An overexpressed cytochrome P450 CYP439A1v3 confers deltamethrin resistance in Laodelphax striatellus Fallén (Hemiptera: Delphacidae)

Deltamethrin resistance in Laodelphax striatellus had been associated with its oxidative detoxification by overexpression of four cytochrome P450 monooxygenases like CYP353D1v2, CYP6FU1, CYP6AY3v2, and CYP439A1v3. The first three P450s have been validated for insecticide‐metabolizing capability and only CYP6FU1 was found to degrade deltamethrin. In this study, an investigation was conducted to confirm the capability of CYP439A1v3 to degrade deltamethrin. The CYP439A1v3 was first expressed in Sf9 cell line and its recombinant enzyme was tested for metabolic activity against different insecticides using substrate depletion assay combined with metabolite identification. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and carbon monoxide (CO)‐difference spectra analysis showed that the intact cytochrome P450 protein was successfully expressed. Tests with probe substrates proved its enzyme activity, as p‐nitroanisole, ethoxycoumarin, and ethoxyresorufin were preferentially metabolized (specific activity 7.767 ± 1.22, 1.325 ± 0.37, and 0.355 ± 0.37 nmol/min per mg of protein, respectively) while only luciferin‐HEGE was not. In vitro incubation of the recombinant CYP439A1v3 protein with deltamethrin revealed hydroxylation by producing hydroxydeltamethrin. On the contrary, no metabolite/metabolism was seen with nonpyrethroid insecticide, including imidacloprid, buprofezin, chlorpyrifos, and fipronil. To the best of our knowledge, this is the first study to link a CYP450 from family 439 to confer pyrethroid resistance to L. striatellus. This finding should help in the design of appropriate insecticide resistance management for control of this strain of L. striatellus.     

Induction of P450 monooxygenases in the German cockroach,Blattella germanica L

The cytochrome P450 monooxygenases are an important metabolic system whose level of activity can be influenced by several dietary constituents. We examined the effects of six known P450 inducers on the levels of total cytochromes P450, cytochrome b(5), and six monooxygenase activities in adult German cockroaches. In addition, the levels of CYP6L1 and CYP9E2 mRNA were also investigated. Phenobarbital treatment resulted in increases in total cytochromes P450 and metabolism of three resorufin analogues, but not CYP6L1 nor CYP9E2 mRNA. There was no significant effect of the other five inducers on any of the monooxygenase parameters we measured. In comparison with other insects, the German cockroach seems unusually refractory to most inducing agents.     

亚致死剂量马拉硫磷和西维因对飞蝗P450基因表达研究

采用实时定量PCR技术检测飞蝗Locusta migratoria细胞色素P450基因CYP4G62、CYP6EL1和CYP9AQ1经马拉硫磷和西维因不同亚致死剂量和不同时间处理后mRNA的表达水平。结果表明,经马拉硫磷不同亚致死剂量LD10、LD30和LD50处理后,飞蝗CYP4G62和CYP6EL1均在高剂量LD50被显著诱导,分别为对照的3.03和2.0倍,而在低剂量LD10无显著性差异。CYP9AQ1在3个亚致死剂量下表达量均显著提高,且增量随处理浓度增加而降低。经西维因3个亚致死剂量处理后,除在LD30这一浓度下引起CYP4G62表达提高外,其它的P450基因均无显著性差异。选择2种农药的LD15对飞蝗进行点滴处理,分别检测6、12、24和48 h基因的相对表达。经马拉硫磷LD15处理后,除CYP6EL1在12 h和CYP9AQ1在24 h表达量显著降低外,其它各时间点基因表达均无显著差异。经西维因处理不同时间后,3个P450基因的相对表达量均无显著性差异。综合上述结果说明马拉硫磷对3个细胞色素P450基因有一定的诱导作用,而西维因对其无诱导作用。     

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THREE NEW FULL‐LENGTH cDNAs OF CYTOCHROME P450 (CYP6N3v1‐v3)*FROM AEDES ALBOPICTUS**

Abstract The three new full‐length cDNA sequences including the complete 5′‐and 3′‐ untranslated regions (UTR) coding for cytochrome P450s from Aedes albopictus have been obtained. The P450 proteins deduced from the nucleotide sequences shared 58.6% ‐ 62.4% amino acid identity with CYP6N1 and CYP6N2 from Anopheles gambiae, and 99% with each other. The three new complete sequences have been submitted and named as CYP6N3v1, CYP6N3v2 and CYP6N3v3 by the P450 Nomenclature Committee. The original cDNAs were obtained by rapid amplification of cDNA ends (RACE) approach with several pairs of gene specific primers based on the cDNA fragment previously obtained from deltamethrin‐resistant strain of Ae. albopictus. Further analysis showed that the three new sequences are present in both resistant strain and susceptible strain and might be effectively translated. In addition, the 5′‐ and 3′‐UTRs were compared between the CYP6N3vl‐v3 and other known insect P450s. The multiplicity of trans‐lational control of insect P450 genes was discussed.     

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.     

Adult specific expression and induction of cytochrome P450tpr in house flies

Cytochrome P450_tpr is a xenobiotic metabolizing P450 that is found in house flies (Musca domestica). To better understand the regulation of cytochrome P450_tpr, the effects of 21 potential monooxygenase inducers were examined for their ability to induce total cytochromes P450 and cytochrome P450_tpr levels in adult flies. Six compounds caused induction of total cytochromes P450 per mg protein in adult susceptible (CS) house flies: ethanol (1.6-fold), phenobarbital in food (1.5-fold) or water (1.5-fold), naphthalene (1.3-fold), DDT (1.3-fold), xanthotoxin (1.4-fold), and α-pinene (1.2-fold). Six compounds were found to be inducers of cytochrome P450_tpr: piperonyl butoxide in food (1.9-fold), phenobarbital in food (1.4-fold) and water (3.4-fold), clofibrate (1.3-fold), xanthotoxin (1.3-fold), methohexital (1.3-fold), and isosafrole (1.3-fold). Comparison of our results with house fly P450 6A1 indicates that there are specific inducers for each of these individual P450s as well as compounds that induce both P450s. Total P450s were inducible by PB in CS house fly larvae, but not in LPR larvae. Immunoblotting revealed no detectable P450_tpr in control or PB-treated larvae in either strain. Thus, although total P450s are inducible in the susceptible strain larvae, P450_tpr does not appear to be normally present or inducible with PB in larvae of either strain. Northern blots of phenobarbital (in water) treated CS flies indicated that there was a 4.2-fold increase in the P450_tpr (i.e., CYP6D1) mRNA levels over the untreated flies. In the multiresistant LPR strain there was no apparent induction of CYP6D1 mRNA by phenobarbital. Following phenobarbital induction, the level of CYP6D1 mRNA in the CS strain was about half of the level in the LPR strain. © 1996 Wiley-Liss, Inc.     

Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus‐mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH‐cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ‐cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ‐cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.     

Permethrin resistance variation and susceptible reference line isolation in a field population of the mosquito,Culex quinquefasciatus (Diptera: Culicidae)

This study examines the genetic variations and mechanisms involved in the development of permethrin resistance in individual mosquitoes from a field population of Culex quinquefasciatus, HAmCq^G0, and characterizes susceptible reference lines of mosquitoes with a similar genetic background to the field HAmCq^G0 strain. Six upregulated cytochrome P450 genes, CYP9M10, CYP9J34, CYP6P14, CYP9J40, CYP6AA7, and CYP4C52v1, previously identified as being upregulated in the larvae of resistant HAmCq^G8 mosquitoes were examined in the larvae of 3 strains (susceptible S‐Lab, parental HAmCq^G0 and permethrin‐selected highly resistant HAmCq^G8) and 8 HAmCq^G0 single‐egg raft colonies, covering a range of levels of susceptibility/resistance to permethrin and exhibiting different variations in the expression of A and/or T alleles at the L‐to‐F kdr locus of the sodium channel. The 2 lines with the lowest tolerance to permethrin and bearing solely the susceptible A allele at the L‐to‐F kdr locus of the sodium channels, from colonies Cx_SERC5 and Cx_SERC8, showed lower or similar levels of all 6 of the P450 genes tested compared with the S‐Lab strain, suggesting that these 2 lines could be used as the reference mosquitoes in future studies characterizing insecticide resistance in HAmCq mosquitoes. This study also provides a detailed investigation of the mechanisms involved in insecticide resistance in individuals within a population: individuals with elevated levels of resistance to permethrin all displayed one or more potential resistance mechanisms–either elevated levels of P450 gene expression, or L‐to‐F mutations in the sodium channel, or both.     

Resistance to neonicotinoid insecticides and expression changes of eighteen cytochrome P450 genes in field populations of Bemisia tabaci from Xinjiang,China

The occurrence of Bemisia tabaci poses an increasingly serious threat to cotton and vegetable crops in Xinjiang, China. Currently, neonicotinoid insecticides are commonly used to control the insect, to which resistance is inevitable due to intensive use. However, the resistance status and mechanism of B. tabaci to neonicotinoid insecticides in Xinjiang are poorly understood. Cytochrome P450 monooxygenases represent a key detoxification mechanism in the neonicotinoid resistance of B. tabaci. In this study, the resistance level to imidacloprid and thiamethoxam was investigated using the leaf dipping method in five field populations of B. tabaci from Turpan (TP, two sampling sites), Shache (SC), Hotan (HT) and Yining (YN) in northern and southern Xinjiang. The expression changes of eighteen cytochrome P450 genes from the select B. tabaci populations were determined by real‐time fluorescence quantitative PCR (qPCR). The bioassay revealed that the five populations tested had developed moderate to high levels of resistance to imidacloprid (12.26–46.07‐fold), while the populations remained sensitive to thiamethoxam except for HT, which had a low level of resistance. The qPCR results showed that the expression levels of five P450 genes, CYP4G68, CYP6CM1, CYP303A1‐like, CYP6DZ7 and CYP6DZ4, were significantly higher in some resistant field populations than in the susceptible strain. Resistance to imidacloprid in field populations of B. tabaci might be associated with the increased expression of these five cytochrome P450 genes. The results are useful for further understanding the mechanism of neonicotinoid resistance and will contribute to the management of insecticide‐resistant B. tabaci in Xinjiang.     

Exposure to herbicides reduces larval sensitivity to insecticides in Spodoptera litura (Lepidoptera: Noctuidae)

Herbicides and insecticides are widely used in modern agriculture. It has been reported in various studies that application of insecticides can increase tolerance of herbivorous insects to insecticides. However, limited information exists on susceptibility to insecticides when insects are exposed to herbicides. This study was conducted to investigate the potential impact of the herbicides trifluralin and 2-methyl-4-chlorophenoxyacetic acid sodium salt (MCPA-Na) on the susceptibility of the nocturnal moth Spodoptera litura to the insecticides X-cyhalothrin, phoxim and bifenthrin. We found that larvae exposed to trifluralin or MCPA-Na became significantly less susceptible to both insecticides than nonexposed control larvae. Herbicide-treated larvae did not show altered growth under the used test conditions. However, heads of herbicide-treated larvae showed increased expression of the acetylcholinesterase genes SI Ace I and SI Ace 2. Moreover, the fat body and midgut of herbicide-treated larvae displayed elevated expression of detoxification genes (the carboxylesterase gene SI CarE;the glutathione S-transferase genes SlGSTe2 and SlGSTe3\ the cytochrome P450 monooxygenase genes CYP6B48, CYP9A40 and CYP321B1). The CYP6B48 gene exhibited highest inducibility. In conclusion, the data of this study suggest that exposure of S. litura larvae to herbicides may stimulate detoxification mechanisms that compromise the efficacy of insecticides.