Genetics, genomics and mechanisms responsible for high levels of pyrethroid resistance in Musca domestica.

Insecticide resistance is both economically important and evolutionarily interesting phenomenon. Identification of the mutations responsible for resistance allows for highly sensitive resistance monitoring and allows tools to study the forces (population genetics, fitness costs, etc.) that shape the evolution of resistance. Genes coding for insecticide targets have many well-characterized mutations, but the mutations responsible for enhanced detoxification have proven difficult to identify. We employed multiple strategies to identify the mutations responsible for the extraordinarily high permethrin resistance in the KS17-R strain of house fly (Musca domestica): insecticide synergist assays, linkage analysis, bulk segregant analyses (BSA), transcriptomics and long read DNA (Nanopore) sequencing. The >85,100-fold resistance in KS17-R was partially suppressed by the insecticide synergists piperonyl butoxide and S,S,S-tributylphosphorothionate, but not by diethyl maleate nor by injection. This suggests the involvement of target site insensitivity, CYP-mediated resistance, possibly hydrolase mediated resistance and potentially other unknown factors. Linkage analysis identified chromosomes 1, 2, 3 and 5 as having a role in resistance. BSA mapped resistance loci on chromosomes 3 and 5. The locus on chromosome 3 was centered on the voltage sensitive sodium channel. The locus on chromosome 5 was associated with a duplication of multiple detoxification genes. Transcriptomic analyses and long read DNA sequencing revealed overexpressed CYPs and esterases and identified a complex set of structural variants at the chromosome 5 locus.

New insights into immune genes and other expanded gene families of the house fly, Musca domestica, from an improved whole genome sequence.

The house fly, Musca domestica, is a pest of livestock, transmits pathogens of human diseases, and is a model organism in multiple biological research areas. The first house fly genome assembly was published in 2014 and has been of tremendous use to the community of house fly biologists, but that genome is discontiguous and incomplete by contemporary standards. To improve the house fly reference genome, we sequenced, assembled, and annotated the house fly genome using improved techniques and technologies that were not available at the time of the original genome sequencing project. The new genome assembly is substantially more contiguous and complete than the previous genome. The new genome assembly has a scaffold N50 of 12.46 Mb, which is a 50-fold improvement over the previous assembly. In addition, the new genome assembly is within 1% of the estimated genome size based on flow cytometry, whereas the previous assembly was missing nearly one-third of the predicted genome sequence. The improved genome assembly has much more contiguous scaffolds containing large gene families. To provide an example of the benefit of the new genome, we used it to investigate tandemly arrayed immune gene families. The new contiguous assembly of these loci provides a clearer picture of the regulation of the expression of immune genes, and it leads to new insights into the selection pressures that shape their evolution.

Selection and characterization of spinetoram resistance in field collected Drosophila melanogaster.

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.

Frequencies and distribution of kdr and Ace alleles that cause insecticide resistance in house flies in the United States.

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.

Selection for, and characterization of, fluralaner resistance in the house fly, Musca domestica.

House flies, Musca domestica (L), are the mechanical vector of >100 human and animal pathogens, including those that are antibiotic-resistant. Given that house flies are associated closely with human and livestock activity, they present medical and veterinary health concerns. Although there are numerous strategies for control of house fly populations, chemical control has been favored in many facilities. Products with pyrethroid active ingredients have been used predominantly for >35 years in space sprays. As a result, strong selection for pyrethroid resistance has led to reduced control of many populations. Reliance on a limited number of insecticides for decades has created fly control problems necessitating the discovery and formulation of new control chemistries. Fluralaner is a relatively new insecticide and acaricide (first reported in 2010), belonging to the isoxazoline class. These insecticides target the glutamate- and gamma-aminobutyric acid-gated (GABA) chloride channels, which is a different mode of action from other insecticides used against house flies. Although is it not currently registered for house fly control in the United States, previous work has shown that fluralaner is highly toxic to house flies and that there was limited cross-resistance found in laboratory strains having high levels of resistance to other insecticides. Herein, we characterized the time and age dependency of fluralaner toxicity, detected cross-resistance in populations from across the United States, and selected a highly resistant (>11,000-fold) house fly strain. We found that the fluralaner LD50 of 18-24 h old flies was 2-fold higher than for 5-6 d old flies. This appears to be due to more rapid penetration of fluralaner into the 5-6 d old flies. Fluralaner resistance was inherited as an intermediate to incompletely dominant trait and was mapped to chromosomes 5 and 3. Resistance could be suppressed to 7-fold with piperonyl butoxide, suggesting that cytochrome P450 (CYP)-mediated detoxification was a major mechanism of resistance. Decreased penetration was also demonstrated as a mechanism of resistance. The utility of fluralaner for house fly control is discussed.

