Sequencing of Voltage Gated Sodium Channel α-Subunit Gene Fragments From CIN-1
In previous studies, we reported that bed bugs collected from the Cincinnati, OH, area (CIN-1) showed very high resistance (>12,765-fold) to deltamethrin relative to the susceptible strain, Fort Dix (FD-1) (Romero et al., 2007). To determine if the mutations in the voltage-gated sodium channel α-subunit gene are responsible for observed deltamethrin resistance in CIN-1 population, 14 primers were designed to clone the fragments I to V that include 12 reported mutations including V419L and L925I, which had been confirmed to be responsible for sodium channel insensitivity to pyrethroids in insects (Dong, 2007; Fig. 1; Table 1). No mutations were detected in the five fragments of CIN-1 voltage-gated sodium channel α-subunit gene, suggesting mutations in this gene are not responsible for the deltamethrin resistance observed in the CIN-1 population.
Determining a Causal Link Between Haplotypes and Deltamethrin Resistance
To identify if there is a causal link between two identified mutations, V419L and L925I in sodium channel α-subunit gene and deltamethrin resistance in bed bug populations, residual bioassays and sequencing of sodium channel α-subunit gene fragments containing amino acids of two identified mutations were performed for 17 bed bug populations. A discriminating dose (0.13 mg/cm2) of deltamethrin (Romero et al., 2007) was used to evaluate the resistant status of 17 bed bug populations. Bioassay results showed that three populations, FD-1, GA-1, and LA-1 (two of which were long-maintained laboratory colonies), are susceptible to deltamethrin. The other 14 populations showed moderate to high resistance to deltamethrin (Table 1). Sequencing of para gene fragments containing two amino acids for identified mutations showed that neither of the two mutations is present in the para gene fragments amplified using the DNA of bed bugs from FD-1, GA-1, and LA-1 populations susceptible to deltamethrin. In addition, two populations resistant to deltamethrin (CIN-1 and CIN-3) also showed neither of these two mutations; this group is designated as Haplotype A. Only L925I mutation but not V419L mutation, is present in three resistant populations (LEX-1, TRO-1, and DOV-1; designated as Haplotype B). Both V419L and L925I mutations are identified in nine other resistant populations (Haplotype C).
These results suggest a potential causal link. Haplotypes B (with mutation L925I) and C (with mutations L925I and V419L) are resistant to deltamethrin. We have not characterized any populations with only the V419L mutation (Haplotype D). Although Haplotype A is likely pyrethroid-sensitive, bed bugs possessing this haplotype could also be resistant by a mechanism that does not involve target site insensitivity. For example, two populations from the Cincinnati area (CIN-1 and CIN-3) are HaplotypeA, but are nonetheless resistant to deltamethrin. These two populations showed neither V419L nor L925I mutation in the voltage-gated sodium channel α-subunit gene. Additionally, CIN-1 contained none of the 12 voltage-gated sodium channel α-subunit gene mutations shown to be involved in pyrethroid resistance. Recent studies showed that the inhibitors of P450 reduce deltamethrin resistance in CIN-1 (Romero et al., 2009). Therefore, we hypothesized that P450-mediated metabolic detoxification may be a mechanism responsible for deltamethrin resistance observed in this population. Studies are underway in our laboratory to determine the mechanism of resistance in CIN-1.
Examining Geographic Distribution of Knockdown Resistance in Bed Bug Populations
We developed ASPCR primers for two mutations, V419L and L925I, identified as responsible for deltamethrin resistance in the NY-BB population. To determine if the ASPCR primers developed for these two mutations are reliable to detect mutations in bed bug populations, we performed ASPCR using the genomic DNA isolated from 17 bed bug populations. Similar to the results observed by sequencing, ASPCR also detected neither mutation in five populations (Haplotype A; FD-1, GA-1, LA-1, CIN-1 and CIN-3), only L925I but not V419L in three resistant populations (Haplotype B; LEX-1, TRO-1, and DOV-1), and both V419L and L925I mutations in the other nine resistant populations (Haplotype C) (Fig. 2). Thus, ASPCR consistently confirmed results observed by sequencing, suggesting that ASPCR could be used to monitor resistance in bed bug populations.
Figure 2. 1st PCR and ASPCR results for fragments I (A) and III (B) amplified using DNA isolated from 17 bed bug populations. The 1st PCR fragments were amplified using allele-independent primer pairs, BBParaF1/BBParaR1 (A) and BBParaF3/BBParaR3 (B). The ASPCR fragments were amplified using allele-specific primer pairs, BBParaF1-AS/BBParaR1 (A) and BBParaF3-AS/BBParaR3 (B). The Arabic numbers on the top of the gel pictures are the sample numbers as shown in Table 2. The same DNA template was used for both A and B. S, Susceptible; R, Resistant.
