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.

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.

All resistance alleles are not equal: the high fitness cost of super-kdr in the absence of insecticide.

BACKGROUND: Mutations in the voltage-sensitive sodium channel are an important mechanism of resistance to pyrethroid insecticides. In Musca domestica, common resistance alleles are kdr, super-kdr and kdr-his. The levels of resistance that these alleles confer is known, but the fitness of these alleles relative to each other and to susceptible alleles is unknown. We used crosses from congenic strains of M. domestica to establish populations with known allele frequencies and then examined the changes in allele and genotype frequencies over 25 generations under laboratory conditions. RESULTS: There was a significant fitness cost for the super-kdr allele, which decreased from the starting frequency of 0.25 to 0.05 after 25 generations. The fitness of the kdr, kdr-his and susceptible alleles were similar. The greatest change in genotype frequency was seen for the super-kdr/super-kdr genotype, which was no longer detected after 25 generations. CONCLUSION: The fitness cost associated with the super-kdr allele is consistent with previous reports and appears to be a factor in helping to restrain high levels of resistance in field populations (the super-kdr allele confers higher levels of resistance than kdr or kdr-his). It is known that the relative costs of different alleles are environmentally dependent, but our results also demonstrate that the relative fitness of given alleles depends on which alleles are present in a given population, as previous pairwise comparisons of allele fitness do not exactly match (except for super-kdr) the results obtained using this four allele study. © 2020 Society of Chemical Industry.

Fitness studies of insecticide resistant strains: lessons learned and future directions.

The evolution of insecticide resistance is generally thought to be associated with a fitness cost in the absence of insecticide exposure. However, it is not clear how these fitness costs manifest or how universal this phenomenon is. To investigate this, we conducted a literature review of publications that studied fitness costs of insecticide resistance, selected papers that met our criteria for scientific rigor, and analyzed each class of insecticides separately as well as in aggregate. The more than 170 publications on fitness costs of insecticide resistance show that in 60% of the experiments there is a cost to having resistance, particularly for measurements of reversion of resistance and reproduction. There were differences between classes of insecticides, with fitness costs seen less commonly for organochlorines. There was considerable variation in the experiments performed. We suggest that future papers will have maximum value to the community if they quantitatively determine resistance levels, identify the resistance mechanisms present (and the associated mutations), have replicated experiments, use related strains (optimally congenic with the resistance mutation introgressed into different genetic backgrounds) and measure fitness by multiple metrics. Studies on the fitness costs of insecticide resistance will continue to enlighten our understanding of the evolutionary process and provide valuable information for resistance management. © 2021 Society of Chemical Industry.