Lilly, D. G., K. Dang, C. E. Webb, and S. L. Doggett. 2018. Are Australian field-collected strains of Cimex lectularius and Cimex hemipterus (Hemiptera: Cimicidae) resistant to deltamethrin and imidacloprid as revealed by topical assay? Austral Entomology. 57: 77–84. doi: 10.1111/aen.12268
“Resistance profiling on Cimex lectularius and Cimex hemipterus in Australia has previously revealed the existence of resistance to pyrethroid insecticides and that multiple resistant mechanisms exist in field populations. To further investigate insecticide resistance in modern field populations of bed bugs, a program to collect and screen field specimens was initiated. Over 50 bed bug strains (a mixture that included both C. lectularius and C. hemipterus) have been collected from across Australian, with 35 subsequently colonised (31 C. lectularius and 4 C. hemipterus) for laboratory-based insecticide resistance testing. Bed bugs were exposed topically to pyrethroids at a discriminating dose of 2.5 g/L deltamethrin in acetone applied at a rate of 1 µL/bug and, for neonicotinoids, a dose of 0.1 g/L of imidacloprid applied at a rate of 1 µL/bug. Mortality was recorded 24 h post application. Twenty adult bed bugs of mixed sex and age in two replicates of 10 were used for the bioassays, plus controls. Results indicated a broad spread in the frequency of resistance against deltamethrin in C. lectularius, represented by a range of mortality values between 15 and 100%. Mortality was predominantly uniform with C. lectularius against imidacloprid at ≥95%, except for several recently collected strains that returned between 70 and 85%, indicating that a degree of tolerance may be developing in select strains. Three of four C. hemipterus strains returned mortality values at or below 10%, with the remainder recording 75% mortality, thereby indicating pyrethroid resistance is similarly advanced in this species. Normal susceptibility (≥95% mortality) was otherwise evident in all four C. hemipterus strains to imidacloprid. These findings complement concurrent research, which indicates that variable patterns of resistance are present across the country and around the world. The results have profound implications for product manufacturers and registration authorities in ensuring that a product is as effective at controlling bed bugs in the field as the laboratory.”
Ashbrook, A. R., M. E. Scharf, G. W. Bennett, and A. D. Gondhalekar. 2017. Detection of reduced susceptibility to chlorfenapyr- and bifenthrin-containing products in field populations of the bed bug (Hemiptera: Cimicidae). Journal of Economic Entomology. 110: 1195–1202. doi: 10.1093/jee/tox070
“Insecticide resistance is a major impediment for effective control of Cimex lectularius L. Previous resistance detection studies with bed bugs have focused on certain pyrethroid, neonicotinoid, organochlorine, organophosphate, and carbamate insecticides. Within the pyrethroid class, resistance studies have mostly been limited to deltamethrin, lambda-cyhalothrin, and alpha- and beta-cyfluthrin. The goal of this study was to develop diagnostic concentration bioassays for assessing bed bug susceptibility levels to chlorfenapyr- and bifenthrin-containing products. First, glass vial and filter paper bioassay methods were compared for their utility in susceptibility monitoring. Statistical comparison of toxicity data between bioassays indicated that the vial assay was less confounded by assay substrate effects, required less insecticide, and was faster, especially for chlorfenapyr. Next, using vial diagnostic concentrations (LC99) for each insecticide, 10 laboratory-adapted field strains and the Harlan lab-susceptible strain were screened for susceptibility to chlorfenapyr and bifenthrin. The results of this study reveal recent bed bug susceptibility levels to certain chlorfenapyr- and bifenthrin-containing products. Reduced susceptibility was detected in three and five field strains to chlorfenapyr and bifenthrin, respectively. Detection of reduced susceptibility suggests that certain strains may be segregating toward greater chlorfenapyr and bifenthrin resistance. These results merit continuous resistance monitoring efforts to detect chlorfenapyr and bifenthrin susceptibility shifts. Additionally, to reduce insecticide selection pressures and delay resistance development, adoption of integrated bed bug control strategies that combine chemical and nonchemical methods is recommended.”
Dang, K., S. L. Doggett, G. Veera Singham, and C.-Y. Lee. 2017. Insecticide resistance and resistance mechanisms in bed bugs, Cimex spp. (Hemiptera: Cimicidae). Parasites & Vectors. 10: 318. doi: 10.1186/s13071-017-2232-3
“The worldwide resurgence of bed bugs [both Cimex lectularius L. and Cimex hemipterus (F.)] over the past two decades is believed in large part to be due to the development of insecticide resistance. The transcriptomic and genomic studies since 2010, as well as morphological, biochemical and behavioral studies, have helped insecticide resistance research on bed bugs. Multiple resistance mechanisms, including penetration resistance through thickening or remodelling of the cuticle, metabolic resistance by increased activities of detoxification enzymes (e.g. cytochrome P450 monooxygenases and esterases), and knockdown resistance by kdr mutations, have been experimentally identified as conferring insecticide resistance in bed bugs. Other candidate resistance mechanisms, including behavioral resistance, some types of physiological resistance (e.g. increasing activities of esterases by point mutations, glutathione S-transferase, target site insensitivity including altered AChEs, GABA receptor insensitivity and altered nAChRs), symbiont-mediated resistance and other potential, yet undiscovered mechanisms may exist. This article reviews recent studies of resistance mechanisms and the genes governing insecticide resistance, potential candidate resistance mechanisms, and methods of monitoring insecticide resistance in bed bugs. This article provides an insight into the knowledge essential for the development of both insecticide resistance management (IRM) and integrated pest management (IPM) strategies for successful bed bug management.”
