三种杀虫剂亚致死剂量对草地贪夜蛾细胞色素P450基因表达的影响

【目的】为明确杀虫剂亚致死剂量对草地贪夜蛾Spodoptera frugiperda细胞色素P450基因表达的影响。【方法】本研究采用叶片浸渍法测定了3种杀虫剂[氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和苏云金杆菌Bacillus thuringiensis(Bt)]对草地贪夜蛾2龄幼虫的毒力,以及通过实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)技术测定了这3种杀虫剂亚致死剂量(LC_(10))处理后48 h时草地贪夜蛾2龄幼虫16个P450基因的表达量。【结果】氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和Bt对草地贪夜蛾2龄幼虫的LC_(10)值分别为0.931, 0.283和1 089.688 mg/L。2龄幼虫受LC_(10)氯虫苯甲酰胺胁迫后,13个P450基因(CYP4G75,CYP6AB12,CYP6B50,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP337B5,CYP9A59,CYP9A58,CYP6AE44及CYP6AE43)表达上调,其中CYP6AE44表达量为对照的34.60倍;2龄幼虫受LC_(10)甲维盐胁迫后,11个P450基因(CYP4G75,CYP6AB12,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP337B5,CYP9A58,CYP6AE44及CYP6AE43)表达上调,其中CYP321B1表达量为对照的28.70倍;2龄幼虫受LC_(10)Bt胁迫后,11个P450基因(CYP4G75,CYP6AB12,CYP6AN4,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP337B5,CYP6AE44及CYP6AE43)表达上调,其中CYP6AE44表达量为对照的40.80倍。【结论】草地贪夜蛾2龄幼虫的多个P450基因受这3种杀虫剂亚致死剂量处理后表达上调,其中CYP4G75,CYP6AB12,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP321B5,CYP6AE44及CYP6AE43均能被这3种杀虫剂诱导表达。     

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

Background

Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results

The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion

Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.     

Pinpointing P450s associated with pyrethroid metabolism in the dengue vector, Aedes aegypti: developing new tools to combat insecticide resistance

Background

Pyrethroids are increasingly used to block the transmission of diseases spread by Aedes aegypti such as dengue and yellow fever. However, insecticide resistance poses a serious threat, thus there is an urgent need to identify the genes and proteins associated with pyrethroid resistance in order to produce effective counter measures. In Ae. aegypti, overexpression of P450s such as the CYP9J32 gene have been linked with pyrethroid resistance. Our aim was to confirm the role of CYP9J32 and other P450s in insecticide metabolism in order to identify potential diagnostic resistance markers.

Methodology/Principal Findings

We have expressed CYP9J32 in Escherichia coli and show that the enzyme can metabolize the pyrethroids permethrin and deltamethrin. In addition, three other Ae. aegypti P450s (CYP9J24, CYP9J26, CYP9J28) were found capable of pyrethroid metabolism, albeit with lower activity. Both Ae. aegypti and Anopheles gambiae P450s (CYP''s 6M2, 6Z2, 6P3) were screened against fluorogenic and luminescent substrates to identify potential diagnostic probes for P450 activity. Luciferin-PPXE was preferentially metabolised by the three major pyrethroid metabolisers (CYP9J32, CYP6M2 and CYP6P3), identifying a potential diagnostic substrate for these P450s.

Conclusions/Significance

P450s have been identified with the potential to confer pyrethroid resistance in Ae.aegypti. It is recommended that over expression of these enzymes should be monitored as indicators of resistance where pyrethroids are used.     

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.     

Adaptation of Musca domestica L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

Background

The housefly, Musca domestica, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain.

Results

We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F₁, F₁₃ and F₂₉ to test how gene expression changes following laboratory adaption. Four (CYP6A1, CYP6A36, CYP6D3, CYP6G4) of five investigated P450 genes adapted to breeding by decreasing expression. CYP6D1 showed higher female expression in F₂₉ than in F₁. For males, about half of the genes accessed in the global gene expression were up-regulated in F₁₃ and F₂₉ in comparison with the F₁ population. In females, 60% of the genes were up-regulated in F₁₃ in comparison with F₁, while 33% were up-regulated in F₂₉. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F₁₃ flies in comparison with F₁. Gene expression then increased from F₁₃ to F₂₉ in males and decreased further in females.

Conclusion

The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains.     

Cytochrome P450 diversification and hostplant utilization patterns in specialist and generalist moths: Birth,death and adaptation

Across insect genomes, the size of the cytochrome P450 monooxygenase (CYP) gene superfamily varies widely. CYPome size variation has been attributed to reciprocal adaptive radiations in insect detoxification genes in response to plant biosynthetic gene radiations driven by co‐evolution between herbivores and their chemically defended hostplants. Alternatively, variation in CYPome size may be due to random “birth‐and‐death” processes, whereby exponential increase via gene duplications is limited by random decay via gene death or transition via divergence. We examined CYPome diversification in the genomes of seven Lepidoptera species varying in host breadth from monophagous (Bombyx mori) to highly polyphagous (Amyelois transitella). CYPome size largely reflects the size of Clan 3, the clan associated with xenobiotic detoxification, and to some extent phylogenetic age. Consistently across genomes, families CYP6, CYP9 and CYP321 are most diverse and CYP6AB, CYP6AE, CYP6B, CYP9A and CYP9G are most diverse among subfamilies. Higher gene number in subfamilies is due to duplications occurring primarily after speciation and specialization (“P450 blooms”), and the genes are arranged in clusters, indicative of active duplicating loci. In the parsnip webworm, Depressaria pastinacella, gene expression levels in large subfamilies are high relative to smaller subfamilies. Functional and phylogenetic data suggest a correlation between highly dynamic loci (reflective of extensive gene duplication, functionalization and in some cases loss) and the ability of enzymes encoded by these genes to metabolize hostplant defences, consistent with an adaptive, nonrandom process driven by ecological interactions.     