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The distribution of V419L and L925I mutations in 93 additional bed bug populations collected from dwellings across the United States was examined by ASPCR. Out of the 93 populations screened by ASPCR, 12 showed neither mutation (Haplotype A), 42 showed L925I but not V419L mutation (Haplotype B), and 36 showed both mutations (Haplotype C) (Table 3). In addition, in three populations only V419L but not L925I mutation was detected and designated as Haplotype D. Since mutation V419L was shown as one of the two mutations present in the deltamethrin-resistant NY-BB colony (Yoon et al., 2008), the populations with V419L mutation (Haplotype D) may be resistant to deltamethrin but we have no bioassay data to support this hypothesis. Mapping the geographical distribution of two identified mutations in sodium channel α-subunit gene showed that populations collected from 9 of the 17 states showed 100% resistant bed bug populations (with haplotypes B, C, or both B and C; Fig. 3). Five other states showed more than 50% populations with resistance to deltamethrin (haplotypes B, C, and/or D; Fig. 3).
Figure 3. The geographic distribution of kdr mutations in the bed bug populations collected from the United States. The pies show the haplotype composition of all population samples in each state. The number under each pie is the number of populations collected from each state.
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It is interesting that the presence of even a single mutation, L925I, could confer significant deltamethrin resistance (Table 2). The frequency of a single mutation L925I (Haplotype B) is 0.43 in 110 populations tested. This is even higher than the frequency (0.41) of Haplotype C containing both mutations. The frequency of occurrence of a single mutation V419L (haplotype D) is very low (around 0.027). These data support the important role of L925I mutation in deltamethrin resistance. Even though L925I mutation is present in two haplotypes (B and C), these two haplotypes have a somewhat different geographic distribution (Fig. 3). Haplotype C is predominantly present in the northeastern part of the United States and occurs very infrequently in the south. In contrast, haplotype B is present in both northern and southern states. Interestingly, haplotype D is present in very limited regions and co-occurrs with haplotype A.
Among the 17 states represented, samples collected from only one state (IL) and District of Columbia showed no mutations in the kdr gene (only single sample per region was collected). More than half of the states (9 out of 17) showed haplotypes B or C or both B and C that confer resistance to deltamethrin. These data indicate that deltamethrin resistance conferred by target-site insensitivity of sodium channel is widely distributed in the bed bug populations across the United States. It is also interesting that the predominance of resistance-conferring haplotypes B and C is higher in the northeastern states than in the southern states.
In the 1940s and 1950s, DDT was commonly used to control bed bug infestations (Boase, 2001). Subsequently, a high degree of resistance to DDT was reported in many strains (Johnson and Hill, 1948; Gratz, 1959; Mallis and Miller, 1964; Nagem and Williams, 1992). Since pyrethroids and chlorinated hydrocarbons share a similar mode of action, cross-resistance may occur between these two classes of insecticides, and, therefore, could explain why bed bugs have developed resistance to pyrethroids so quickly. As the primary target of pyrethroids and DDT, the voltage-gated sodium channel was subjected to selection since the 1940s when DDT was first introduced (ffrench-Constant et al., 2004). Therefore, the kdr-mediated resistance mechanism is well suited for studying the origins of pyrethroid resistance (Anstead et al., 2005). Use of pyrethroids and pyrethroid-treated bed nets in different parts of the world for controlling mosquitoes may have also contributed to pyrethroid resistance in bed bugs (Myamba et al., 2002). Our study was not designed to look for the point of origin or introduction of pyrethroid resistance into bed bug populations. However, because at least three factors (two kdr mutations and one likely P450) are implicated and these are found in independent samples, it is likely that resistance will have multiple geographic origins. Further studies with more populations and additional markers are required to reach a final conclusion on the origins of bed bug resistance in the United States.
In conclusion, the current study is the first effort to investigate the distribution and extent of kdr mutations in bed bug populations across the United States. In addition, we established a causal link between haplotypes and deltamethrin resistance in bed bug populations and analyzed the geographic distribution of these mutations. The major finding of this report is the discovery that target site–based mutations are the main reason for reported pyrethroid resistance in bed bug populations from across the United States. About 88% of the bed bug populations tested showed target-site mutation(s) and most likely are resistant to deltamethrin. The remaining 12% of populations may be susceptible to pyrethroids or resistant to them through some other mechanism such as increased activity of detoxifying enzymes such as P450. Our study will serve as baseline data to study the origin of pyrethroid-resistant bed bug populations. In addition, this study has some important practical applications. ASPCR primers developed in this study can be used for monitoring the target-site mutations in bed bug populations. This type of monitoring will help in the development of improved bed bug population management strategies. For example, currently, several pest-management companies add additives such as piperonyl butoxide to synthetic pyrethroid formulations to treat resistant populations of bed bugs. If the monitoring program determines that the population being treated contains target-site mutations (as we predict based upon data included in this report), then the treatment strategy should include an insecticide that functions through a target site other than the sodium channel protein. Thus, the studies reported here will help to conduct resistance monitoring in bed bug populations leading to improvement in bed bug management strategies.