Fountain, T., M. Ravinet, R. Naylor, K. Reinhardt, and R. K. Butlin. 2016. A linkage map and QTL analysis for pyrethroid resistance in the bed bug Cimex lectularius. G3 (Bethesda). 6: 4059–4066. doi: 10.1534/g3.116.033092
“The rapid evolution of insecticide resistance remains one of the biggest challenges in the control of medically and economically important pests. Insects have evolved a diverse range of mechanisms to reduce the efficacy of the commonly used classes of insecticides, and finding the genetic basis of resistance is a major aid to management. In a previously unstudied population, we performed an F2 resistance mapping cross for the common bed bug, Cimex lectularius, for which insecticide resistance is increasingly widespread. Using 334 SNP markers obtained through RAD-sequencing, we constructed the first linkage map for the species, consisting of 14 putative linkage groups (LG), with a length of 407 cM and an average marker spacing of 1.3 cM. The linkage map was used to reassemble the recently published reference genome, facilitating refinement and validation of the current genome assembly. We detected a major QTL on LG12 associated with insecticide resistance, occurring in close proximity (1.2 Mb) to a carboxylesterase encoding candidate gene for pyrethroid resistance. This provides another example of this candidate gene playing a major role in determining survival in a bed bug population following pesticide resistance evolution. The recent availability of the bed bug genome, complete with a full list of potential candidate genes related to insecticide resistance, in addition to the linkage map generated here, provides an excellent resource for future research on the development and spread of insecticide resistance in this resurging pest species.”
Lilly, D.G., S. L. Latham, C.E. Webb, S. L. Doggett. 2016. Cuticle thickening in a pyrethroid-resistant strain of the common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae). Plos One. 11(4):1–13. doi: 10.1371/journal.pone.0153302
“Thickening of the integument as a mechanism of resistance to insecticides is a well recognised phenomenon in the insect world and, in recent times, has been found in insects exhibiting pyrethroid-resistance. Resistance to pyrethroid insecticides in the common bed bug, Cimex lectularius L., is widespread and has been frequently inferred as a reason for the pest’s resurgence. Overexpression of cuticle depositing proteins has been demonstrated in pyrethroid-resistant bed bugs although, to date, no morphological analysis of the cuticle has been undertaken in order to confirm a phenotypic link. This paper describes examination of the cuticle thickness of a highly pyrethroid-resistant field strain collected in Sydney, Australia, in response to time-to-knockdown upon forced exposure to a pyrethroid insecticide. Mean cuticle thickness was positively correlated to time-to-knockdown, with significant differences observed between bugs knocked-down at 2 hours, 4 hours, and those still unaffected at 24 hours. Further analysis also demonstrated that the 24 hours survivors possessed a statistically significantly thicker cuticle when compared to a pyrethroid-susceptible strain of C. lectularius. This study demonstrates that cuticle thickening is present within a pyrethroid-resistant strain of C. lectularius and that, even within a stable resistant strain, cuticle thickness will vary according to time-to-knockdown upon exposure to an insecticide. This response should thus be considered in future studies on the cuticle of insecticide-resistant bed bugs and, potentially, other insects.”
Lilly, D. G., K. Dang, C. E. Webb, and S. L. Doggett. 2016. Evidence for metabolic pyrethroid resistance in the common bed bug (Hemiptera: Cimicidae). Journal of Economic Entomology. 109: 1364–1368. doi: 10.1093/jee/tow041
“Resistance to insecticides, especially the pyrethroids, in the common bed bug, Cimex lectularius L., has been well-documented. However, the presence and relative contribution of metabolic detoxifying microsomal oxidases and hydrolytic esterases to the observed resistance has yet to be fully elucidated. This is due, in part, to the absence of a simple bioassay procedure that appropriately isolates esterases from potentially competing oxidases. Recently, an analogue of piperonyl butoxide (PBO) was developed, EN16/5-1 (6-[2-(2-butoxyethoxy)ethoxymethyl]-5-propyl-2,3-dihydrobenzofuranby), which inhibits esterases but has limited efficacy against the oxidases, whereas PBO inhibits both. The opportunity is now available to use both synergists via established bioassay methodologies and to screen for the potential presence of oxidase- or esterase-derived pyrethroid resistance in insecticide-resistant insects, including bed bugs. In the present study, EN16/5-1 and PBO were assayed in conjunction with deltamethrin against four field strains of C. lectularius collected from independent geographic locations across Australia. All strains expressed a high degree of resistance to deltamethrin and significant inhibition of the observed resistance with preexposure to PBO. Nonsignificant differences between the cumulative mortality values for PBO and EN16/5-1 were then observed in two of the four bed bug strains, which indicate that detoxifying esterases are conferring substantially to the observed resistance in those strains. This study is the first to provide evidence that metabolic detoxification in the form of both hydrolytic esterases and microsomal oxidases is a major contributing factor to pyrethroid resistance in C. lectularius.”
