New Introductions, Spread of Existing Matrilines, and High Rates of Pyrethroid Resistance Result in Chronic Infestations of Bed Bugs (Cimex lectularius L.) in Lower-Income Housing

Infestations of the common bed bug (Cimex lectularius L.) have increased substantially in the United States in the past 10-15 years. The housing authority in Harrisonburg, Virginia, conducts heat-treatments after bed bugs are detected in a lower-income housing complex, by treating each infested unit at 60°C for 4-6 hours. However, a high frequency of recurrent infestations called into question the efficacy of this strategy. Genetic analysis using Bayesian clustering of polymorphic microsatellite loci from 123 bed bugs collected from 23 units from May 2012 to April 2013 in one building indicated that (a) 16/21 (73%) infestations were genetically similar, suggesting ineffective heat-treatments or reintroductions from within the building or from a common external source, followed by local spread of existing populations; and (b) up to 5 of the infestations represented new genotypes, indicating that 5 new populations were introduced into this building in one year, assuming they were not missed in earlier screens. There was little to no gene flow among the 8 genetic clusters identified in the building. Bed bugs in the U.S. often possess one or both point mutations in the voltage-gated sodium channel, termed knockdown resistance (kdr), from valine to leucine (V419L) and leucine to isoleucine (L925I) that confer target-site resistance against pyrethroid insecticides. We found that 48/121 (40%) bed bugs were homozygous for both kdr mutations (L419/I925), and a further 59% possessed at least one of the kdr mutations. We conclude that ineffective heat treatments, new introductions, reintroductions and local spread, and an exceptionally high frequency of pyrethroid resistance are responsible for chronic infestations in lower-income housing. Because heat treatments fail to protect from reintroductions, and pesticide use has not decreased the frequency of infestations, preventing new introductions and early detection are the most effective strategies to avoid bed bug infestations in multistory apartment buildings

Frequency of <i>kdr</i> genotypes from bed bugs collected from individual apartment units.

<p>Frequency of <i>kdr</i> genotypes from bed bugs collected from individual apartment units.</p

Number of microsatellite alleles present in each of the genetic clusters identified in BAPS.

<p>Number of microsatellite alleles present in each of the genetic clusters identified in BAPS.</p

Genetic clustering of samples using the Bayesian method implemented in the program BAPS.

<p>The numbers refer to the sample numbers. The width of each band is proportional to the number of individuals in each sample. Samples collected at a hotel (sample 31) and homeless shelter (sample 32) are included in the analysis.</p

Primers used to amplify <i>kdr</i> genes.

<p>Primers used to amplify <i>kdr</i> genes.</p

Number of microsatellite alleles present in each sample.

<p>Number of microsatellite alleles present in each sample.</p

Genetic clusters and degree of admixture identified by the Bayesian clustering method implemented in the program BAPS using only samples collected from the apartment building.

<p>(A) The 8 clusters identified in the apartment samples; each individual is shown by a single narrow bar. (B) Admixture of individuals within each cluster with the height of each color proportional to the degree of admixture. Sample 38 was not included in the admixture analysis because it was placed in its own cluster and the minimum population size used in the admixture analysis was 3 individuals. Cluster colors and numbers correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117805#pone.0117805.g005" target="_blank">Fig 5</a>.</p

Units and dates in an apartment complex from which bed bugs were collected from May 2012 to April 2013.

<p>Consecutive sample numbers 11 & 12, 16 & 17, 29 & 30, 39 & 40, and 43 & 44 represent two sites in the same apartment from which bed bugs were collected on the same date. The number of bed bugs in each sample (<i>n</i>) is provided after the sample number. Sample 24 was an internal laboratory colony and is not shown. Samples 31 and 32 were collected at a hotel and homeless shelter, respectively, and were used as outgroups. Because all five floors have the same layout, a common floorplan is provided to help visualize the proximity of units to one another.</p

Gene flow diagram among clusters showing the relative amount of ancestry among clusters.

<p>The colors for each cluster correspond to the colors shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117805#pone.0117805.g004" target="_blank">Fig 4B</a>. Only samples represented by at least three individuals were included in the admixture analysis.</p

<i>kdr</i> Diploid genotypes following PCR amplification of apartment samples.

<p>(A) The frequency of each genotype is presented in the bottom right corner of each gel image. The two brightest bands in the molecular weight ladder denote 500 and 1000 basepairs. “Hom” denotes homozygous, and “Het” denotes heterozygous. Two genotypes–Hom V419/Hom I925 and Het V419L/Hom L925 –were not represented in any of the collections and are therefore not shown. (B) Frequency of 419 or 925 susceptible (V419/L925) or <i>kdr</i> mutant (L419/I925) alleles from bed bugs collected in an apartment complex.</p