The Orco gene involved in recognition of host plant volatiles and sex pheromone in the chive maggot Bradysia odoriphaga.

The insect olfactory recognition system plays a crucial role in the feeding and reproductive behaviors of insects. The odorant receptor co-receptor (Orco), as an obligatory chaperone, is critical for odorant recognition by way of forming heteromeric complexes with conventional odorant receptors (ORs). To investigate the biological functions of Orco in perceiving host plant volatiles and sex pheromone, the Orco gene was identified from the chive maggot Bradysia odoriphaga transcriptome data. Multiple sequence alignment reveals that BodoOrco exhibits an extremely high sequence identity with Orcos from other dipteran insects. The expression of BodoOrco is significantly higher in adults than in larvae and pupae, and the BodoOrco gene is primarily expressed in the antennae of both sexes. Furthermore, the Y-tube assay indicated that knockdown of BodoOrco leads to significant reductions in B. odoriphaga adults' response to all tested host plant volatiles. The dsOrco-treated unmated male adults show less attraction to unmated females and responded slowly compared with dsGFP control group. These results indicated that BodoOrco is involved in recognition of sex pheromone and host plant volatiles in B. odoriphaga and has the potential to be used as a target for the design of novel active compounds for developing ecofriendly pest control strategies.

Odorant-binding Protein 10 From Bradysia odoriphaga (Diptera: Sciaridae) Binds Volatile Host Plant Compounds.

Bradysia odoriphaga (Diptera: Sciaridae) is a major insect pest of seven plant families including 30 commercial crops in Asia. The long-term use of chemical pesticides leads to problems such as insect resistance, environmental issues, and food contamination. Against this background, a novel pest control method should be developed. In insects, odorant-binding proteins (OBPs) transport odor molecules, including pheromones and plant volatiles, to olfactory receptors. Here, we expressed and characterized the recombinant B. odoriphaga OBP BodoOBP10, observing that it could bind the sulfur-containing compounds diallyl disulfide and methyl allyl disulfide with Ki values of 8.01 µM and 7.00 µM, respectively. Homology modeling showed that the BodoOBP10 3D structure was similar to that of a typical OBP. Both diallyl disulfide and methyl allyl disulfide bound to the same site on BodoOBP10, mediated by interactions with six hydrophobic residues Met70, Ile75, Thr89, Met90, Leu93, and Leu94, and one aromatic residue, Phe143. Furthermore, silencing BodoOBP10 expression via RNAi significantly reduced the electroantennogram (EAG) response to diallyl disulfide and methyl allyl disulfide. These findings suggest that BodoOBP10 should be involved in the recognition and localization of host plants.

Integration analysis of PacBio SMRT- and Illumina RNA-seq reveals P450 genes involved in thiamethoxam detoxification in Bradysia odoriphaga.

The sciarid fly Bradysia odoriphaga is a serious pest of Chinese chive (Liliaceae). Neonicotinoid insecticides including thiamethoxam have been used for B. odoriphaga control. However, thiamethoxam resistance in B. odoriphaga has developed in recent years. To identify potential genes involved in detoxification metabolism of thiamethoxam in B. odoriphaga, a PacBio single-molecule real-time (SMRT) transcriptome sequencing and Illumina RNA-seq analysis on thiamethoxam treated B. odoriphaga were performed to explore differentially expressed genes in B. odoriphaga. After SMRT sequencing, analysis of Illumina RNA-Seq data showed a total of 172 differentially expressed genes (DEGs) after thiamethoxam treatment, among which eight upregulated DEGs were P450 genes that may be related to thiamethoxam metabolism. The qRT-PCR results of the eight up-regulated P450 unigenes after thiamethoxam treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of the eight upregulated P450 transcripts followed by insecticide bioassay was conducted, and three P450 unigenes were verified to be related to thiamethoxam detoxification in B. odoriphaga. This study provides new information about the P450 genes involved in thiamethoxam detoxification in B. odoriphaga.