Lilly, D. G., C. E. Webb, and S. L. Doggett. 2016. Evidence of tolerance to silica-based desiccant dusts in a pyrethroid-resistant strain of Cimex lectularius (Hemiptera: Cimicidae). Insects. 7: 1–12. doi: 10.3390/insects7040074
“Insecticide resistance in bed bugs (Cimex lectularius and Cimex hemipterus) has become widespread, which has necessitated the development of new IPM (Integrated Pest Management) strategies and products for the eradication of infestations. Two promising options are the diatomaceous earth and silica gel-based desiccant dusts, both of which induce dehydration and eventual death upon bed bugs exposed to these products. However, the impact of underlying mechanisms that confer resistance to insecticides, such as cuticle thickening, on the performance of these dusts has yet to be determined. In the present study, two desiccant dusts, CimeXa Insecticide Dust (silica gel) and Bed Bug Killer Powder (diatomaceous earth) were evaluated against two strains of C. lectularius; one highly pyrethroid-resistant and one insecticide-susceptible. Label-rate doses of both products produced 100% mortality in both strains, albeit over dissimilar time-frames (3-4 days with CimeXa vs. 14 days with Bed Bug Killer). Sub-label rate exposure to CimeXa indicated that the pyrethroid-resistant strain possessed a degree of tolerance to this product, surviving 50% longer than the susceptible strain. This is the first study to suggest that mechanisms conferring resistance to pyrethroids, such as cuticular thickening, may have potential secondary impacts on non-synthetic insecticides, including desiccant dusts, which target the bed bug’s cuticle.”
Romero, A., and T. D. Anderson. 2016. High levels of resistance in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), to neonicotinoid insecticides. Journal of Medical Entomology. doi: 10.1093/jme/tjv253
“The rapid increase of bed bug populations resistant to pyrethroids demands the development of novel control tactics. Products combining pyrethroids and neonicotinoids have become very popular for bed bug control in the United States, but there are concerns about evolution of resistance to these compounds. Laboratory assays were used to measure the toxicity of topical applications of four neonicotinoids to a susceptible population and three pyrethroid-resistant populations. Activity of esterases, glutathione S-transferases, and cytochrome P450s of all strains was also evaluated. High levels of resistance to four neonicotinoids, acetamiprid, imidacloprid, dinotefuran, and thiamethoxam, relative to the susceptible Fort Dix population, were detected in populations collected from human dwellings in Cincinnati and Michigan. Because activity of detoxifying enzymes was increased in these two populations, our results suggest that these enzymes have some involvement in neonicotinoid resistance, but other resistance mechanisms might be involved as well. Detection of high levels of resistance to neonicotinoids further limits the options for chemical control of bed bugs.”
Campbell, C. E., and D. D. Miller. 2015. Insecticide resistance in eggs and first instars of the bed bug, Cimex lectularius (Hemiptera: Cimicidae). Insects. 6(1), 122-132. doi:10.3390/insects6010122
“Two strains of the common bed bug, Cimex lectularius L., eggs and first instars collected from pyrethroid-resistant adults were evaluated for insecticide resistance and compared to a susceptible strain. Dose-response bioassays were conducted using two insecticide formulations (Temprid: imidacloprid/β-cyfluthrin, and Transport: acetamiprid/ bifenthrin). The lethal concentration (LC50) for the two resistant egg strains exposed to imidacloprid/β-cyfluthrin ranged from 3 to 5-fold higher than susceptible strain eggs. Resistant strain eggs dipped into formulations of acetamiprid/bifenthrin had LC50 values which were significantly greater (39 to 1,080-fold) than susceptible strain eggs. Similar to eggs, resistant strain first instars exposed to residual applications of imidacloprid/β-cyfluthrin had LC50 values ranging from 121 to 493-fold greater than susceptible strain first instars. When resistant strain first instars were treated with acetamiprid/bifenthrin, they had LC50 values that were 99 to >1,900-fold greater than susceptible strain first instars. To determine differences between egg and first instar resistance, stage resistance ratios (SRR) were compared between the two stages. There was little difference between the egg and first instar stages, indicated by small SRR values ranging from 1.1 to 10.0. This study suggests that insecticide resistance is expressed early during bed bug development.”
