Multiple cis-acting elements involved in up-regulation of a cytochrome P450 gene conferring resistance to deltamethrin in smal brown planthopper, Laodelphax striatellus (Fallén)

Insect Biochemistry and Molecular Biology 78 (2016) 20e28

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Multiple cis-acting elements involved in up-regulation of a cytochrome P450 gene conferring resistance to deltamethrin
n) in smal brown planthopper, Laodelphax striatellus (Falle Jian Pu a, Haina Sun a, Jinda Wang a, Min Wu a, Kangxu Wang a, Ian Denholm b, Zhaojun Han a, * a Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Jiangsu, The Key Laboratory of Monitoring and Management of Plant Diseases and Insects, Ministry of Agriculture, Nanjing, 210095, Jiangsu, China b Biological and Environmental Sciences Department, University of Hertfordshire, Hatfield, Hertfordshire, United Kingdom

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 April 2016 Received in revised form 2 August 2016 Accepted 26 August 2016 Available online 31 August 2016

As well as arising from single point mutations in binding sites or detoxifying enzymes, it is likely that insecticide resistance mechanisms are frequently controlled by multiple genetic factors, resulting in resistance being inherited as a quantitative trait. However, empirical evidence for this is still rare. Here we analyse the causes of up-regulation of CYP6FU1, a monoxygenase implicated in resistance to deltamethrin in the rice pest Laodelphax striatellus. The 50 -flanking region of this gene was cloned and sequenced from individuals of a susceptible and a resistant strain. A luminescent reporter assay was used to evaluate different 50 -flanking regions and their fragments for promoter activity. Mutations enhancing promoter activity in various fragments were characterized, singly and in combination, by site mutation recovery. Nucleotide diversity in flanking sequences was greatly reduced in deltamethrin-resistant insects compared to susceptible ones. Phylogenetic sequence analysis found that CYP6FU1 had five different types of 50 -flanking region. All five types were present in a susceptible strain but only a single type showing the highest promoter activity was present in a resistant strain. Four cis-acting elements were identified whose influence on up-regulation was much more pronounced in combination than when present singly. Of these, two were new transcription factor (TF) binding sites produced by mutations, another one was also a new TF binding site alternated from an existing one, and the fourth was a unique transcription start site. These results demonstrate that multiple cis-acting elements are involved in up-regulating CYP6FU1 to generate a resistance phenotype. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Laodelphax striatellus Monooxygenase Gene expression Promoter activity Transcription factor Selective sweep

1. Introduction The evolution of insecticide resistance threatens human welfare through its impact on crop protection and disease transmission (Denholm et al., 2002). Mechanisms of insecticide resistance include reduced cuticular penetration, enhanced detoxification, enhanced excretion of insecticides, and target site insensitivity (Liu et al., 2006). Point mutations in a coding region can reduce the sensitivity of insecticide target sites (Feyereisen et al., 2015; Ffrench-Constant et al., 2000; Kasai et al., 2014; Marichal et al., 1999; Liu et al., 2005; Tay et al., 2015; Weill et al., 2003; Williamson et al., 1996) and

* Corresponding author. E-mail address: [email protected] (Z. Han). http://dx.doi.org/10.1016/j.ibmb.2016.08.008 0965-1748/© 2016 Elsevier Ltd. All rights reserved.

sometimes enhance the activity of detoxification enzymes (Amichot et al., 2004; Ibrahim et al., 2015; Russell et al., 2011; Schuler and Berenbaum, 2013). However, in several cases resistance genes without related mutations in their coding sequences have been shown to be constitutively over-expressed in insecticideresistant strains. Examples include the monooxygenases CYP6A1 and CYP6D1 in the housefly Musca domestica (Feyereisen, 2012), CYP6G1 in the fruitfly Drosophila melanogaster (Daborn et al., 2002), CYP6P3 in the mosquito Anopheles gambiae (Djouaka et al., 2008), CYP6CY3 in the aphid Myzus persicae (Puinean et al., 2010) and CYP6AY1 in the brown planthopper Nilaparvata lugens (Ding et al., 2013). Such over-expression can result in cross-resistance within and between different classes of insecticides. For example, overexpression of CYP6G1 in the midgut, malpighian tubules and fat body of D. melanogaster confers resistance to four unrelated insecticides (DDT, lufenuron, nitenpyram and diazinon) (Chung et al.,

