Insect Cytochrome P450 Research Articles

Function analysis of CYP321A9 from Spodoptera frugiperda (Lepidoptera: Noctuidae) associated with emamectin benzoate, and a novel insecticide, cyproflanilide detoxification.

The fall armyworm, Spodoptera frugiperda, is an invasive agricultural pest that is a serious threat to agricultural production and global food security. Chemical control is the most effective method for preventing outbreaks of S. frugiperda. However, insecticide resistance often develops as a result of prolonged pesticide use, and the molecular mechanisms involved in insecticide resistance remain unclear. Insect cytochrome P450 monooxygenases play an important role in the detoxification of insecticides and insecticide resistance in Lepidoptera. In our study, the LC50 of a novel insecticide (cyproflanilide) and a conventional insecticide (emamectin benzoate) for S. frugiperda second-instar larvae were 7.04 and 1.61 mg/L, respectively. Furthermore, CYP321A9 expression was upregulated in larvae exposed to these insecticides. Additionally, knockdown of CYP321A9 by feeding larvae with dsRNA for 72 h significantly increased the mortality of S. frugiperda exposed to emamectin benzoate and cyproflanilide by 23.33% and 7.78%, respectively. Our results indicate that CYP321A9 may play an important role in the detoxification of emamectin benzoate and cyproflanilide in S. frugiperda. Our findings provide a basis to better understand the mechanisms of insecticide resistance and contribute to the control of S. frugiperda.

Functional roles of two novel P450 genes in the adaptability of Conogethes punctiferalis to three commonly used pesticides.

Introduction: Insect cytochrome P450 (CYP450) genes play important roles in the detoxification and metabolism of xenobiotics, such as plant allelochemicals, mycotoxins and pesticides. The polyphagous Conogethes punctiferalis is a serious economic pest of fruit trees and agricultural crops, and it shows high adaptability to different living environments. Methods: The two novel P450 genes CYP6CV1 and CYP6AB51 were identified and characterized. Quantitative real-time PCR (qRT-PCR) technology was used to study the expression patterns of the two target genes in different larval developmental stages and tissues of C. punctiferalis. Furthermore, RNA interference (RNAi) technology was used to study the potential functions of the two P450 genes by treating RNAi-silenced larvae with three commonly used pesticides. Results: The CYP6CV1 and CYP6AB51 genes were expressed throughout various C. punctiferalis larval stages and in different tissues. Their expression levels increased along with larval development, and expression levels of the two target genes in the midgut were significantly higher than in other tissues. The toxicity bioassay results showed that the LC50 values of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin on C. punctiferalis larvae were 0.2028μg/g, 0.0683μg/g and 0.6110mg/L, respectively. After treating with different concentrations of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin (LC10, LC30, LC50), independently, the relative expressions of the two genes CYP6CV1 and CYP6AB51 were significantly induced. After the dsRNA injection, the expression profiles of the two CYP genes were reduced 72.91% and 70.94%, respectively, and the mortality rates of the larvae significantly increased when treated with the three insecticides independently at LC10 values. Discussion: In the summary, after interfering with the CYP6CV1 and CYP6AB51 in C. punctiferalis, respectively, the sensitivity of C. punctiferalis to chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin was significantly increased, indicating that the two CYP6 genes were responsible for the adaptability of C. punctiferalis to the three chemical insecticides in C. punctiferalis. The results from this study demonstrated that CYP6CV1 and CYP6AB51 in C. punctiferalis play crucial roles in the detoxification of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin.

Activation of CncC pathway is involved in the regulation of P450 genes responsible for clothianidin resistance in Bradysia odoriphaga.