Dang, K. ,C. S. Toi, D. G Lilly, C. Y. Lee, R. Naylor, A. Tawatsin, E. U. Thavara, et al. 2015. Identification of putative kdr mutations in the tropical bed bug, Cimex hemipterus (Hemiptera: Cimicidae). Pest Management Science. 71: 1015-1020. doi: 10.1002/ps.3880
“BACKGROUND: Bed bugs [both Cimex hemipterus (F.) and Cimex lectularius L.] are highly resistant to pyrethroids worldwide. An important resistance mechanism known as ‘knockdown resistance’ (kdr) is caused by genetic point mutations on the voltage-gated sodium channel (VGSC) gene. Previous studies have identified two point mutations (V419L and L925I) on the VGSC gene in C. lectularius that are responsible for kdr-type resistance. However, the kdr mutations in C. hemipterus have not been investigated. RESULTS: Four novel mutations, L899V (leucine to valine), M918I (methionine to isoleucine), D953G (aspartic acid to glycine) and L1014F (leucine to phenylalanine), were identified in the domain II region of the C. hemipterus VGSC gene. This region has been widely investigated for the study of kdr-type resistance to pyrethroids in other insect pests. The V419L and L925I kdr mutations as previously identified in C. lectularius were not detected in C. hemipterus. CONCLUSION: M918I and L1014F are considered to be probable kdr mutations and may play essential roles in kdr-type resistance to pyrethroids in C. hemipterus. Further studies are under way in the authors’ laboratory to determine the non-kdr-type resistance mechanisms in C. hemipterus.”
DeVries, Z. C., W. R. Reid, S. A. Kells, and A. G. Appel. 2015. Effects of starvation on deltamethrin tolerance in bed bugs, Cimex lectularius L. (Hemiptera: Cimicidae). Insects. 6: 102–111. doi: 10.3390/insects6010102
“Bed bugs, Cimex lectularius L., are a major pest in the urban environment. Their presence often results in physical, psychological, and financial distress of homeowners and apartment dwellers. Although many insecticide bioassays have been performed on this pest, little attention has been paid to bed bug feeding status, which is closely linked to metabolism, molting, and mass. Therefore, we evaluated the toxicity of topically applied deltamethrin on insecticide susceptible adult male bed bugs fed 2 d, 9 d, and 21 d prior to testing. When toxicity was evaluated on a “per-bug” basis, there was no difference between 2 d [LD50 = 0.498 (0.316 – 0.692) ng.bug-1] and 9 d [LD50 = 0.572 (0.436 – 0.724) ng.bug-1] starved bugs, while 21 d starved bugs had a significantly lower LD50 [0.221 (0.075 – 0.386) ng.bug-1]. When toxicity was evaluated in terms of body mass, 9 d starved bugs had the highest LD50 values [0.138 (0.102 – 0.176) ng.mg-1], followed by 2 d starved bugs [0.095 (0.060 – 0.134) ng.mg-1], and then 21 d starved bugs [0.058 (0.019-0.102) ng.mg-1]; the LD50 values of 2 d and 9 d starved bugs were significantly different from 21 d starved bugs. These results indicate that feeding status plays an important role in the toxicity of deltamethrin. In addition, the lack of differences between 2 d and 9 d starved bugs indicate that the blood meal itself has little impact on tolerance, but rather it is some physiological change following feeding that confers increased tolerance to bed bugs.”
Doggett, S. L., K. Dang, W. Bu, and D. G. Lilly. 2015. Simple, rapid and cost-effective technique for the detection of pyrethroid resistance in bed bugs, Cimex spp. (Hemiptera: Cimicidae). Austral Entomology. 54: 191–196. doi: 10.1111/aen.12109
“A ready to use d-allethrin impregnated mat (‘Mortein Odourless Mozzie Zapper’, Reckitt Benckiser) was used to detect pyrethroid resistance in bed bugs, Cimex spp. Bed bugs of different species, stages and strains were confined on the mat and knockdown measured over 24 h. The insecticide-susceptible ‘Monheim’ strain of Cimex lectularius was significantly (P < 0.001) more sensitive than the insecticide-resistant ‘Sydney’ strain of C. lectularius and the ‘North Queensland’ strain of C. hemipterus. Based on the results, a protocol to rapidly diagnose pyrethroid resistance is suggested whereby, unless 100% of 10 bed bugs (irrespective of the life stage) are knocked down after 1 h exposure to the d-allethrin impregnated mat at room temperature of 23 ± 1°C, the population can be considered resistant. This protocol was validated using six Australian field-collected strains of C. lectularius, which were known to possess ‘kdr’-type resistance mechanisms, of which there was no mortality at 1 h exposure. Thus, the protocol developed in this study can be used to monitor pyrethroid resistance in bed bug populations and should assist pest managers in determining the most appropriate control strategies for managing bed bug infestations.”