Identification and Pathogenicity of a New Entomopathogenic Fungus, Mucor hiemalis (Mucorales: Mucorales), on the Root Maggot, Bradysia odoriphaga (Diptera: Sciaridae).

Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae), the Chinese chive root maggot, is a destructive pest of Allium vegetables and flowers that causes severe losses in northern China. Novel biological control technologies are needed for controlling this pest. We identified a new entomopathogenic fungus isolated from infected B. odoriphaga larvae and evaluated the susceptibility of the biological stages of B. odoriphaga and the effects of temperature on fungus growth and pathogenicity. Based on morphological characteristics and molecular phylogeny, the fungus was identified as Mucor hiemalis BO-1 (Mucorales: Mucorales). This fungus had the strongest virulence to B. odoriphaga larvae followed by eggs and pupae, while B. odoriphaga adults were not susceptible. A temperature range of 18-28°C was optimum for the growth and sporulation of M. hiemalis BO-1 and virulence to B. odoriphaga larvae. At 3 and 5 d after inoculation with 105 spores/ml at 23°C, the survival rates were 24.8% and 4.8% (2nd instar larvae), respectively, and 49.6% and 12.8% (4th instar larvae), respectively. The potted plant trials confirmed that M. hiemalis BO-1 exerted excellent control efficiency against B. odoriphaga larvae, and the control exceeded 80% within 5 d when the spore concentration applied exceeded 107 spores/ml. In conclusion, these findings supported the hypotheses that this fungus could serve as an effective control agent against B. odoriphaga larvae and is worth being further tested to determine its full potential as a biocontrol agent.

Characterization and functional analysis of two acetylcholinesterase genes in Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae).

Two acetylcholinesterase genes (Boace1 and Boace2) were cloned from Bradysia odoriphaga, a devastating soil pest that mainly damages Chinese chives. The Boace1 encodes BoAChE1 protein consisting of 696 amino acid residues, while Boace2 encodes BoAChE2 containing 638 amino acids. Phylogenetic analysis showed that Boace1 and Boace2 are appeared to be distinct clusters. The gene expression patterns at different development stages and various body parts tissues were examined, and their biological functions were characterized by RNA interference and analog docking prediction. The results showed that both Boace genes were expressed in all developmental stages and examined tissues. The transcript level of Boace2 was significantly higher than Boace1 in all tested samples, and Boace1 was found most abundant in the head while Boace2 was highly expressed in the fat body of B. odoriphaga. The silencing of Boace1 and Boace2 significantly decreased the AChE activity of 36.6% and 14.8% separately, and increased the susceptibility of B. odoriphaga to phoxim, with 60.8% and 44.7% mortality. Besides, overexpression and gene duplication of Boace1 were found in two field resistant populations, and two major mutations, A319S and G400V, were detected in Boace1. Moreover, the docking results revealed that BoAChE1 had a higher affinity towards organophosphorus than BoAChE2. It is concluded that Boace2 is the most abundant ace type in B. odoriphaga, while both Boace play vital roles. Boace1 might play a major neurological function and more likely be the prime target for insecticides, while Boace2 might play some important unidentified roles.

Comparative transcriptome analysis of differentially expressed genes in Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) at different acute stress temperatures.

The gnat, Bradysia odoriphaga Yang et Zhang, is an important underground pest in Asia. B. odoriphaga differ in heat and cold tolerance and exhibit quite different developmental strategies. To understand the underlying mechanisms, we sequenced and compared the transcriptome of B. odoriphaga under 40 °C (a stressful high temperature), 25 °C, and 4 °C (a stressful low temperature) for 1 h. We found that metabolism- and ribosome-related genes were modulated. In high temperature (40 °C), heat shock protein (HSP) genes, detoxication genes, metabolism genes, protein turnover genes, and stress signal transduction genes were differentially expressed. In low temperature (4 °C), genes related with heat shock protein (HSP) and detoxication were differentially expressed. Our study increases our understanding of the complex molecular mechanisms involved in the responses of B. odoriphaga to acute temperature stress and provides a potential strategy for pest management.

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in Bradysia odoriphaga.

Bradysia odoriphaga is a destructive insect pest, damaging more than 30 crop species. Nicotinic acetylcholine receptors (nAChRs) mediating fast excitatory transmission in the central nervous system in insects are the molecular targets of some economically important insecticides including imidacloprid, which has been widely used to control B. odoriphaga in China since 2013. However, the clear characterization about nAChRs in B. odoriphaga is still unknown. Hence, our objective is to identify and characterize the nAChR gene family in B. odoriphaga based on the transcriptome database and sequence, phylogenetic and expression profiles analysis. In this study, we cloned seven nAChR subunit genes from B. odoriphaga, including Boα1, Boα2, Boα3, Boα7, Boα8, Boß1 and Boß3. Sequence analysis revealed that the seven nAChR subunits of B. odoriphaga shared the typical structural features with Drosophila melanogaster nAChR α1 subunit, including an extracellular N-terminal domain containing six functional loops (loop A-F), a signature Cys-loop with two disulfide bond-forming cysteines separated by 13 amino acid residues, and four typical transmembrane helices (TM1-TM4) in the C-terminal region. Phylogenetic analysis suggested that seven nAChR subunit genes in B. odoriphaga are evolutionarily conserved among four model insects, including D. melanogaster, Bombyx mori, Apis mellifera and Tribolium castaneum. Meanwhile, nAChR α4, α5, α6 and ß2 subunit genes may potentially exist in B. odoriphaga, which need further study. Furthermore, quantitative real-time PCR analysis revealed the specific expression pattern of nAChR subunits in three body parts including head, thorax and abdomen, and developmental expression pattern of nAChR subunits throughout the B. odoriphaga life cycle. These results provided necessary information for further investigating the diverse functions of nAChRs in B. odoriphaga.

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.

Sublethal effects of chlorfenapyr on the life table parameters, nutritional physiology and enzymatic properties of Bradysia odoriphaga (Diptera: Sciaridae).

Bradysia odoriphaga (Diptera: Sciaridae) is the major pest affecting Chinese chive production. Chlorfenapyr is a halogenated pyrrole-based pro-insecticide that is currently used to control insects and mites on a variety of crops. In the present study, fourth-instar larvae of B. odoriphaga were exposed to chlorfenapyr at LC1, LC20 and LC50 concentrations. The developmental duration of the treated larvae was not significantly different, but fecundity was significantly increased in the LC1 and LC20 treatment groups compared with the control group. The population parameters of the LC1 treatment group were increased significantly, whereas those of the LC50 treatment group were reduced significantly compared with the control. The food consumption by larvae and pupal weight were significantly increased under the LC1 treatment and decreased under the LC50 treatment compared with the control. Moreover, chlorfenapyr decreased the lipid, carbohydrate and trehalose contents significantly, whereas the total protein content was increased compared with the control. Additionally, the activities of protease, lipase and trehalase were significantly decreased. Chlorfenapyr treatment for 24 h also induced the activities of glutathione S-transferase (GST), carboxylesterase (CarE) and O-demethylation. The results of this study suggest that low lethal concentrations of chlorfenapyr can affect oviposition, population development, the activities of digestion and detoxification enzymes, and nutrient accumulation in B. odoriphaga. This study provides valuable information for the assessment and rational application of chlorfenapyr for effective control of this pest.

Detection of insecticide resistance in Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) in China.

Bradysia odoriphaga Yang et Zhang is a destructive insect pest of Chinese chives. To understand the current status of insecticide resistance of B. odoriphaga in China, the sensitivity variation of eight field populations to six commonly used insecticides, including chlorpyrifos, phoxim, imidacloprid, thiamethoxam, clothianidin and beta-cypermethrin were evaluated. The results showed that almost all the tested B. odoriphaga populations had developed moderate to high resistance to chlorpyrifos and phoxim. There were different resistance levels found in the eight field populations among the three neonicotinoids, imidacloprid, thiamethoxam and clothianidin. Imidacloprid was very effective against B. odoriphaga in most tested populations except those from Yangzhou (10.35-fold) and Tangshan (14.56-fold). While four populations kept susceptible to thiamethoxam, the other four populations showed decreased susceptibility or low resistance. To clothianidin, five populations displayed moderate resistance, two populations displayed low resistance, and one population exhibited susceptibility, respectively. All the tested populations were resistance to beta-cypermethrin, the highest resistance was found in the Tangshan population with a resistance ratio of 172.56-fold. The results of this study provided valuable information for choosing insecticides for control and integrated resistance management of B. odoriphaga.

Lethal and sublethal effects of the chitin synthesis inhibitor chlorfluazuron on Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae).

Bradysia odoriphaga Yang and Zhang is the primary insect pest that affects Chinese chive in northern China. Nevertheless, very few studies have been conducted on the use of chitin synthesis inhibitors (CSIs) for the control of B. odoriphaga. Here, lethal and sublethal effects of the CSI chlorfluazuron on B. odoriphaga were studied to explore the use for integrated pest management (IPM) of B. odoriphaga. A contact and ingestion toxicity bioassay showed that chlorfluazuron was more active against B. odoriphaga than three other CSIs, with a 72h LC50 of 0.1593mg/L. Treatment with the LC50 dose of chlorfluazuron decreased both the intrinsic and finite rates of increase of B. odoriphaga, in addition to reproduction rate, survival rate, and fecundity, and the mean generation time, total preovipositional period and larval development duration were shortened, compared with those of the control and LC10 groups. The mean generation time, total preovipositional period and larval development duration were all also markedly decreased by treatment with chlorfluazuron at the LC10. Furthermore, chlorfluazuron inhibited the feeding of the final instar larvae for a short period. Glutathione S-transferase and microsomal mixed function oxidase activities increased after exposure to the chemical. These results showed that chlorfluazuron at the sublethal LC50 treatment inhibited B. odoriphaga population growth, whereas the danger of causing rapid population growth by using a lower sublethal concentration was demonstrated with the sublethal LC10 treatment. Therefore, chlorfluazuron should be used with caution in an IPM program for B. odoriphaga.

Using Next-Generation Sequencing to Detect Differential Expression Genes in Bradysia odoriphaga after Exposure to Insecticides.

Bradysia odoriphaga (Diptera: Sciaridae) is the most important pest of Chinese chive. Insecticides are used widely and frequently to control B. odoriphaga in China. However, the performance of the insecticides chlorpyrifos and clothianidin in controlling the Chinese chive maggot is quite different. Using next generation sequencing technology, different expression unigenes (DEUs) in B. odoriphaga were detected after treatment with chlorpyrifos and clothianidin for 6 and 48 h in comparison with control. The number of DEUs ranged between 703 and 1161 after insecticide treatment. In these DEUs, 370-863 unigenes can be classified into 41-46 categories of gene ontology (GO), and 354-658 DEUs can be mapped into 987-1623 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expressions of DEUs related to insecticide-metabolism-related genes were analyzed. The cytochrome P450-like unigene group was the largest group in DEUs. Most glutathione S-transferase-like unigenes were down-regulated and most sodium channel-like unigenes were up-regulated after insecticide treatment. Finally, 14 insecticide-metabolism-related unigenes were chosen to confirm the relative expression in each treatment by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The results of qRT-PCR and RNA Sequencing (RNA-Seq) are fairly well-established. Our results demonstrate that a next-generation sequencing tool facilitates the identification of insecticide-metabolism-related genes and the illustration of the insecticide mechanisms of chlorpyrifos and clothianidin.

Effects of the microbial secondary metabolite benzothiazole on the nutritional physiology and enzyme activities of Bradysia odoriphaga (Diptera: Sciaridae).