Insect cytochrome P450 monooxygenases (P450s) play a key role in the detoxification metabolism of insecticides and their overexpression is often associated with insecticide resistance. Our previous research showed that the overexpression of four P450 genes is responsible for clothianidin resistance in B. odoriphaga. In this study, we characterized another P450 gene CYP6FV21 associated with clothianidin resistance. However, the molecular basis for the overexpression of P450 genes in clothianidin resistant strain remains obscure in B. odoriphaga. In this study, the CYP6FV21 gene was significantly overexpressed in the clothianidin resistant (CL-R) strain. Clothianidin exposure significantly increased the expression level of CYP6FV21. Knockdown of CYP6FV21 significantly increased the susceptibility of B. odoriphaga larvae to clothianidin. The transcription factor Cap 'n' Collar isoform-C (CncC) was highly expressed in the midgut of larvae in B. odoriphaga. The expression level of CncC was higher in the CL-R strain compared with the susceptible (SS) strain. Clothianidin exposure caused reactive oxygen species (ROS) accumulation and significantly increased the expression level of CncC. Knockdown of CncC cause a significant decrease in the expression of CYP3828A1 and CYP6FV21 and P450 enzyme activity, and leads to a significant increase in mortality after exposure to LC30 clothianidin. After treatment with CncC agonist curcumin, the P450 activity and the expression levels of CYP3828A1 and CYP6FV21 significantly increased, and larval sensitivity to clothianidin decreased. The ROS scavenger NAC treatment significantly inhibited the expression levels of CncC, CYP3828A1 and CYP6FV21 in response to clothianidin exposure and increased larval sensitivity to clothianidin. Taken together, these results indicate that activation of the CncC pathway by the ROS burst plays a critical role in clothianidin resistance by regulating the expression of CYP3828A1 and CYP6FV21 genes in B. odoriphaga. This study provides more insight into the mechanisms underlying B. odoriphaga larval resistance to clothianidin.

RNAi-mediated silencing of AccCYP6k1 revealed its role in the metabolic detoxification of Apis cerana cerana

Insect cytochrome P450 monooxygenases (P450s or CYPs) perform important functions in the metabolic detoxification of both endogenous and exogenous substrates. However, the mechanism of action of the P450 genes in bees is unclear. In this study, we investigated the effects of AccCYP6k1 on the metabolism and detoxification of Apis cerana cerana. Spatiotemporal expression profiling revealed that the expression of AccCYP6k1 was the highest in foragers (A15) and was mainly expressed in the leg, midgut and head. RT–qPCR results showed that AccCYP6k1 exhibited different expression patterns following exposure to xenobiotics. In addition, silencing AccCYP6k1 increased the pesticides sensitivity and affected the detoxification system and antioxidant process of A. cerana cerana. In brief, the induced expression of AccCYP6k1 is related to the resistance of A. cerana cerana, while knockdown AccCYP6k1 affect the pesticides resistance and metabolic detoxification system of A. cerana cerana. These findings not only support the theoretical basis of metabolic detoxification in bees but also provide a better understanding of P450-mediated resistance to pesticides in insects.

Disruption of CYP6DF1 and CYP6DJ2 increases the susceptibility of Dendroctonus armandi to (+)-α-pinene

Bark beetles rely on detoxifying enzymes to resist the defensive oleoresin terpenes of the host tree. Insect cytochrome P450 (CYPs) plays a key role in the detoxification of plant allelochemicals and pesticides. CYP6 family is unique to Insecta, and its biochemical function is basically related to catabolize heterologous substances. In this study, two Dendroctonus armandi CYP6 genes, CYP6DF1 and CYP6DJ2, were characterized. Spatiotemporal expression profiling revealed that CYP6DF1 and CYP6DJ2 expressions were higher in larvae and adult stages of D. armandi than in egg and pupae stages, and that two genes predominantly expressed in brain, midgut, fat body, or Malpighian tubules. Moreover, CYP6DF1 and CYP6DJ2 expressions were significantly induced after exposure to (+)-α-pinene. Importantly, silencing CYP6DF1 and CYP6DJ2 significantly inhibited the CYP activity and increased the mortality in the adults fumigated with (+)-α-pinene. Additionally, piperonyl butoxide exposure to adults also increase the sensitivity after treatment with (+)-α-pinene, which led to a significant reduction of the CYP activity, resulting a significant increase in adult mortality. These results suggest that the CYP6 family plays a key role in determining the susceptibility of D. armandi to (+)-α-pinene, which may have implications for the development of novel therapeutics to control this important pest.