Donahue, W. A., M. W. Donahue, B. E. Vinson, A. T. Showler, W. L. A. Osbrink, and L. Hui. 2015. Knockdown and lethal effects of eight commercial nonconventional and two pyrethroid insecticides against moderately permethrin-resistant adult bed bugs, Cimex lectularius (L.) (Hemiptera: Cimicidae). Biopesticides International. 11: 108–117.
“The common bed bug, Cimex lectularius (L.) (Hemiptera: Cimicidae) is undergoing a rapid resurgence in the United States during the last decade which has created a notable pest management challenge largely because the pest has developed resistance against DDT, organophosphates, carbamates, and pyrethroids, the latter class of insecticide being most commonly used today. Eight nonconventional insecticides Orange Guard (a.i., d-limonene), Natria Home Pest Control (a.i., soy bean oil and eugenol), RestAsure (a.i., sodium laurel sulfate, sodium chloride, and potassium sorbate), CedarCide (a.i. cedar oil), Essentria Broadcast Insecticide (a.i., 2-phenethyl propionate, rosemary oil, and peppermint oil), EcoSmart Organic Home Pest Control (a.i., 2-phenethyl propionate, clove oil, rosemary oil, peppermint oil, and thyme oil), Cirkil (a.i., neem oil) and CimeXa (a.i., silica gel) were compared with two pyrethorids Bonide Bedbug Killer (a.i.,permethrin) and D-Force (a.i.,deltamethrin) as positive controls, and water for direct contact spray knockdown and lethal effects in the laboratory over 4 days. Orange Guard, CedarCide, Essentria, EcoSmart, and Cirkil provided extensive knockdown within 15 min (recovery was, at most, negligible), and caused 80 to 100% mortality within a day making them as effective as the two pyrethroids. CimeXa did not cause appreciable knockdown, but nearly complete mortality was achieved within a day. Product effects in terms of active ingredients and factors that might increase and decrease product effectiveness, such as cimicid aggregation behavior and residual effects, are discussed.”
Hardstone, M. C., J. P. Strycharz, J. Kim, I.-K. Park, K. S. Yoon, Y. J. Ahn, L. C. Harrington, S. H. Lee, and J. M. Clark. 2015. Development of multifunctional metabolic synergists to suppress the evolution of resistance against pyrethroids in insects that blood feed on humans. Pest Management Science. 71: 842–849. doi: 10.1002/ps.3856
“Pyrethroids are the insecticides of choice when exposure to humans is likely, such as occurs in vector and public‐health‐related control programs. Unfortunately, the pyrethroids share a common resistance mechanism with dichlorodiphenyltrichloroethane (DDT), knockdown resistance (kdr), and prior extensive use of DDT has predisposed the pyrethroids to cross‐resistance via kdr. Given the widespread occurrence of kdr, the use of synergists with pyrethroids is considered to be prudent to guard against the selection of multiply resistant insects. 3‐Phenoxybenzyl hexanoate (PBH) was synthesized as a multifunctional pyrethroid synergist that, besides being a surrogate substrate for sequestration/hydrolytic carboxylesterases, now also functions as a substrate for oxidative xenobiotic metabolism. The addition of PBH to permethrin‐treated females of the ISOP450 strain of Culex pipiens quinquefasciatus resulted in a threefold increase in synergism, as judged by the synergistic ratio. Similarly, PBH synergized the action of deltamethrin sixfold on females of the common bed bug, Cimex lectularius, and was 2.8‐fold more synergistic than piperonyl butoxide (PBO). PBH synergized the action of both type I and type II pyrethroids in a mosquito vector (Cx. p. quinquefasciatus) and in a public‐health pest, C. lectularius, respectively, indicating a broad spectrum of action on blood‐feeding insects. PBH appears to have residual properties similar to permethrin and is itself non‐toxic, unlike PBO, and therefore should be compatible with existing pyrethroid formulations used for insecticide‐treated nets and home/residential sprays.”
Koganemaru, R. 2015. Reduced cuticular penetration as a contributor to insecticide resistance in the common bed bug, Cimex lectularius L. (doctoral dissertation).
