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Knockdown of NADPH-cytochrome P450 reductase increases the susceptibility to carbaryl in the migratory locust, Locusta migratoria
Accepted Manuscript Knockdown of NADPH-cytochrome P450 reductase increases the susceptibility to carbaryl in the migratory locust, Locusta migratoria Xueyao Zhang, Junxiu Wang, Jiao Liu, Yahong Li, Xiaojian Liu, Haihua Wu, Enbo Ma, Jianzhen Zhang PII:
S0045-6535(17)31368-1
DOI:
10.1016/j.chemosphere.2017.08.157
Reference:
CHEM 19855
To appear in:
ECSN
Received Date: 9 May 2017 Revised Date:
22 August 2017
Accepted Date: 25 August 2017
Please cite this article as: Zhang, X., Wang, J., Liu, J., Li, Y., Liu, X., Wu, H., Ma, E., Zhang, J., Knockdown of NADPH-cytochrome P450 reductase increases the susceptibility to carbaryl in the migratory locust, Locusta migratoria, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.08.157. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Submitted to Chemosphere:
Knockdown
of
NADPH-cytochrome
P450
reductase
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increases the susceptibility to carbaryl in the migratory locust, Locusta migratoria
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Xueyao Zhang#, Junxiu Wang#, Jiao Liu, Yahong Li, Xiaojian Liu, Haihua Wu,
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Enbo Ma* and Jianzhen Zhang*
Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi, China
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#These authors contributed equally to this work.
*Corresponding author: Enbo Ma, [email protected] Jianzhen Zhang, [email protected]
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Tel: +86-0351-7018871
Background: NADPH-cytochrome P450 reductase (CPR) plays important roles in cytochrome P450-mediated metabolism of endogenous and exogenous compounds, and participates in cytochrome P450-related detoxification of insecticides. However, the CPR from Locusta migratoria has not been well characterized and its function is still undescribed.
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Results: The full-length of CPR gene from Locusta migratoria (LmCPR) was cloned by RT-PCR based on transcriptome information. The membrane anchor region, and 3
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conserved domains (FMN binding domain, connecting domain, FAD/NADPH binding domain) were analyzed by bioinformatics analysis. Phylogenetic analysis showed that LmCPR was grouped in the Orthoptera branch and was more closely related to the
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CPRs from hemimetabolous insects. The LmCPR gene was ubiquitously expressed at all developmental stages and was the most abundant in the fourth-instar nymphs and the
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lowest in the egg stage. Tissue-specific expression analysis showed that LmCPR was higher expressed in ovary, hindgut, and integument. The CPR activity was relatively higher in Malpighian tubules and integument. Silencing of LmCPR obviously reduced
migratoria to carbaryl.
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the enzymatic activity of LmCPR, and enhanced the susceptibility of Locusta
Conclusion: These results suggest that LmCPR contributes to the susceptibility of L.
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migratoria to carbaryl and could be considered as a novel target for pest control.
Key words: Locusta migratoria, NADPH-cytochrome P450 reductase, carbaryl, RNAi
1 INTRODUCTION
The oriental migratory locust (Locusta migratoria) is not only one of the most destructive agricultural pests in the world, but also a good research model for insect
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molecular biology and toxicology (Zhang et al., 2014). In past decades, the frequent application of insecticides has inevitably led to insecticide resistance in some natural
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populations of L. migratoria (Yang et al., 2009). Cytochrome P450 monooxygenase (CYP) is a member of the hemoprotein family, and it catalyzes a series of crucial biological reactions in all living organisms from
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bacteria to humans (Feyereisen, 2011). The most common reaction catalyzed by CYP is the monooxygenase reaction, which introduces oxygen into various reducing substrates
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(Sezutsu et al., 2013). In insects, CYPs participate in the detoxification of insecticides, drugs, and plant secondary metabolites and play a key role in metabolizing ecdysteroids, juvenile hormones, and fatty acids (Feyereisen, 2006). Our previous studies suggested
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that CYP409A1 and CYP408B1 (Guo et al., 2012), CYP9A subfamily (Guo et al., 2015; Zhu et al., 2016) and CYP6FF1 were involved in the detoxification of pyrethroid pesticides, and CYP6FD2 and CYP6FE1 were associated with the metabolism of
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carbaryl in L. migratoria (Guo et al., 2016).
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The reaction of microsomal CYPs depends on electron transfer from NADPH through FAD and FMN to the ferrum atom of the prosthetic heme group in the CYP by its electron transfer partner, NADPH-Cytochrome P450 Reductase (Masters and Okita, 1980) (CPR; Fig. 2). Generally speaking, each insect genome possesses only 1 CPR gene. Since the first insect CPR was cloned from Musca domestica (Koener et al., 1993), more than 20 insect CPRs have been identified, and some upregulated CPRs were
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further proved to participate in insecticide resistance in several species, such as Tetranychus cinnabarinus (Shi et al., 2015), Laodelphax striatellus (Zhang et al., 2016),
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and Bactrocera dorsalis (Huang et al., 2015). Furthermore, RNAi-mediated silencing of insect CPR genes enhanced insecticide-induced mortality in Cimex lectularius (Zhu et al., 2012) and Nilaparvata lugens (Liu et al., 2015b). For its essential role in the
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CYPs/CPR electron cycle, insect CPRs were considered as a novel target for the development of synergists (Zhu et al., 2012). However, there is no report on the
insecticide-induced mortality.
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sequence and biological function of CPR from L. migratoria and whether it is related to
In the present study, a full-length cDNA of the CPR from L. migratoria (LmCPR)
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was cloned and characterized. The developmental and spatial expression patterns of LmCPR were examined by RT-qPCR. Tissue distribution of CPR activity was determined by spectrophotometrical method at 550 nm. The transcription of LmCPR
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was silenced by injection of LmCPR-specific dsRNA, which depressed CPR enzymatic
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activity. Then, a bioassay was performed to compare the carbaryl susceptibility of LmCPR-silenced and control L. migratoria. This study will help to elucidate the role of CPRs in CYP-mediated metabolic detoxification of carbaryl in L. migratoria.
2 MATERIAL AND METHODS 2.1 Insect cultures and treatments
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L. migratoria eggs were purchased from Insect Protein Co., Ltd. (Cangzhou, China). The eggs were hatched in sandy soil, and instar nymphs were reared on fresh leaves of
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Setaria viridis (L.) at 30 ± 1 °C under a 14 h:10 h light: dark cycle. Adults were bred under the same light and temperature conditions, and were supplied with fresh leaves of
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Setaria viridis (L.) and wheat bran.
2.2 Reagents and assay kits
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The pEASY-Blunt Zero cloning kit and Fastpfu DNA polymerase were obtained from Transgen Biotech Co. Ltd. (Beijing, China). RevertAid H minus reverse transcriptase was purchased from Fermentas (MA, USA). SYBR Green Real-time PCR
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Master Mix was obtained from Toyobo (Osaka, Japan). dNTPs, TRIzol, and the gel extraction kit were obtained from TaKaRa Bio Group (Dalian, China). The T7 RiboMAx Express RNAi System was from Promega (WI, USA). The Cytochrome c reductase
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(NADPH) assay kit was obtained from Sigma-Aldrich (MO, USA). All other reagents
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were of the highest grade commercially available.
2.3 Synthesis of cDNA and cloning of LmCPR Total RNA, which used to clone full-length LmCPR, was extracted from nymphs and adults of L. migratoria by the TRIzol method, according to the manufacturer’s protocol. The quality and concentration of the extracted RNA was determined with a
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Nanodrop2000. The first-strand cDNA was synthesized from 4 µg of total RNA by RevertAid H minus reverse transcriptase in a reaction containing dNTPs, RNase inhibitor,
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and oligo(dT)18. Based on the L. migratoria transcriptome data, a pair of full-length primers for RT-PCR was designed and is shown in Table 1. The RT-PCR was performed with FastPfu DNA polymerase. The PCR product was recovered with the gel extraction
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kit and ligated into the pEASY Blunt Zero vector, which was then transformed into Trans-T1 competent cells and sequenced in both directions by Sangon Biotech (Shanghai,
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China).