Functional validation of CYP304A1 associated with haedoxan A detoxification in Aedes albopictus by RNAi and transgenic drosophila.

Insect cytochrome P450 monooxygenases play important roles in the detoxification metabolism of endogenous and exogenous compounds. Haedoxan A (HA) from Phryma leptostachya L. is a highly efficient natural pesticide used to control houseflies and mosquitos. CYP4C21 and CYP304A1 were previously demonstrated to be transcriptionally increased in Aedes albopictus in response to HA exposure, but their involvement in HA metabolism is unknown. Our data showed that CYP304A1 expression levels in A. albopictus were highest in third-instar larvae, and the expression level of CYP4C21 decreased significantly with the growth of instars, with the lowest occurring in the pupal stage. Compared with the control, the silencing of CYP304A1 and CYP4C21 genes by chitosan nanoparticle-mediated RNA interference could deplete 58.2% and 54.0% of the expression of corresponding genes, respectively. The bioassay data showed that knocking down the expression of CYP304A1 increased the mortality of A. albopictus when exposed to HA at LC30 and LC50 doses, but did not significantly increase mortality after silencing CYP4C21. Our data demonstrated that CYP304A1, but not CYP4C21, may be involved in HA detoxification. Moreover, the resistance ratio of CYP304A1 overexpressing flies was approximately 2-fold higher than that of the control line. The metabolized product of HA by CYP304A1 needs to be further confirmed by in vitro expression. This finding showed that inducibility was not always linked to detoxifying capabilities, and enhanced our understanding of the molecular basis of HA metabolic detoxification in A. albopictus. © 2022 Society of Chemical Industry.

Knockdown of CYP6CR2 and CYP6DE5 reduces tolerance to host plant allelochemicals in the Chinese white pine beetle Dendroctonus armandi

Bark beetles rely on detoxifying enzymes to resist the defensive terpenoids of the host tree. Insect cytochrome P450 (CYPs) plays a key role in the detoxification of pesticides and plant allelochemicals. CYP6 family is unique to Insecta, and its biochemical function is basically related to the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of two CYP6 genes from Chinese white pine beetle, Dendroctonus armandi. Spatiotemporal expression profiling revealed that the expression of CYP6CR2 and CYP6DE5 was higher in larval and adult stages of D. armandi than that in other developmental stages, and that two genes predominantly expressed in brain, midgut, fat body, Malpighian tubules or hemolymph. The expression of CYP6CR2 and CYP6DE5 was significantly induced after feeding on the phloem of Pinus armandii and exposure to six stimuli [(±)- α -pinene, (−)-α-pinene, (−)-β-pinene, (+)-3-carene, (±)-limonene and turpentine]. Importantly, silencing CYP6CR2 and CYP6DE5 separately could increase the sensitivity, led to a significant reduction of the activity of P450, resulting a significant increase in adult mortality after treatment with terpenoids. The comprehensive results of this study showed that in the process of host selection and colonization, the functions of CYPs were mainly to hydrolyze the chemical defense of the host and degrade odor molecules. These findings may help to develop new treatments to control this important pest.

