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BACKGROUND: Spinosyns are a group of naturally occurring and semi-synthetic insecticides with widespread utility in agriculture, including organic production systems. One example is spinetoram (Delegate), which is the only registered insecticide in New York State (for control of Drosophila melanogaster in vineyards) to which vinegar flies have not yet evolved high levels of resistance. However, low levels of resistance have been found in vineyard populations of D. melanogaster, and a highly resistant strain was obtained after only five selections (in the laboratory). We identified the nAChR α6 mutation (G275A) responsible for the resistance and developed a rapid, high-throughput assay for resistance. RESULTS: Surveys of collections made in 2023 show low levels of the resistance allele in four populations. A correlation was observed between vineyard use of spinetoram and frequency of the resistance allele, but not between county-wide use of spinosyns and frequency of the resistance allele. CONCLUSIONS: One of the sites we monitored was previously surveyed in 2019 and the frequency of the resistance allele detected in 2023 had increased. Implications of these findings to resistance management of D. melanogaster are discussed. © 2024 Society of Chemical Industry.

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Aedes aegypti is the vector of viruses such as chikungunya, dengue, yellow fever and Zika that have a critical impact on human health. Control of adult mosquitoes is widely done using pyrethroids, but resistance has reduced the effectiveness of this class of insecticides. Resistance to pyrethroids in mosquitoes is commonly due to mutations in the voltage-gated sodium channel (Vgsc) gene (these mutations are known as knockdown resistance, kdr). In the Americas and the Caribbean, the most common kdr alleles are 410L+1016I+1534C and 1534C. In this study, we conducted a population cage experiment to evaluate changes in the allele and genotype frequencies of the 410L+1016I+1534C allele by crossing two congenic strains; one carrying the 410L+1016I+1534C and another with the 1534C allele. Changes in allele frequencies were measured over 10 generations in the absence of insecticide exposure. We also applied one cycle of selection with deltamethrin at F9 to evaluate the changes in allele and genotype frequencies. Our findings indicate that fitness costs were higher with the 410L+1016I+1534C allele, relative to the 1534C allele, in the absence of deltamethrin exposure, but that the 410L+1016I+1534C allele provides a stronger advantage when exposed to deltamethrin relative to the 1534C allele. Changes in genotype frequencies were not in Hardy-Weinberg equilibrium and could not be explained by drift. Our results suggest the diametrically opposed fitness costs in the presence and absence of insecticides is a reason for the variations in frequencies between the 410L+1016I+1534C and 1534C alleles in field populations.

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House flies (Musca domestica L) are nuisances and vectors of pathogens between and among humans and livestock. Population suppression has been accomplished for decades with pyrethroids and acetylcholinesterase (AChE) inhibitors, but recurrent selection has led to increased frequency of alleles conferring resistance to those two classes of active ingredients (Geden et al., 2021). A common mechanism of resistance to both classes involves an altered target site (mutations in Voltage gated sodium channel (Vgsc) for pyrethroids or in Ace for AChE inhibitors). As part of ongoing efforts to understand the origin, spread and evolution of insecticide resistance alleles in house fly populations, we sampled flies in 11 different US states, sequenced, and then estimated frequencies of the Vgsc and Ace alleles. There was substantial variation in frequencies of the four common knockdown resistance alleles (kdr (L1014F), kdr-his (L1014H), super-kdr (M918T + L10414F) and 1B (T929I + L1014F) across the sampled states. The kdr allele was found in all 11 states and was the most common allele in four of them. The super-kdr allele was detected in only six collections, with the highest frequencies found in the north, northeast and central United States. The kdr-his allele was the most common allele in PA, NC, TN and TX. In addition, a novel super-kdr-like mutation in mutually exclusive exon 17a was found. The overall frequencies of the different Ace alleles, which we name based on the amino acid present at the mutation sites (V260L, A316S, G342A/V and F407Y), varied considerably between states. Five Ace alleles were identified: VAGF, VAVY, VAGY, VAAY and VSAY. Generally, the VSAY allele was the most common in the populations sampled. The susceptible allele (VAGF) was found in all populations, ranging in frequency from 3% (KS) to 41% (GA). Comparisons of these resistance allele frequencies with those previously found suggests a dynamic interaction between the different alleles, in terms of levels of resistance they confer and likely fitness costs they impose in the absence of insecticides.

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Insecticides are commonly employed in vineyards to control vinegar flies and limit sour rot disease. Widespread resistance to available insecticides is having a negative impact on managing Drosophila melanogaster populations, rendering control of sour rot more difficult. An insecticide registered for use in vineyards to which resistance is not yet widespread (at least in New York and Missouri) is spinetoram. Spinetoram targets the nicotinic acetylcholine receptor α6, and mutations in α6 have been associated with resistance in some insects. Our goals were to select for a spinetoram resistant strain of D. melanogaster (starting with field collected populations), characterize the resistance, and identify the mutation responsible. After five selections a strain (SpinR) with >190-fold resistance was obtained. Resistance could not be overcome by insecticide synergists, suggesting an altered target site was involved. We cloned and sequenced the α6 allele from the spinetoram resistant strain and identified a mutation causing a glycine to alanine change at amino acid 301 (equivalent position to the G275E mutation found in some spinosad/spinetoram resistant insects). This mutation was found at low levels in field populations, but increased with each selection until it became homozygous in SpinR. We discuss how the identification of the spinetoram resistance mutation can be used for resistance management.

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The cosmopolitan mosquito Aedes aegypti is a vector of harmful arboviruses. Pyrethroid insecticides are used to reduce adult populations and prevent the spread of disease. Pyrethroids target the insect voltage-gated sodium channel (VGSC). Collectively, mutations in Vgsc that confer resistance are referred to as knock-down resistance or kdr. There are numerous kdr mutations found in A. aegypti Vgsc, and there is co-occurrence of some mutations. Full-length cDNA sequences have identified nine known kdr (e.g., 1534C) alleles. The 1534C allele is among the most common kdr alleles, but allele frequencies between populations vary considerably. We used the 1534C:RK strain, which has the 1534C (kdr) allele in the genetic background of the insecticide susceptible Rockefeller (ROCK) strain, and conducted population cage experiments to assess the potential intrinsic fitness cost of the 1534C allele relative to the susceptible allele (F1534) in the ROCK strain. Individuals were genotyped across generations using allele specific PCR. A fitness cost of the 1534C allele was detected across seven generations of mosquitos reared in the absence of insecticide selection pressure. The decrease in allele frequency was not due to drift. Comparison of our results to previous studies suggests that the magnitude of the fitness cost of kdr alleles in the absence of insecticide is disconnected from the level of resistance they confer, and that the fitness costs of different kdr alleles can be variable.

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