Identification of a novel cytochrome P450 CYP3356A1 linked with insecticide detoxification in Bradysia odoriphaga.

BACKGROUND: Cytochrome P450 monooxygenases play an important role in the metabolic detoxification of insecticides in insect pests. However, little is known about the role of a specific P450 gene and its responses to insecticide exposure in Bradysia odoriphaga, a major pest in Chinese chive production. RESULTS: In this study, a novel P450 gene, CYP3356A1, was cloned from Bradysia odoriphaga. The full-length cDNA sequence of CYP3356A1 is 2153 bp and its open reading frame (ORF) encodes 508 amino acids. Quantitative real time PCR(qRT-PCR) analyses in different tissues showed that CYP3356A1 expression was the highest in the Malpighian tubule. Moreover, among the different developmental stages of the insect, the highest expression of CYP3356A1 was found in fourth-instar larvae. Expression of CYP3356A1 was upregulated by treatment with imidacloprid, thiamethoxam, and ß-cypermethrin at median lethal concentrations (LC50 ). RNA interference (RNAi)-mediated silencing of CYP3356A1 significantly increased mortality by 36.90%, 25.17%, and 36.73 when fourth-instar B. odoriphaga larvae were exposed to imidacloprid, thiamethoxam, and ß-cypermethrin, respectively, at the LC50 dose. CONCLUSION: These results demonstrate that CYP3356A1 is related to the detoxification of imidacloprid, thiamethoxam, and ß-cypermethrin in B. odoriphaga. Moreover, the study also increased our understanding of the molecular mechanisms of insecticide detoxification in this pest insect. © 2018 Society of Chemical Industry.

Endogenous insensitivity to the Orco agonist VUAA1 reveals novel olfactory receptor complex properties in the specialist fly Mayetiola destructor.

Insect olfactory receptors are routinely expressed in heterologous systems for functional characterisation. It was recently discovered that the essential olfactory receptor co-receptor (Orco) of the Hessian fly, Mayetiola destructor (Mdes), does not respond to the agonist VUAA1, which activates Orco in all other insects analysed to date. Here, using a mutagenesis-based approach we identified three residues in MdesOrco, located in different transmembrane helices as supported by 3D modelling, that confer sensitivity to VUAA1. Reciprocal mutations in Drosophila melanogaster (Dmel) and the noctuid moth Agrotis segetum (Aseg) Orcos diminish sensitivity of these proteins to VUAA1. Additionally, mutating these residues in DmelOrco and AsegOrco compromised odourant receptor (OR) dependent ligand-induced Orco activation. In contrast, both wild-type and VUAA1-sensitive MdesOrco were capable of forming functional receptor complexes when coupled to ORs from all three species, suggesting unique complex properties in M. destructor, and that not all olfactory receptor complexes are "created" equal.

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.

Resistance of Garlic Cultivars to Bradysia odoriphaga and Its Correlation with Garlic Thiosulfinates.

Garlic, a widely cultivated global vegetable crop, is threatened by the underground pest Bradysia odoriphaga in China. Previous reports indicated that garlic essential oil, of which the dominant components are sulfides or thiosulfinates, exhibits insecticidal activity against pests. However, it is unclear whether the resistance of garlic to B. odoriphaga is related to thiosulfinates. Here, we compared the resistance of 10 garlic cultivars at various growth stages to B. odoriphaga by field investigation and indoor life-table data collection. Furthermore, the relationship between thiosulfinates content and resistance, as well as the toxicity of garlic oil and allicin against B. odoriphaga larvae was determined. Field surveys demonstrated that the garlic cultivars Qixian and Cangshan possessed the highest resistance, while Siliuban and Yishui were the most sensitive. When reared on Qixian, B. odoriphaga larval survival and fecundity declined by 26.2% and 17.7% respectively, but the development time was prolonged by 2.8 d compared with Siliuban. A positive correlation was detected between thiosulfinates content and resistance. Furthermore, garlic oil and allicin exhibited strong insecticidal activity. We screened out 2 pest-resistant cultivars, for which thiosulfinate content was highest. Additionally, the insecticidal activity displayed by sulfides and allcin suggests their potential for exploitation as botanical insecticides.