Decade Long Upsurge In Target Site Pyrethroid Resistance In Bed Bug Populations In The United States

Over the past three decades, the bed bug Cimex lectularius has resurged as a prominent indoor pest on a global scale. Knockdown-associated insecticide resistance (kdr) involving the voltage-gated sodium channel, targeted by organochlorine and pyrethroid insecticides, was rst reported in C. lectularius within a few years of the widespread use of Dichlorodiphenyltrichloroethane (DDT) and has been implicated as a signicant factor contributing to the species recent resurgence. Since then, selection with pyrethroid insecticides has intensied, yet little is known regarding its short-term impacts on the frequency of kdr-associated mutations. Here, we report temporal changes in the frequencies of three kdr-associated mutations in C. lectularius populations collected across the United States from two time periods, sampled approximately a decade apart. Results reveal a signicant increase in the frequencies of kdr-associated mutations over this period, and absence of the insecticide-susceptible genotype in recent collections. Furthermore, a signicant transition towards infestations possessing multiple kdr-associated mutations was observed. These results suggest that the persistent use of pyrethroid insecticides over the past decade continues to impose strong selection pressure on C. lectularius populations, driving the proliferation of kdr-associated mutations. They demonstrate that, if unabated, strong anthropogenic selection can drive the rapid evolution of adaptive traits. temporal and turnover of report temporal changes in the frequency of the three kdr-associated mutations from populations of C. collected States at two time periods approximately apart. both the number of infestations exhibiting kdr-associated mutations, and the number of adaptive mutations per individual bed bug would increase over time, due to selection imposed by the frequent and widespread use of pyrethrin and pyrethroid insecticides. in the development of management for the control of C. of with pesticides.


Introduction
Globally, urban areas are undergoing rapid expansion and are increasing in density (Homer et al. 2020 One group of insecticides, the sodium channel modulators, which includes the organochlorides (e.g., dichloro-diphenyl trichloroethane [DDT]), pyrethrins, and pyrethroids, alter the activity of para-type voltage-gated sodium channels (VGSCs), which are necessary for neuronal function. These insecticides maintain the sodium channels in an activated state, preventing cellular repolarization, resulting in paralysis and death (Soderlund and Knipple 2003). Nonsynonymous mutations within the VGSC gene have been identi ed that reduce kdr-associated target-site sensitivity to these insecticides across a variety of arthropod pests (Milani 1956 Australia (Dang et al. 2015). However, we are unaware of any studies that assessed temporal changes in the frequencies of kdr-associated mutations at any landscape level. Although it would be most instructive to track the frequency of kdr mutations over time within the same populations, this design is particularly challenging with residential infestations due to their ephemerality and turnover of residents. Here, we report temporal changes in the frequency of the three kdrassociated mutations from populations of C. lectularius collected across the United States at two time periods approximately 10-years apart. We hypothesized that both the number of infestations exhibiting kdr-associated mutations, and the number of adaptive mutations per individual bed bug would increase over time, due to selection imposed by the frequent and widespread use of pyrethrin and pyrethroid insecticides.  Fig. 1a) and were made by pest management companies (electronic supplementary material, Table S3). Upon collection, specimens were preserved in 100% ethanol and stored at -20°C until DNA extraction. Genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (QIAGEN, Germantown, MD). , which results from population foundation by a single gravid female or small group of highly related individuals, the genetic diversity and hence kdr-associated mutation pro le of a given population can be determined through the sequencing of a single individual. Therefore, each population is represented here by a single specimen. Two genomic fragments that contain the three kdr-associated mutations were PCR-ampli ed and sequenced using primer combinations BBParaF1/BBParaR1 (V419L) and BBParaF3/BBParaR3 (L925I, I936F), following the methods of Holleman et al. Genotype frequencies were compared using Chi-square tests. Additionally, the overall homozygous genotype frequencies of populations present in the same U.S. states across both sample periods were compared.