“The Common bed bug, Cimex lectularius L., suddenly reappeared in developed countries in the past 15 years. The factor contributing to the sudden resurgence of the bed bugs is insecticide resistance. In this study, we investigated the mechanisms of reduced cuticular penetration type insecticide resistance in bed bugs. First, we determined and compared the lethal dosage (LD50) of a pyrethroid insecticide using topical and injection application. The resistant strain not only had significantly greater resistance ratios, but also demonstrated significantly greater penetration resistance ratios. This provided the evidence of the reduced cuticular penetration in bed bugs. Second, we determined the levels of gene transcription (CPR-type cuticle protein genes) using real-time quantitative polymerase chain reaction (qRT-PCR). We identified 62 putative bed bug cuticle protein-encoding contigs based on the presence of the Chitin-binding 4 (CB4) domain. Based on the qRT-PCR analysis of the mRNAs, we found many of the genes were up-regulated in the resistant strain suggesting thickening of the cuticle or increasing the cuticular proteins might be involved in the reduced cuticular penetration. Third, we identified and described the cuticular proteins using the matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometry (MALDI- TOF/TOF). The total of 265 peptides were identified, among which 206 belonged to one of 50 confidently identified proteins. We identified the CPRL, CPF, CPFL, TWDL, and CPAP1 family proteins. The profile of the cuticular proteins between the resistant and the susceptible strains bed bugs were almost identical. Fourth, we determined and compared the cuticular thickness using Scanning Electron Microscopy (SEM). We found statistical differences of the cuticular thickness among different strains (populations), however, correlation between the levels of insecticide resistance and cuticular thickness were not found. Finally, we identified and described bed bug cuticular hydrocarbon profiles using Gas-Chromatography and Mass-Spectrometry (GC-MS). The total of 87 compounds in addition to n-alkanes were extracted and identified. There were no correlation found with the concentration and the levels of insecticide resistance. However, several additional compounds exhibited the correlation between the concentration of the compounds and the levels of insecticide resistance. Overall, we found three lines of evidence to support reduced cuticular penetration as a mechanism of insecticide resistance in some bed bug populations. This study provides additional evidence of the reduced cuticular penetration type resistance in bed bugs.”
Lilly, D. G., R. C. Russell, C. E. Webb, S. L. Doggett, M. P. Zalucki, and C. J. Orton. 2015. Confirmation of insecticide resistance in Cimex lectularius Linnaeus ( Hemiptera: Cimicidae) in Australia. Austral Entomology. 54: 96–99. doi: 10.1111/aen.12098
“Insecticide resistance in the common bed bug, Cimex lectularius Linnaeus has been suspected in Australia with anecdotal reports of poor product performance. To investigate this possibility, LD50 values were determined via topical application of technical grade permethrin, deltamethrin, bendiocarb, pirimiphos-methyl and imidacloprid serially diluted in acetone to a suspected resistant field-collected strain and a susceptible laboratory strain. All compounds tested against the susceptible strain were efficacious. However, for the field strain, only pirimiphos-methyl and imidacloprid showed high levels of activity. Resistance was confirmed in the field-collected strain to the pyrethroids and bendiocarb, but not to pirimiphos-methyl or imidacloprid. Resistance factors (“resistant” LD50/susceptible LD50) for each compound were: permethrin ≈ 1.235 million, deltamethrin ≈ 370 000, bendiocarb ≈ 250, pirimiphos-methyl = 2.6, imidacloprid = 2.6. Bendiocarb, permethrin and deltamethrin all failed to return greater than 60% mortality at the maximum dose of 100 μg/μ L. This research has significant operational implications for bed bug control and the registration process of new products in Australia.”
Dang, K., C. S. Toi, D. G. Lilly, W. Bu, and S. L. Doggett. 2014. Detection of knockdown resistance mutations in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), in Australia. Pest Management Science. 71(7):914-922. doi: 10.1002/ps.3861
“Pyrethroid resistance in the common bed bug, Cimex lectularius L., has been reported worldwide. An important resistance mechanism is via knockdown resistance (kdr) mutations, notably V419L and L925I. Information regarding this kdr-type resistance mechanism is unknown in Australia. This study aims to examine the status of kdr mutations in Australian C. lectularius strains.”
Gordon, J. R., M. H. Goodman, M. F. Potter, and K. F. Haynes. 2014. Population variation in and selection for resistance to pyrethroid-neonicotinoid insecticides in the bed bug. Scientific Reports. 4:3836. doi: 10.1038/srep03836
“Pyrethroid resistance in bed bugs, Cimex lectularius, has prompted a change to combination products that include a pyrethroid and a neonicotinoid. Ten populations of bed bugs were challenged with two combination products (Temprid SC and Transport GHP). Susceptibility of these populations varied, with the correlated response of the two products indicating cross resistance. We imposed selection on three populations using label rate Temprid, and then reared progeny from unselected and selected strains. All selected strains were significantly less susceptible to Temprid SC than unselected strains. Temprid selected strains were also less susceptible to Transport. The pyrethroid component of Temprid showed a significantly higher LD50 in selected strains, but susceptibility to the neonicotinoid remained unchanged. Taken together these results indicate resistance to combination insecticides is present in field populations at levels that should be of concern, and that short-term selection affecting existing variance in susceptibility can quickly increase resistance.”