2.4 Bioinformatics analysis of LmCPR gene
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The predicted open reading frame (ORF) and protein sequence of LmCPR was deduced from the LmCPR gene by DNA translate (http://web.expasy.org/translate/). The theoretical isoelectric point and molecular weight of LmCPR were predicted by
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ProtParam (http://web.expasy.org/protparam/). The amino acid sequences of various
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insect CPRs were aligned using Muscle software. Then, a phylogenetic tree was constructed by the neighbor-joining method using MEGA 6.0 software (Tamura et al., 2013), with 1000 bootstrap replicates.
2.5 Reverse transcription quantitative PCR analysis The primers used for the RT-qPCR analysis are shown in Table 1. The RT-qPCR
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assay was performed on an ABI 7300 real-time PCR system in a final volume of 20 µL, containing 4 µL of cDNA,10 µL of 2× SYBR Green Real-time PCR Master Mix (with
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ROX), 1.5 µL of each RT-qPCR primer (10 pmol/µL), and 3 µL of DNase-free water. The PCR started with a denaturation step at 94 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 61 °C for 31 s. For the
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tissue-specific expression profiles, total RNA samples were prepared from the brain, foregut, midgut, gastric caecum, hindgut, fat bodies, Malpighian tubules, ovary, spermary,
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hemolymph, integument and muscles from the fifth-instar nymphs of L. migratoria. For the stage-specific expression profiles, the eggs, the first-, second-, third-, fourth- and fifth-instar nymphs and adults were collected for RNA extraction. EF1α, RpL32 and
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Hsp70 were selected as the reference genes. All data were analyzed by one-way ANOVA, Tukey’s HSD test. Relative transcript levels were determined by using the double standard curve method. The amplification efficiency of the primer used for RT-qPCR was
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higher than 0.95. The experiment was repeated with five biological replicates.
2.6 Tissue distribution of CPR activity The foregut, midgut, hindgut, gastric caecum, Malpighian tubules, fat bodies, integument, muscles, spermary, ovary, hemolymph and brain were dissected from fifth-instar nymphs of L. migratoria. Then the dissected samples were homogenized on ice with homogenization buffer (0.1 mol/L sodium phosphate, pH 7.6, containing 1
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mmol/L EDTANa2, 1 mmol/L phenylmethylsulphonyl fluoride, 0.1 mmol/L dithiothreitol, 1 mmol/L leupeptin and 20% glycerol). Then, the homogenates were centrifuged at
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12,000 g for 15 min at 4 oC. The supernatants were used for CPR activity assay. The CPR activity was measured at 550 nm with a microplate spectrophotometer, according to cytochrome P450 reductase assay kit instructions (Vermilion and Coon, 1974). Briefly,
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the 1-mL reaction mix contained 950 µL of 36 µmol/L cytochrome c (0.3 mol/L potassium phosphate buffer, pH 7.8), 50 µL of protein supernatant, and 100 µL of 0.85
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mg/L NADPH. One unit of CPR was defined as the amount of enzyme required to reduce 1 µmole of oxidized cytochrome c in the presence of 100 µmol/L NADPH per minute at room temperature. The protein concentration was measured by the BCA method (Smith
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et al., 1985) using bovine serum albumin as a standard. The experiment was repeated with three biological replicates. All data were analyzed by one-way ANOVA, Tukey’s
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HSD test.
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2.7 Silencing of LmCPR by RNAi The LmCPR and green fluorescent protein (GFP) genes were amplified by PCR using dsRNA primers containing the T7 RNA polymerase promoter. The primers used for RNAi analysis are shown in Table 1. The PCR fragments containing T7 promoter sequences on both ends were purified with the gel extraction kit and used as templates to synthesize dsRNA with the T7 RiboMAX Express RNAi System. The concentration of
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the dsRNA was determined with a Nanodrop2000. Then, the dsRNA was dissolved in ddH2O to a final concentration of 1.5 µg/µL. Approximately 3 µg of the dsRNA were
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injected into the abdomen between the second and third segments of third-instar nymphs on the second day. To determine the suppression level of LmCPR transcripts, cDNAs were synthesized from total RNA isolated from whole nymphs at 24, 48 and 72 hours
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after LmCPR dsRNA injection. β-actin, EF1α and Hsp70 were selected as the reference genes. The amplification efficiency of the primer used for RT-qPCR was higher than 0.95.
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The experiment was repeated with five biological replicates. All data were analyzed by Student’s t-test.
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2.8 CPR activity assay after silencing of LmCPR
The third-instar nymphs were homogenized on ice with potassium phosphate buffer (0.3 mol/L, pH 7.8) at 24 and 48 hours after dsRNA injection, and then homogenates
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were centrifuged at 10,000 g for 10 min at 4 oC. The supernatants were used for CPR
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activity assay. The CPR activity was measured at 550 nm with a microplate spectrophotometer, as described in section 2.6. The protein concentration of the enzyme solutions was determined by the Bradford method using bovine serum album as the standard (Noble and Bailey, 2009). The experiment was repeated with three biological replicates. All data were analyzed by Student’s t-test.
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2.9 Bioassays with carbaryl after RNAi To determine the effect of gene silencing on carbaryl-induced mortality, carbaryl
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was dissolved in acetone, and 3 µL of the carbaryl solutions (10 µg/mL and 15 µg/mL) were dropped on the abdomen of the locust at 24 hours after dsRNA injection. Mortality was determined at 24 hours after carbaryl exposure. Mean and standard errors for each
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concentration were performed in five independent bioassays. All data were analyzed by
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Student’s t-test.
3 RESULTS
3.1 Cloning and sequence analysis of LmCPR
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In our previous study, a transcriptome database of L. migratoria was constructed by using mixture mRNA from whole bodies at all developmental stages (Zhao et al., 2017). Based on the transcriptome data and a keyword search of the annotation database, only
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one candidate CPR gene was identified by keyword search of annotation database.
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The full-length cDNA sequence of LmCPR was obtained by RT-PCR. The 2043 bp cDNA sequence has been submitted to GenBank (accession number KX765501). The cDNA sequence of LmCPR was searched against the locust genome by local BLAST. The LmCPR gene was at least 42 kb in length and was composed of 14 introns and 15 exons (Fig. 1 and Fig. S1). The length of the introns ranged from 91 bp to more than 8800 bp. LmCPR encoded a deduced protein containing 680 amino acids. The predicted pI
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and MW of the deduced protein were 5.49 and 77.1 kDa, respectively. It shared the highest amino acid identity with CPR from Oxya chinensis (96.33%), followed by
LmCPR.
However,
a
hydrophobic
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Pediculus humanus corporis (75.81%; Table S1). No signal peptide was identified in transmembrane
motif
(20L-39L:
LGAVDILLLAALLALAVWYL) was predicted in the N-terminal region of LmCPR, and
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may be involved in the localization of LmCPR on the endoplasmic reticulum.
As shown in Fig. 1 and Fig. 2, LmCPR was mainly composed of three conserved
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domains, including the FMN binding domain, connecting domain, and FAD/NADPH binding domain. The membrane anchor region was encoded by the second exon, while the FMN binding domain, connecting domain, FAD/NADPH binding domain were
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encoded by exons 3-6, 7-10, 7, 10, 11 and 12-15, respectively (Fig. 1). The FAD binding domain was interrupted by connecting domain, and the two domains were intertwined in
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linear sequence (Wang et al., 1997).
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3.2 Phylogenetic relationships among insect CPRs A phylogenetic tree was generated by MEGA6 using the neighbor-joining method based on the amino acid sequences of LmCPR and 22 other insect CPRs. The result showed that the insect CPRs from the same insect order were clustered into same branch with high bootstrap support (Fig. 3). The LmCPR shares the relatively higher sequence identity with the CPRs from Oxya chinensis (OcCPR) and Pediculus humanus corporis
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(PhCPR; Table S1). Therefore, LmCPR and OcCPR were first grouped into the Orthoptera branch, and then grouped with PhCPR, followed by the CPRs from Hemiptera
insects.
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3.3 Distribution of LmCPR mRNA and LmCPR activity.
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and Coleoptera, indicating closer evolutionary relationship with CPRs in hemimetabolous
LmCPR was detected in all tested tissues. Higher mRNA expression was found in
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the ovary, integument, and hindgut, whereas lower expression was detected in the hemolymph, muscles, and midgut (Fig. 4A). It is worthy to note that the CPR activity was relatively higher in Malpighian tubules and integument and lower in the spermary,
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ovary and hemolymph (Fig. 4B). Pearson correlation analysis (Fig. 4C) showed that there was no significant correlation (r=0.137, P=0.642) between the gene expression pattern of LmCPR and the distribution of CPR activity among all the 12 tissues.
