Michael F. Potter

Insecticide resistance in the bed bug: a factor in the pest's sudden resurgence?

Abstract Infestations of the bed bug, Cimex lectularius L. (Heteroptera: Cimicidae), are increasing around the world at an alarming rate and have become a major public health concern. The evolution of insecticide resistance could be a primary factor in explaining this resurgence. Extremely high levels of resistance to two pyrethroid insecticides, deltamethrin and &lgr;-cyhalothrin, relative to a susceptible colony, were detected in populations collected from human dwellings in Kentucky and Ohio. Offspring of a cross between a resistant and susceptible colony had intermediate susceptibility. Evaluations of populations from across the United States indicate that resistance to pyrethroid insecticides is already widespread. Without the development of new tactics for bed bug management, further escalation of this public health problem should be expected.

Widespread distribution of knockdown resistance mutations in the bed bug, Cimex lectularius (Hemiptera: Cimicidae), populations in the United States

We previously reported high deltamethrin resistance in bed bugs, Cimex lectularius, collected from multiple areas of the United States (Romero et al., 2007). Recently, two mutations, the Valine to Leucine mutation (V419L) and the Leucine to Isoleucine mutation (L925I) in voltage-gated sodium channel alpha-subunit gene, had been identified to be responsible for knockdown resistance (kdr) to deltamethrin in bed bugs collected from New York (Yoon et al., 2008). The current study was undertaken to investigate the distribution of these two kdr mutations in 110 bed bug populations collected in the United States. Out of the 17 bed bug populations that were assayed for deltamethrin susceptibility, two resistant populations collected in the Cincinnati area and three deltamethrin-susceptible lab colonies showed neither of the two reported mutations (haplotype A). The remaining 12 populations contained L925I or both V419L and L925I mutations in voltage-gated sodium channel alpha-subunit gene (haplotypes B&C). In 93 populations that were not assayed for deltamethrin susceptibility, 12 contained neither of the two mutations (haplotype A) and 81 contained L925I or V419L or both mutations (haplotypes B-D). Thus, 88% of the bed bug populations collected showed target-site mutations. These data suggest that deltamethrin resistance conferred by target-site insensitivity of sodium channel is widely spread in bed bug populations across the United States.

Bed bugs evolved unique adaptive strategy to resist pyrethroid insecticides

Recent advances in genomic and post-genomic technologies have facilitated a genome-wide analysis of the insecticide resistance-associated genes in insects. Through bed bug, Cimex lectularius transcriptome analysis, we identified 14 molecular markers associated with pyrethroid resistance. Our studies revealed that most of the resistance-associated genes functioning in diverse mechanisms are expressed in the epidermal layer of the integument, which could prevent or slow down the toxin from reaching the target sites on nerve cells, where an additional layer of resistance (kdr) is possible. This strategy evolved in bed bugs is based on their unique morphological, physiological and behavioral characteristics and has not been reported in any other insect species. RNA interference-aided knockdown of resistance associated genes showed the relative contribution of each mechanism towards overall resistance development. Understanding the complexity of adaptive strategies employed by bed bugs will help in designing the most effective and sustainable bed bug control methods.

RNA Interference of NADPH-cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius

Background NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. Methodology/Principal Findings The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. Conclusions/Significance These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

Behavioral Responses of the Bed Bug to Insecticide Residues

ABSTRACT The recent resurgence of bed bugs, Cimex lectularius L. (Heteroptera: Cimicidae), has increased the demand for information about effective control tactics. Several studies have focused on determining the susceptibility of bed bug populations to insecticides. However, behavioral responses of bed bugs to insecticide residues could influence their efficacy. The behavioral responses of bed bugs to deltamethrin and chlorfenapyr, two commonly used insecticides for bed bug control in the United States, were evaluated. In two-choice tests, grouped insects and individual insects avoided resting on filter paper treated with deltamethrin. Insects did not avoid surfaces treated with chlorfenapyr. Harborages, containing feces and eggs and treated with a deltamethrin-based product, remained attractive to individuals from a strain resistant to pyrethroids. Video recordings of bed bugs indicated that insects increased activity when they contacted sublethal doses of deltamethrin. Insecticide barriers of chlorfenapyr or deltamethrin did not prevent bed bugs from reaching a warmed blood source and acquiring blood meals. We discuss the impact of these responses on bed bug control practices.

Evaluation of piperonyl butoxide as a deltamethrin synergist for pyrethroid-resistant bed bugs.

ABSTRACT An understanding of the mechanisms of insecticide resistance in the bed bug, Cimex lectularius L., has the potential to lead to new approaches for the control of resistant populations. We used the cytochrome P450 monooxygenase (P450) inhibitor piperonyl butoxide (PBO) to assess the role of P450s in deltamethrin resistance in three field-collected bed bug strains, LA-1, CIN-1 and WOR-1. In addition, we exposed two highly resistant strains, CIN-1 and WOR-1 (resistance ratio [RR] >2,500-fold), to dry residues of piperonyl butoxide-synergized pyrethroid formulations to determine the utility of synergism by PBO. Piperonyl butoxide synergized deltamethrin in all three strains, but its impact was variable. The synergistic ratio varied from 40 in CIN-1 to 176 in WOR-1. Because the resistance ratio for each strain after piperonyl butoxide treatment was 174 and 39, respectively, our results suggest that P450s have some involvement in deltamethrin resistance, but other resistance mechanisms must be involved as well. No significant synergistic effect of formulated deltamethrin was observed with the addition of synergized Pyrethrins or formulated piperonyl butoxide in the CIN-1 strain, but synergism occurred in the WOR-1 strain. Addition of PBO to pyrethroids is not a comprehensive solution to pyrethroid resistance because strains vary in both overall resistance level and the proportion of that resistance attributable to P450s.