Conservation of the voltage-sensitive sodium channel protein within the Insecta.

The voltage-sensitive sodium channel (VSSC) is essential for the generation and propagation of action potentials. VSSC kinetics can be modified by producing different splice variants. The functionality of VSSC depends on features such as the voltage sensors, the selectivity filter and the inactivation loop. Mutations in Vssc conferring resistance to pyrethroid insecticides are known as knockdown resistance (kdr). We analysed the conservation of VSSC in both a broad scope and a narrow scope by three approaches: (1) we compared conservation of sequences and of differential exon use across orders of the Insecta; (2) we determined which kdr mutations were possible with a single nucleotide mutation in nine populations of Aedes aegypti; and (3) we examined the individual VSSC variation that exists within a population of Drosophila melanogaster. There is an increasing amount of transcript diversity possible from Diplura towards Diptera. The residues of the voltage sensors, selectivity filter and inactivation loop are highly conserved. The majority of exon sequences were >88.6% similar. Strain-specific differences in codon constraints exist for kdr mutations in nine strains of A. aegypti. Three Vssc mutations were found in one population of D. melanogaster. This study shows that, overall, Vssc is highly conserved across Insecta and within a population of an insect, but that important differences do exist.

Life and Death at the Voltage-Sensitive Sodium Channel: Evolution in Response to Insecticide Use.

The voltage-sensitive sodium channel (VSSC) is a critical component of the insect nervous system. Pyrethroids and DDT are insecticides that have been widely used, and they kill insects by perturbations of the VSSC. Decades of insecticide use selected for mutations in Vssc that give rise to resistance in almost all pest insects. However, the mutations responsible for the resistance are not always the same, and some unusual patterns have emerged. This review focuses on what pyrethroid/DDT selection has done, in terms of Vssc changes that have occurred, using four well-studied species as examples of the differences that have evolved. Information is provided about the mutations that occur, potential pathways by which alleles with multiple mutations arose, the relative fitness of the alleles, the levels of resistance conferred, and the geographic distribution of the mutations. The lessons learned and exciting new areas of research are discussed.

Drosophila melanogaster as a powerful tool for studying insect toxicology.

Insecticides are valuable and widely used tools for the control of pest insects. Despite the use of synthetic insecticides for >50 years, we continue to have a limited understanding of the genes that influence the key steps of the poisoning process. Major barriers for improving our understanding of insecticide toxicity have included a narrow range of tools and/or a large number of candidate genes that could be involved in the poisoning process. Herein, we discuss the numerous tools and resources available in Drosophila melanogaster that could be brought to bear to improve our understanding of the processes determining insecticide toxicity. These include unbiased approaches such as forward genetic screens, population genetic methods and candidate gene approaches. Examples are provided to showcase how D. melanogaster has been successfully used for insecticide toxicology studies in the past, and ideas for future studies using this valuable insect are discussed.

Insecticide resistance monitoring of house fly populations from the United States.

Insecticide resistance in house fly populations is a major problem faced by livestock producers worldwide. A survey of insecticide resistance levels and pyrethroid resistance allele frequencies in the United States was conducted in 2008-09, but little is known about how resistance levels have changed over the last 10 years. In addition, new target-site pyrethroid resistance alleles that confer high levels of resistance have been recently identified in the voltage-sensitive sodium channel, and their frequencies in field populations are unknown. Our aim in this study was to reassess the resistance status of house flies from select locations in the United States by examining resistance levels against commonly used insecticides and frequencies of known resistance alleles. House flies were collected from animal production facilities in five different states between 2016 and 2018. Resistance levels to three insecticides (permethrin, tetrachlorvinphos, and methomyl), representing three classes of insecticides (pyrethroids, organophosphates and carbamates) varied geographically and were lowest in the population collected from New Mexico, intermediate in the population collected from Utah, and greatest in the population from Kansas. The recently identified 1B pyrethroid resistance allele increased dramatically in frequency compared to previous reports, most notably in populations from Kansas and Maryland, indicating that it may already be widespread around the United States. Based on comparison with historical data, the population collected from Kansas represents one of the most highly permethrin resistant populations ever sampled. If the alleles responsible for this level of resistance spread, pyrethroids may be of limited use for house fly control in the United States in the near future.

CYP-mediated resistance and cross-resistance to pyrethroids and organophosphates in Aedes aegypti in the presence and absence of kdr.

Aedes aegypti thrives in urban environments and transmits several debilitating human viral diseases. Thus, our ability to control this mosquito species in endemic areas is of utmost importance. The use of insecticides, mostly pyrethroids and organophosphates (OPs), has long been the primary means of controlling A. aegypti, but widespread insecticide resistance has emerged. The two main mechanisms of pyrethroid resistance in A. aegypti are CYP-mediated detoxification and mutations in the target site, voltage-sensitive sodium channel (Vssc), referred to as knockdown resistance (kdr). Knowledge about the contributions and interactions of these mechanisms to resistance is important for the understanding of the molecular and evolutionary basis of insecticide resistance, and to determine the effectiveness of insecticides. In this study, we address two aims: 1) determine the patterns of CYP-mediated cross-resistance to pyrethroid and OP insecticides, both in the presence and absence of kdr (S989P + V1016G), and 2) determine whether the interaction between the two mechanisms yields a greater than, less than, or additive effect on resistance. We tested seven pyrethroids and four OPs against three congenic strains of A. aegypti: ROCK (susceptible), CYP:ROCK (CR) (resistant due to CYP-mediated detoxification without kdr), and CYP + KDR:ROCK (CKR) (resistant due to both CYPs and kdr), and compared these to the congenic KDR:ROCK strain that was previously reported. We found that resistance ratios (RRs) were variable between pyrethroids and strains, ranging from 6.2- to 42-fold for CR, and 70- to 261-fold for CKR. In general, we found that CYP-mediated resistance alone contributes less to resistance than kdr. The effect of the combined mechanisms on resistance was significantly greater than additive for all pyrethroids except (1R)-trans-fenfluthrin. CYP-mediated pyrethroid resistance conferred cross-resistance to both methyl paraoxon and fenitrothion, and negative cross-resistance to methyl parathion and naled. Based on our results, we recommend that etofenprox and cyfluthrin be avoided for A. aegypti control in areas where these two resistance mechanisms are prevalent.

Using genomic data to study insecticide resistance in the house fly, Musca domestica.

The house fly, Musca domestica, is a major pest at livestock facilities throughout the world. Insecticides have been the most common control strategy for flies, but many populations have evolved resistance. The speed by which we are able to identify the mutations responsible for resistance has been a major challenge for the development of high throughput resistance monitoring assays as new insecticides are introduced for control. This is particularly true for mutations that cause trans regulation of a gene, which then results in resistance. In this paper we take advantage of the conserved homology of dipteran chromosomes to assign 3069 genes to chromosomes. Of these, 234 were of toxicological interest (CYPs, esterases/hydrolases, glutathione S-transferases (GSTs) and target sites). The chromosomal location of genes known from linkage analysis studies matched the location predicted by homology mapping in ten out of ten cases, indicating a high reliability of our approach. The CYPs, esterases/hydrolases and GSTs were not randomly distributed throughout the genome. They clustered on chromosomes, but the pattern was different between the CYPs, esterases/hydrolases and GSTs. Examples are provided for how the availability of the house fly genome, combined with an ability to assign genes to chromosomes, will help to accelerate research in house flies.