Results
Only samples with unambiguous sequences for both PCR fragments were analyzed in this study. Of the 161 populations sampled between 2005 and 2009, genotypes L925I and L925I&V419L accounted for 36% and 50% of the populations, respectively ( Table 2 and Fig. 1a). The wild type ('susceptible') genotype was found in 2.5% of the populations. Approximately 10% of samples screened were found to be L925I&V419L het , while the I936F, L925I het &I936F het , and I936F het genotypes each comprised < 0.1% of the remaining infestations ( Table 2 and Fig. 1a). In contrast, genotype L925I&V419L predominated in samples collected between 2018 and 2019, accounting for 84% of the populations, while genotypes L925I (~ 5%), L925I het (~ 1%), L925I&V419L het (~ 9%), and L925I het &V419L (~ 1%) comprised the remaining populations ( Table 2 and Fig. 1a). The wild type and I936F genotypes were not detected in the 2018-2019 populations. Temporally, there were signi cant differences in the frequencies of all homozygous [wild type, p < 0.05; L925I, p < 0.001; L925I&V419L, p < 0.001), but not the heterozygous genotypes and I936F, likely due to their low frequency across datasets (Table 2). Comparisons conducted using only populations sampled in the same U.S. states in both timeframes again revealed signi cant differences in the frequencies of all homozygous genotypes (electronic supplementary material, Table S4).

Discussion
By surveying 394 distinct populations of C. lectularius across the U.S., collected from two time-periods separated by approximately a decade, this study represents the most comprehensive screening to date of kdr-associated mutation frequencies for the species. To our knowledge, this is also the rst temporal comparison of kdr mutation frequencies in any urban pest insect in the U.S., outside of house ies collected from animal production facilities (Freeman et al.  (Fig. 1b). This is likely in response to persistent selection driven by the long-term use of pyrethroid insecticides.
Prior bioassay-based research in C. lectularius has linked genotypes L925I and L925I&V419L to increased levels of pyrethroid resistance (Zhu et al. 2010), including > 12,765-fold deltamethrin resistance (Romero et al. 2007), when compared to a susceptible wild type population. Furthermore, the presence of multiple kdr mutations has been associated with elevated levels of pyrethroid resistance in other pest insects (Soderlund 2008; Abdalla et al. 2014). Thus, we presume that a higher proportion of the bed bugs collected in 2018-2019 would exhibit greater resistance to pyrethroids than those collected a decade earlier.
Comparable ndings of temporal change in kdr mutations have been reported in mosquito populations exposed to the prolonged use of insecticides. Congruent with the results presented here, several long-term studies (> 10 years) showed an increase in kdr mutation frequencies in populations of Anopheles gambiae (Mathias et al. 2011) (Tancredi et al. 2020). A signi cant increase was also reported for the 1b resistance allele in house ies collected over a 10-year period from animal production facilities across ve U.S. States (Freeman et al. 2019). While these studies span different regional pest management practices, the trend of increasing kdr mutation frequencies is consistent. However, a unique characteristic of C. lectularius is the high level of intra-population inbreeding, and an apparent lack of signi cant levels of gene ow (Booth et al , this study reports the zygosity of the kdrassociated mutations detected. This information may be relevant for control, for while the level of resistance exhibited by C. lectularius heterozygous for a given mutation is unclear, it has been suggested that heterozygotes may show incomplete dominance (Romero et al. 2007). Heterozygotes may therefore exhibit intermediate levels of resistance between wild type and homozygous mutants, although this has yet to be con rmed. If kdr exhibits incomplete dominance in C. lectularius, heterozygotes would be expected in areas undergoing genotype replacement from wild type to resistant, either through population spread or continued pyrethroid applications. However, bed bugs heterozygous for kdr mutations would also be expected to succumb to control more rapidly than homozygous mutant individuals. In both time periods sampled, L925I&V419F het was the most common heterozygous genotype observed, representing 89% (2005-2009) and 78% (2018-2019) of the heterozygous individuals. Given the short generation time of C. lectularius, high fecundity, and lack of obvious inbreeding depression (Fountain et al. 2014), the transition within a population to the double homozygous mutant (L925I&V419L) would be expected to occur rapidly following selection by insecticide treatment.
In summary, the signi cant temporal shift towards increased frequencies of kdr mutations reveals how anthropogenic selection can in uence the evolution of a species of public health, veterinary, and economic signi cance, and suggests that indoor pest insects may represent ideal model systems to study the effects of anthropogenic in uences on the evolution of urban species. Furthermore, these ndings are practically signi cant in the development of management strategies for the control of C. lectularius populations and those of other urban and indoor pest species commonly controlled with pesticides.