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2007; Daborn et al., 2007), and the CYP6M2 enzyme of A. gambiae can metabolize both DDT and pyrethroids (Mitchell et al., 2012). Constitutive over-expression can arise from gene amplification or from up-regulation of the expression of single genes. Gene amplification was first reported for a carboxylesterase in the peachpotato aphid M. persicae (Field et al., 1988). Other examples include MdGST-3 in M. domestica resistant to organophosphates (Zhou and Syvanen, 1997), CYP6P4 and CYP6P9 in the mosquito Anopheles funestus resistant to pyrethroids (Wondji et al., 2009) and CYP6CY3 in M. persicae resistant to neonicotinoids (Puinean et al., 2010). Cases of up-regulation without amplification have proved more challenging to address. Both cis- and trans-acting factors have been implicated in influencing expression (Bhaskara et al., 2008; Brown et al., 2005; Feyereisen et al., 2015; Kalsi and Palli, 2015; Misra et al., 2013; Sun et al., 2014; Wan et al., 2014). Cis-acting regulation of resistance-conferring monooxygenases can be achieved through insertions/deletions (indels) or mutations in their promoter region, such as a transposable element insertion in the 50 -untranslated region (50 UTR) of the CYP6G1 gene in D. melanogaster (Daborn et al., 2002), a 15-bp insertion in the 50 UTR of the CYP6D1 allele in M. domestica (Gao and Scott, 2006), mutations alternating TF binding site in the 50 -promoter core region of CYP6A2 in D. melanogaster (Wan et al., 2014), and a single nucleotide polymorphism (SNP) in the promoter region of CYP6AY1 in Nilaparvata lugens (Pang et al., 2014). Examples of trans-acting factors include over-expression of CYP6A2 and CYP6A8, linked on chromosome 2 of D. melanogaster but regulated by a repressor locus on chromosome 3 (Maitra et al., 2000), and of CYP6A1 on chromosome 5 in M. domestica but regulated by a semi-dominant factor on chromosome 2 (Carino et al., 1992, 1994; Cohen et al., 1994). However, in most cases only one mutation affecting regulation has been identified whereas resistance due to enhanced detoxification has frequently been assumed to be a quantitative trait, potentially under the control of multiple genetic determinants (Feyereisen et al., 2015; Karasov et al., 2010; Menozzi et al., 2004; Morton, 1993; Via, 1986; Zhong et al., 2013).
n) The small brown planthopper, Laodelphax striatellus (Falle (Hemiptera: Delphacidae) (hereafter referred to as SBPH) is one of the most destructive pests of agriculture in Asia (Kisimoto, 1989). Long-term reliance on chemical control has selected for resistance to multiple classes of insecticide including organophosphates, carbamates, pyrethroids and neonicotinoids (LiHua et al., 2008). Our recent analyses showed that strong resistance to deltamethrin (a pyrethroid) in SBPH was due to enhanced detoxification rather than target site insensitivity, and that five monooxygenase genes and one esterase gene were over-expressed in a resistant strain. One monooxygenase (CYP6FU1) was 16-fold overexpressed without gene amplification (Xu et al., 2013). Here we report on cloning the 50 -flanking region of CYP6FU1 from both resistant and susceptible strains, and compare fragments for their promoter activity using a luminescent reporter assay. Results demonstrate an abundance of transcription-related elements, SNPs and fragment indels in the 50 -flanking region. At least four mutations contribute to constitutive over-expression of the CYP6FU1 gene. 2. Materials and methods 2.1. Insect strains The two SBPH strains (JHS and JH-del) used in this study were derived from the same field population and reared on rice seedlings in an insectary at a temperature of 26  C, a relative humidity of 70%, and a photoperiod of 16L:8D. The JHS strain had been reared without exposure to insecticide and was susceptible to