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In view of the CPR activity was affected by many factors, and moreover no
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significant correlation was observed between the mRNA of LmCPR and the enzymatic activity of CPR among different tissues, the subsequent developmental expression experiments were focused on the mRNA level of LmCPR. We examined the mRNA expression level of LmCPR by RT-qPCR, and it was detected from the egg stage to the adult (Fig. 5). LmCPR expression level was the lowest in the egg stage, gradually and significantly increased from the first- to fourth-instar stage (P < 0.05), and was slightly
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decreased in fifth-instar nymphs and adults, but the differences were not significant. When exposed to carbaryl, the mRNA level of LmCPR cannot be induced by carbaryl at
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10 µg/ml and 22 µg/ml (Fig. S2).
3.4 Knockdown of LmCPR by RNAi
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To determine the efficiency of the RNAi, the expression level of LmCPR in third-instar nymphs was analyzed by RT-qPCR. The mRNA level of LmCPR decreased
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by 71.2% at 24 hours after LmCPR dsRNA injection and further declined by 78.6% and 80.6% at 48 and 72 hours after dsRNA injection, respectively, when compared to the dsGFP control (Fig. 6A). This result suggests that the RNA interference effect of the
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LmCPR dsRNA was retained for at least 72 hours; and subsequent experiments were limited to 48 hours. To further detect the influence of LmCPR knockdown, we detected CPR activity by the colorimetric method at 550 nm. The results showed that the CPR
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activity of L. migratoria was obviously decreased 18.4% and 27.2% at 24 and 48 hours
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after LmCPR dsRNA injection (Fig. 6B). It is reasonable that the down-regulation of CPR activity is consistent with the interference of LmCPR gene, because there was only one candidate LmCPR gene in locust genome. The lower decline in CPR enzymatic activity when compared to the decrease in LmCPR mRNA level, may be due to one or more factors, including a low degradation rate of CPR enzyme, or a contribution of other enzymes to cytochrome c reduction, for instance cyanide-suppressible mitochondrial
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cytochromes that were present in the crude homogenates.
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3.5 The sensitivity to carbaryl after LmCPR knockdown Knockdown of LmCPR in the nymphs of L. migratoria significantly increased its sensitivity to carbaryl. When treated with 10 µg and 15 µg carbaryl, the mortality of
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third-instar nymphs was 17.5% and 37.5%, respectively, in the LmCPR-knockdown group, whereas it was 7.5% and 21.67% in the control group, respectively (Fig. 7). These
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results showed that silencing of LmCPR has an obviously impact on the susceptibility of L. migratoria to carbaryl.
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4 DISCUSSIONS
Insect CYPs are known to play an essential role in insecticide resistance and the detoxification of exogenous compounds (Feyereisen, 2011). As an indispensable part of
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CYPs/CPR system, insect CPR transfers electrons from NADPH to CYPs (Masters and
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Okita, 1980; Liu et al., 2015a). Further research to characterize insect CPR will help to reveal the function of CYPs/CPR system in the detoxification of insecticides and other xenobiotics and facilitate the development of novel targets for insecticides. In this study, we cloned the full-length cDNA of the CPR gene from L. migratoria (LmCPR). Bioinformatics analysis showed that this enzyme consists of an N-terminal membrane anchor and FMN binding domain, connecting domain, and FAD/NADPH
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binding domain. The FAD binding domain and connecting domain are intertwined in linear sequence (Wang et al., 1997). The FAD/NADPH binding domain is located near
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the C-terminal end, while the FMN binding domain is situated in the N-terminal region, and the two domains are interconnected through random coils. The N-terminal membrane anchor functions to anchor the protein to the endoplasmic membrane, and makes the
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other parts of LmCPR face the cytoplasmic side for the CYPs, which is essential for electron transfer between CPR and CYPs (Laursen et al., 2011). Within the FAD/NADPH
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binding domain, the FAD binding motif, which comprises 3 amino acids (R459, Y460, and S461), is highly conserved in insects. The catalytic center of LmCPR, which comprised 4 conserved residues (S462, C632, D677, and W679), is considered to be
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indispensable in the hydride transfer cycle. The flexibility of the random coils possibly participates in the conformational plasticity of FMN domain, which facilitates the interaction between FMN and various CYPs (Laursen et al., 2011).
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CYP activity depends on the electrons from NADPH, which is transferred by insect
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CPR. Therefore, tissue- and stage-specific expression of LmCPR reflects certain CYP activities. The data showed that LmCPR expression level was higher in ovary, integument, and hindgut. The CPR activity was higher in Malpighian tubules and integument. It is worthy to note that the higher levels of both CPR activity and mRNA of LmCPR gene in integument. This result was similar to that of the CPRs in Drosophila melanogaster (Qiu et al., 2012), Anopheles gambiae (Lycett et al., 2006; Balabanidou et al., 2016). These
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insect CPRs may function to the biosynthesis of lipids for the insect cuticle. Our results indicated that LmCPR expression level was the lowest in the egg stage and higher during
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the later developmental stages. This phenomenon was similar to the expression pattern of CPR in T. cinnabarinus (Shi et al., 2015) and C. lectularius (Zhu et al., 2012). The distinct expression patterns of LmCPR in a variety of developmental stages and various
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tissues suggests that LmCPR can adapt to the different demands of LmCYPs for driving endogenous and exogenous metabolic processes, which may improve the adaptability of
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L. migratoria to internal and external stresses.
Several studies have shown that upregulation of insect CPRs is related to insecticide resistance. The mRNA level of CPR from Plutella xylostella in a β-cypermethrin resistant
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strain was 12-fold higher than that in a susceptible strain (Chen and Zhang, 2015). Compared to a susceptible stain, the expression level of CPR from T. cinnabarinus (Shi et al., 2015) was significantly enhanced (3.12-fold) in a fenpropathrin-resistant strain. In
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contrast, knockdown of the CPR from Anopheles gambiae increased susceptibility to
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permethrin (Lycett et al., 2006). RNA interference of NlCPR enhanced susceptibility to β-cypermethrin and imidacloprid (Liu et al., 2015b). Our results suggest that LmCPR was higher expressed in the ovary, hindgut, and integument. Knockdown of LmCPR can decrease the enzymatic activity of LmCPR, and enhanced the susceptibility of L. migratoria to carbaryl. These findings could be explained by 3 possible mechanisms. First, the CYPs/CPR complex is involved in the biosynthesis of lipids for the insect
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cuticle; therefore, knockdown of these components possible altered cuticle penetration. RNA interference of CYP4G1 or CPR from D. melanogaster blocked hydrocarbon
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biosynthesis in oenocytes (Qiu et al., 2012). Silencing of CYP4G102 in L. migratoria resulted in loss of cuticular waterproofing (Yu et al., 2016). In a resistant strain of An. gambiae, upregulation of CYP4G16 led to a thicker epicuticle and slower insecticide
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uptake (Balabanidou et al., 2016). Therefore, it is worth to note that LmCPR are higher expressed in integument, and knockdown of LmCPR possible influences the activity of
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integument CYPs and cuticle penetration.
Second, the CYPs/CPR system is known to directly participate in insecticide metabolism. The CYP6Z1 from An. gambiae can metabolize DDT (Chiu et al., 2008).
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The CYP6M2, CYP6P3, CYP6P9b, and CYP9J32 from mosquito can metabolize pyrethroids (David et al., 2013), while CYP6Z2 and CYP6Z8 play key roles in the clearance of the pyrethroid metabolites (Chandor-Proust et al., 2013). These
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detoxification reactions all depend on the electron transferred from NADPH by insect
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CPRs. Knockdown of LmCPR gene, can not only decrease the mRNA level of LmCPR, but also possible depressed the detoxification ability of CYPs/CPR system against carbaryl in Locusta migratoria. Third, CPR also can transfer electron to other electron receptors, such as haem oxygenase and cytochrome b5, and indirectly increase the tolerance to insecticides in insect (Guzov et al., 1996; Murataliev et al., 2008; Liu et al., 2015b).
