP9J34, and CYP9M10 isolated from C. quinquefasciatus were strongly correlated with levels of resistance to permethrin in the larval stage, with the highest expression levels identified in the most RR, suggesting the importance of CYP6AA7, CYP9J40, CYP9J34, and CYP9M10 in permethrin resistance of larva mosquitoes (Hardstone et al. 2010; Liu et al. 2011b). The over expression of CYP9M10 has also been reported in a resistant C. quinquefasciatus mosquito strain in Japan (Komagata et al. 2010) and has been linked with pyrethroid resistance in C. quinquefasciatus (Itokawa et al. 2010; Xu et al. 2005). Recent studies (Zhu et al. 2008b) indicated that several P450 genes were up-regulated in insecticide resistant house flies through a similar induction mechanism. However some researchers considered that a strain of D. melanogaster may not have the CYP6A1 gene insertion and that CYP6A1 may not have to be over expressed for DDT resistance to occur (Kuruganti et al. 2007). The results from this study supported that 22 cytochrome P450 genes were up-regulated and 24 cytochrome P450 genes were down-regulated in RR mites. CYP389A6 (log2 ratio [RR/SS] = 12.14) and CYP307A2 (log2 ratio [RR/SS] = -11.079) were the most up- and down-regulated genes, respectively, and are likely associated with resistance of P. citri to hexythiazox.
Cytochrome P450 gene expression differences between hexythiazoxsusceptible and hexythiazox-resistant Panonychus citri strains.
However, these gene expression differences were shown later to be negative to some extent by quantitative RT-PCR (Fig. 3). The amplification bias between Illumina sequencing and qRT- PCR was the major driving force to vary the amplification difference between the RS and SS strains; meanwhile the RPKM and the 2-δδCT method were 2 different ways to analyze gene differential expression generated from Illumina sequencing and qRT-PCR. The results of qRT-PCR indicated that no obvious difference could be observed in the hexythiazox-resistance line, and the expression difference of CYPs was not closely related to resistance. Therefore, the primary goal of this study was to provide baseline information for future research on P. citri. The resistance ratios of mites to various pesticides have grown so dramatically that researchers would be perplexed by these differences between mites and insects. In establishing the molecular action and resistance mechanisms of these and other pesticides, a limiting factor has been the lack of genetic systems and accompanying genomic resources needed for efficient identification of resistance mutations that suggest molecular mechanisms (Heckel 2003). For these reasons, other researchers employed variety of methods, such as genome sequencing, functional genomics and RNAi methods to uncover the molecular mechanisms of deltamethrin resistance in the T. castaneum QTC279 strain (Zhu et al. 2010). Subsequently bulk segregant analysis (BSA) mapping, SNP detection and complementation assays were used to identify a locus for monogenic, recessive resistance to etoxazole in T. urticae (Van Leeuwen et al. 2012). These and other methods will need to be employed to more fully understand the genetic basis of chemical resistance in the red mite and other members of the Acari.
This research was supported by the National Science and Technology Support Programme (2012BAD19B06), the National Sparking Plan Project (2014GA811008), the Chongqing Scientific Research Project (cstc2014fazktjcsf80031), the Special Foundation of Chongqing Key Laboratory of Citrus, China Scholarship Council (CSC) (No.  3009) and the TECHNO II Erasmus Mundus Programme. The first 2 authors contributed equally to this paper.