RESUMEN
The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith; Lepidoptera: Noctuidae) is an invasive agricultural pest with a global distribution, causing major crop losses annually. Its control strategies largely rely on chemical insecticides and transgenic crops expressing Bacillus thuringiensis insecticidal proteins (Cry and Vip toxins); however, the development of high resistance poses a significant issue. The ATP-binding cassette transporter C2 (ABCC2) has been linked to Cry toxin pore formation, acting as a receptor of some Cry toxins. Recently detected mutations in the SfABCC2 gene in extracellular loop 4 (ECL4) have been associated with Bt toxin resistance in FAW. In the present study, we expressed the SfABCC2 gene in Drosophila melanogaster, a species normally unaffected by the Bt toxins. We demonstrate that susceptibility can be introduced by the ectopic and tissue-specific expression of wildtype SfABCC2. Next, we introduced mutations into ECL4-both individually and in combination-that have been recently described in Brazilian FAW and functionally validated by toxicity bioassays against the foliar Bt product Xentari. Our results provide an efficient demonstration of the suitability of transgenic Drosophila for validating FAW ABCC2 resistance mutations in ECL4 against Bt toxins, and potential cross-resistance issues between closely related proteins that use ABCC2.
Asunto(s)
Bacillus thuringiensis , Insecticidas , Animales , Spodoptera/fisiología , Toxinas de Bacillus thuringiensis/metabolismo , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Insecticidas/farmacología , Bacillus thuringiensis/genética , Animales Modificados Genéticamente , Endotoxinas/genética , Endotoxinas/farmacología , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacología , Proteínas Hemolisinas/metabolismo , Mutación , Resistencia a los Insecticidas/genética , Plantas Modificadas Genéticamente/metabolismo , Larva/genéticaRESUMEN
BACKGROUND: High-levels of etoxazole resistance have not yet been frequently reported in Panonychus citri. Although a highly resistant strain was discovered in 2014, etoxazole resistance has not become a significant problem in areas of citrus production in Japan. A target site mutation in chitin synthase 1 (CHS1), I1017F, is a major etoxazole-resistance factor in Tetranychus urticae. To investigate the mechanisms of etoxazole resistance and the dispersal of resistance genes, we analyzed target-site mutations in a highly resistant strain and their geographical distribution in Japan. RESULTS: High-level etoxazole resistance was completely recessive. The I1017F mutation was detected in CHS1 of the highly resistant strain, and its frequency was correlated with the hatchability of eggs treated with etoxazole. Sequencing and variant frequency analyses of local populations by quantitative polymerase chain reaction revealed that I1017F is restricted to the Ariake Sea area of Kyushu Island. Although a new nonsynonymous substitution, S1016L, accompanied by I1017F was found in CHS1 of the highly resistant strain, CRISPR/Cas9 engineering of flies showed that S1016L had no effect on the etoxazole resistance conferred by I1017F. CONCLUSION: I1017F is a major target site mutation that confers high-level etoxazole resistance on P. citri. Dispersion of I1017F possibly was suppressed as a result of the completely recessive inheritance of resistance together with low gene flow between local populations. © 2022 Society of Chemical Industry.
Asunto(s)
Acaricidas , Citrus , Tetranychidae , Acaricidas/farmacología , Animales , Quitina Sintasa/genética , Japón , Mutación , Oxazoles , Tetranychidae/genéticaRESUMEN
The putative synergistic action of target-site mutations and enhanced detoxification in pyrethroid resistance in insects has been hypothesized as a major evolutionary mechanism responsible for dramatic consequences in malaria incidence and crop production. Combining genetic transformation and CRISPR/Cas9 genome modification, we generated transgenic Drosophila lines expressing pyrethroid metabolizing P450 enzymes in a genetic background along with engineered mutations in the voltage-gated sodium channel (para) known to confer target-site resistance. Genotypes expressing the yellow fever mosquito Aedes aegypti Cyp9J28 while also bearing the paraV1016G mutation displayed substantially greater resistance ratio (RR) against deltamethrin than the product of each individual mechanism (RRcombined: 19.85 > RRCyp9J28: 1.77 × RRV1016G: 3.00). Genotypes expressing Brassicogethes aeneus pollen beetle Cyp6BQ23 and also bearing the paraL1014F (kdr) mutation, displayed an almost multiplicative RR (RRcombined: 75.19 ≥ RRCyp6BQ23: 5.74 × RRL1014F: 12.74). Reduced pyrethroid affinity at the target site, delaying saturation while simultaneously extending the duration of P450-driven detoxification, is proposed as a possible underlying mechanism. Combinations of target site and P450 resistance loci might be unfavourable in field populations in the absence of insecticide selection, as they exert some fitness disadvantage in development time and fecundity. These are major considerations from the insecticide resistance management viewpoint in both public health and agriculture.