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In conclusion, the present study provides preliminary information on the sequence, phylogeny and expression pattern of LmCPR in L. migratoria. Silencing of LmCPR
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obviously reduced the enzymatic activity of LmCPR, and enhanced the susceptibility of L. migratoria to carbaryl. Further researches are needed to demonstrates the essential role of the LmCPR in CYPs-mediated detoxification of insecticides and other physiological
ACKNOWLEDGEMENTS
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functions in L. migratoria.
This work was supported by the National Natural Science Foundation of China (31320103921, 31101463, 31402020) and the Shanxi Province Science Foundation
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709-717. Guo, Y.Q., Wu, H.H., Zhang, X.Y., Ma, E., Guo, Y.P., Zhu, K.Y., Zhang, J.Z., 2016. RNA interference of cytochrome P450 CYP6F subfamily genes affects susceptibility to different insecticides in Locusta migratoria. Pest Management Science 72, 2154-2165. Guo, Y.Q., Zhang, X.Y., Wu, H.H., Yu, R.R., Zhang, J.Z., Zhu, K.Y., Guo, Y.P., Ma, E.B., 2015. Identification and functional analysis of a cytochrome P450 gene CYP9AQ2 involved in deltamethrin detoxification from Locusta migratoria. Pesticide Biochem Physiol 122, 1-7. Guzov, V.M., Houston, H.L., Murataliev, M.B., Walker, F.A., Feyereisen, R., 1996. Molecular Cloning, Overexpression in Escherichia coli, Structural and Functional Characterization of House Fly Cytochrome b5. Journal of Biological Chemistry 271, 26637-26645. Huang, Y., Lu, X.P., Wang, L.L., Wei, D., Feng, Z.J., Zhang, Q., Xiao, L.F., Dou, W., Wang, J.J., 2015. Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion. Sci Rep 5, 18394. Koener, J.F., Cariño, F.A., Feyereisen, R., 1993. The cDNA and deduced protein sequence of house fly NADPH-cytochrome P450 reductase. Insect Biochemistry and Molecular Biology 23, 439-447. Laursen, T., Jensen, K., Møller, B.L., 2011. Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450. BBA-Proteins Proteom 1814, 132-138. Liu, N.N., Li, M., Gong, Y.H., Liu, F., Li, T., 2015a. Cytochrome P450s – Their expression, regulation, and role in insecticide resistance. Pestic Biochem Phys 120, 77-81. Liu, S., Liang, Q.M., Zhou, W.W., Jiang, Y.D., Zhu, Q.Z., Yu, H., Zhang, C.X., Gurr, G.M., Zhu, Z.R., 2015b. RNA interference of NADPH–cytochrome P450 reductase of the rice brown planthopper, Nilaparvata lugens, increases susceptibility to insecticides. Pest Manag Sci 71, 32-39. Lycett, G.J., McLaughlin, L.A., Ranson, H., Hemingway, J., Kafatos, F.C., Loukeris, T.G., Paine, M.J.I., 2006. Anopheles gambiae P450 reductase is highly expressed in oenocytes and in vivo knockdown increases permethrin susceptibility. Insect Mol Biol 15, 321-327. Masters, B.S.S., Okita, R.T., 1980. The history, properties, and function of NADPH-cytochrome P-450 reductase. Pharmacology & Therapeutics 9, 227-244. Murataliev, M.B., Guzov, V.M., Walker, F.A., Feyereisen, R., 2008. P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis. Insect Biochemistry and Molecular Biology 38, 1008-1015. Noble, J.E., Bailey, M.J.A., 2009. Chapter 8 Quantitation of Protein. in: Richard, R.B., Murray, P.D. (Eds.). Methods in Enzymology. Academic Press, pp. 73-95. Qiu, Y., Tittiger, C., Wicker-Thomas, C., Le Goff, G., Young, S., Wajnberg, E., Fricaux, T., Taquet, N., Blomquist, G.J., Feyereisen, R., 2012. An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis. P Natl Acad Sci USA 109, 14858-14863. Sezutsu, H., Le Goff, G., Feyereisen, R., 2013. Origins of P450 diversity. Philos T R Soc B 368, 20120428. Shi, L., Zhang, J., Shen, G.M., Xu, Z.F., Wei, P., Zhang, Y.C., Xu, Q., He, L., 2015. Silencing NADPH-cytochrome P450 reductase results in reduced acaricide resistance in Tetranychus cinnabarinus (Boisduval). Sci Rep 5, 15581. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150, 76-85. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30, 2725-2729. Vermilion, J.L., Coon, M.J., 1974. Highly purified detergent-solubilized NADPH-cytochrome P450 reductase from phenobarbital-induced rat liver microsomes. Biochem Bioph Res Co 60, 1315-1322. Wang, M., Roberts, D.L., Paschke, R., Shea, T.M., Masters, B.S.S., Kim, J.-J.P., 1997. Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes. Proceedings of the National Academy of Sciences 94, 8411-8416. Yang, M.L., Zhang, J.Z., Zhu, K.Y., Xuan, T., Liu, X.J., Guo, Y.P., Ma, E.B., 2009. Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen). Arch Insect Biochem 71, 3-15. Yu, Z.T., Zhang, X.Y., Wang, Y., Wen, Moussian, B., Zhu, K.Y., Li, S., Ma, E.B., Zhang, J.Z., 2016. LmCYP4G102: An oenocyte-specific cytochrome P450 gene required for cuticular waterproofing in the migratory locust, Locusta migratoria. Scientific Reports 6, 29980. Zhang, X.Y., Wang, J.X., Zhang, M., Qin, G.H., Li, D.Q., Zhu, K.Y., Ma, E.B., Zhang, J.Z., 2014. Molecular Cloning, Characterization and Positively Selected Sites of the Glutathione S-Transferase Family from Locusta migratoria. PLoS ONE 9, e114776. Zhang, Y.L., Wang, Y.M., Wang, L.H., Yao, J., Guo, H.F., Fang, J.C., 2016. Knockdown of NADPH-cytochrome P450 19
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reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén). Pestic Biochem Phys 127, 21-27. Zhao, X., Gou, X., Qin, Z., Li, D., Wang, Y., Ma, E., Li, S., Zhang, J., 2017. Identification and expression of cuticular protein genes based on Locusta migratoria transcriptome. Scientific Reports 7, 45462. Zhu, F., Sams, S., Moural, T., Haynes, K.F., Potter, M.F., Palli, S.R., 2012. RNA Interference of NADPH-Cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius. PLoS ONE 7, e31037. Zhu, W.Y., Yu, R.R., Wu, H.H., Zhang, X.Y., Liu, Y.M., Zhu, K.Y., Zhang, J.Z., Ma, E.B., 2016. Identification and characterization of two CYP9A genes associated with pyrethroid detoxification in Locusta migratoria. Pesticide Biochem Physiol 132, 65-71.
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Table 1 Primer used in this study Name
5'-3' sequence
Full-length
CPR-Full FW
ATGGAGGCAGAGGCAGGCAACG
RT-PCR
CPR-Full RV
TCAGCTCCATACATCTGAAGAAT
CPR-FW
TTACCCACCTGCTACAAAAGAA
CPR-RV
CACAACAATGTCATGCACATCA
Hsp70-FW
CTGGTGTGCTCATTCAGGTAT
Hsp70-RV
TCGTGGGGCAGGTGGTATT
EF1α-FW
AGCCCAGGAGATGGGTAAAG
EF1α-RV
CTCTGTGGCCTGGAGCATC
RPL32-FW
ACTGGAAGTCTTGATGATGCAG
RPL32-RV
CTGAGCCCGTTCTACAATAGC
β-actin-FW
CGAAGCACAGTCAAAGAGAGGTA
β-actin-RV
GCTTCAGTCAAGAGAACAGGATG
RNAi template
CPR-RNAi FW
taatacgactcactatagggTATGAAATGGGTCTTGGGGA
PCR
CPR-RNAi RV
taatacgactcactatagggGGGTGCTTCTTCGTTGACTC
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RT-qPCR
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Application
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Legends of Figures Figure 1. Exon-intron organization of the LmCPR gene. The exons are shown as colored boxes, and the introns are indicated by
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dark lines. The corresponding conserved domains of LmCPR are shown below.
