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 first temporal comparison of kdr mutation frequencies in any urban pest insect in the U.S., outside of house flies collected from animal production facilities (Freeman et al. 2019) and mosquitoes (Mathias et al. 2011; Abdalla et al. 2014; Linss et al. 2014; Aguirre-Obando et al. 2016; Foster et al. 2016; Ismail et al. 2018; Amoudji et al. 2019; Biduda et al. 2019; Mandeng et al. 2019; Tancredi et al. 2020; Baltzegar et al. 2021). Comparing our homozygous populations from 2005–2009 (N = 143) to those of Zhu et al. (2010) [N = 93] revealed significant differences in kdr genotype frequencies (wild type: χ2(1, n = 236) = 9.11, p < 0.05; L925I: χ2(1, n = 236) = 0.45, p > 0.05; L925I&V419L: χ2(1, n = 236) = 7.25, p < 0.05; V419L: χ2(1, n = 236) = 4.67, p < 0.05), supporting the lower baseline frequency of kdr mutations present in the U.S. in 2006–2007 (Zhu et al. 2010), and a reduction in wild type and increase in L925I&V419L by 2008–2009 when the majority of our data were collected. Prior to our study, the most recent study conducted in the U.S. reported kdr genotype frequencies for populations collected across Midwestern states between 2014 and 2015 (Holleman et al. 2018), with the frequencies of L925I (~ 21%) and L925I&V419L (~ 76%) intermediate between the data sets presented here (Fig. 1b), as expected for a shift towards increased resistance. Thus, our results, when viewed together with those of Zhu et al. (2010) and Holleman et al (2018), reveal a significant increase in the frequency of kdr mutations over approximately a decade, with a shift towards multiple kdr mutations per population (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 findings 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) and Aedes aegypti (Linns et al. 2014; Baltzegar et al. 2021), from different geographic regions. Recently, Baltzegar et al (2021), documented the complete replacement of the wild type pyrethroid-susceptible genotype, with either single or double kdr-mutants in Ae. Aegypti mosquitos sampled in Peru, with the replacement occurring over a period of 13 years following the introduction of a city-wide pyrethroid insecticide control strategy. Similarly, the malaria vector, An. arabiensis, was shown to respond to strong selection for deltamethrin resistance over four years, which was correlated with the L1014F kdr mutation (Ismail et al. 2018). Verhaeghen et al. (2010) documented a five-year increase in kdr mutations in An. gambiae from multiple geographic locations, and similar region-specific fluctuations in kdr mutation frequencies have been shown in Ae. albopictus at multiple time intervals (Tancredi et al. 2020). A significant increase was also reported for the 1b resistance allele in house flies collected over a 10-year period from animal production facilities across five 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 significant levels of gene flow (Booth et al. 2012, 2015, 2018; Saenz et al. 2012; Fountain et al. 2014). As a result, resistance alleles rapidly propagate and are then spread in a fixed-state through propagule transfer. While there is still uncertainty surrounding the fitness costs of resistance in C. lectularius (Polanco et al. 2011; Gordon et al. 2015), the frequency and distribution of kdr mutations and the prevalence of the species suggests this may be minimal.
The zygosity of kdr mutations and its impact on insecticide resistance adds additional complexity. For example, in the house fly, Musca domestica, mutations exhibit a recessive pattern of inheritance (Rinkevich et al. 2012). In contrast, polygenic modes of inheritance have been documented in the codling moth, Cydia pomonella (Bouviet et al. 2001), with both recessive and incomplete dominance described. Unlike Zhu et al. (2010), this study reports the zygosity of the kdr-associated 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 confirmed. 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&V419Fhet 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 significant temporal shift towards increased frequencies of kdr mutations reveals how anthropogenic selection can influence the evolution of a species of public health, veterinary, and economic significance, and suggests that indoor pest insects may represent ideal model systems to study the effects of anthropogenic influences on the evolution of urban species. Furthermore, these findings are practically significant 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.