Evaluation and optimization of the protocols for measuring cytochrome P450 activity in aphids

Insect cytochrome P450 plays major roles in detoxification of phytotoxin and insecticides. However, determination of P450 activity in aphids has variable success and there is no reliable method yet. In this study, we found that homogenizing the green peach aphid, Myzus persicae, in the 96-well microplate resulted in significantly higher P450 activities than those in Eppendorf tube. Homogenizing aphids in Eppendorf tube released uncharacterized compounds that inhibited aphids and pig liver P450 activities, whereas aphids homogenized in the microplate may not be completely ground and thus released fewer such inhibitors. Then, the microplate homogenization method was optimized as follows: one or two aphids were placed in one well of the 96 well-microplate and ground in phosphate buffer using pipette tips for 20 cycles, followed by addition of 7-ethoxycoumarin, and then incubated for 1 h at room temperature, after which glycine buffer-ethanol mixture was added to stop the reaction. This method is also suitable for the pea aphid, Acyrthosiphon pisum, and the bird cherry‐oat aphid, Rhopalosiphum padi. These results highlight the importance of considering inhibitory effects of endogenous compounds in insects on their P450 activities and provide one possible method to reduce these inhibitory effects.

The Role of Cytochrome P450s in Insect Toxicology and Resistance.

Insect cytochrome P450 monooxygenases (P450s) perform a variety of important physiological functions, but it is their role in the detoxification of xenobiotics, such as natural and synthetic insecticides, that is the topic of this review. Recent advances in insect genomics and postgenomic functional approaches have provided an unprecedented opportunity to understand the evolution of insect P450s and their role in insect toxicology. These approaches have also been harnessed to provide new insights into the genomic alterations that lead to insecticide resistance, the mechanisms by which P450s are regulated, and the functional determinants of P450-mediated insecticide resistance. In parallel, an emerging body of work on the role of P450s in defining the sensitivity of beneficial insects to insecticides has been developed. The knowledge gained from these studies has applications for the management of P450-mediated resistance in insect pests and can be leveraged to safeguard the health of important beneficial insects.

The role of cytochrome P450-mediated detoxification in insect adaptation to xenobiotics.

Insect cytochrome P450 monooxygenases (P450s) are well known to be involved in metabolic detoxification of xenobiotics, such as phytochemicals, insecticides and environmental pollutants. Enhanced metabolic detoxification is closely associated with the constitutive overexpression and induction of P450s. In general, multiple insect P450s are co-responsible for xenobiotic detoxification. Considering the capacity of P450s to respond to a wide range of xenobiotics, synergistic interactions between natural and synthetic xenobiotics and P450-mediated cross-tolerance/resistance are ubiquitous. Recent studies have indicated that both transcription factors and signaling pathways are involved in the regulation of P450 genes in xenobiotic responses. This article reviews our current understanding of P450-mediated detoxification in insect adaptation to xenobiotics and highlights recent progress in the molecular basis of P450 regulation.

Xenobiotic transcription factors CncC and maf regulate expression of CYP321A16 and CYP332A1 that mediate chlorpyrifos resistance in Spodoptera exigua

Insect cytochrome P450 s (P450 s) are associated with the metabolic detoxification of toxic xenobiotics and their constitutive upregulation is often associated with resistance to natural and synthetic toxicants. The P450 s CYP321A16 and CYP332A1 are constitutively overexpressed in an insecticide-resistant strain of beet armyworm, Spodoptera exigua. However, the function and upstream regulation of these two P450 s remain unknown. Here, we investigated the function of CYP321A16 and CYP332A1 in resistance to the organophosphate insecticide, chlorpyrifos and their regulation by the transcription factors CncC and Maf. Transgenic strains of Drosophila melanogaster expressing CYP321A16 or CYP332A1 showed higher levels of tolerance to chlorpyrifos than the control flies with the same genetic background. Furthermore, recombinant CYP321A16 and CYP332A1 proteins metabolized chlorpyrifos. Analysis of the putative promoter sequences of the genes coding for CYP321A16 and CYP332A1 revealed conserved CncC/Maf binding sites. Transfection of luciferase reporter plasmids containing the promoter of CYP450 gene together with CncC and Maf expression plasmids significantly enhanced the activity of the reporter. Promoter truncation identified a site in the promoter of CYP321A16 that is critical for the CncC/Maf binding. These data demonstrate that resistance to chlorpyrifos in S. exigua is conferred by the combined action of CYP321A16 and CYP332A1 and uncovered their regulation by the transcription factors CncC and Maf.