Asunto(s)
Resistencia a los Insecticidas , Insecticidas/química , Aedes , Animales , Escarabajos , Sistema Enzimático del Citocromo P-450/genética , Mosquitos Vectores , PiretrinasRESUMEN
The citrus red mite, Panonychus citri, is a major pest on citrus all around the world. Mitochondrial Electron Transport Inhibitors of complex I (METI-I) acaricides such as fenpyroximate have been used extensively to control P. citri populations, which resulted in multiple reports of METI-I resistant populations in the field. In this study, biochemical and molecular mechanisms of fenpyroximate resistance were investigated in P. citri. Seven populations were collected from Northern provinces of Iran. Resistance ratios were determined and reached up to 75-fold in comparison to a fenpyroximate susceptible population. Cross-resistance to two additional METI-I acaricides, pyridaben and tebufenpyrad, was detected. PBO synergism experiments, in vivo enzyme assays and gene expression analysis suggest a minor involvement of cytochrome P450 monooxygenases in fenpyroximate resistance, which is in contrast with many reported cases for the closely related Tetranychus urticae. Next, we determined the frequency of a well-known mutation in the target-site of METI-Is, the PSST subunit, associated with METI-I resistance. Indeed, the H92R substitution was detected in a highly fenpyroximate resistant P. citri population. Additionally, a new amino acid substitution at a conserved site in the PSST subunit was detected, A94V, with higher allele frequencies in a moderately resistant population. Marker-assisted back-crossing in a susceptible background confirmed the potential involvement of the newly discovered A94V mutation in fenpyroximate resistance. However, introduction of the A94V mutation in the PSST homologue of D. melanogaster using CRISPR-Cas9 did not result in fenpyroximate resistant flies. In addition, differences in binding curves between METI-Is and complex I measured directly, in isolated transgenic and wildtype mitochondria preparations, could not be found.
Asunto(s)
Acaricidas , Citrus , Tetranychidae , Animales , Drosophila melanogaster , IránRESUMEN
The acaricidal compounds pyridaben, tebufenpyrad and fenpyroximate are frequently used in the control of phytophagous mites such as Tetranychus urticae, and are referred to as Mitochondrial Electron Transport Inhibitors, acting at the quinone binding pocket of complex I (METI-I acaricides). Because of their very frequent use, resistance evolved fast more than 20 years ago, and is currently wide-spread. Increased activity of P450 monooxygenases has been often associated with resistance, but target-site based resistance mechanisms were never reported. Here, we report the discovery of a mutation (H92R) in the PSST homologue of complex I in METI-I resistant T. urticae strains. The position of the mutation was studied using the high-resolution crystal structure of Thermus thermophilus, and was located in a stretch of amino acids previously photo-affinity labeled by fenpyroximate. Selection experiments with a strain segregating for the mutant allele, together with marker-assisted back-crossing of the mutation in a susceptible background, confirmed the involvement of the mutation in METI-I resistance. Additionally, an independent genetic mapping approach; QTL analysis identified the genomic region of pyridaben resistance, which included the PSST gene. Last, we used CRISPR-Cas9 genome editing tools to introduce the mutation in the Drosophila PSST homologue.
Asunto(s)
Acaricidas , Proteínas de Artrópodos/genética , Resistencia a Medicamentos/genética , Complejo I de Transporte de Electrón/genética , Tetranychidae/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , ARN/genética , ARN/metabolismo , Alineación de Secuencia , Tetranychidae/crecimiento & desarrolloRESUMEN
Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin-an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro.