Functional study on candidate regulators mediating the expression of CYP6B7 induced by fenvalerate in a susceptible strain of Helicoverpa armigera.

Transcription factors play an important role in regulating the expression of detoxification genes (e.g. P450s) that confer insecticide resistance. Our previous study identified a series of candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may be related to fenvalerate-induced expression of CYP6B7 in a field HDTJ strain of H. armigera. Whether these CAPs can mediate the transcript of CYP6B7 induced by fenvalerate in a susceptible HDS strain of H. armigera remains unknown. Further study showed that the expression levels of multiple CAPs were significantly induced by fenvalerate in HDS strain. Knockdown of CAP19 [fatty acid synthase-like (FAS)], CAP22 [polysaccharide biosynthesis domain-containing protein 1 (PBDC1)], CAP24 [5-formyltetrahydrofolate cycloligase (5-FCL)], CAP30 [peptidoglycan recognition protein LB-like (PGRP)] and CAP33 [NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 (NDUFA11)] resulted in significant inhibition of CYP6B7 and some other P450 genes expression; meanwhile, the sensitivity of HDS strain larvae to fenvalerate was significantly increased. In addition, PBDC1, PGRP and NDUFA11, either alone or in combination, could significantly enhance the activity of CYP6B7 promoter in HDS strain, as well as the expression level of CYP6B7 gene in Sf9 cells line. These results suggested that PBDC1, PGRP and NDUFA11 may be involved in the transcript regulation of key detoxifying genes in response to fenvalerate in HDS strain of H. armigera.

An Africa-wide genomic evolution of insecticide resistance in the malaria vector Anopheles funestus involves selective sweeps, copy number variations, gene conversion and transposons.

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improving control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweeps, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes.

A cluster of CYP6 gene family associated with the major quantitative trait locus is responsible for the pyrethroid resistance in Culex pipiens pallen.

The emergence and rapid spread of insecticide resistance in several mosquito species has become a significant obstacle in management of mosquito-borne diseases, including deltamethrin resistance in Culex pipiens pallens. Previous study identified a major deltamethrin resistance quantitative trait locus (DR-6) that alone explained 62% of the genetic variance. In this study, the marker L4B1.102 and L4B1.175 associated with the DR-6 were characterized. We searched for potential candidate genes in the flank region of two markers in the genome sequence and showed that a cluster of CYP6 cytochrome P450 genes (CYP6BB4, CYP6BB3, CYP6CC2, CYP6P14, CYP6BZ2, CYP6AA9, CYP6AA8, CYP6AA7) was in the vicinity of DR-6. Significant differences in the expression of these P450s in the larval and adult stages were identified in the resistant strains compared with the susceptible strain. For CYP6AA9 and CYP6BB4, the correlation analysis showed a highly positive correlation between relative gene expression quantification and the resistance level in different strains. Knockdown of CYP6BB4 increased the sensitivity of mosquitoes to deltamethrin. We identified that the deltamethrin resistance was in a cluster of CYP6 genes in C. pipiens pallens, and CYP6BB4 may play a significant role in the development of deltamethrin resistance.

Functional characterization of CYP6A51, a cytochrome P450 associated with pyrethroid resistance in the Mediterranean fruit fly Ceratitis capitata.

Overexpression of the cytochrome P450 monooxygenase CYP6A51 has been previously associated with pyrethroid resistance in the Mediterranean fruit fly (medfly) Ceratitis capitata, an important pest species worldwide; however, this association has not been functionally validated. We expressed CYP6A51 gene in Escherichia coli and produced a functional enzyme with preference for the chemiluminescent substrate Luciferin-ME EGE. In vitro metabolism assays revealed that CYP6A51 is capable of metabolizing two insecticides that share the same mode of action, λ-cyhalothrin and deltamethrin, whereas no metabolism or substrate depletion was observed in the presence of spinosad or malathion. We further expressed CYP6A51 in vivo via a GAL4/UAS system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. Toxicity bioassays indicated that CYP6A51 confers knock-down resistance to both λ-cyhalothrin and deltamethrin. Detection of CYP6A51 - associated pyrethroid resistance in field populations may be important for efficient Insecticide Resistance Management (IRM) strategies.

Metabolism of selected model substrates and insecticides by recombinant CYP6FD encoded by its gene predominately expressed in the brain of Locusta migratoria.

The migratory locust, Locusta migartoria, is a major agricultural insect pest and its resistance to insecticides is becoming more prevalent. Cytochrome P450 monooxygenases (CYPs) are important enzymes for biotransformations of various endogenous and xenobiotic substances. These enzymes play a major role in developing insecticide resistance in many insect species. In this study, we heterologously co-expressed a CYP enzyme (CYP6FD1) and cytochrome P450 reductase (CPR) from L. migartoria in Sf9 insect cells. The recombinant enzymes were assayed for metabolic activity towards six selected model substrates (luciferin-H, luciferin-Me, luciferin-Be, luciferin-PFBE, luciferin-CEE and 7-ethoxycoumarin), and four selected insecticides (deltamethrin, chlorpyrifos, carbaryl and methoprene). Recombinant CYP6FD1 showed activity towards 7-ethoxycoumarin and luciferin-Me, but no detectable activity towards the other luciferin derivatives. Furthermore, the enzyme efficiently oxidized deltamethrin to hydroxydeltamethrin through an aromatic hydroxylation in a time-dependent manner. However, the enzyme did not show any detectable activity towards the other three insecticides. Our results provide direct evidence that CYP6FD1 is capable of metabolizing deltamethrin. This work is a step towards a more complete characterization of the catalytic capabilities of CYP6FD1 and other xenobiotic metabolizing CYP enzymes in L. migratoria.

Overexpression of CYP6ER1 associated with clothianidin resistance in Nilaparvata lugens (Stål).

The brown planthopper, Nilaparvata lugens (Stål), is one of the most economically important rice pests in Asia and has become resistant to various kinds of insecticides, including neonicotinoid insecticides. In this study, an N. lugens clothianidin-resistant (CLR) strain and a susceptible (CLS) strain were established, and the potential resistance mechanisms of N. lugens to clothianidin were elucidated. The cross-resistance studies showed that the clothianidin-resistant strain exhibited cross-resistance to most neonicotinoid insecticides, especially nitenpyram (99.19-fold) and dinotefuran (77.68-fold), while there was no cross-resistance to chlorpyrifos (1.79-fold). The synergism assays and the activities of the detoxification enzymes were performed, and we found that a cytochrome P450 conferred the clothianidin resistance. Two P450 genes (CYP6ER1 and CYP6AY1) were found to be significantly overexpressed in the CLR strain compared with the CLS strain based on qRT-PCR. In addition, the knockdown of CYP6ER1 by RNA interference dramatically increased the toxicity of clothianidin against N. lugens. These data demonstrated that the overexpression of CYP6ER1 could contribute to clothianidin resistance in N. lugens. Our findings will help to improve the design of effective resistance management strategies to control brown planthoppers.

Identification of a cytochrome P450 CYP6AB60 gene associated with tolerance to multi-plant allelochemicals from a polyphagous caterpillar tobacco cutworm (Spodoptera litura).

Generalist phytophagous insects adapt to adventurous chemical environment in a wide variety of host plants by extraordinary detoxifying metabolic abilities. However, how polyphagous insect cope with the diversity of plant defenses remains largely unknown and only a few counter-defense genes detoxifying a wide range of toxic secondary metabolites have been well characterized. Here, we identify a cytochrome P450 gene (CYP6AB60) from tobacco cutworm (Spodoptera litura) in response to three different plant's defense metabolites. After being exposed to artificial diet supplemented with coumarin (COU), xanthotoxin (XAN) or tomatine (TOM), activities of P450 and CYP6AB60 transcript levels in both midgut and fat body tissues were significantly increased. Developmental expression analysis revealed that CYP6AB60 was expressed highly during the larval stages, and tissue distribution analysis showed that CYP6AB60 was expressed extremely high in the midgut, which correspond to the physiological role of CYP6AB60 from S. litura larvae in response to plant allelochemicals. Furthermore, when larvae are injected with double-stranded RNA (dsRNA) specific to CYP6AB60, levels of this transcript in the midgut and fatbody decrease and the negative effect of plant's defense metabolites on larval growth is magnified. These data demonstrate that the generalist insect S. litura might take advantage of an individual detoxificative gene CYP6AB60 to toxic secondary metabolites from different host plants. The CYP6AB60 can be a potential gene to carry out RNAi-mediated crop protection against the major polyphagous pest S. litura in the future.

Transcription Factors AhR/ARNT Regulate the Expression of CYP6CY3 and CYP6CY4 Switch Conferring Nicotine Adaptation.

Nicotine is one of the most toxic secondary plant metabolites in nature and it is highly toxic to herbivorous insects. The overexpression of CYP6CY3 and its homologous isozyme CYP6CY4 in Myzus persicae nicotianae is correlated with nicotine tolerance. The expanded (AC)n repeat in promoter is the cis element for CYP6CY3 transcription. These repeat sequences are conserved in the CYP6CY3 gene from Aphis gossypii and the homologous P450 genes in Acyrthosiphon pisum. The potential transcriptional factors that may regulate CYP6CY3 were isolated by DNA pulldown and sequenced in order to investigate the underlying transcriptional regulation mechanism of CYP6CY3. These identified transcriptional factors, AhR and ARNT, whose abundance was highly correlated with an abundance of the CYP6CY3 gene, were validated. RNAi and co-transfection results further confirm that AhR and ARNT play a major role in the transcriptional regulation of the CYP6CY3 gene. When the CYP6CY3 transcript is destabilized by AhR/ARNT RNAi, the transcription of the CYP6CY4 is dramatically up-regulated, indicating a compensatory mechanism between the CYP6CY3 and CYP6CY4 genes. Our present study sheds light on the CYP6CY3 and CYP6CY4 mediated nicotine adaption of M. persicae nicotianae to tobacco. The current studies shed light on the molecular mechanisms that underlie the genotypic and phenotypic changes that are involved in insect host shifts and we conclude that AhR/ARNT regulate the expression of CYP6CY3 and CYP6CY4 cooperatively, conferring the nicotine adaption of M. persicae nicotianae to tobacco.

Cytochrome P450 CYP6EV11 in Chironomus kiiensis Larvae Involved in Phenol Stress.

Phenol is one of the organic pollutants which can cause water environment pollution. It is not only enriched in aquatic organisms but is also a serious threat to human health. Chironomus kiiensis is very sensitive to the contaminants in water and its cytochrome P450s are usually chosen as biomarkers for water pollution. To examine whether CYP6EV11 plays a role in the oxidative metabolism of phenol, we measured the silencing efficiency of CYP6EV11 and evaluated larval susceptibility to sublethal phenol levels by RNA interference (RNAi) technology. The results showed that the transcription of CYP6EV11 was found significantly up-regulated when the 4th instar C.kiiensis larvae were exposed to three doses of phenol. However, the transcriptional levels of CYP6EV11 were significantly suppressed by 92.7% in the 4th instar C. kiiensis larvae soaked in dsCYP6EV11 compared with those soaked in dsGFP for 6 h. The CYP6EV11 expression and mortality of the 4th instar C. kiiensis larvae with CYP6EV11 silencing were mostly decreased under phenol stress. Therefore, the CYP6EV11 gene may be used as a molecular biomarker for earlier warning and monitoring for water pollution.

Genomic exaptation enables Lasius niger adaptation to urban environments.

BACKGROUND: The world is rapidly urbanizing, and only a subset of species are able to succeed in stressful city environments. Efficient genome-enabled stress response appears to be a likely prerequisite for urban adaptation. Despite the important role ants play in the ecosytem, only the genomes of ~13 have been sequenced so far. Here, we present the draft genome assembly of the black garden ant Lasius niger - the most successful urban inhabitant of all ants - and we compare it with the genomes of other ant species, including the closely related Camponotus floridanus. RESULTS: Sequences from 272 M Illumina reads were assembled into 41,406 contigs with total length of 245 MB, and N50 of 16,382 bp, similar to other ant genome assemblies enabling comparative genomic analysis. Remarkably, the predicted proteome of L. niger is significantly enriched relative to other ant genomes in terms of abundance of domains involved in nucleic acid binding, DNA repair, and nucleotidyl transferase activity, reflecting transposable element proliferation and a likely genomic response. With respect to environmental stress, we note a proliferation of various detoxification genes, including glutatione-S-transferases and those in the cytochrome P450 families. Notably, the CYP9 family is highly expanded with 19 complete and 21 nearly complete members - over twice as many compared to other ants. This family exhibits the signatures of strong directional selection, with eleven positively selected positions in ligand-binding pockets of enzymes. Gene family contraction was detected for several components of the olfactory system, accompanied by instances of both directional selection and relaxation. CONCLUSIONS: Our results suggest that the success of L. niger in urbanized areas may be the result of fortuitous coincidence of several factors, including the expansion of the CYP9 cytochrome family due to coevolution with parasitic fungi, the diversification of DNA repair systems as an answer to proliferation of retroelements, and the reduction of olfactory system and behavioral preadaptations from non-territorial subdominant life strategies found in natural environments. Diversification of cytochromes and DNA repair systems along with reduced odorant communication are the basis of L. niger pollutant resistance and polyphagy, while non-territorial and mobilization strategies allows more efficient exploitation of large but patchy food sources.

Transcription factor aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator is involved in regulation of the xenobiotic tolerance-related cytochrome P450 CYP6DA2 in Aphis gossypii Glover.

The cotton aphid, Aphis gossypii, is one of the most economically important agricultural pests worldwide as it is polyphagous and resistant to many classes of insecticides. Overexpression of the cytochrome P450 monooxygenase (P450) CYP6DA2 has previously been found to be associated with gossypol and spirotetramat tolerance in the cotton aphid. In the present study, the elements located in the promoter region (-357:-343; -250:-241; -113:-104) of CYP6DA2 were shown to control promoter activity, and gossypol induction was observed. We hypothesized that the expression of CYP6DA2 is subject to transcriptional regulation. To investigate the underlying mechanism, we assessed two transcription factors, aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT), and found that the abundance of AhR was highly correlated with CYP6DA2 abundance. RNA interference of AhR or ARNT significantly decreased the levels of the target gene as well as those of its counterpart, and both dramatically repressed CYP6DA2 expression. Cotransfection of the ARNT, AhR, or AhR plus ARNT and CYP6DA2 promoter constructs elevated CYP6DA2 promoter activity, with the AhR plus ARNT cotransfection being the most effective. Thus, these elements located in the promoter were responsible for CYP6DA2 transcription, and CYP6DA2 expression was regulated by the transcription factors AhR and ARNT.

CYP6B6 is involved in esfenvalerate detoxification in the polyphagous lepidopteran pest, Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera, is a polyphagous pest that has a strong capacity to evolve resistance against various classes of insecticides. Cytochrome P450 enzymes have been suspected involved in pyrethroid metabolism and resistance in this pest. However, how many and which P450s are involved in pyrethroid metabolism is largely unknown. In this study, CYP6B6 and NADPH-cytochrome P450 reductase (HaCPR) from H. armigera were successfully co-expressed in Escherichia coli. Incubation of esfenvalerate with the recombinant CYP6B6-HaCPR monooxygenase complex revealed that CYP6B6 was able to transform esfenvalerate into 4'-hydroxy fenvalerate. Kcat and Km values for the formation of 4'-hydroxyfenvalerate by the E. coli-produced CYP6B6 were determined to be 1.65±0.11min-1 and 4.10±0.84µM respectively. Our results demonstrate that CYP6B6 has the ability to hydroxylate esfenvalerate, thus plays a role in fenvalerate detoxification.

Yeast one-hybrid screening the potential regulator of CYP6B6 overexpression of Helicoverpa armigera under 2-tridecanone stress.

In insect, the cytochrome P450 plays a pivotal role in detoxification to toxic allelochemicals. Helicoverpa armigera can tolerate and survive in 2-tridecanone treatment owing to the CYP6B6 responsive expression, which is controlled by some regulatory DNA sequences and transcription regulators. Therefore, the 2-tridecanone responsive region and transcription regulators of the CYP6B6 are responsible for detoxification of cotton bollworm. In this study, we used yeast one-hybrid to screen two potential transcription regulators of the CYP6B6 from H. armigera that respond to the plant secondary toxicant 2-tridecanone, which were named Prey1 and Prey2, respectively. According to the NCBI database blast, Prey1 is the homology with FK506 binding protein (FKBP) of Manduca sexta and Bombyx mori that belongs to the FKBP-C superfamily, while Prey2 may be a homology of an unknown protein of Papilio or the fcaL24 protein homology of B. mori. The electrophoretic mobility shift assays revealed that the FKBP of prokaryotic expression could specifically bind to the active region of the CYP6B6 promoter. After the 6th instar larvae of H. armigera reared on 2-tridecanone artificial diet, we found there were similar patterns of CYP6B6 and FKBP expression of the cotton bollworm treated with 10 mg g-1 2-tridecanone for 48 h, which correlation coefficient was the highest (0.923). Thus, the FKBP is identified as a strong candidate for regulation of the CYP6B6 expression, when the cotton bollworm is treated with 2-tridecanone. This may lead us to a better understanding of transcriptional mechanism of CYP6B6 and provide very useful information for the pest control.

MiR-285 targets P450 (CYP6N23) to regulate pyrethroid resistance in Culex pipiens pallens.

MicroRNAs play critical roles in post-transcriptional regulation of gene expression, which participate in the modulation of almost all of the cellular processes. Although emerging evidence indicates that microRNAs are related with antineoplastic drugs resistance, whether microRNAs are responsible for insecticide resistance in mosquitos is poorly understood. In this paper, we found that miR-285 was significantly upregulated in the deltamethrin-resistant strain of Culex pipiens pallens, and overexpression miR-285 through microinjection increased mosquito survival rate against deltamethrin treatement. Using bioinformatic software, quantitative reverse transcription PCR, luciferase reporter assay and microinjection approaches, we conformed that CYP6N23 was the target of miR-285. Lower expression of CYP6N23 was observed in the deltamethrin-resistant strain. While, mosquito mortality rate was decreased after downregulating expression of CYP6N23 by dsRNA against CYP6N23 or miR-285 mimic microinjection. These findings revealed that miR-285 could target CYP6N23 to regulate pyrethroid resistance, providing new insights into mosquito insecticide resistance surveillance and control.

CYP6P9-Driven Signatures of Selective Sweep of Metabolic Resistance to Pyrethroids in the Malaria Vector Anopheles funestus Reveal Contemporary Barriers to Gene Flow.

Pyrethroid resistance in major malaria vectors such as Anopheles funestus threatens malaria control efforts in Africa. Cytochrome P450-mediated metabolic resistance is best understood for CYP6P9 genes in southern Africa in An. funestus. However, we do not know if this resistance mechanism is spreading across Africa and how it relates to broader patterns of gene flow across the continent. Nucleotide diversity of the CYP6P9a gene and the diversity pattern of five gene fragments spanning a region of 120 kb around the CYP6P9a gene were surveyed in mosquitoes from southern, eastern and central Africa. These analyses revealed that a Cyp6P9a resistance-associated allele has swept through southern and eastern Africa and is now fixed in these regions. A similar diversity profile was observed when analysing genomic regions located 34 kb upstream to 86 kb downstream of the CYP6P9a locus, concordant with a selective sweep throughout the rp1 locus. We identify reduced gene flow between southern/eastern Africa and central Africa, which we hypothesise is due to the Great Rift Valley. These potential barriers to gene flow are likely to prevent or slow the spread of CYP6P9-based resistance mechanism to other parts of Africa and would to be considered in future vector control interventions such as gene drive.