Mamidala, P., P. Mittapelly, S. C. Jones, P. M. Piermarini, and O. Mittapalli. 2013. Molecular characterization of genes encoding inward rectifier potassium (Kir) channels in the bed bug (Cimex lectularius). Comparative Biochemistry and Physiology–B: Biochemistry and Molecular Biology. 164: 275–279. doi: 10.1016/j.cbpb.2013.02.002
“The molecular genetics of inward-rectifier potassium (Kir) channels in insects is poorly understood. To date, Kir channel genes have been characterized only from a few representative dipterans (i.e., fruit flies and mosquitoes). The goal of the present study was to characterize Kir channel cDNAs in a hemipteran, the bed bug (Cimex lectularius). Using our previously reported bed bug transcriptome (RNA-seq), we identified two cDNAs that encode putative Kir channels. One was a full-length cDNA that encodes a protein belonging to the insect ‘Kir3’ clade, which we designate as ‘ClKir3’. The other was a partial cDNA that encodes a protein with similarity to both the insect ‘Kir1’ and ‘Kir2’ clades, which we designate as ‘ClKir1/2’. Quantitative real-time PCR analysis revealed that ClKir1/2 and ClKir3 exhibited peak expression levels in late-instar nymphs and early-instar nymphs, respectively. Furthermore, ClKir3, but not ClKir1/2, showed tissue-specific expression in Malpighian tubules of adult bed bugs. Lastly, using an improved procedure for delivering double-stranded RNA (dsRNA) to male and female bed bugs (via the cervical membrane) we demonstrate rapid and systemic knockdown of ClKir3 transcripts. In conclusion, we demonstrate that the bed bug possesses at least two genes encoding Kir channels, and that RNAi is possible for at least Kir3, thereby offering a potential approach for elucidating the roles of Kir channel genes in bed bug physiology.”
Rinkevich, F. D., Y. Du, and K. Dong. 2013. Diversity and convergence of sodium channel mutations involved in resistance to pyrethroids. Pesticide Biochemistry and Physiology. 106: 93–100. doi: 10.1016/j.pestbp.2013.02.007
“Pyrethroid insecticides target voltage-gated sodium channels, which are critical for electrical signaling in the nervous system. The intensive use of pyrethroids in controlling arthropod pests and disease vectors has led to many instances of pyrethroid resistance around the globe. In the past two decades, studies have identified a large number of sodium channel mutations that are associated with resistance to pyrethroids. The purpose of this review is to summarize both common and unique sodium channel mutations that have been identified in arthropod pests of importance to agriculture or human health. Identification of these mutations provides valuable molecular markers for resistance monitoring in the field and helped the discovery of the elusive pyrethroid receptor site(s) on the sodium channel.”
Davies, T. G. E., L. M. Field, and M. S. Williamson. 2012. The re-emergence of the bed bug as a nuisance pest: implications of resistance to the pyrethroid insecticides. Medical and Veterinary Entomology. 26: 241–254.
“A global resurgence of bed bugs (Hemiptera: Cimicidae) has led to renewed scientific interest in these insects. The current bed bug upsurge appears to have started almost synchronously in the late 1990 s in Europe, the U.S.A. and Australia. Several factors have led to this situation, with resistance to applied insecticides making a significant contribution. With a growing number of insecticides (DDT, carbamates, organophosphates etc.) being no longer available as a result of regulatory restrictions, the mainstay chemistry used for bed bug control over the past few decades has been the pyrethroid insecticides. With reports of increasing tolerance to pyrethroids leading to control failures on the rise, containing and eradicating bed bugs is proving to be a difficult task. Consequently, several recent studies have focused on determining the mode of action of pyrethroid resistance in bed bug populations sourced from different locations. Correct identification of the factor(s) responsible for the increasing resistance is critical to the development of effective management strategies, which need to be based, wherever possible, on firm scientific evidence. Here we review the literature on this topic, highlighting the mechanisms thought to be involved and the problems currently faced by pest control professionals in dealing with a developing pandemic.”
Mamidala, P., A. J. Wijeratne, S. Wijeratne, K. Kornacker, B. Sudhamalla, L. J. River-Vega, A. Hoelmer, T. Meulia, S. C. Jones, and O. Mittapalli. 2012. RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug. BMC Genomics. 13:6. doi: 10.1186/1471-2164-13-6
“BACKGROUND: Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance.
“RESULTS: We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid.
“CONCLUSIONS: We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius.”
Zhu, F., S. Sams, T. Moural, K. F. Haynes, M. F. Potter, and S. R. Palli. 2012. RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius. PLoS One. 7: e31037. doi: 10.1371/journal.pone.0031037
“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.”
Adelman, Z. N., K. A. Kilcullen, R. Koganemaru, M. A. E. Anderson, T. D. Anderson, and D. M. Miller. 2011. Deep sequencing of pyrethroid-resistant bed bugs reveals multiple mechanisms of resistance within a single population. PLoS One. 6: e26228. doi: 10.1371/journal.pone.0026228
“A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD50 bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.”
Bai, X., P. Mamidala, S. P. Rajarapu, S. C. Jones, and O. Mittapalli. 2011. Transcriptomics of the bed bug (Cimex lectularius). PLoS One. 6: 1–10.
Molecular techniques were used to identify potential genes involved in insecticide resistance in the bed bug. Genes for cytochrome P450 monoxygenases were more highly expressed in resistant bed bugs than susceptible bed bugs.
