Cross-resistance and biochemical mechanisms of abamectin resistance in the B-type Bemisia tabaci

Abstract:  To understand the risk of resistance and the possible mechanisms of resistance to abamectin in B-type Bemisia tabaci (Gennadius) better, a resistant strain of B. tabaci was selected in the laboratory and cross-resistance pattern and resistance mechanisms to abamectin were investigated. The NJ-Abm strain of B. tabaci was derived from a field population (NJ) collected in Nanjing, China in 2002 with 18 generations of selection with abamectin in the laboratory. Compared with the unselected NJ strain, the selected NJ-Abm strain developed 14.5-fold resistance to abamectin and showed significant cross-resistance to emamectin benzoate (4.4-fold) and imidacloprid (3.4-fold), but no cross-resistance to fipronil. The oxidase inhibitor piperonyl butoxide (PBO) and glutathione S -transferase inhibitor diethyl maleate (DEM) produced significant synergism on abamectin in the NJ-Abm strain (with synergistic ratios of 3.9- and 4.1-fold respectively); however, the esterase inhibitor triphenyl phosphate (TPP) did not act synergistically with abamectin. Biochemical analysis confirmed that P450 monooxygenase activity and glutathione S -transferase activity of the NJ-Abm strain were elevated to 2.1- and 2.0-fold, respectively, compared with that of the NJ strain. This indicated that enhanced metabolism mediated by P450 monooxygenase and glutathione S -transferase is likely to be involved in abamectin resistance and cross-resistance to imidacloprid and emamectin benzoate in the NJ-Abm strain.     

橘全爪螨对双甲脒的抗性选育及其P450基因的表达差异分析

为了对双甲脒进行抗性风险评估, 弄清P450基因在橘全爪螨Panonychus citri抗药性中的作用, 在室内用双甲脒对橘全爪螨进行了抗性选育和交互抗性研究, 同时分析了橘全爪螨双甲脒抗性和敏感品系P450基因表达差异。经过12代抗性选育, 获得了橘全爪螨双甲脒抗性品系, 与敏感品系比较, 橘全爪螨对双甲脒的抗性倍数达到26.32倍。抗性风险评估表明, 橘全爪螨对双甲脒抗性遗传力h²为0.148。螺螨酯、 丁醚脲、 炔螨特和三唑锡对抗性品系的LC₅₀分别为敏感品系的16.85, 4.98, 2.13和2.05倍, 表明双甲脒抗性品系对螺螨酯、 丁醚脲、 炔螨特和三唑锡具有明显的交互抗性。阿维菌素、 苯丁锡、 哒螨灵、 矿物油对抗性品系LC₅₀分别为敏感品系的1.10, 1.21, 0.67和0.99倍, 表明双甲脒抗性品系对上述4种药剂没有显著的交互抗性。基因差异性分析发现, 抗性品系中有16条P450基因发生了上调, 27条P450基因发生了下调, 其中CYP389A6上调倍数最高［log₂ratio (RS/SS)=11.526］, CYP389A2下调倍数最高［log2ratio(RS/SS) =-12.683］, 由此推断, CYP389A6上调和CYP389A2下调可能是橘全爪螨对双甲脒产生抗性的重要原因。     

Expression of Xenobiotic Metabolizing Cytochrome P450 Genes in a Spinosad-Resistant Musca domestica L. Strain

Background

Spinosad is important in pest management strategies of multiple insect pests. However, spinosad resistance is emerging in various pest species. Resistance has in some species been associated with alterations of the target-site receptor, but in others P450s seems to be involved. We test the possible importance of nine cytochrome P450 genes in the spinosad-resistant housefly strain 791spin and investigate the influence of spinosad on P450 expression in four other housefly strains.

Results

Significant differences in P450 expression of the nine P450 genes in the four strains after spinosad treatment were identified in 40% of cases, most of these as induction. The highly expressed CYP4G2 was induced 6.6-fold in the insecticide susceptible WHO-SRS females, but decreased 2-fold in resistant 791spin males. CYP6G4 was constitutively higher expressed in the resistant strain compared to the susceptible strain. Furthermore, CYP6G4 gene expression was increased in susceptible WHO-SRS flies by spinosad while the expression level did not alter significantly in resistant fly strains. Expression of CYP6A1 and male CYP6D3 was constitutively higher in the resistant strain compared to the susceptible. However, in both cases male expression was higher than female expression.

Conclusion

CYP4G2, CYP6A1, CYP6D3 and CYP6G4 have expressions patterns approaching the expectations of a hypothesized sex specific spinosad resistance gene. CYP4G2 fit requirements of a spinosad resistance gene best, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin. Differential expression of P450s between sexes is more a rule than an exception. Noteworthy differences between spinosad influenced expression of P450 genes between a field population and established laboratory strains were shown.     

Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> infection

Background  

Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines.     

Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen,but not its corresponding enol

Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase). In this study, we investigated two genetically distant spider mite strains with high resistance to spirodiclofen. Despite the strong resistance levels to spirodiclofen (up to 680-fold), only limited cross-resistance with other members of this group such as spiromesifen and spirotetramat could be detected. Amplification and sequencing of the ACCase gene from resistant and susceptible strains did not reveal common non-synonymous mutations, and expression levels of ACCase were similar in both resistant and susceptible strains, indicating the absence of target-site resistance. Furthermore, we collected genome-wide expression data of susceptible and resistant T. urticae strains using microarray technology. Analysis of differentially expressed genes revealed a broad response, but within the overlap of two resistant strains, several cytochrome P450s were prominent. Quantitative PCR confirmed the constitutive over-expression of CYP392E7 and CYP392E10 in resistant strains, and CYP392E10 expression was highly induced by spirodiclofen. Furthermore, stage specific expression profiling revealed that expression levels were not significantly different between developing stages, but very low in eggs, matching the age-dependent resistance pattern previously observed. Functional expression of CYP392E7 and CYP392E10 confirmed that CYP392E10 (but not CYP392E7) metabolizes spirodiclofen by hydroxylation as identified by LC–MS/MS, and revealed cooperative substrate binding and a K_m of 43 μM spirodiclofen. CYP392E10 also metabolizes spiromesifen, but not spirotetramat. Surprisingly, no metabolism of the hydrolyzed spirodiclofen-enol metabolite could be detected. These findings are discussed in the light of a likely resistance mechanism.     

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 7-ethoxycoumarin-O-deethylase from malathion resistant and susceptible strains of Drosophila melanogaster

Mixed-function oxidase activity in a D. melanogaster strain carrying at least two closely linked malathion resistance genes on chromosome 3 was compared with the susceptible Canton S strain. The kinetics of O-deethylation of 7-ethoxycoumarin (7-ECD activity) with respect to pH, temperature, substrate and cofactor (NADPH) affinities and the response to metal salts of both strains were similar. The resistant strain had approx. 5-fold greater 7-ECD specific activity, and a parallel increase in total cytochrome P-450 content. Developmental stage, sex and nutritional state affected Drosophila 7-ECD activity. The intestine, fat body and Malpighian tubules contained the largest 7-ECD specific activity. Both susceptible and resistant strains had similar patterns of 7-ECD expression and differed only in total activity. In addition to more cytochrome P-450, the resistant strain had increased amounts of two microsomal, heme-staining polypeptides (M_r = 50 and 54 K after SDS-PAGE). The results suggest that the genetic change in the resistant strain involves the regulation of the Drosophila cytochrome P-450 system.     

Status of Resistance of <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases

Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC₅₀) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l^?1) and acetamiprid (4.96 to 865 mg l^?1). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR?=?205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.     

甜菜夜蛾对虫酰肼的抗性选育、风险评估及交互抗性

虫酰肼是目前防治甜菜夜蛾Spodoptera exigua（Hübner）的一种主要杀虫剂,为评估其抗性风险,在室内进行了抗性筛选和交互抗性的研究。采用饲料感染法,在甜菜夜蛾饲养75代期间用虫酰肼筛选62代,与起始种群相比抗性上升39.2倍,与室内敏感品系相比抗性上升141.3倍。在筛选的早、中、后期,现实遗传力h²分别为0.1075（F₀～F₂₅）、0.2780（F₂₆～F₅₀）和0.0538（F₅₁～F₇₅）,整个筛选62代现实遗传力为0.1556。抗性种群筛选43次（F55）后停止用药筛选,饲养21代后,与敏感品系相比,抗性水平由63.5倍下降到21倍,抗性下降3倍。说明甜菜夜蛾具有对虫酰肼产生抗性的风险,且抗性衰退缓慢,短期内很难恢复到敏感水平。交互抗性测定结果表明,上述室内选育的抗性品系对甲氧虫酰肼具有71.4倍的高水平交互抗性,对阿维菌素具有13.1倍的中等水平交互抗性,对甲维盐、茚虫威和呋喃虫酰肼分别具有7.0、8.4和4.7倍的低水平交互抗性,但对溴虫腈交互抗性不明显（1.9倍）。结果提示： 间断交替使用虫酰肼可以延缓抗性的发展,但除溴虫腈外,虫酰肼和其他几种新型杀虫剂之间的轮用可能不是甜菜夜蛾抗性治理的有效策略。     