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deltamethrin. The JH-del strain had been selected to exhibit >1000-fold resistance to deltamethrin, as previously described (Xu et al., 2013). 2.2. DNA/RNA extraction and cDNA synthesis Insects were ground in liquid nitrogen, and total RNA was extracted using the Trizol extraction kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. The extraction was digested using DNase I (Takara, Japan) in order to eliminate genomic DNA contamination, and the total RNA integrity and concentration were checked by NanoDrop ND-1000 (Thermo Scientific, Waltham, MA, USA). The first-strand cDNA was synthesized with PrimeScript™ reverse transcriptase (Takara, Japan) according to the manufacturer's instructions and stored at 20  C. Genomic DNA was extracted from SBPH individuals using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. 2.3. Cloning and sequencing the 50 -flanking regions SBPH genomic DNAs were digested with four different restriction enzymes provided by the Universal Genome Walker Kit as instructed by the manufacturer (Clontech, Palo Alto, CA, USA) to generate four pools of blunt-end fragments of SBPH genomic DNAs. For each pool, the genomic DNA fragments were ligated to Genome Walker adaptors with T4 DNA ligase (referred to as a genome walker library). The adaptor ligated DNA fragments in these libraries were amplified by polymerase chain reaction (PCR) with LATaq polymerase (Takara, Japan). Two specific antisense primers PCYP6FU1-1 and PCYP6FU1-2 were designed based on the CYP6FU1 gene sequence (KC161438) in NCBI GenBank, and two sense primers AP1 and AP2 were based on sequences of the adaptor (Table S1). Subsequent gene-walking was performed using the specific antisense primers PCYP6FU1-3 and PCYP6FU1-4 designed based on the front round of gene-walking (Table S1). Finally, specific primers CYP6FU1-DNA-F and CYP6FU1-DNA-R were designed spanning all the lined 50 -flanking region (Table S1), and end-to-end PCR was performed to confirm the whole sequence. The PCR products were cloned into the PEASY-T3-Clone vectors (TransGen Biotech, China) and sequenced by Invitrogen Life Technologies, Shanghai, China. All the sequences have been deposited in GenBank (Accession nos: KU645913- KU645927, KU601751). The putative TF binding sites in 50 -flanking regions were identified by website programs http://www.cbrc.jp/research/db/TFSEARCH.html and http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?
et al., 2003; Messeguer et al., 2002). The sedirDB¼TF_8.3 (Farre quences from resistant and susceptible individuals were compared by using GeneDoc software. The nucleotide polymorphism index was calculated using DnaSP v5 (Librado and Rozas, 2009). A phylogenetic tree was constructed by MEGA 5.0 applying the maximum likelihood method (Tamura et al., 2011). 2.4. Cloning 50 UTR sequences in resistant and susceptible strains For confirmation of the function of the transcription start sites found in 50 -flanking regions, rapid amplification of cDNA ends (RACE) was employed to clone the 50 UTR of CYP6FU1. The cDNA template prepared from a pooled sample containing 15 resistant and 15 susceptible individuals was synthesized with SMART™ kit (Clontech, USA). Two specific primers CYP6FU1-50 GSP1 and CYP6FU1-50 GSP2 were designed for nested PCR based on the CYP6FU1 gene sequence (KC161438) in NCBI GenBank (Table S1). After two rounds of PCR amplification, PCR products were cloned into the PEASY-T3-Clone vectors (TransGen Biotech, China) and

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sequenced. To investigate the frequency of a special 50 UTR allele in the susceptible and resistant strains, cDNA templates were prepared with the total RNAs extracted from nine individuals of JH-del and 10 individuals of JHS, respectively, and the gene-specific primers CYP6FU1-50 F, CYP6FU1-50 R were designed (Table S1) and used to clone this allele. 2.5. Promoter activity analysis of the 50 -flanking region sequences A luminescent reporter assay was employed to evaluate the 50 flanking region sequences for their promoter activity. 50 -flanking region sequences (DNA50 -D1, DNA50 -S2 and DNA50 -S6)were amplified using primers 6FU1-F(-1515) and 6FU1-R(-12) and inserted into the firefly luciferase reporter vector PGL3-Basic (Promega, Madison, WI, USA) between the XhoI and KpnI restriction sites to form the different promoter-PGL3 plasmids for testing. The luminescent reporter assays followed manufacturer's instructions. Drosophila S2 cells were cultured at 27  C in HyQ SFXinsect cell culture medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Gaithersburg, MD, USA). The cells were kept at a density of 0.5  106 cells/ml in 24-well tissue culture plates for 12 h before transfection. After removal of culture medium, the cells were transfected with a mixture of 8.5 fmole promoter-PGL3 plasmid, 0.375 fmole internal control plasmid (pRL-CMV vector, Promega) and 2.88 ml of Cellfectin reagent (Invitrogen) in the HyQ SFX-insect medium without FBS. After 3.5 h, the transfection mixture was replaced with 500 ml fresh HyQ SFX-insect medium containing 10% FBS. Cells were harvested after 48 h post transfection culture and lysed in 1  passive lysis buffer (Promega). Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Each experiment was replicated three times. 2.6. Localization and identification of cis-acting elements To localize and identify mutations responsible for up-regulation, fragments of the 50 -flanking region of CYP6FU1 from both resistant and susceptible strains were compared for their promoter activity using the reporter assay described above. Based on the whole flanking sequence from 1515 to 12 (as numbered relative to the beginning of the open reading frame), progressive 50 deletion fragments 1269~12, 1113~12, 1000~12, 906~12, 826~12, 531~12, 396~12, 299~12 and 186~12 were constructed as promoter sequences using the common reverse primer 6FU1-R(-12) and each of the forward primers 6FU1-F(1269), 6FU1-F(-1113), 6FU1-F(-1000), 6FU1-F(-906), 6FU1-F(-826), 6FU1-F(-531), 6FU1-F(-396), 6FU1-F(-299), and 6FU1-F(-186) (Fig. S1), and tested to see if the cut fragments 1269~1114, 1113~1001, 1000~907, 906~827, 826~532, 531~397, 396~300, 299~187 and 186~12 contribute to enhancing promoter activity. Once localized, the contributing fragments were checked to identify mutations by comparing the major flanking region from the resistant and susceptible strain. Candidate mutations were recovered and sequences with and without these mutations were tested to identify cis-acting mutations with an up-regulation function. The recovery mutation constructs (P(-1515/-12)D-d1, P(-396/-12)D-M1, P(-396/-12)D-M2, P(396/-186)D-M3 and P(-396/-12)D-M1M2M3) were made using the corresponding mutation-introducing primers(Fig. S1). The recovery mutation constructs P(-1515/-12)D-d1d2 and P(-1515/-12) D-d1d2d3 were made by overlap extension PCR as described by Horton and Pease (1991). The primer pairs and templates used are listed in S2 Table. The fragments cloned for promoter constructs are shown in S1 Figure, and the primer sequences used are listed in S1