Two novel house fly Vssc mutations, D600N and T929I, give rise to new insecticide resistance alleles.

The house fly, Musca domestica, is a serious pest because it transmits a large diversity of human and veterinary diseases. Insecticides, particularly pyrethroids, are commonly used to control house flies. However, the evolution of pyrethroid resistance has reduced the effectiveness of these insecticides. A major mechanism of resistance to pyrethroids is target site insensitivity caused by the mutations in the voltage-sensitive sodium channel (Vssc) gene (e.g. kdr [L1014F] and super-kdr [M918T+L1014F]). Recently, two novel Vssc alleles, super-kdr+D600N and kdr+T929I were detected in a field collected resistant house fly population in Kansas, USA in 2013. To determine the levels of resistance that these new alleles confer to pyrethroids, we isolated strains having the unique Vssc alleles, but being otherwise congenic to the susceptible strain, aabys. We compared levels of resistance conferred to 14 pyrethroids and determined the inheritance of resistance to 8 pyrethroids. Our results revealed that super-kdr+D600N conferred higher levels of resistance to seven pyrethroids relative to super-kdr, and kdr+T929I showed super-kdr-like levels of resistance in house flies. Our results are compared with previous studies and reveal that addition of T929I to the kdr mutation (L1014F) increased resistance to all pyrethroids (except etofenprox), and enhanced resistance by ~1000-fold to acrinathrin and flumethrin. The implications of these results on the evolution of resistance are discussed.

Is Multifactorial Sex Determination in the House Fly, Musca domestica (L.), Stable Over Time?

Sex determination pathways evolve rapidly, usually because of turnover of master regulatory genes at the top of the developmental pathway. Polygenic sex determination is expected to be a transient state between ancestral and derived conditions. However, polygenic sex determination has been observed in numerous animal species, including the house fly, Musca domestica House fly males carry a male-determining factor (M) that can be located on any chromosome, and an individual male may have multiple M factors. Females lack M and/or have a dominant allele of the Md-tra gene (Md-tra D ) that acts as a female-determining locus even in the presence of multiple copies of M. We found the frequency and linkage of M in house flies collected in Chino, CA (USA) was relatively unchanged between 1982 and 2014. The frequency of females with Md-tra D in the 2014 collection was 33.6% (n = 140). Analysis of these results, plus previously published data, revealed a strong correlation between the frequencies of Md-tra D and multiple M males, and we find that these populations are expected to have balanced sex ratios. We also find that fitness values that allow for the invasion and maintenance of multiple sex determining loci suggest that sexually antagonistic selection could be responsible for maintaining polygenic sex determination in house fly populations. The stability over time and equilibrium frequencies within populations suggest the house fly polygenic sex determination system is not in transition, and provide guidance for future investigations on the factors responsible for the polymorphism.

Pyrethroid resistance in Aedes aegypti and Aedes albopictus: Important mosquito vectors of human diseases.

Aedes aegypti and A. albopictus mosquitoes are vectors of important human disease viruses, including dengue, yellow fever, chikungunya and Zika. Pyrethroid insecticides are widely used to control adult Aedes mosquitoes, especially during disease outbreaks. Herein, we review the status of pyrethroid resistance in A. aegypti and A. albopictus, mechanisms of resistance, fitness costs associated with resistance alleles and provide suggestions for future research. The widespread use of pyrethroids has given rise to many populations with varying levels of resistance worldwide, albeit with substantial geographical variation. In adult A. aegypti and A. albopictus, resistance levels are generally lower in Asia, Africa and the USA, and higher in Latin America, although there are exceptions. Susceptible populations still exist in several areas of the world, particularly in Asia and South America. Resistance to pyrethroids in larvae is also geographically widespread. The two major mechanisms of pyrethroid resistance are increased detoxification due to P450-monooxygenases, and mutations in the voltage sensitive sodium channel (Vssc) gene. Several P450s have been putatively associated with insecticide resistance, but the specific P450s involved are not fully elucidated. Pyrethroid resistance can be due to single mutations or combinations of mutations in Vssc. The presence of multiple Vssc mutations can lead to extremely high levels of resistance. Suggestions for future research needs are presented.

Pyrethroid resistance in Culex pipiens mosquitoes.

Mosquitoes within the Culex pipiens complex are widely distributed and important in the transmission of many human diseases. Insecticides, pyrethroids in particular, remain a mainstay for control of these important vectors. In this paper we review what is known about the levels, mechanisms and fitness costs of pyrethroid resistance in Cx. pipiens. Pyrethroid resistance in Cx. pipiens is a global problem, and resistance ratios of up to 7000-fold have been found in larvae of field collected mosquitoes. However, there is considerable variation between populations, indicating significant geographic heterogeneity of the resistance. The two major mechanisms of resistance to pyrethroids in Culex are mutations in Vssc (target site insensitivity) and overexpression of cytochrome P450(s) (increased detoxification). The most frequently reported Vssc mutation is L1014F (i.e. kdr), which has been found throughout the world. The L1014S mutation has been found in Cx. p. pallens from Japan and China, and in Cx. p. pipiens from China. The L1014C mutation has only been reported for Cx. p. pipens molestus from China and the V1016G mutation has only been reported from Saudi Arabia. Studies on the P450s of Cx. pipiens have identified several that are overexpressed (measured as transcript levels) in pyrethroid resistant strains. CYP9M10 is consistently overexpressed in pyrethroid resistant Cx. pipiens from at least seven countries, suggesting this P450 might be of global importance in resistance. Both CYP9M10-mediated pyrethroid resistance and kdr have fitness costs in the absence of insecticides under certain environmental conditions. Research needs and future directions are discussed.

Genetics and mechanisms of imidacloprid resistance in house flies.

Imidacloprid is the most widely used neonicotinoid insecticide against house flies, which are major pests at animal production facilities worldwide. However, cases of both physiological and behavior resistance have been reported. Recently, physiological resistance to imidacloprid was found in the United States (California and Florida). However, no studies have been undertaken to characterize this resistance in house flies from the United States. Three imidacloprid selections of a strain originally collected from Florida increased the level of resistance, ultimately resulting in a strain that had 2300-fold resistance in females and 130-fold in males. Imidacloprid resistance was not overcome with piperonyl butoxide (PBO) suggesting that resistance is not due to detoxification by cytochrome P450s. Resistance was mapped to autosomes 3 and 4. There was⩾100-fold cross-resistance to acetamiprid and dinotefuran, but no cross-resistance to spinosad. The resistance in this imidacloprid selected population was unstable and declined over a period of several months. The significance of these results to management of imidacloprid resistance in the field, and potential mechanisms of resistance involved, are discussed.

The α6 nicotinic acetylcholine receptor subunit of Frankliniella occidentalis is not involved in resistance to spinosad.

Insects evolve resistance which constrains the sustainable use of insecticides. Spinosyns, a class of environmentally-friendly macrolide insecticides, is not an exception. The mode of inheritance and the mechanisms of resistance to spinosad (the most common spinosyn insecticide) in Frankliniella occidentalis (Western flower thrips, WFT) were investigated in this study. Resistance (170,000-fold) was autosomal and completely recessive. Recent studies showed that deletion of the nicotinic acetylcholine receptor α6 subunit gene resulted in strains of Drosophila melanogaster, Plutella xylostella and Bactrocera dorsalis that are resistant to spinosad, indicating that nAChRα6 subunit maybe important for the toxic action of this insecticide. Conversely, a G275E mutation of this subunit in F. occidentalis was recently proposed as the mechanism of resistance to spinosad. We cloned and characterized nAChRα6 from three susceptible and two spinosad resistant strains from China and the USA. The Foα6 cDNA is 1873bp and the open reading frame is 1458bp which encodes 485 amino acid residues with a predicted molecular weight of 53.5-kDa, the 5' and 3' UTRs are 121 and 294bp, respectively. There was no difference in the cDNA sequence between the resistant and susceptible thrips, suggesting the G275E mutation does not confer resistance in these populations. Ten isoforms of Foα6, arising from alternative splicing, were isolated and did not differ between the spinosad-susceptible and resistant strains. Quantitative real time PCR analysis showed Foα6 was highly expressed in the first instar larva, pupa and adult, and the expression levels were 3.67, 2.47, 1.38 times that of the second instar larva. The expression level was not significantly different between the susceptible and resistant strains. These results indicate that Foα6 is not involved in resistance to spinosad in F. occidentalis from China and the USA.