An independent occurrence of the chimeric P450 enzyme CYP337B3 of Helicoverpa armigera confers cypermethrin resistance in Pakistan

The increasing resistance level of insect pest species is a major concern to agriculture worldwide. The cotton bollworm, Helicoverpa armigera, is one of the most important pest species due to being highly polyphagous, geographically widespread, and resistant towards many chemical classes of insecticides. We previously described the mechanism of fenvalerate resistance in Australian populations conferred by the chimeric cytochrome P450 monooxygenase CYP337B3, which arose by unequal crossing-over between CYP337B1 and CYP337B2. Here, we show that this mechanism is also present in the cypermethrin-resistant FSD strain from Pakistan. The Pakistani and the Australian CYP337B3 alleles differ by 18 synonymous and three nonsynonymous SNPs and additionally in the length and sequence of the intron. Nevertheless, the activity of both CYP337B3 proteins is comparable. We demonstrate that CYP337B3 is capable of metabolizing cypermethrin (trans- and especially cis-isomers) to the main metabolite 4'-hydroxycypermethrin, which exhibits no intrinsic toxicity towards susceptible larvae. In a bioassay, CYP337B3 confers a 7-fold resistance towards cypermethrin in FSD larvae compared to susceptible larvae from the Australian TWB strain lacking CYP337B3. Linkage analysis shows that presence of CYP337B3 accounts for most of the cypermethrin resistance in the FSD strain; up-regulation of other P450s in FSD plays no detectable role in resistance. The presence or absence of CYP337B3 can be easily detected by a simple PCR screen, providing a powerful tool to rapidly distinguish resistant from susceptible individuals in the field and to determine the geographical distribution of this resistance gene. Our results suggest that CYP337B3 evolved twice independently by unequal crossing-over between CYP337B2 and two different CYP337B1 alleles.     

亚致死剂量毒死蜱对飞蝗细胞色素P450酶活性及基因表达的影响

【目的】研究有机磷杀虫剂毒死蜱对飞蝗体内细胞色素P450的影响。【方法】采用酶活力测定法和实时定量PCR技术分别研究了毒死蜱3种亚致死剂量(LD_(10)、LD_(30)和LD_(50))处理飞蝗3龄幼虫24 h后,体内细胞色素P450酶活性及CYP409A1和CYP408B1基因表达量的变化。【结果】不同亚致死剂量毒死蜱处理引起细胞色素P450活性显著性降低,分别为对照组的0.68、0.50和0.62倍。同时通过mRNA水平表达的差异比较显示,飞蝗的两个P450基因CYP409A1和CYP408B1的表达受到抑制,均出现表达量减少的现象。【结论】某些细胞色素P450基因表达受不同亚致死剂量毒死蜱的抑制而使酶的量被降低,从而造成飞蝗整体细胞色素P450酶活性的下降。     

Permethrin Induction of Multiple Cytochrome P450 Genes in Insecticide Resistant Mosquitoes,Culex quinquefasciatus

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.     

Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450_bisd), ferredoxin (Fd_bisd), and ferredoxin reductase (Red_bisd). We demonstrated that P450_bisd and Fd_bisd are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd_bisd revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450_bisd, in the presence of Fd_bisd, Red_bisd, and NADH, was able to convert BPA. The K_m and k_cat values for BPA degradation were 85 ± 4.7 μM and 3.9 ± 0.04 min⁻¹, respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450_bisd might exhibit strict specificity. Two BPA degradation products of the P450_bisd system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.     

CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d''Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.