Table. The luciferase activity was tested in S2 cells as described previously. 2.7. Joint effect of the cis-acting mutations The effect of accumulating cis-acting mutations was investigated using promoter constructs with the Type1 sequence intact or with different combinations of the mutations already identified. The recovery mutation constructs P(-1515/-12)D-d2 and P(1515/-12)D-d2d3 were made using the primer pairs 6FU1-F(1515)L/6FU1-R(-12) from template P(-1515/-12)D-d1d2 and 6FU1-F(-1515)L/6FU1-R(-12) from template P(-1515/-12)Dd1d2d3, respectively. P(-1515/-12)D-dele100, P(-1515/-12)D-M2, P(-1515/-12)D-M2dele100, P(-1515/-12)D-d3dele100, P(-1515/12)D-d2dele100, P(-1515/-12)D-d3M2, P(-1515/-12)D-d2d3M2, P(-1515/-12)D-d2d3dele100, P(-1515/-12)D-d2M2dele100, P(1515/-12)D-d3M2dele100 and P(-1515/-12)D-d2d3M2dele100 were made by overlap extension PCR using PrimeSTAR Max DNA Polymerase (Takara, Japan) for high fidelity. The primer pairs and templates used are listed in S2 Table. All fragments cloned for promoter constructs are shown in S1 Figure, and the primer sequences used are listed in S1 Table. The luciferase activity was tested in S2 cells as described previously. 3. Results 3.1. 50 -flanking region sequences of CYP6FU1 cloned from resistant and susceptible strains Sixteen DNA templates prepared from eight resistant and eight susceptible individuals were used to amplify the 50 -flanking region of CYP6FU1 and yield 16 corresponding sequences (Fig. S2). The nucleotide polymorphism index for this region (0.057) was much higher than that of the nearby coding regions (0.031). A total of 316 SNP and fragment indel sites were found, and the sequence length varied from 1497 to 1533 bp. Furthermore, the 50 -flanking region abounded with functional elements, including 6 transcription start sites, named from the far 50 -end as Inr1~Inr6, and 37 putative TF binding sites (Fig. S2). Phylogenetic analysis showed the 16 cloned sequences to cluster into 5 different groups (Fig. 1). Within each group the sequence

Fig. 1. Phylogenetic relationship of the 50 -flanking regions. The phylogenetic tree was inferred using maximum likelihood. Bootstrap values in percent from 1000 replicates are shown.

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similarity was higher than 99%, whereas between groups it was less than 90% (Table S3). Furthermore, sequences within the same group shared the same transcriptional start sites and had similar TF binding sites, but these varied between groups (Fig. S2). The five groups representing five different flanking region types were designated as Type 1 to 5. Type 1, which consisted of all eight sequences cloned from resistant insects and one from the susceptible strain (S1), was about 1515 bp in length with four transcriptional start sites (Inr1, 2, 5 and 6) and 20 TFs, including AP-1 (activator protein 1), BR-C (Broad-Complex), Dfd (Deformed), Dl (Dorsal), EcR (ecdysone receptor), GATA binding site, Hb (Hunchback), Nrf2 (NFE2-related factor 2), OCT (Octamer binding protein), Ubx (Ultrabithorax) and XBP-1(X-box binding protein 1) (Fig. 2). Type 2 consisted of four sequences, all cloned from susceptible insects. These sequences were about 1497 bp in length and had five transcriptional start sites (Inr2, 3, 4, 5 and 6). Type 2 lacked seven of the TF binding sites present in Type 1, but contained five other ones resulting from base and fragment deletions, insertions and substitutions. The other three sequences were each represented by a single insect from the susceptible strain. Type 3 had three transcriptional start sites (Inr2, 5 and 6), Type 4 had an additional three (Inr4, 5 and 6), and Type 5 had four (Inr2, 4, 5 and 6). Their TF sites also differed (see Fig. S2). Whereas all resistant insects conformed to the same flanking sequence (Type 1), individuals from the susceptible strain exhibited all five types, with Type 2 being the most frequent. The eight sequences cloned from susceptible insects had a nucleotide polymorphism index of 0.090, 312 nucleotide polymorphism sites and three to five different transcription start sites. The eight sequences cloned from resistant insects had a much lower nucleotide polymorphism index (0.001), only six nucleotide polymorphism sites and the same four transcription start sites. These changes among the 50 -flanking regions and between the resistant and susceptible strain could result from insecticide selection and influence their promoter activity. Sequences representing Types 1, 2 and 3 (DNA50 -D1, DNA50 -S2 and DNA50 -S6) all showed significant promoter activity (Fig. 3), but expression of the reporter gene driven by the Type 1 flanking sequence (from the resistant strain) was significantly higher (2.4 and 3.7-fold, respectively) than those driven by Type 2 and 3 sequences. This supports a hypothesis that over-expression of