Silencing of Cytochrome P450 in Spodoptera frugiperda (Lepidoptera: Noctuidae) by RNA Interference Enhances Susceptibility to Chlorantraniliprole.

Fall armyworm, Spodoptera frugiperda (Smith), has caused significant losses for crop production in China. The fall armyworm is mainly controlled by the chemical insecticides, whereas the frequent application of insecticides would result in the resistance development. Insect cytochrome P450 monooxygenases play an essential part in the detoxification of insecticides. In this study, five P450 genes were selected to determine the role in response to insecticides by RNA interference (RNAi). Developmental expression pattern analysis revealed that S. frugiperda CYP321A8, CYP321A9, and CYP321B1 were highest in second-instar larvae among developmental stages, with 2.04-, 3.39-, and 8.58-fold compared with eggs, whereas CYP337B5 and CYP6AE44 were highest in adult stage, with 16.3- and 10.6-fold in comparison of eggs, respectively. Tissue-specific expression pattern analysis exhibited that CYP321A8, CYP321B1, and CYP6AE44 were highest in the midguts, with 3.56-, 3.33-, and 3.04-fold compared with heads, whereas CYP321A9 and CYP337B5 were highest in wings, with 3.07- and 3.36-fold compared with heads, respectively. RNAi was also conducted to explore detoxification effects of the five P450 genes on chlorantraniliprole. The second-instar larvae became more sensitive to chlorantraniliprole with a higher mortality rate than the control, after silencing CYP321A8, CYP321A9, and CYP321B1, respectively. These findings strongly supported our viewpoint that CYP321A8, CYP321A9, and CYP321B1 may play a critical role in insecticide detoxification. It will provide a basis for further study on regulation of P450 genes and the management of S. frugiperda.

CYP303A1 has a conserved function in adult eclosion in Locusta migratoria and Drosophila melanogaster

Insect cytochrome P450 monooxygenases (CYPs) play essential roles in both xenobiotic metabolism and developmental processes. However, the exact physiological function of many CYP genes remains largely unknown. Screening the expression of the CYP genes from the CYP2 and mitochondrial CYP clans of Drosophila melanogaster revealed that Cyp303a1 is highly expressed in the pupal stage. Knockdown of CYP303A1 transcripts by RNAi using the Gal4/UAS system with a ubiquitous driver (tubulin-Gal4) in Drosophila or by dsRNA injection in the last nymph stage of Locusta migratoria resulted in severe defects in eclosion and lethality during and after adult emergence. In Drosophila, tissue-specific RNAi of Cyp303a1 with a wing-specific driver (MS1096-Gal4) revealed that Cyp303a1 was essential for wing extension. Stage-specific RNAi of Cyp303a1 using Gal80ts for thermal-dependent-suppression found that the expression of Cyp303a1 at the middle pupal stage was absolutely required. Meanwhile, Cyp303a1 mutants exhibited more than 80% lethality at the late embryonic development stages. Embryonic lethality of the Cyp303a1 mutants was fully rescued by the ubiquitous overexpression of exogenous Cyp303a1. Taken together, we conclude that Cyp303a1 is indispensable for embryonic development and adult eclosion in D. melanogaster, the latter role being conserved over 400 million years of insect evolution.

CYP4BN6 and CYP6BQ11 mediate insecticide susceptibility and their expression is regulated by Latrophilin in Tribolium castaneum.