Deltamethrin Resistance Mechanisms in Aedes aegypti Populations from Three French Overseas Territories Worldwide

Background

Aedes aegypti is a cosmopolite mosquito, vector of arboviruses. The worldwide studies of its insecticide resistance have demonstrated a strong loss of susceptibility to pyrethroids, the major class of insecticide used for vector control. French overseas territories such as French Guiana (South America), Guadeloupe islands (Lesser Antilles) as well as New Caledonia (Pacific Ocean), have encountered such resistance.

Methodology/Principal Findings

We initiated a research program on the pyrethroid resistance in French Guiana, Guadeloupe and New Caledonia. Aedes aegypti populations were tested for their deltamethrin resistance level then screened by an improved microarray developed to specifically study metabolic resistance mechanisms. Cytochrome P450 genes were implicated in conferring resistance. CYP6BB2, CYP6M11, CYP6N12, CYP9J9, CYP9J10 and CCE3 genes were upregulated in the resistant populations and were common to other populations at a regional scale. The implication of these genes in resistance phenomenon is therefore strongly suggested. Other genes from detoxification pathways were also differentially regulated. Screening for target site mutations on the voltage-gated sodium channel gene demonstrated the presence of I1016 and C1534.

Conclusion /significance

This study highlighted the presence of a common set of differentially up-regulated detoxifying genes, mainly cytochrome P450 genes in all three populations. GUA and GUY populations shared a higher number of those genes compared to CAL. Two kdr mutations well known to be associated to pyrethroid resistance were also detected in those two populations but not in CAL. Different selective pressures and genetic backgrounds can explain such differences. These results are also compared with those obtained from other parts of the world and are discussed in the context of integrative research on vector competence.     

Characterization of multiple <Emphasis Type="Italic">CYP9A</Emphasis> genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed, focusing mainly on gene duplication. All four CYP9A subfamily members from the silkworm, Bombyx mori, were cloned by RT-PCR and designated CYP9A19–CYP9A22 by the P450 Nomenclature Committee. They each contain an open reading frame of 1,593 bp in length and encode a putative polypeptide of 531 amino acids. Both nucleic acid and amino acid sequences share very high identities with one another. The typical motifs of insect cytochrome P450, including the heme-binding region, helix-C, helix-I, helix-K, and PERF, show high sequence conservation among the multiple proteins. Alignment with their cDNA sequences revealed that these paralogues share identical gene structures, each comprising ten exons and nine introns of variable sizes. The locations of their introns (all nine introns follow the GT–AG rule) are absolutely conserved. CYP9A19, CYP9A20, and CYP9A21 form a tandem cluster on chromosome 17, whereas CYP9A22 is separated from the cluster by four tandem alcohol-dehydrogenase-like genes. Their phylogenetic relationships and structural comparisons indicated that these paralogues arose as the results of gene duplication events. RT-PCR detected their mRNAs in different “first line of defense” tissues, as well as in several other organs, suggesting diverse functions. Tissue-selective expression also indicates their functional divergence. The identified CYP9A genes have not yet been found outside the Lepidoptera, and are probably unique to the Lepidoptera. They show high sequence and structural similarities to each other, indicating that the Lepidoptera-specific P450s may be of functional importance. This analysis constitutes the first report of the clustering, spatial organization, and functional divergence of P450 in the silkworm.     

CYP175A1 from<Emphasis Type="Italic"> Thermus thermophilus</Emphasis> HB27, the first β-carotene hydroxylase of the P450 superfamily

The biological function of thermostable P450 monooxygenase CYP175A1 from Thermus thermophilus HB27 was studied by functional complementation in Escherichia coli. The gene product of CYP175A1 added hydroxyl groups to both rings of -carotene to form zeaxanthin (,-carotene-3,3-diol) in E. coli, which produces -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. In addition, spectroscopic methods revealed that E. coli carrying CYP175A1 and the cDNA of the Haematococcus pluvialis carotene ketolase was able to synthesise hydroxyechinenone. The predicted amino acid sequence of the enzyme from T. thermophilus does not show substantial similarity with other known -carotene hydroxylases, but 41% with the cytochrome P450 monooxygenase from Bacillus megaterium (CYP102A1, P450 BM3). It is concluded that CYP175 A1 represents a new type of -carotene hydroxylase of the P450 superfamily.     

Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b₅. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA). No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6-, 16-, and 21-hydroxyprogesterone. Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450. Based on the V_max/K_m values for P450 3A9 using either 21-hydroxprogesterone or 6-hydroxyprogesterone as a substrate, 4-pregnen-6, 21-diol-3, 20-dione may be formed either by 6-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.     

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.     

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.     

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.