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Fig. 3. Expression of the reporter gene in PGL3 constructs with different flanking sequences as promoters. Type 1(DNA50 -D1), Type 2(DNA50 -S2) and Type 3(DNA50 -S6) were cloned into the firefly luciferase reporter vector PGL3-Basic plasmid, respectively. The relative luciferase activities are corrected for transfection efficiency with the Renilla luciferase pRL-CMV plasmid and are presented as ratios to the activity driven by promoter DNA50 -S2. The data and error bars represent the mean and standard errors from at least three independent experiments. Values sharing the same letter are not significantly different at P < 0.05 (Duncan tests).

CYP6FU1 in the resistant strain results from an enhanced promoter activity of the Type 1 flanking region. 3.2. Localization of cis-acting elements in the type 1 flanking region Different fragments of the Type 1 flanking region 1515~1114, 1113~827, 826~532, 531~397, 396~187 and 186~12 were compared for promoter activity, with those from Type 2 flanking region as controls. Results suggest that the elements up-regulating CYP6FU1 reside within two fragments of the Type 1 flanking region, -1515~-827 and 396~-187 (Fig. 4). When these fragments were cut from the promoter as seen from the constructs P(-1515/-12) to P(-826/-12) and from P(-396/12) to P(-186/-12), Type 1 fragments showed a significant decrease in promoter activity, unlike the corresponding Type 2 fragments. The sequence from 826 to 397 may have silencers, but not enhancers. When this fragment was cut, as shown from P(-826/-12) to

Fig. 2. Sequence analysis of the 50 -flanking region (DNA5′-D1) of the cytochrome P450 gene CYP6FU1. The nucleotides are numbered relative to the translation start site indicated by þ1, with sequence upstream of it preceded by “-”. The transcription start sites (Inr) as determined and other putative transcription factor (TF) binding sites are underlined.

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start site because of the dramatic difference between the constructs PGL3-Basic and P(-186/-12). Sequence checking found that all five transcription start sites other than Inr 1 are located in P(-186/-12) (Fig. 6C). However, resistance-associated enhancers are unlikely to be located within this fragment, which only differed slightly in activity between the Type 1 and Type 2 flanking regions. Thus, the cis-elements influencing expression appeared to be located in the two fragments, 1515~827 and 396~187.

3.3. Identification of the cis-acting elements

Fig. 4. 50 -end deletion analysis of the flanking regions of CYP6FU1 for their promoter activity. Different fragments of Type 1 (DNA50 -D1) from the resistant strain (black bars) and Type 2 (DNA50 -S2) from the susceptible strain (white bars) were compared. All the fragment constructs are named with ‘‘P’’ as start letter, followed by a pair of parentheses that contain two numerals, separated by a dash(/), to specify the 50 and 30 positions of the corresponding promoter fragment. ‘‘D’’ and ‘‘S’’ indicate the fragment cloned from DNA50 -D1 and DNA50 -S2, respectively. The luciferase activities are corrected for transfection efficiency with the Renilla luciferase pRL-CMV plasmid and presented as ratios to the activity of the promoter P(-1515/-12) from Type 2 (DNA50 -S2). The data and error bars represent the means and standard errors from at least three independent experiments. Values sharing the same letter are not significantly different at P < 0.05 (Duncan tests).