Many insect cytochrome P450 proteins (CYPs) are involved in the metabolic detoxification of exogenous compounds such as plant toxins and insecticides. Tribolium castaneum, the red flour beetle, is a major agricultural pest that damages stored grains and cereal products. With the completion of the sequencing of its genome, two T. castaneum species-specific CYP genes, CYP4BN6, and CYP6BQ11, were identified. However, it is unknown whether the functions of most CYPs are shared by TcCYP4BN6 and TcCYP6BQ11, and the upstream regulatory mechanism of these two CYPs remains elusive. QRT-PCR analysis indicated that TcCYP4BN6 and TcCYP6BQ11 were both most highly expressed at the late pupal stage and were mainly observed in the head and gut, respectively, of adults. Moreover, the transcripts of these two CYPs were significantly induced by dichlorvos and carbofuran, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to these two insecticides. Intriguingly, knockdown of the latrophilin (lph) gene, which has been reported to be related to the insecticide susceptibility, reduced the expression of TcCYP4BN6 and TcCYP6BQ11 after insecticide treatment, suggesting that these two CYP genes are regulated by lph to participate in insecticide susceptibility in T. castaneum. These results shed new light on the function and mechanism of CYP genes associated with insecticide susceptibility and could facilitate research on appropriate and sustainable pest control management. © 2019 Society of Chemical Industry.

Characterization of the Cytochrome P450 Gene CYP305A1 of the Cotton Aphid (Hemiptera: Aphididae) and Its Responsive Cis-Elements to Plant Allelochemicals.

Insect cytochrome P450 monooxygenases play an important role in plant allelochemical detoxification. In this study, a full-length gene CYP305A1 of the P450 Clan 2 family was cloned from Aphis gossypii Glover, and its promoter was identified and characterized. The transcript level of CYP305A1 and its promoter activity were significantly induced by two plant allelochemicals, gossypol and 2-tridecanone. Furthermore, the 5'-end promoter region from -810 to +62 bp was demonstrated to be essential for basal transcriptional activity of CYP305A1, and the promoter region from -810 to -581 bp was shown as an essential plant allelochemical responsive element and had a cis-element 5'-CACACTA-3' as the binding site of aryl hydrocarbon receptor. Interestingly, there was an identical overlapping region of 1,094 bp between CYP305A1 promoter and the venom protease gene. When the expression of CYP305A1 gene was knocked down by RNA interference with CYP305A1 dsRNA, the expression of the venom protease gene was decreased. However, the knockdown of the expression of the venom protease gene did not affect the CYP305A1 expression. These results provide important insights for understanding the functions of P450 genes and the regulatory mechanism of P450 gene expressions in the resistance of Aphis gossypii Glover to plant allelochemicals.

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.

Identification and functional analysis of a cytochrome P450 gene involved in imidacloprid resistance in Bradysia odoriphaga Yang et Zhang

Insect cytochrome P450 monooxygenases played an important role in detoxifying insecticides which potentially contributed to the metabolic resistance to insecticides. Bradysia odoriphaga, as a major pest of Chinese chive, was reported to be highly tolerant to neonicotinoid insecticides imidacloprid. In this study, a novel P450 gene, CYP6FV12, was cloned from B. odoriphaga. The full-length cDNA sequence of CYP6FV12 is 2520 bp long and its open reading frame (ORF) encodes 519 amino acids. Quantitative real-time PCR showed that the highest expression of CYP6FV12 was observed in fourth-instar larvae, which is 154.32-fold higher than that of eggs. Highest expression of CYP6FV12 was observed in the midgut, followed by fat body, which was 13.67 and 5.42-fold higher than that in cuticle, respectively. The expression of CYP6FV12 was significantly up-regulated in B. odoriphaga larvae after exposed to imidacloprid at the concentrations of 10, 30, 50, and 70 mg/L. Moreover, RNAi mediated silencing of CYP6FV12 increased mortality by 28.62% when the fourth-instar larvae were treated with imidacloprid. This is the first systematic study on isolated P450s gene involved in imidacloprid resistance in B. odoriphaga and increased our understanding of the molecular mechanisms of insecticide detoxification in this pest insect.