Bradysia odoriphaga (Diptera: Sciaridae) is the major pest that damages Chinese chive production. As a volatile compound derived from microbial secondary metabolites, benzothiazole has been determined to possess fumigant activity against B. odoriphaga. However, the mechanism of action of benzothiazole is not well understood. In the present study, fourth-instar larvae of B. odoriphaga were exposed to LC10 and LC30 of benzothiazole. Sublethal concentrations (LC10 and LC30) of benzothiazole significantly reduced the food consumption of the larvae on the second day after treatment (2 DAT). However, there were no significant changes in pupal weight among the different treatments. We also measured the protein, lipid, carbohydrate, and trehalose contents and the digestive enzyme activities of the larvae, and the results suggest that benzothiazole reduced the nutrient accumulation and decreased the digestive enzyme activities of B. odoriphaga. In addition, the activity of glutathione S-transferase was significantly decreased at 6h after treatment with benzothiazole, whereas general esterase activities were significantly increased at 6 and 24h after treatment. The results of this study indicate that benzothiazole interferes in the normal food consumption and digestion process by decreasing the activities of digestive enzymes. These results provide valuable information for understanding the toxicity of benzothiazole and for exploring volatile compound for the control of this pest.

Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae).

The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga.

Effects of Temperature on the Age-Stage, Two-Sex Life Table of Bradysia odoriphaga (Diptera: Sciaridae).

The demographics of Bradysia odoriphaga were examined with an age-stage, two-sex life table that was developed at 15, 20, 25, and 30°C under controlled conditions. The intrinsic rate of increase and mean generation time were 0.0574 and 48.08 d at 15°C, 0.1175 and 30.09 d at 20°C, 0.1369 and 26.31 d at 25°C, and 0.1247 and 26.55 d at 30°C, respectively. The gross reproductive rate (GRR), the net reproductive rate (R0), and the pre-adult survivorship (la) were consistent with the relationship R0

Biological Activity of trans-2-Hexenal Against Bradysia odoriphaga (Diptera: Sciaridae) at Different Developmental Stages.

trans-2-Hexenal, one of the C6 green leaf volatiles, is potentially useful for the control of Bradysia odoriphaga Yang et Zhang. In this study, the biological activity of trans-2-hexenal on B. odoriphaga was assessed in the laboratory. trans-2-Hexenal was observed to kill B. odoriphaga in different developmental stages at a relatively low concentration under fumigation. The respiration rate in the male treatment group decreased from 131.44 to 4.07 nmol/g · min with a prolonged fumigation time, while the respiration rate in females decreased from 128.82 to 24.20 nmol/g · min. Male adults exhibited a more sensitive electroantennogram response at 0.05-500 µl/ml at the dose of 10.0 µl than female adults. Moreover, trans-2-hexenal had a repellent effect on adults based on the results with a Y-tube olfactometer at 10.0 µl, as shown by the deterrent rate of male and female adults with 96.67% and 98.33%, respectively. The results showed that trans-2-hexenal had good biological activity in different developmental stages of B. odoriphaga, which could reduce the need for, and risks associated with, the use of traditional insecticides and enable nonharmful management.

Life table study of the effects of sublethal concentrations of thiamethoxam on Bradysia odoriphaga Yang and Zhang.

Bradysia odoriphaga Yang and Zhang (chive gnat) is the major insect pest affecting Chinese chive in Northern China. In order to explore the integrated control of B. odoriphaga, sublethal effects of the neonicotinoid insecticide thiamethoxam were studied. The standard contact and stomach bioassay method was used to assess the effects of sublethal (LC5 and LC20) concentrations of thiamethoxam on the demographic parameters of B. odoriphaga, and data were interpreted based on the age-stage, two-sex life table theory. After thiamethoxam treatment, the intrinsic and finite rates of increase, net reproduction rate, survival rate, and reproductive value were all markedly decreased, while the mean generation time, total preovipositional period, and larval and pupal duration were prolonged, compared with controls. The intrinsic rates of increase dropped from 0.1775/day to 0.1502-0.1136/day. Following LC5 and LC20 treatments, net reproduction rate dropped from 61.75 offspring/individual (control) to 43.36 and 20.75 offspring/individual, respectively. Sublethal concentrations of thiamethoxam decreased the developmental rate of laboratory populations of B. odoriphaga, suggesting that such doses may be useful in integrated pest management strategies.