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Figure 2. The reaction mechanism of CPR/CYPs system.
Figure 3. Phylogenetic tree of insect CPR genes.
Phylogenetic tree of insect CPR genes was constructed using the neighbor-joining method and tested by the bootstrap method with 1000
Data S1.
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replications. The sequences used for constructing the tree are shown in
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Figure 4. Tissue-distribution of LmCPR mRNA and LmCPR activity.
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A, Tissue-specific expression patterns of LmCPR. The data are the mean ± SD of 5 biological repeats. B. The activity of LmCPR among different tissues from 5th instar nymph of L. migratoria. The data are the mean ± SD of 3 biological repeats. C. Pearson correlation analysis between gene expression level of LmCPR and activity distribution of LmCPR.
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foregut (FG), midgut (MG), hindgut (HG), gastric caecum (GC), Malpighian tubules (MT), fat bodies (FB), spermary (SP), ovary (OV), muscles (MU), integument (IN), hemolymph (HM), and brain (BR).
ANOVA and Tukey’s HSD test.
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Small letters indicate significant differences (p < 0.05), according to
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Figure 5. Developmental expression patterns of LmCPR.
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EG (eggs), first (1st), second (2nd), third (3rd), fourth (4th), and fifth (5th) instar nymphs and adults (AD).
Small letters indicate significant differences (p < 0.05), according to ANOVA and Tukey’s HSD test. The data are the mean ± SD of 5
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biological repeats.
Figure 6. The effect of RNAi of LmCPR on mRNA expression and
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enzymatic activity.
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A, The mRNA levels of LmCPR after injection of LmCPR-specific dsRNA was evaluated by RT-qPCR. B, The enzymatic activity of LmCPR after dsRNA injection. dsGFP, third-instar nymphs injected with dsRNA against green fluorescent protein; dsCPR, third-instar nymphs injected with dsRNA against LmCPR. The data are the mean ± SD of at least 3 biological repeats. One asterisk on the error bar indicates significant differences (p <
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0.05).
Figure 7. The susceptibility of the locusts to carbaryl after LmCPR
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silencing dsGFP, third-instar nymphs injected with dsRNA against green fluorescent protein; dsCPR, third-instar nymphs injected with dsRNA
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against LmCPR. The data are the mean ± SD of at least 3 biological
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repeats. One asterisk on the error bar indicates significant differences (p < 0.05).
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Figure S1. Genome structure of LmCPR gene.
Figure S2. The expression level of LmCPR exposed to carbaryl. For induction effect of carbaryl on LmCPR genes, the third-instar
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nymphs were topically applied 3 µL solution onto the abdomen between
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the second and third sterna of each nymph for 24 hours. The two carbaryl solutions, (10 mg/ml and 22 mg/ml carbaryl), were in distilled in cold acetone, which served as a control. The normalization factors for induction effect generated by the combination of three best reference genes, EF1α, RpL32 and Rp49. Small letters indicate significant differences (p < 0.05), according to ANOVA and Tukey’s HSD test. The data are the mean ± SD of 5 biological repeats.
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Highlights
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1. The 2043 bp cDNA sequence of CPR gene from Locusta migratoria was cloned by RT-PCR.
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2. Higher mRNA expression was found in the antenna, ovary, integument, and hindgut.
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3. Silencing of LmCPR reduced the enzymatic activity of LmCPR.
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4. Silencing of LmCPR enhanced the susceptibility of Locusta migratoria to carbaryl.
S0045-6535(17)31368-1
DOI:
10.1016/j.chemosphere.2017.08.157
Reference:
CHEM 19855
To appear in:
ECSN
Received Date: 9 May 2017 Revised Date:
22 August 2017
Accepted Date: 25 August 2017
Please cite this article as: Zhang, X., Wang, J., Liu, J., Li, Y., Liu, X., Wu, H., Ma, E., Zhang, J., Knockdown of NADPH-cytochrome P450 reductase increases the susceptibility to carbaryl in the migratory locust, Locusta migratoria, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.08.157. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Submitted to Chemosphere:
Knockdown
of
NADPH-cytochrome
P450
reductase
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increases the susceptibility to carbaryl in the migratory locust, Locusta migratoria
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Xueyao Zhang#, Junxiu Wang#, Jiao Liu, Yahong Li, Xiaojian Liu, Haihua Wu,
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Enbo Ma* and Jianzhen Zhang*
Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi, China
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#These authors contributed equally to this work.
*Corresponding author: Enbo Ma, [email protected] Jianzhen Zhang, [email protected]
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Tel: +86-0351-7018871
Background: NADPH-cytochrome P450 reductase (CPR) plays important roles in cytochrome P450-mediated metabolism of endogenous and exogenous compounds, and participates in cytochrome P450-related detoxification of insecticides. However, the CPR from Locusta migratoria has not been well characterized and its function is still undescribed.
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Results: The full-length of CPR gene from Locusta migratoria (LmCPR) was cloned by RT-PCR based on transcriptome information. The membrane anchor region, and 3
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conserved domains (FMN binding domain, connecting domain, FAD/NADPH binding domain) were analyzed by bioinformatics analysis. Phylogenetic analysis showed that LmCPR was grouped in the Orthoptera branch and was more closely related to the
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CPRs from hemimetabolous insects. The LmCPR gene was ubiquitously expressed at all developmental stages and was the most abundant in the fourth-instar nymphs and the
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lowest in the egg stage. Tissue-specific expression analysis showed that LmCPR was higher expressed in ovary, hindgut, and integument. The CPR activity was relatively higher in Malpighian tubules and integument. Silencing of LmCPR obviously reduced
migratoria to carbaryl.
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the enzymatic activity of LmCPR, and enhanced the susceptibility of Locusta
Conclusion: These results suggest that LmCPR contributes to the susceptibility of L.
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migratoria to carbaryl and could be considered as a novel target for pest control.
Key words: Locusta migratoria, NADPH-cytochrome P450 reductase, carbaryl, RNAi
1 INTRODUCTION
The oriental migratory locust (Locusta migratoria) is not only one of the most destructive agricultural pests in the world, but also a good research model for insect
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molecular biology and toxicology (Zhang et al., 2014). In past decades, the frequent application of insecticides has inevitably led to insecticide resistance in some natural
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populations of L. migratoria (Yang et al., 2009). Cytochrome P450 monooxygenase (CYP) is a member of the hemoprotein family, and it catalyzes a series of crucial biological reactions in all living organisms from
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bacteria to humans (Feyereisen, 2011). The most common reaction catalyzed by CYP is the monooxygenase reaction, which introduces oxygen into various reducing substrates
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(Sezutsu et al., 2013). In insects, CYPs participate in the detoxification of insecticides, drugs, and plant secondary metabolites and play a key role in metabolizing ecdysteroids, juvenile hormones, and fatty acids (Feyereisen, 2006). Our previous studies suggested
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that CYP409A1 and CYP408B1 (Guo et al., 2012), CYP9A subfamily (Guo et al., 2015; Zhu et al., 2016) and CYP6FF1 were involved in the detoxification of pyrethroid pesticides, and CYP6FD2 and CYP6FE1 were associated with the metabolism of
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carbaryl in L. migratoria (Guo et al., 2016).
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The reaction of microsomal CYPs depends on electron transfer from NADPH through FAD and FMN to the ferrum atom of the prosthetic heme group in the CYP by its electron transfer partner, NADPH-Cytochrome P450 Reductase (Masters and Okita, 1980) (CPR; Fig. 2). Generally speaking, each insect genome possesses only 1 CPR gene. Since the first insect CPR was cloned from Musca domestica (Koener et al., 1993), more than 20 insect CPRs have been identified, and some upregulated CPRs were
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further proved to participate in insecticide resistance in several species, such as Tetranychus cinnabarinus (Shi et al., 2015), Laodelphax striatellus (Zhang et al., 2016),
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and Bactrocera dorsalis (Huang et al., 2015). Furthermore, RNAi-mediated silencing of insect CPR genes enhanced insecticide-induced mortality in Cimex lectularius (Zhu et al., 2012) and Nilaparvata lugens (Liu et al., 2015b). For its essential role in the
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CYPs/CPR electron cycle, insect CPRs were considered as a novel target for the development of synergists (Zhu et al., 2012). However, there is no report on the
insecticide-induced mortality.