1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology.

1-Aminobenzotriazole (1-ABT) is a pan-specific, mechanism-based inactivator of the xenobiotic metabolizing forms of cytochrome P450 in animals, plants, insects, and microorganisms. It has been widely used to investigate the biological roles of cytochrome P450 enzymes, their participation in the metabolism of both endobiotics and xenobiotics, and their contributions to the metabolism-dependent toxicity of drugs and chemicals. This review is a comprehensive evaluation of the chemistry, discovery, and use of 1-aminobenzotriazole in these contexts from its introduction in 1981 to the present.

Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides

Insect cytochrome P450 monooxygenases (CYPs or P450s) play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura, has developed resistance to a wide range of insecticides. In the present study, a novel P450 gene, CYP321B1, was cloned from S. litura. The function of CYP321B1 was assessed using RNA interference (RNAi) and monitoring resistance levels for three commonly used insecticides, including chlorpyrifos, β-cypermethrin and methomyl. The full-length complementary DNA sequence of CYP321B1 is 1814 bp long with an open reading frame of 1488 bp encoding 495 amino acid residues. Quantitative reverse-transcriptase polymerase chain reaction analyses during larval and pupal development indicated that CYP321B1 expression was highest in the midgut of fifth-instar larvae, followed by fat body and cuticle. The expression of CYP321B1 in the midgut was up-regulated by chlorpyrifos, β-cypermethrin and methomyl with both lethal concentration at 15% (LC15 ) (50, 100 and 150 μg/mL, respectively) and 50%(LC50 ) dosages (100, 200 and 300 μg/mL, respectively). Addition of piperonyl butoxide (PBO) significantly increased the toxicity of chlorpyrifos, β-cypermethrin and methomyl to S. litura, suggesting a marked synergism of the three insecticides with PBO and P450-mediated detoxification. RNAi-mediated silencing of CYP321B1 further increased mortality by 25.6% and 38.9% when the fifth-instar larvae were exposed to chlorpyrifos and β-cypermethrin, respectively, at the LC50 dose levels. The results demonstrate that CYP321B1 might play an important role in chlorpyrifos and β-cypermethrin detoxification in S. litura.

Cloning and Expression of Multiple Cytochrome P450 Genes: Induction by Fipronil in Workers of the Red Imported Fire Ant (Solenopsis invicta Buren).

Both exogenous and endogenous compounds can induce the expression of cytochrome P450 genes. The insect cytochrome P450 genes related to insecticide resistance are likely to be expressed as the “first line of defense” when challenged with insecticides. In this study, four cytochrome P450 genes, SinvCYP6B1, SinvCYP6A1, SinvCYP4C1, and SinvCYP4G15, were firstly isolated from workers of the red imported fire ant (Solenopsis invicta) through rapid amplification of cDNA ends (RACE) and sequenced. The fipronil induction profiles of the four cytochrome P450 genes and the two previously isolated CYP4AB1 and CYP4AB2 were characterized in workers. The results revealed that the expression of SinvCYP6B1, SinvCYP6A1, CYP4AB2, and SinvCYP4G15, increased 1.4-fold and 1.3-fold more than those of acetone control, respectively, after 24 h exposure to fipronil at concentrations of 0.25 μg mL−1 (median lethal dose) and 0.56 μg mL−1 (90% lethal dose), while no significant induction of the expression of CYP4AB1 and SinvCYP4C1 was detected. Among these genes, SinvCYP6B1 was the most significantly induced, and its maximum expression was 3.6-fold higher than that in acetone control. These results might suggest that multiple cytochrome P450 genes are co-up-regulated in workers of the fire ant through induction mechanism when challenged with fipronil. These findings indicated that cytochrome P450 genes play an important role in the detoxification of insecticides and provide a theoretical basis for the mechanisms of insecticide metabolism in the fire ant.