Impacts of climate change and host plant availability on the potential distribution of Bradysia odoriphaga (Diptera: Sciaridae) in China.

BACKGROUND: Chinese chives (Allium tuberosum Rottler ex Sprengel) are favored by consumers because of its delicious taste and unique fragrance. Bradysia odoriphaga (Diptera: Sciaridae) is a main pest that severely harms Chinese chives and other Liliaceae's production. Climate change may change the future distribution of B. odoriphaga in China. In this study, the CLIMEX was employed to project the potential distribution of B. odoriphaga in China, based on China's historical climate data (1987-2016) and forecast climate data (2021-2100). RESULTS: Bradysia odoriphaga distributed mainly between 19.8° N-48.3° N and 74.8° E-134.3° E, accounting for 73.25% of the total mainland area of China under historical climate conditions. Among them, the favorable and highly favorable habitats accounted for 30.64% of the total potential distribution. Under future climate conditions, B. odoriphaga will be distributed mainly between 19.8° N-49.3° N and 73.8° E-134.3° E, accounting for 84.89% of China's total mainland area. Among them, the favorable and highly favorable habitats will account for 35.23% of the total potential distribution, indicating an increase in the degree of fitness. Areas with relatively appropriate temperature and humidity will be more suitable for the survival of B. odoriphaga. Temperature was a more important determinant of the climatic suitability of the pest B. odoriphaga than humidity. Host plants (Liliaceae) availability also had impact on climate suitability in some regions. CONCLUSIONS: These projected potential distributions will provide supportive information for monitoring and early forecasting of pest outbreaks, and to reduce future economic and ecological losses. © 2024 Society of Chemical Industry.

Functional Characterization of CYP6QE1 and CYP6FV21 in Resistance to λ-Cyhalothrin and Imidacloprid in Bradysia odoriphaga.

Cytochrome P450 monooxygenases (P450s) belong to a family of metabolic enzymes that are involved in the detoxification of insecticides. In this study, our bioassay results showed that a field-collected population of Bradysia odoriphaga displayed a moderate resistance to λ-cyhalothrin and imidacloprid. Compared to susceptible population, CYP6QE1 and CYP6FV21 were significantly overexpressed in the field population. The expression of CYP6QE1 and CYP6FV21 was more abundant in the third and fourth larval stages, and CYP6QE1 and CYP6FV21 were most highly expressed in the midgut and Malpighian tubules. Exposure to λ-cyhalothrin and imidacloprid significantly increased the expression levels of CYP6QE1 and CYP6FV21. Furthermore, the silencing of CYP6QE1 and CYP6FV21 significantly increased the susceptibility of B. odoriphaga larvae to λ-cyhalothrin and imidacloprid. The overexpression of CYP6QE1 and CYP6FV21 significantly enhanced the tolerance of transgenic Drosophila melanogaster lines to λ-cyhalothrin and imidacloprid. In addition, molecular docking revealed that these two P450 proteins have strong binding affinity toward λ-cyhalothrin and imidacloprid insecticides. Taken together, these results indicate that the overexpression of CYP6QE1 and CYP6FV21 is responsible for resistance to λ-cyhalothrin and imidacloprid in B. odoriphaga.

Transcription Factors AhR and ARNT Regulate the Expression of CYP6SX1 and CYP3828A1 Involved in Insecticide Detoxification in Bradysia odoriphaga.

Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to ß-naphthoflavone (BNF) significantly increased the expression of BoAhR, BoARNT, CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR/BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1.