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sequence and biological function of CPR from L. migratoria and whether it is related to
In the present study, a full-length cDNA of the CPR from L. migratoria (LmCPR)
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was cloned and characterized. The developmental and spatial expression patterns of LmCPR were examined by RT-qPCR. Tissue distribution of CPR activity was determined by spectrophotometrical method at 550 nm. The transcription of LmCPR
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was silenced by injection of LmCPR-specific dsRNA, which depressed CPR enzymatic
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activity. Then, a bioassay was performed to compare the carbaryl susceptibility of LmCPR-silenced and control L. migratoria. This study will help to elucidate the role of CPRs in CYP-mediated metabolic detoxification of carbaryl in L. migratoria.
2 MATERIAL AND METHODS 2.1 Insect cultures and treatments
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L. migratoria eggs were purchased from Insect Protein Co., Ltd. (Cangzhou, China). The eggs were hatched in sandy soil, and instar nymphs were reared on fresh leaves of
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Setaria viridis (L.) at 30 ± 1 °C under a 14 h:10 h light: dark cycle. Adults were bred under the same light and temperature conditions, and were supplied with fresh leaves of
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Setaria viridis (L.) and wheat bran.
2.2 Reagents and assay kits
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The pEASY-Blunt Zero cloning kit and Fastpfu DNA polymerase were obtained from Transgen Biotech Co. Ltd. (Beijing, China). RevertAid H minus reverse transcriptase was purchased from Fermentas (MA, USA). SYBR Green Real-time PCR
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Master Mix was obtained from Toyobo (Osaka, Japan). dNTPs, TRIzol, and the gel extraction kit were obtained from TaKaRa Bio Group (Dalian, China). The T7 RiboMAx Express RNAi System was from Promega (WI, USA). The Cytochrome c reductase
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(NADPH) assay kit was obtained from Sigma-Aldrich (MO, USA). All other reagents
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were of the highest grade commercially available.
2.3 Synthesis of cDNA and cloning of LmCPR Total RNA, which used to clone full-length LmCPR, was extracted from nymphs and adults of L. migratoria by the TRIzol method, according to the manufacturer’s protocol. The quality and concentration of the extracted RNA was determined with a
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Nanodrop2000. The first-strand cDNA was synthesized from 4 µg of total RNA by RevertAid H minus reverse transcriptase in a reaction containing dNTPs, RNase inhibitor,
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and oligo(dT)18. Based on the L. migratoria transcriptome data, a pair of full-length primers for RT-PCR was designed and is shown in Table 1. The RT-PCR was performed with FastPfu DNA polymerase. The PCR product was recovered with the gel extraction
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kit and ligated into the pEASY Blunt Zero vector, which was then transformed into Trans-T1 competent cells and sequenced in both directions by Sangon Biotech (Shanghai,
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2.4 Bioinformatics analysis of LmCPR gene
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The predicted open reading frame (ORF) and protein sequence of LmCPR was deduced from the LmCPR gene by DNA translate (http://web.expasy.org/translate/). The theoretical isoelectric point and molecular weight of LmCPR were predicted by
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ProtParam (http://web.expasy.org/protparam/). The amino acid sequences of various
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insect CPRs were aligned using Muscle software. Then, a phylogenetic tree was constructed by the neighbor-joining method using MEGA 6.0 software (Tamura et al., 2013), with 1000 bootstrap replicates.
2.5 Reverse transcription quantitative PCR analysis The primers used for the RT-qPCR analysis are shown in Table 1. The RT-qPCR
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assay was performed on an ABI 7300 real-time PCR system in a final volume of 20 µL, containing 4 µL of cDNA,10 µL of 2× SYBR Green Real-time PCR Master Mix (with
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ROX), 1.5 µL of each RT-qPCR primer (10 pmol/µL), and 3 µL of DNase-free water. The PCR started with a denaturation step at 94 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 61 °C for 31 s. For the
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tissue-specific expression profiles, total RNA samples were prepared from the brain, foregut, midgut, gastric caecum, hindgut, fat bodies, Malpighian tubules, ovary, spermary,
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hemolymph, integument and muscles from the fifth-instar nymphs of L. migratoria. For the stage-specific expression profiles, the eggs, the first-, second-, third-, fourth- and fifth-instar nymphs and adults were collected for RNA extraction. EF1α, RpL32 and
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Hsp70 were selected as the reference genes. All data were analyzed by one-way ANOVA, Tukey’s HSD test. Relative transcript levels were determined by using the double standard curve method. The amplification efficiency of the primer used for RT-qPCR was
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higher than 0.95. The experiment was repeated with five biological replicates.
2.6 Tissue distribution of CPR activity The foregut, midgut, hindgut, gastric caecum, Malpighian tubules, fat bodies, integument, muscles, spermary, ovary, hemolymph and brain were dissected from fifth-instar nymphs of L. migratoria. Then the dissected samples were homogenized on ice with homogenization buffer (0.1 mol/L sodium phosphate, pH 7.6, containing 1
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mmol/L EDTANa2, 1 mmol/L phenylmethylsulphonyl fluoride, 0.1 mmol/L dithiothreitol, 1 mmol/L leupeptin and 20% glycerol). Then, the homogenates were centrifuged at
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12,000 g for 15 min at 4 oC. The supernatants were used for CPR activity assay. The CPR activity was measured at 550 nm with a microplate spectrophotometer, according to cytochrome P450 reductase assay kit instructions (Vermilion and Coon, 1974). Briefly,
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the 1-mL reaction mix contained 950 µL of 36 µmol/L cytochrome c (0.3 mol/L potassium phosphate buffer, pH 7.8), 50 µL of protein supernatant, and 100 µL of 0.85
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mg/L NADPH. One unit of CPR was defined as the amount of enzyme required to reduce 1 µmole of oxidized cytochrome c in the presence of 100 µmol/L NADPH per minute at room temperature. The protein concentration was measured by the BCA method (Smith
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et al., 1985) using bovine serum albumin as a standard. The experiment was repeated with three biological replicates. All data were analyzed by one-way ANOVA, Tukey’s
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HSD test.
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2.7 Silencing of LmCPR by RNAi The LmCPR and green fluorescent protein (GFP) genes were amplified by PCR using dsRNA primers containing the T7 RNA polymerase promoter. The primers used for RNAi analysis are shown in Table 1. The PCR fragments containing T7 promoter sequences on both ends were purified with the gel extraction kit and used as templates to synthesize dsRNA with the T7 RiboMAX Express RNAi System. The concentration of
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the dsRNA was determined with a Nanodrop2000. Then, the dsRNA was dissolved in ddH2O to a final concentration of 1.5 µg/µL. Approximately 3 µg of the dsRNA were
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injected into the abdomen between the second and third segments of third-instar nymphs on the second day. To determine the suppression level of LmCPR transcripts, cDNAs were synthesized from total RNA isolated from whole nymphs at 24, 48 and 72 hours
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after LmCPR dsRNA injection. β-actin, EF1α and Hsp70 were selected as the reference genes. The amplification efficiency of the primer used for RT-qPCR was higher than 0.95.
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The experiment was repeated with five biological replicates. All data were analyzed by Student’s t-test.
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2.8 CPR activity assay after silencing of LmCPR
The third-instar nymphs were homogenized on ice with potassium phosphate buffer (0.3 mol/L, pH 7.8) at 24 and 48 hours after dsRNA injection, and then homogenates
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were centrifuged at 10,000 g for 10 min at 4 oC. The supernatants were used for CPR
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activity assay. The CPR activity was measured at 550 nm with a microplate spectrophotometer, as described in section 2.6. The protein concentration of the enzyme solutions was determined by the Bradford method using bovine serum album as the standard (Noble and Bailey, 2009). The experiment was repeated with three biological replicates. All data were analyzed by Student’s t-test.
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2.9 Bioassays with carbaryl after RNAi To determine the effect of gene silencing on carbaryl-induced mortality, carbaryl
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was dissolved in acetone, and 3 µL of the carbaryl solutions (10 µg/mL and 15 µg/mL) were dropped on the abdomen of the locust at 24 hours after dsRNA injection. Mortality was determined at 24 hours after carbaryl exposure. Mean and standard errors for each
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concentration were performed in five independent bioassays. All data were analyzed by
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Student’s t-test.
