Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.     

Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

Adult, 20-week-old, rats from a Danish bromadiolone-resistant strain of rats (Rattus norvegicus) over-express the cytochrome P450 genes Cyp2e1, Cyp3a2 and Cyp3a3 upon bromadiolone exposure. Furthermore, adult female rats of this strain over-express the Cyp2c13 gene and suppress Cyp2c12, while males over-express the Cyp2a1 gene. The altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared expression profiles from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant rats. The P450-3a2 and -3a3 isoforms were proposed to be of higher importance in adult male resistance than in pre-pubertal resistance. In contrast, the P450-2c13 and -3a2 isoforms were proposed to be more important in sexual immature female resistance, while the P450-2e1 and -3a3 isoforms were suggested to play a more significant role in adult female resistance.     

Induction of cytochrome P450 monooxygenase activity in the two-spotted spider mite Tetranychus urticae and its influence on acaricide toxicity

Detoxification by cytochrome P450 monooxygenases is an important mechanism involved in pesticide resistance in insects and mites. The activity of these enzymes can be induced by a variety of chemicals. The aim of this study was to evaluate the effect of six P450 inducers (phenobarbital, barbital, 3-methylcholanthrene, geraniol, isosafrole, pentamethylbenzene), known to have an inducing activity in insects and mammals, on the O-deethylation activity in the two-spotted spider mite Tetranychus urticae. Treatment with barbital, phenobarbital and geraniol resulted in a dose-dependent increase in activity. Neither 3-methylcholanthrene, isosafrole nor pentamethylbenzene were effective inducers. Time course studies showed that induction by geraniol and barbital started rapidly within a period of 1-4 h after initiation of the treatment, while maximal activity was reached within 4 and 48 h, respectively. In addition, it was shown that induction with xenobiotic compounds can alter the monooxygenase-mediated acaricide tolerance in a susceptible strain of T. urticae. Although barbital induced higher levels of P450 activity, geraniol proved to be a better compound to decrease toxicity of the tested acaricides.     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64).     

Regulation of cytochrome P450 expression in Drosophila: Genomic insights

Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers/induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action.     

Spontaneous ultraweak photon emission from rice (Oryza sativa L.) and paddy weeds treated with a sulfonylurea herbicide

All living organisms spontaneously generate ultraweak photon emissions, which originate from biochemical reactions in cells. Current research uses the ultraweak photon emissions from organisms as a novel tool to investigate the physiological states of plants. In this study, we found ultraweak photon emissions from leaf segments of rice and several paddy weed species treated with a sulfonylurea herbicide. There is a definite difference in photon emissions among plant species, and rice (Oryza sativa), barnyardgrass (Echinochloa crus-galli) and Cyperus serotinus showed extremely strong enhancement of photon emissions. Photon emissions from these three species treated with sulfonylurea herbicide were suppressed when the leaf segments were treated with the cytochrome P450 monooxygenase (P450) inhibitors, piperonyl butoxide and malathion. These results suggest that P450 inhibitors affect the ultraweak photon emissions from plants.     

The effects of Artemisia annua L. and Achillea millefolium L. crude leaf extracts on the toxicity, development, feeding efficiency and chemical activities of small cabbage Pieris rapae L. (Lepidoptera: Pieridae)

The biological effects of two important medicinal plants, Artemisia annua L. and Achillea millefolium (L.) (viz, mortality, growth, and feeding indices as well as enzyme and non-enzymatic activities) were studied on small white Pieris rapae L a deleterious pest of cruciferous plants under controlled conditions (16:8 h L:D at 25 ± 1 °C and 65 ± 5% RH). The LC₅₀ and LC₂₅ values were 9.387% and 3.645% for A. annua L. and 4.19% and 1.69% for A. millefolium (L.), respectively. At the lowest concentration (0.625%), the deterrency was 29.826% and 44.185% for A. annua L. and A. millefolium (L.), respectively. Feeding indices were variously affected with changes in a number of parameters and an increase in larval and pupal duration. The activity level of alkaline phosphatase increased sharply while alanin and aspartate aminotransferases showed a sharp decrease. For non-enzymatic compounds, the amount of glucose and uric acid increased, but total protein and cholesterol decreased. These results indicate that these two medicinal plants might possess potential secondary metabolites that may be useful for controlling potential insect pests.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Isolation and functional characterization in yeast of CYP72A18, a rice cytochrome P450 that catalyzes (ω-1)-hydroxylation of the herbicide pelargonic acid

Cytochrome P450 proteins play important roles in plant herbicide selectivity. Here, we demonstrate metabolism of the herbicide pelargonic acid by CYP72A18, a novel cytochrome P450 isolated from the rice Oryza sativa L. cv. Nipponbare. The CYP72A18 cDNA was cloned from rice and heterologously expressed in Saccharomyces cerevisiae AH22 cells from the alcohol dehydrogenase (ADH1) promoter. Microsomes isolated from recombinant yeast cells contained the CYP72A18, which was found to catalyze the (ω-1)-hydroxylation of the herbicide pelargonic acid. We also show that (ω-1)-hydroxypelargonic acid has reduced herbicide activity against rice seedlings. Based on these results, we suggest that CYP72A18 participates in the detoxification of the herbicide pelargonic acid in rice plants.     

RNAi介导的棉铃虫细胞色素P450酶系几种组分基因沉默对高效氯氰菊酯毒力的影响

为进一步明确细胞色素P450 CYP6B7、细胞色素b5(cytochrome b5,Cyt-b5)和细胞色素P450还原酶(cytochrome P450 reductase,CPR)在棉铃虫Helicoverpa armigera (Hübner)对拟除虫菊酯类药剂抗性中的作用,通过RNA干扰技术沉默了抗氰戊菊酯棉铃虫中3种组分的基因,并测定基因沉默后高效氯氰菊酯对棉铃虫的毒力变化.结果显示,经单个CYP6B7或CYP6 B7与CPR、Cyt-b5基因的双链RNA注射处理后12 ～ 48 h,棉铃虫幼虫死亡率在19.4％ ～ 92.5％之间,明显高于空白对照处理的10.47％ ～ 62.87％;高效氯氰菊酯对基因沉默后幼虫的LD50值随着时间的延长而逐渐下降,12～48 h的LD50值在0.97～2.97 μg/头之间,而空白对照处理为2.27 ～ 3.57μg/头.研究表明,细胞色素P450 CYP6B7,或CYP6B7与CPR、Cyt-b5基因的沉默,可提高高效氯氰菊酯对抗性棉铃虫的毒力,进一步证明了CYP6B7、CPR和Cyt-b5在棉铃虫对菊酯类药剂抗性中起着重要作用.     

Cytochrome P450 monooxygenase-mediated permethrin resistance confers limited and larval specific cross-resistance in the southern house mosquito, Culex pipiens quinquefasciatus

The cytochrome P450-dependent monooxygenases (P450s) are an important enzymatic system that metabolizes xenobiotics (e.g., pesticides), as well as endogenous compounds (e.g., hormones). P450-mediated metabolism can result in detoxification of insecticides such as pyrethroids, or can be involved in the bioactivation and detoxification of insecticides such as organophosphates. We isolated (from the JPAL strain) a permethrin resistant strain (ISOP450) of Culex pipiens quinquefasciatus, having 1300-fold permethrin resistance using standard backcrossing procedures. ISOP450 is highly related to the susceptible lab strain (SLAB) and the high resistance to permethrin is due solely to P450-mediated detoxification. This is the first time in mosquitoes that P450 monooxygenase involvement in pyrethroid resistance has been isolated and studied without the confounding effects of kdr. Resistance in ISOP450 is incompletely dominant (D = +0.3), autosomally linked, and monofactorally inherited. It is expressed in the larvae, but not in adults. Cross-resistance to pyrethroids lacking a 3-phenoxybenzyl moiety (tetramethrin, fenfluthrin, bioallethrin, and bifenthrin) ranged from 1.5- to 12-fold. ISOP450 had only limited (6.6- and 11-fold) cross-resistance to 3-phenoxybenzyl pyrethroids with an α-cyano group (cypermethrin and deltamethrin, respectively). Examination of cross-resistance patterns to organophosphate insecticides in ISOP450 showed an 8-fold resistance to fenitrothion, while low, but significant, levels of negative cross-resistance were found for malathion (RR = 0.84), temephos (RR = 0.73), and methyl-parathion (RR = 0.55). The importance and uniqueness of this P450 mechanism in insecticide resistance is discussed.     

Effects of Artemisia annua L. (Asteracea) on nutritional physiology and enzyme activities of elm leaf beetle, Xanthogaleruca luteola Mull. (Coleoptera: Chrysomellidae)

Methanolic extract of Artemisia annua L., a weed collected around paddy fields in north of Iran, was investigated for its toxic effects on: feeding, growth, fecundity, fertility including the biochemical characteristics of elm leaf beetle Xanthogaleruca luteola Mull. The toxicity tests indicate that insecticidal effect was 2.4-fold higher in 3rd instar larvae than in adults. The LC₅₀ was 48% and 43.77% of a methanolic leaf extract at 24 and 48 h, respectively. The feeding deterrence was highest at 10% concentration and lowest at 0.625% for 3rd instar larvae and adults during the first 24 h and decreased thereafter. Larval duration significantly increased at higher concentrations. Larval treatment with 10% methanolic extract resulted in deformed adults (adultoids) with drastic changes in gross morphology of female reproductive system. Food digestion and assimilation significantly decreased in adults. Larval duration and adult fecundity and fertility were also significantly reduced. Twenty-four hours after treating 3rd instar larva with the extract the levels of glucose, protein, urea, uric acid, α-amylase, alkaline phosphatase, alanine amino transferase and aspartate amino transferase significantly changed. However, at 48 h the extract lost its potency. The extract did not effect the level of cholesterol in treated larva. These results indicate that Artemisia annua L. extract contains inhibitors of key metabolic pathways that may be useful in future control of the elm leaf beetle.