P(-396/-12), the promoter activity increased, slightly for the Type 2 flanking region but significantly for Type 1. The key factors responsible for the major promoter activity of both DNA50 -D1 and DNA50 -S2 may lie within the 186 bp fragment near the translation

To narrow down the location of possible cis-elements, shorter fragments were analysed within the region from 1515 to 827 and from 396 to 187. As shown in Fig. 5A, the full Type 1 flanking region was deleted from 1515 to 1269, 1113, 1000, 906 and 826, and only the deletion from 1515 to 1269 caused a significant decrease in promoter activity. The remaining further deletions to 1113, -1000, -906 and 826 did cause a small but non-significant successive decrease in activity. When the sequences of Type 1 and Type 2 were compared, three mutation sites designated as d1, d2 and d3 were identified in the fragment from 1515 to 1269 (Fig. 5C). The first two were both nucleotide substitutions (G/C at 1479 and A/G at 1368), whereas d3 involved a nucleotide substitution (T/A at 1292) and a short fragment insertion (AGAAG between 1287 and 1283). The mutation changes at the d2 and d3 sites led to the appearance of two TF binding sites (XBP-1 and Dl) in the Type 1 sequence. To characterize these three alterations further, P(-1515/-12)D was selected as standard and three site mutation constructs P(-

Fig. 5. Functional analysis of the promoter activity for the fragment between -1515 to -826. (A) Luciferase analysis of progressive 50 deletion constructs from 1515 to 826 in Type1 flanking region. (B) The luciferase (Luc) activities of recovery mutation constructs in the region from 1515 to 1269 in Type1 flanking region. The names (d1, d2 and d3) of three mutation sites are added after hyphen in the name of corresponding construct. The average relative luciferase activity and standard errors of three independent experiments are presented. Bars marked with different letters are significantly different at p < 0.05 (Duncan tests). (C) The sequences of Type 1 (DNA50 -D1) and Type 2(DNA50 -S2) were compared. Three mutation positions are marked by the letter and number above the corresponding nucleotides. Two putative TF binding sites are underlined. The arrow and number indicate the boundaries of the 50 deletion.

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Fig. 6. Functional analysis of the 50 -flanking region fragment from -396 to -12 for promoter activities. (A) Luminescent reporter assay of the progressive 50 deletion constructs from 396 to 186 in Type1 flanking region. (B) The luciferase (Luc) activities of recovery mutation constructs in the region from 396 to 299 in Type1 flanking region. Bars marked with different letters are significantly different at p < 0.05 (Duncan tests). (C) The sequences of Type 1 (DNA50 -D1) and Type 2(DNA50 -S2) were compared. Three mutation regions (M1, M2 and M3), two putative cis-acting elements and six transcriptional start sites (Inr) are marked under the corresponding nucleotides. The arrow and number indicate the left and right boundaries of the promoter constructs used in A and B.

1515/-12)D-d1, P(-1515/-12)D-d1d2 and P(-1515/-12)D-d1d2d3 were generated to recover these mutations one by one. Activity tests with these constructs demonstrated that d1 recovery did not change promoter activity (Fig. 5B). However, d2 and d3 recovery both led to a significant decrease in activity, implying that the two TF binding sites resulting from mutation at these sites could significantly up-regulate the expression of CYP6FU1. The Type 1 and Type 2 flanking regions had a different sequence structure for the fragment 396~187 (Fig. 6C). The most obvious difference was the 100bp insertion from 294 to 195, present in Type 1, but absent in Type 2. The other differences were designated as three small regions M1 (374~365), M2 (352~342) and M3 (308~300). Furthermore, the change at M2 led to the alteration of two TF binding sites (Dfd in Type 2 and BR-C in Type 1). Thus, a similar approach to that described above was adopted to locate possible cis-elements in this region. The construct P(-299/-12)D showed similar activity to P(186/-12)D, but much lower activity than P(-396/-12)D (Fig. 6A). Thus, positive cis-elements might reside in the region from 396 to 299. Because only three site alterations (M1, M2 and M3) occurred in this region, four constructs P(-396/-12) D-M1, P(-396/-12)D-M2, P(-396/-12)D-M3 and P(-396/-12)DM1M2M3 were generated to recover the three mutations individually and collectively. Recovery of M1 and M3 had no significant effect on promoter activity, whereas M2 recovery and M1M2M3 recovery led to a significant decrease in activity. Furthermore, the effects of M2 recovery alone and M1M2M3 recovery were similar, indicated that the mutation at M2 changing Dfd binding site to BR-C, could increase the expression level of CYP6FU1 (Fig. 6B).