3 RESULTS
3.1 Cloning and sequence analysis of LmCPR
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In our previous study, a transcriptome database of L. migratoria was constructed by using mixture mRNA from whole bodies at all developmental stages (Zhao et al., 2017). Based on the transcriptome data and a keyword search of the annotation database, only
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one candidate CPR gene was identified by keyword search of annotation database.
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The full-length cDNA sequence of LmCPR was obtained by RT-PCR. The 2043 bp cDNA sequence has been submitted to GenBank (accession number KX765501). The cDNA sequence of LmCPR was searched against the locust genome by local BLAST. The LmCPR gene was at least 42 kb in length and was composed of 14 introns and 15 exons (Fig. 1 and Fig. S1). The length of the introns ranged from 91 bp to more than 8800 bp. LmCPR encoded a deduced protein containing 680 amino acids. The predicted pI
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and MW of the deduced protein were 5.49 and 77.1 kDa, respectively. It shared the highest amino acid identity with CPR from Oxya chinensis (96.33%), followed by
LmCPR.
However,
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Pediculus humanus corporis (75.81%; Table S1). No signal peptide was identified in transmembrane
motif
(20L-39L:
LGAVDILLLAALLALAVWYL) was predicted in the N-terminal region of LmCPR, and
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may be involved in the localization of LmCPR on the endoplasmic reticulum.
As shown in Fig. 1 and Fig. 2, LmCPR was mainly composed of three conserved
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domains, including the FMN binding domain, connecting domain, and FAD/NADPH binding domain. The membrane anchor region was encoded by the second exon, while the FMN binding domain, connecting domain, FAD/NADPH binding domain were
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encoded by exons 3-6, 7-10, 7, 10, 11 and 12-15, respectively (Fig. 1). The FAD binding domain was interrupted by connecting domain, and the two domains were intertwined in
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linear sequence (Wang et al., 1997).
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3.2 Phylogenetic relationships among insect CPRs A phylogenetic tree was generated by MEGA6 using the neighbor-joining method based on the amino acid sequences of LmCPR and 22 other insect CPRs. The result showed that the insect CPRs from the same insect order were clustered into same branch with high bootstrap support (Fig. 3). The LmCPR shares the relatively higher sequence identity with the CPRs from Oxya chinensis (OcCPR) and Pediculus humanus corporis
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(PhCPR; Table S1). Therefore, LmCPR and OcCPR were first grouped into the Orthoptera branch, and then grouped with PhCPR, followed by the CPRs from Hemiptera
insects.
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3.3 Distribution of LmCPR mRNA and LmCPR activity.
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and Coleoptera, indicating closer evolutionary relationship with CPRs in hemimetabolous
LmCPR was detected in all tested tissues. Higher mRNA expression was found in
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the ovary, integument, and hindgut, whereas lower expression was detected in the hemolymph, muscles, and midgut (Fig. 4A). It is worthy to note that the CPR activity was relatively higher in Malpighian tubules and integument and lower in the spermary,
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ovary and hemolymph (Fig. 4B). Pearson correlation analysis (Fig. 4C) showed that there was no significant correlation (r=0.137, P=0.642) between the gene expression pattern of LmCPR and the distribution of CPR activity among all the 12 tissues.
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In view of the CPR activity was affected by many factors, and moreover no
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significant correlation was observed between the mRNA of LmCPR and the enzymatic activity of CPR among different tissues, the subsequent developmental expression experiments were focused on the mRNA level of LmCPR. We examined the mRNA expression level of LmCPR by RT-qPCR, and it was detected from the egg stage to the adult (Fig. 5). LmCPR expression level was the lowest in the egg stage, gradually and significantly increased from the first- to fourth-instar stage (P < 0.05), and was slightly
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decreased in fifth-instar nymphs and adults, but the differences were not significant. When exposed to carbaryl, the mRNA level of LmCPR cannot be induced by carbaryl at
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10 µg/ml and 22 µg/ml (Fig. S2).
3.4 Knockdown of LmCPR by RNAi
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To determine the efficiency of the RNAi, the expression level of LmCPR in third-instar nymphs was analyzed by RT-qPCR. The mRNA level of LmCPR decreased
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by 71.2% at 24 hours after LmCPR dsRNA injection and further declined by 78.6% and 80.6% at 48 and 72 hours after dsRNA injection, respectively, when compared to the dsGFP control (Fig. 6A). This result suggests that the RNA interference effect of the
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LmCPR dsRNA was retained for at least 72 hours; and subsequent experiments were limited to 48 hours. To further detect the influence of LmCPR knockdown, we detected CPR activity by the colorimetric method at 550 nm. The results showed that the CPR
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activity of L. migratoria was obviously decreased 18.4% and 27.2% at 24 and 48 hours
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after LmCPR dsRNA injection (Fig. 6B). It is reasonable that the down-regulation of CPR activity is consistent with the interference of LmCPR gene, because there was only one candidate LmCPR gene in locust genome. The lower decline in CPR enzymatic activity when compared to the decrease in LmCPR mRNA level, may be due to one or more factors, including a low degradation rate of CPR enzyme, or a contribution of other enzymes to cytochrome c reduction, for instance cyanide-suppressible mitochondrial
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cytochromes that were present in the crude homogenates.
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3.5 The sensitivity to carbaryl after LmCPR knockdown Knockdown of LmCPR in the nymphs of L. migratoria significantly increased its sensitivity to carbaryl. When treated with 10 µg and 15 µg carbaryl, the mortality of
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third-instar nymphs was 17.5% and 37.5%, respectively, in the LmCPR-knockdown group, whereas it was 7.5% and 21.67% in the control group, respectively (Fig. 7). These
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results showed that silencing of LmCPR has an obviously impact on the susceptibility of L. migratoria to carbaryl.
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4 DISCUSSIONS
Insect CYPs are known to play an essential role in insecticide resistance and the detoxification of exogenous compounds (Feyereisen, 2011). As an indispensable part of
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CYPs/CPR system, insect CPR transfers electrons from NADPH to CYPs (Masters and
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Okita, 1980; Liu et al., 2015a). Further research to characterize insect CPR will help to reveal the function of CYPs/CPR system in the detoxification of insecticides and other xenobiotics and facilitate the development of novel targets for insecticides. In this study, we cloned the full-length cDNA of the CPR gene from L. migratoria (LmCPR). Bioinformatics analysis showed that this enzyme consists of an N-terminal membrane anchor and FMN binding domain, connecting domain, and FAD/NADPH
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binding domain. The FAD binding domain and connecting domain are intertwined in linear sequence (Wang et al., 1997). The FAD/NADPH binding domain is located near
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the C-terminal end, while the FMN binding domain is situated in the N-terminal region, and the two domains are interconnected through random coils. The N-terminal membrane anchor functions to anchor the protein to the endoplasmic membrane, and makes the
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other parts of LmCPR face the cytoplasmic side for the CYPs, which is essential for electron transfer between CPR and CYPs (Laursen et al., 2011). Within the FAD/NADPH
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binding domain, the FAD binding motif, which comprises 3 amino acids (R459, Y460, and S461), is highly conserved in insects. The catalytic center of LmCPR, which comprised 4 conserved residues (S462, C632, D677, and W679), is considered to be
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indispensable in the hydride transfer cycle. The flexibility of the random coils possibly participates in the conformational plasticity of FMN domain, which facilitates the interaction between FMN and various CYPs (Laursen et al., 2011).
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CYP activity depends on the electrons from NADPH, which is transferred by insect
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CPR. Therefore, tissue- and stage-specific expression of LmCPR reflects certain CYP activities. The data showed that LmCPR expression level was higher in ovary, integument, and hindgut. The CPR activity was higher in Malpighian tubules and integument. It is worthy to note that the higher levels of both CPR activity and mRNA of LmCPR gene in integument. This result was similar to that of the CPRs in Drosophila melanogaster (Qiu et al., 2012), Anopheles gambiae (Lycett et al., 2006; Balabanidou et al., 2016). These
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insect CPRs may function to the biosynthesis of lipids for the insect cuticle. Our results indicated that LmCPR expression level was the lowest in the egg stage and higher during
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the later developmental stages. This phenomenon was similar to the expression pattern of CPR in T. cinnabarinus (Shi et al., 2015) and C. lectularius (Zhu et al., 2012). The distinct expression patterns of LmCPR in a variety of developmental stages and various
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tissues suggests that LmCPR can adapt to the different demands of LmCYPs for driving endogenous and exogenous metabolic processes, which may improve the adaptability of
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L. migratoria to internal and external stresses.