Efficacy of two pyrethroid insecticides applied as barrier treatments for managing mosquito (Diptera: Culicidae) populations in suburban residential properties

Abstract Increased threat of mosquito-borne disease coupled with decreased tolerance of nuisance mosquitoes has opened a market for pest management professionals to offer mosquito control services for homeowners. A pest management professional applied bifenthrin (0.08%) and lambda-cyhalothrin (0.1%) at their maximum label concentrations as barrier treatments. We tested treatments residual efficacy in reducing adult mosquito populations and compared these chemicals against a water control at 24 residential properties (eight replications by three treatments). Mosquito populations were measured on each property by using five methods: CO2-baited Centers for Disease Control (CDC) light traps (without a light), human landing rates, CDC gravid traps, ovitraps, and sweep nets. Populations were monitored weekly for 2 wk before treatment and 8 wk posttreatment. Additionally, to confirm residual efficacy of each insecticide, a randomly treated leaf underwent a no-choice bioassay with laboratory-reared Aedes albopictus (Skuse). Trap collections were dominantly Aedes albopictus and Culex pipiens L. Both insecticidal treatments significantly reduced Aedes spp. lambda-Cyhalothrin- and bifenthrin-treated sites had 89.5 and 85.1% fewer Ae. albopictus bites than the untreated control, respectively. Ae. albopictus bioassay results showed significant residual efficacy for both insecticides up to 6 wk posttreatment. There were no significant differences between properties treated with the two insecticides. In contrast, Culex spp. were not reduced by either insecticidal treatment. Our study indicated that barrier sprays applied to low-lying vegetation do not properly target adult daytime resting sites for Culex mosquitoes but that they can reduce Aedes mosquitoes. Perhaps by treating upper tree canopies Culex spp. abundance may be reduced.

Circadian rhythm of spontaneous locomotor activity in the bed bug, Cimex lectularius L.

Bed bugs must avoid detection when finding hosts and returning to hidden harborages. Their stealthy habits include foraging when hosts are asleep. Characteristics of spontaneous locomotor activity rhythm of bed bugs with different feeding histories were studied. In the absence of host stimuli, adults and nymphs were much more active in the dark than in the light. The onset of activity in the scotophase commenced soon after lights-off. The free-running period (tau) for all stages was longer in continuous darkness (DD) than in continuous light (LL). The lengthening of tau in DD is an exception for the circadian rule that predicts the opposite in nocturnal animals. Activity in all stages was entrained to reverse L:D regimes within four cycles. Short-term starved adults moved more frequently than recently fed adults. While bed bugs can survive for a year or more without a blood meal, we observed a reduction in activity in insects held for five weeks without food. We suggest that bed bugs make a transition to host-stimulus dependent searching when host presence is not predictable. Such a strategy would enable bed bugs to maximize reproduction when resources are abundant and save energy when resources are scarce.

Evaluation of chlorfenapyr for control of the bed bug, Cimex lectularius L.

BACKGROUND The presence of bed bug populations resistant to pyrethroids demands the development of new control tactics, including the use of insecticides with new modes of action. Insecticides that disrupt oxidative phosphorylation in insect mitochondria can be an option. Laboratory assays were used to measure the toxicity of chlorfenapyr to susceptible strains and two strains highly resistant to pyrethroids. The effectiveness of two chlorfenapyr-based formulations was compared, and behavioral responses of bed bugs to dry residues of aerosol sprays were evaluated. RESULTS Chlorfenapyr was effective against all bed bug strains, killing them at a similar rate, regardless of their susceptibility status to pyrethroids. Dry residues aged for 4 months were as toxic as fresh dry residues. The aerosol formulation had contact activity and caused faster mortality than a water-based formulation. Bed bugs did not avoid resting on surfaces treated with aerosol. CONCLUSION Chlorfenapyr is an option for controlling pyrethroid-resistant bed bugs. While it does not cause quick knockdown, its long residual activity and no avoidance behavior of bed bugs to dry residues appear to make this insecticide suitable for bed bug control. A faster insecticidal effect is obtained with the aerosol formulation, suggesting greater bioavailability of the toxicant.

Entrapment of bed bugs by leaf trichomes inspires microfabrication of biomimetic surfaces

Resurgence in bed bug infestations and widespread pesticide resistance have greatly renewed interest in the development of more sustainable, environmentally friendly methods to manage bed bugs. Historically, in Eastern Europe, bed bugs were entrapped by leaves from bean plants, which were then destroyed; this purely physical entrapment was related to microscopic hooked hairs (trichomes) on the leaf surfaces. Using scanning electron microscopy and videography, we documented the capture mechanism: the physical impaling of bed bug feet (tarsi) by these trichomes. This is distinct from a Velcro-like mechanism of non-piercing entanglement, which only momentarily holds the bug without sustained capture. Struggling, trapped bed bugs are impaled by trichomes on several legs and are unable to free themselves. Only specific, mechanically vulnerable locations on the bug tarsi are pierced by the trichomes, which are located at effective heights and orientations for bed bug entrapment despite a lack of any evolutionary association. Using bean leaves as templates, we microfabricated surfaces indistinguishable in geometry from the real leaves, including the trichomes, using polymers with material properties similar to plant cell walls. These synthetic surfaces snag the bed bugs temporarily but do not hinder their locomotion as effectively as real leaves.