Activation of CncC pathway by ROS burst regulates cytochrome P450 CYP6AB12 responsible for λ-cyhalothrin tolerance in Spodoptera litura.

Frequent insecticide use poses an environmental hazard and also selects for insecticide tolerance. Increased metabolic detoxification by cytochrome P450 monooxygenases (P450s) is the most common mechanism of insecticide tolerance. However, the underlying regulatory mechanisms remain unknown. We studied the midgut-specific P450 gene, CYP6AB12, associated with λ-cyhalothrin tolerance. Its regulatory pathway was investigated in the tobacco cutworm, Spodoptera litura (Fabricius). P450 activities and CYP6AB12 transcript levels increased after λ-cyhalothrin exposure. Inhibiting P450 activities with piperonyl butoxide and silencing CYP6AB12 by double-stranded RNA (dsRNA) injection decreased larval tolerance to λ-cyhalothrin. λ-Cyhalothrin exposure induced the expression of the cap 'n' collar isoform C (CncC) and muscle aponeurosis fibromatosis (Maf), increased hydrogen peroxide (H2O2) contents and elevated antioxidant enzyme activities. CncC knockdown by dsRNA feeding suppressed CYP6AB12 expression and decreased larval tolerance to λ-cyhalothrin. In contrast, application of the CncC agonist curcumin induced CYP6AB12 expression and enhanced insecticide tolerance. Ingestion of the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced H2O2 accumulation, suppressed the expression of CncC, Maf and CYP6AB12 and led to increased larval susceptibility to λ-cyhalothrin. The results demonstrate that in S. litura, λ-cyhalothrin induces cytochrome P450 CYP6AB12 via elicitation of the ROS burst and activation of the CncC pathway.

Alcohol dehydrogenase 5 of Helicoverpa armigera interacts with the CYP6B6 promoter in response to 2-tridecanone.

Alcohol dehydrogenase 5 (ADH5) is a member of medium-chain dehydrogenase/reductase family and takes part in cellular formaldehyde and S-nitrosoglutathione metabolic network. 2-tridecanone (2-TD) is a toxic compound in many Solanaceae crops to defend against a variety of herbivory insects. In the broader context of insect development and pest control strategies, this study investigates how a new ADH5 from Helicoverpa armigera (HaADH5) regulates the expression of CYP6B6, a gene involved in molting and metamorphosis, in response to 2-TD treatment. Cloning of the HaADH5 complementary DNA sequence revealed that its 1002 bp open reading frame encodes 334 amino acids with a predicted molecular weight of 36.5 kD. HaADH5 protein was purified in the Escherichia coli Transetta (pET32a-HaADH5) strain using a prokaryotic expression system. The ability of HaADH5 protein to interact with the 2-TD responsive region within the promoter of CYP6B6 was confirmed by an in vitro electrophoretic mobility shift assay and transcription activity validation in yeast. Finally, the expression levels of both HaADH5 and CYP6B6 were found to be significantly decreased in the midgut of 6th instar larvae after 48 h of treatment with 10 mg/g 2-TD artificial diet. These results indicate that upon 2-TD treatment of cotton bollworm, HaADH5 regulates the expression of CYP6B6 by interacting with its promoter. As HaADH5 regulation of CYP6B6 expression may contribute to the larval xenobiotic detoxification, molting and metamorphosis, HaADH5 is a candidate target for controlling the growth and development of cotton bollworm.

Characterization of sulfoxaflor resistance in the brown planthopper, Nilaparvata lugens (Stål).

BACKGROUND: Sulfoxaflor is a new insecticide for controlling Nilaparvata lugens in the field. This study was conducted to investigate the risk of resistance development, the cross-resistance spectrum and the mechanisms of sulfoxaflor resistance in N. lugens. RESULTS: A sulfoxaflor-resistant strain was obtained from a field population by successive selection with sulfoxaflor for 39 generations in the laboratory. Sulfoxaflor-resistant populations showed significant levels of cross-resistance to dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid and cycloxaprid. However, they exhibited only minor or no cross-resistance to isoprocarb, etofenprox, chlorpyrifos, triflumezopyrim and buprofezin. Sulfoxaflor was synergized by the inhibitor piperonyl butoxide (PBO) in the sulfoxaflor-resistant strain (SFX-SEL) with 2.69-fold relative synergistic ratios compared with the unselected strain (UNSEL). Compared with UNSEL, the P450 enzyme activity of SFX-SEL was increased 3.50 times, and eight P450 genes were upregulated more than 2.0-fold in SFX-SEL. RNAi reduced the expression of CYP6ER1 (36.87-fold change) and significantly enhanced the susceptibility of SFX-SEL to sulfoxaflor. CONCLUSION: Resistance development and cross-resistance risk of sulfoxaflor-resistance in N. lugens is evident. The enhanced detoxification of P450 enzymes caused by upregulation of several P450 genes is considered to be the metabolic resistance mechanism. These results suggest that CYP6ER1 might play an important role in sulfoxaflor resistance in N. lugens. © 2018 Society of Chemical Industry.

Knockdown of cytochrome P450 CYP6 family genes increases susceptibility to carbamates and pyrethroids in the migratory locust, Locusta migratoria.

Insect cytochrome P450 monooxygenase (CYP) plays a key role in the detoxification of insecticides. In this study, four cDNA sequences of CYP6 genes were identified and characterized. Transcription levels of LmCYP6HC1 and LmCYP6HCL1 were high in first- and fourth-instar nymph stages, respectively. LmCYP6HN1 was primarily expressed in the egg to third-instar nymph stages, while LmCYP6HQ1 was predominantly expressed in the stages from fourth-instar nymph to the adult. The four CYP6 genes were predominantly distributed in the antenna, brain, fat body, integument, and hemolymph. Piperonyl butoxide exposure inhibited total CYP activity and synergized the toxicity of carbamates and pyrethroids. Knockdown of LmCYP6HL1, LmCYP6HN1, and LmCYP6HQ1 increased nymph mortality following exposure to carbaryl, and silencing of LmCYP6HC1, LmCYP6HL1, LmCYP6HN1, and LmCYP6HQ1 comprehensively raised nymph mortality following exposure to fluvalinate. Knockdown of LmCYP6HL1 or LmCYP6HN1 significantly increased nymph mortality following exposure to cypermethrin or fenvalerate, respectively. These results suggest that the CYP6 family plays a key role in determining the susceptibility of Locusta migratoria to both carbamates and pyrethroids.

The regulation of three new members of the cytochrome P450 CYP6 family and their promoters in the cotton aphid Aphis gossypii by plant allelochemicals.

BACKGROUND: The expression of P450 genes in insects can be induced by plant allelochemicals. To understand the induction mechanisms, we measured the expression profiles of three P450 genes and their promoter activities under the induction of plant allelochemicals. RESULTS: The inducible expression of CYP6CY19 was the highest among three genes, followed by those of CYP6CY22 and CYP6DA1. The regions from -687 to +586 bp of CYP6DA1, from -666 to +140 bp of CYP6CY19 and from -530 to +218 bp of CYP6CY22 were essential for basal transcriptional activity. The cis-elements for plant allelochemicals induction were identified between -193 and +56 bp of CYP6DA1, between -157 and +140 bp of CYP6CY19 and between -108 and +218 bp of CYP6CY22. These promoter regions were found to contain a potential aryl hydrocarbon receptor element binding site with a conservative sequence motif 5'-C/TAC/ANCA/CA-3'. All these four plant allelochemicals were able to induce the expression of these P450 genes. Tannic acid had a better inductive effect than other three plant allelochemicals. CONCLUSIONS: Our study identified the plant allelochemical responsive cis-elements. This provides further research targets aimed at understanding the regulatory mechanisms of P450 genes expression and their interactions with plant allelochemicals in insect pests. © 2018 Society of Chemical Industry.