Several studies have shown that upregulation of insect CPRs is related to insecticide resistance. The mRNA level of CPR from Plutella xylostella in a β-cypermethrin resistant
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strain was 12-fold higher than that in a susceptible strain (Chen and Zhang, 2015). Compared to a susceptible stain, the expression level of CPR from T. cinnabarinus (Shi et al., 2015) was significantly enhanced (3.12-fold) in a fenpropathrin-resistant strain. In
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contrast, knockdown of the CPR from Anopheles gambiae increased susceptibility to
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permethrin (Lycett et al., 2006). RNA interference of NlCPR enhanced susceptibility to β-cypermethrin and imidacloprid (Liu et al., 2015b). Our results suggest that LmCPR was higher expressed in the ovary, hindgut, and integument. Knockdown of LmCPR can decrease the enzymatic activity of LmCPR, and enhanced the susceptibility of L. migratoria to carbaryl. These findings could be explained by 3 possible mechanisms. First, the CYPs/CPR complex is involved in the biosynthesis of lipids for the insect
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cuticle; therefore, knockdown of these components possible altered cuticle penetration. RNA interference of CYP4G1 or CPR from D. melanogaster blocked hydrocarbon
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biosynthesis in oenocytes (Qiu et al., 2012). Silencing of CYP4G102 in L. migratoria resulted in loss of cuticular waterproofing (Yu et al., 2016). In a resistant strain of An. gambiae, upregulation of CYP4G16 led to a thicker epicuticle and slower insecticide
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uptake (Balabanidou et al., 2016). Therefore, it is worth to note that LmCPR are higher expressed in integument, and knockdown of LmCPR possible influences the activity of
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integument CYPs and cuticle penetration.
Second, the CYPs/CPR system is known to directly participate in insecticide metabolism. The CYP6Z1 from An. gambiae can metabolize DDT (Chiu et al., 2008).
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The CYP6M2, CYP6P3, CYP6P9b, and CYP9J32 from mosquito can metabolize pyrethroids (David et al., 2013), while CYP6Z2 and CYP6Z8 play key roles in the clearance of the pyrethroid metabolites (Chandor-Proust et al., 2013). These
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detoxification reactions all depend on the electron transferred from NADPH by insect
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CPRs. Knockdown of LmCPR gene, can not only decrease the mRNA level of LmCPR, but also possible depressed the detoxification ability of CYPs/CPR system against carbaryl in Locusta migratoria. Third, CPR also can transfer electron to other electron receptors, such as haem oxygenase and cytochrome b5, and indirectly increase the tolerance to insecticides in insect (Guzov et al., 1996; Murataliev et al., 2008; Liu et al., 2015b).
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In conclusion, the present study provides preliminary information on the sequence, phylogeny and expression pattern of LmCPR in L. migratoria. Silencing of LmCPR
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obviously reduced the enzymatic activity of LmCPR, and enhanced the susceptibility of L. migratoria to carbaryl. Further researches are needed to demonstrates the essential role of the LmCPR in CYPs-mediated detoxification of insecticides and other physiological
ACKNOWLEDGEMENTS
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functions in L. migratoria.
This work was supported by the National Natural Science Foundation of China (31320103921, 31101463, 31402020) and the Shanxi Province Science Foundation
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Table 1 Primer used in this study Name
5'-3' sequence
Full-length
CPR-Full FW
ATGGAGGCAGAGGCAGGCAACG
RT-PCR
CPR-Full RV
TCAGCTCCATACATCTGAAGAAT
CPR-FW
TTACCCACCTGCTACAAAAGAA
CPR-RV
CACAACAATGTCATGCACATCA
Hsp70-FW
CTGGTGTGCTCATTCAGGTAT
Hsp70-RV
TCGTGGGGCAGGTGGTATT
EF1α-FW
AGCCCAGGAGATGGGTAAAG
EF1α-RV
CTCTGTGGCCTGGAGCATC
RPL32-FW
ACTGGAAGTCTTGATGATGCAG
RPL32-RV
CTGAGCCCGTTCTACAATAGC
β-actin-FW
CGAAGCACAGTCAAAGAGAGGTA
β-actin-RV
GCTTCAGTCAAGAGAACAGGATG
RNAi template
CPR-RNAi FW
taatacgactcactatagggTATGAAATGGGTCTTGGGGA
PCR
CPR-RNAi RV
taatacgactcactatagggGGGTGCTTCTTCGTTGACTC
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Legends of Figures Figure 1. Exon-intron organization of the LmCPR gene. The exons are shown as colored boxes, and the introns are indicated by
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dark lines. The corresponding conserved domains of LmCPR are shown below.
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Figure 2. The reaction mechanism of CPR/CYPs system.
Figure 3. Phylogenetic tree of insect CPR genes.
Phylogenetic tree of insect CPR genes was constructed using the neighbor-joining method and tested by the bootstrap method with 1000
Data S1.
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replications. The sequences used for constructing the tree are shown in
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Figure 4. Tissue-distribution of LmCPR mRNA and LmCPR activity.
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A, Tissue-specific expression patterns of LmCPR. The data are the mean ± SD of 5 biological repeats. B. The activity of LmCPR among different tissues from 5th instar nymph of L. migratoria. The data are the mean ± SD of 3 biological repeats. C. Pearson correlation analysis between gene expression level of LmCPR and activity distribution of LmCPR.
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foregut (FG), midgut (MG), hindgut (HG), gastric caecum (GC), Malpighian tubules (MT), fat bodies (FB), spermary (SP), ovary (OV), muscles (MU), integument (IN), hemolymph (HM), and brain (BR).
ANOVA and Tukey’s HSD test.
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Small letters indicate significant differences (p < 0.05), according to
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Figure 5. Developmental expression patterns of LmCPR.
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EG (eggs), first (1st), second (2nd), third (3rd), fourth (4th), and fifth (5th) instar nymphs and adults (AD).
Small letters indicate significant differences (p < 0.05), according to ANOVA and Tukey’s HSD test. The data are the mean ± SD of 5
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biological repeats.
Figure 6. The effect of RNAi of LmCPR on mRNA expression and
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enzymatic activity.
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A, The mRNA levels of LmCPR after injection of LmCPR-specific dsRNA was evaluated by RT-qPCR. B, The enzymatic activity of LmCPR after dsRNA injection. dsGFP, third-instar nymphs injected with dsRNA against green fluorescent protein; dsCPR, third-instar nymphs injected with dsRNA against LmCPR. The data are the mean ± SD of at least 3 biological repeats. One asterisk on the error bar indicates significant differences (p <
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0.05).
Figure 7. The susceptibility of the locusts to carbaryl after LmCPR
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silencing dsGFP, third-instar nymphs injected with dsRNA against green fluorescent protein; dsCPR, third-instar nymphs injected with dsRNA
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against LmCPR. The data are the mean ± SD of at least 3 biological
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repeats. One asterisk on the error bar indicates significant differences (p < 0.05).
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Figure S1. Genome structure of LmCPR gene.
Figure S2. The expression level of LmCPR exposed to carbaryl. For induction effect of carbaryl on LmCPR genes, the third-instar
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nymphs were topically applied 3 µL solution onto the abdomen between
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the second and third sterna of each nymph for 24 hours. The two carbaryl solutions, (10 mg/ml and 22 mg/ml carbaryl), were in distilled in cold acetone, which served as a control. The normalization factors for induction effect generated by the combination of three best reference genes, EF1α, RpL32 and Rp49. Small letters indicate significant differences (p < 0.05), according to ANOVA and Tukey’s HSD test. The data are the mean ± SD of 5 biological repeats.
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Highlights
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1. The 2043 bp cDNA sequence of CPR gene from Locusta migratoria was cloned by RT-PCR.
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2. Higher mRNA expression was found in the antenna, ovary, integument, and hindgut.
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3. Silencing of LmCPR reduced the enzymatic activity of LmCPR.
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4. Silencing of LmCPR enhanced the susceptibility of Locusta migratoria to carbaryl.