Insecticide resistance in house flies from the United States: resistance levels and frequency of pyrethroid resistance alleles.

Although insecticide resistance is a widespread problem for most insect pests, frequently the assessment of resistance occurs over a limited geographic range. Herein, we report the first widespread survey of insecticide resistance in the USA ever undertaken for the house fly, Musca domestica, a major pest in animal production facilities. The levels of resistance to six different insecticides were determined (using discriminating concentration bioassays) in 10 collections of house flies from dairies in nine different states. In addition, the frequencies of Vssc and CYP6D1 alleles that confer resistance to pyrethroid insecticides were determined for each fly population. Levels of resistance to the six insecticides varied among states and insecticides. Resistance to permethrin was highest overall and most consistent across the states. Resistance to methomyl was relatively consistent, with 65-91% survival in nine of the ten collections. In contrast, resistance to cyfluthrin and pyrethrins + piperonyl butoxide varied considerably (2.9-76% survival). Resistance to imidacloprid was overall modest and showed no signs of increasing relative to collections made in 2004, despite increasing use of this insecticide. The frequency of Vssc alleles that confer pyrethroid resistance was variable between locations. The highest frequencies of kdr, kdr-his and super-kdr were found in Minnesota, North Carolina and Kansas, respectively. In contrast, the New Mexico population had the highest frequency (0.67) of the susceptible allele. The implications of these results to resistance management and to the understanding of the evolution of insecticide resistance are discussed.

A Globally Distributed Insecticide Resistance Allele Confers a Fitness Cost in the Absence of Insecticide in Aedes aegypti (Diptera: Culicidae), the Yellow Fever Mosquito.

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.

Selection for, and characterization of, malathion and zeta-cypermethrin resistance in vineyard-collected Drosophila melanogaster.

BACKGROUND: Drosophila melanogaster is a pest in vineyards because of its role in sour rot disease. Insecticides are commonly used, particularly late in the season, to control D. melanogaster and thus sour rot. Use of insecticides in vineyards and neighboring fruit production systems has led to the evolution of insecticide resistance in D. melanogaster, which is now widespread to commonly used insecticides like zeta-cypermethrin and malathion. Implementation of resistance management strategies is facilitated by an understanding of the mechanisms and genetics underlying the resistance. RESULTS: Starting with a vineyard-collected strain of D. melanogaster (NY18), we selected for a strain that was 1100-fold resistant to zeta-cypermethrin and one that was 40-fold resistant to malathion. Resistance was inherited as an incompletely dominant trait for zeta-cypermethrin. Resistance to malathion was inherited differently between reciprocal crosses. Insecticide bioassays using insecticide synergists found resistance to zeta-cypermethrin was partly suppressible with either piperonyl butoxide or S,S,S-tributylphosphorotrithionate, while resistance to malathion was unchanged by the synergists and mutations in Ace associated with the resistance were found. CONCLUSIONS: Resistance to zeta-cypermethrin is most likely due to enhanced detoxification, while the results with malathion were associated with two Ace alleles. How the newly selected strains can facilitate diagnostic tools for the identification of the mutations causing the resistance is discussed. © 2022 Society of Chemical Industry.