After recovery of all the site mutations, the construct P(-396/12)D-M1M2M3 still showed higher activity than P(-299/-12)D (Fig. 6B), and the latter showed similar activity to P(-186/-12)D and P(-396/-12)S (Fig. 6A). When these four fragments were compared together, it was found that the constructs (P(-396/-12)DM1M2M3) with both the fragment 396~-299 and the 100 bp insertion in fragment 299~186 showed higher activity than those without either fragment (P(-186/-12)D) or with only one of them (P(-396/-12)S and P(-299/-12)D). This implies that these two fragments interact. Sequence checking found a transcription start site existing in the 100 bp insertion (Fig. 6C), and fragment 396 to 299 hosted TF binding sites. The interaction between these two fragments implies that a special transcription start site appearing in 50 -flanking regions could co-operate with sequences up-stream (TF binding sites existed) to up-regulate the expression of CYP6FU1. 3.4. Functional confirmation of transcription start sites in 50 flanking regions The functionality of transcription start sites in 50 -flanking regions was investigated by cloning the 50 UTRs of CYP6FU1 from the cDNA templates containing the resistant and the susceptible strain. A total of six different types of 50 UTR sequences were obtained, which differed in transcription starts and sequence length (Fig. 7). Sequence analysis found all these 50 UTRs were transcribed from the corresponding transcription start sites located in the 50 -flanking regions (Fig. 6C or Fig. S1), demonstrating that all the transcription start sites found in 50 -flanking regions are functional. The longest 50 UTR (50 UTR-1) is transcribed from the transcription start site (Inr 1) found in the 100 bp insertion specific to the

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Fig. 7. 50 UTR of CYP6FU1 mRNA (50 RACE clones) cloned from resistant and susceptible strains. Translation start site (ATG) is indicated by þ1.

Type 1 flanking region. Allele checking found that 7/9 resistant individuals had 50 UTR-1, whereas only 1/10 susceptible ones had it. This implies that though Type 1 flanking region had four different transcription sites, Inr 1 was the most important one influencing transcription of CYP6FU1 mRNA in the resistant strain. 3.5. Joint effect of the cis-acting elements Four mutations in the Type1 flanking region have been implicated in up-regulation of CYP6FU1, including d2 and d3 leading to the appearance of TF binding sites XBP-1 and Dl, respectively, M2 altering a TF binding site from Dfd to BR-C, and Inr 1 representing a unique transcription start site in the Type 1 sequence. We therefore investigated how systematic elimination of different combinations of these mutations affects the promoter activity of the Type 1 sequence. Eliminating any of these four mutations resulted in a significant decrease in promoter activity, this is what we did when identify different cis-acting elements, and eliminating more elements always decreased the promoter activity further. When only one element remained, the promoter activity was not significantly higher than when all elements were absent (Fig. 8A, B). It appears that an accumulation of elements is needed to markedly influence the resistance phenotype. This is consistent with the previous cisacting element identification. Elimination of XBP-1, D1 or BR-C by

recovery resulted in significant influence, and the last two (BR-C and Inr 1) in a short flanking region fragment, P(-396/-12)D, showed obvious joint action. When the 50 -flanking sequences of different types were checked for these four elements, it was found that all variants conforming to Type 1 had all 4 elements, and those of Type 2 had none of them. Type 3 and Type5 had d3 and M2, and Type 4 had only d2 (Fig. S2). This implies that all four elements exist in wild populations of SBPH, and that by bringing them together in the Type 1 sequence, selection with deltamethrin led to substantial over-expression of CYP6FU1. 4. Discussion As well as a serious threat to the sustainability of pest management, insecticide resistance is one of the best-documented examples of micro-evolutionary change in response to environmental challenges (Denholm et al., 2002; Stoddard et al., 2010). Analysis of resistance mechanisms is shedding valuable light on the qualitative and quantitative changes that underpin such adaptations (Denholm and Rowland, 1992; Karasov et al., 2010). Work described in this paper provides a rare demonstration of multiple events being involved in and influencing the up-regulation of a gene CYP6FU1 encoding an enzyme associated with the detoxification of a widely-used pyrethroids insecticide.

Fig. 8. Joint effect of 4 cis-acting mutations in the 50 -flanking region. (A) The luciferase (Luc) activities of recovery mutation constructs in Type1 flanking region. Bars marked with different letters are significantly different at p < 0.05 (Duncan tests). (B) The relative promoter activities were presented as ratios to the activity of Type 1. 0 presents Type 1 was recovered by point mutation to dominate all the four cis-acting elements; 1 presents Type 1 was recovered with only one cis-acting element left (mean of 4 different combinations containing P(-1515/-12)D-d2d3M2, P(-1515/-12)D-d2d3dele100, P(-1515/-12)D-d2M2dele100 and P(-1515/-12)D-d3M2dele100); 2 presents Type 1 was recovered with two cisacting element left (mean of 4 different combinations containing P(-1515/-12)D-M2dele100, P(-1515/-12)D-d3dele100, P(-1515/-12)D-d2dele100 and P(-1515/-12)D-d3M2); 3 presents Type 1 was recovered with three cis-acting element left (mean of 3 different combinations containing P(-1515/-12)D-dele100, P(-1515/-12)D-M2 and P(-1515/-12)D-d2).

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Natural populations adapt constantly to changing environments, with alterations in the amino acid sequence of protein and gene expression providing the main sources of variation upon which natural selection can act. Differences in gene expression are thought to underlie many of the phenotypic differences between species and populations (Glaser-Schmitt et al., 2013; Whitehead and Crawford, 2006; Wray et al., 2003). Comparison of regions within the genome has been one approach for identifying patterns of sequence polymorphism indicative of recent positive selection (Jensen et al., 2007; Stephan, 2010). The gene CYP6FU1 has been proved to be over-expressed by up-regulation in deltamethrinresistant L. striatellus (Xu et al., 2013). Polymorphism analysis of the 50 -flanking region of CYP6FU1 showed a marked difference between susceptible and resistant strains. 50 -flanking regions cloned from a susceptible strain showed 0.090 polymorphism and encompassed all five of the sequence types identified in our study, while those from a resistant strain exhibited only 0.001 polymorphism and all conformed to a single sequence type (Type 1). These results provided initial evidence that positive selection has acted on the regulatory region to increase CYP6FU1 expression in the resistant strain. Much attention has been paid to cis-regulatory elements as they are known to play a key role in the evolution of gene regulation. A well-known example of adaptive cis-regulatory evolution involves the lactase gene (LCT) in humans. SNPs in an upstream cis-acting element are associated with persistent expression of LCT in adults and enable them to digest the milk sugar lactose (Glaser-Schmitt et al., 2013; Ingram et al., 2009). A few elements have also been reported to contribute to insecticide resistance (Daborn et al., 2002; Gao and Scott, 2006; Wan et al., 2014; Pang et al., 2014). In our work, reporter assays showed that the entire flanking region of CYP6FU1 from a resistant strain had higher promoter activity and that four cis-acting mutations contributed to up-regulation. Although these mutations did not up-regulate significantly on their own, their accumulation in same sequence led to substantial up-regulation. Individuals in a deltamethrin-resistant strain consistently had all four mutations whereas susceptible individuals had one or two at most. We hypothesise on this basis that strong and prolonged exposure to deltamethrin has selected for multiple mutations influencing regulation and has led to resistance conferred by over-expression of CYP6FU1 being a quantitative rather than qualitative trait. A knowledge of inheritance characteristics is important for understanding how resistance traits evolve and accumulate in pest populations (Amichot et al., 2004; Hutter et al., 2008; Menozzi et al., 2004; Stephan, 2016). Six start sites were also found to influence transcription of CYP6FU1. The use of multiple promoters and transcriptional start sites is considered to be an important evolutionary mechanism providing flexibility in the regulatory control of gene expression (Ayoubi and Van De Ven, 1996). Previous work reported that a long terminal repeat upstream of the Opitz syndrome gene Mid1 altered the transcription start site and contributed significantly to the level of Mid1 transcripts (Landry et al., 2002). In our study, the insertion Inr 1 (unique to the Type 1 sequence) lost its function when the upstream sequence (with or without the mutations) was deleted. This implies that existing or new TF binding sites in the sequences upstream is essential for normal function. Although nucleotide diversity was greatly reduced in the 50 flanking region of the resistant strain compared with susceptible strain, only four of 316 polymorphism sites in all tested SBPH individuals were confirmed to cause a measurable change in promoter activity. This is indicative of a selective sweep not only increasing the frequency of adaptive mutations, but also affecting genetic variation in areas closely adjacent to these mutations. Previous studies of transposable element (TE) insertion in the

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regulatory region of the ecdysone oxidase (EO) gene in Bombyx mori and an Accord-like element into the 50 region of CYP6G1 in DDT-resistant Drosophila melanogaster have also demonstrated how selective sweeps reduced levels of nucleotide variation around a beneficial mutation (Catania et al., 2004; Sun et al., 2014) - a phenomenon known as ‘hitchhiking’ (Maynard and Haigh, 2007). The size of the genomic region affected depends largely on the selection coefficient and the recombination rate (Catania et al., € tterer and Wiehe, 1999; Wiehe, 1998). 2004; Schlo Acknowledgments This study was supported by the Projects of National Natural Science Foundation of China (Grant No. 31130045), and the Special Fund for Agro-scientific Research in the Public Interest of China (Grant No. 201303017). We thank Yingchuan Pang and Chunqing Zhao (Nanjing Agricultural University, China) for useful discussion. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibmb.2016.08.008. References s, S., Brun-Barale, A., Arthaud, L., Bride, J.M., Berge
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