Cater, Jason; Magee, Danny; Hubbard, Sue Ann; Edwards, Kristine T.; Goddard, J. 2011. Severe infestation of bed bugs in a poultry breeder house. Journal of the American Veterinary Medical Association. 239: 919.
Letter to the Editor regarding severe bed bug infestations in two chicken breeder houses on a farm in the southeastern U.S. Control of the bed bugs was affected by their pyrethroid resistance.
Kilpinen, O., M. Kristensen, and K.-M. V. Jensen. 2011. Resistance differences between chlorpyrifos and synthetic pyrethroids in Cimex lectularius population from Denmark. Parasitology Research. 109: 1461. doi: 10.1007/s00436-011-2423-3
“Bed bug, Cimex lectularius L., populations were investigated for resistance against permethrin and chlorpyrifos in a topical application bioassay, after an initial establishment of a discriminating dose with a susceptible population. For both insecticides, ca. two times the lethal dose LD99 was selected: 2,560 ng of permethrin and 200 ng of chlorpyrifos per bed bug, respectively. Bed bugs were collected from infested homes in Denmark at ten locations and bred in the laboratory. The frequency of permethrin-resistant individuals was high in Danish bed bug populations as susceptible individuals were only found in three of ten populations. In contrast, the frequency of chlorpyrifos-resistant individuals was low in Danish bed bug populations, but resistant individuals were found in five of ten populations. To test the significance of the observed resistance, we performed tarsal contact test with commercially available insecticides. The test indicated that both a permethrin and a deltamethrin product had very low efficacy against the field-collected bed bug populations. Despite the reduced sensitivity to synthetic pyrethroids, all populations tested in the tarsal test on the commercial product with micro-encapsulated chlorpyrifos resulted in close to 100% mortality.”
Mamidala, P., S. C. Jones, and O. Mittapalli. 2011. Metabolic resistance in bed bugs. Insects. 2:36-48. doi: 10.3390/insects2010036
“Blood-feeding insects have evolved resistance to various insecticides (organochlorines, pyrethroids, carbamates, etc.) through gene mutations and increased metabolism. Bed bugs (Cimex lectularius) are hematophagous ectoparasites that are poised to become one of the major pests in households throughout the United States. Currently, C. lectularius has attained a high global impact status due to its sudden and rampant resurgence. Resistance to pesticides is one factor implicated in this phenomenon. Although much emphasis has been placed on target sensitivity, little to no knowledge is available on the role of key metabolic players (e.g., cytochrome P450s and glutathione S-transferases) towards pesticide resistance in C. lectularius. In this review, we discuss different modes of resistance (target sensitivity, penetration resistance, behavioral resistance, and metabolic resistance) with more emphasis on metabolic resistance.”
Mamidala, P., S. P. Rajarapu, S. C. Jones, and O. Mittapalli. 2011. Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius. Journal of Medical Entomology. 48(4):947-951. doi: 10.1603/ME10262
“Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.”
Tawatsin, A., U. Thavara, J. Chompoosri, Y. Phusup, N. Jonjang, C. Khumsawads, P. Bhakdeenuan, P. Sawanpanyalert, P. Asavadachanukorn, M. S. Mulla, P. Siriyasatien, and M. Debboun. 2011. Insecticide resistance in bedbugs in Thailand and laboratory evaluation of insecticides for the control of Cimex hemipterus and Cimex lectularius (Hemiptera: Cimicidae). Journal of Medical Entomology. 48: 1023–1030. doi: 10.1603/ME11003
“Bedbugs are found in many countries around the world, and in some regions they are resistant to numerous insecticides. This study surveyed bedbugs in Thailand and determined their resistance to insecticides. The surveys were carried out in six provinces that attract large numbers of foreign tourists: Bangkok, Chonburi, Chiang Mai, Ubon Ratchathani, Phuket, and Krabi. Bed bugs were collected from hotels and colonized in the laboratory to evaluate their resistance to insecticides. Cimex hemipterus (F.) was found in some hotels in Bangkok, Chonburi, Phuket, and Krabi, whereas Cimex lectularius L. was found only in hotels in Chiang Mai. No bedbugs were found in Ubon Ratchathani. The colonized bedbugs showed resistance to groups of insecticides, including organochlorines (di- chlorodiphenyl trichloroethane, dieldrin), carbamates (bendiocarb, propoxur), organophosphates (malathion, fenitrothion), and pyrethroids (cyfluthrin, deltamethrin, permethrin, lambda -cyhalothrin, etofenprox) in tests using World Health Organization insecticide-impregnated papers. The new insecticides imidacloprid (neonicotinoid group), chlorfenapyr (pyrrole group), and fipronil (phe- nylpyrazole group) were effective against the bedbugs; however, organophosphate (diazinon), carbamates (fenobucarb, propoxur), and pyrethroids (bifenthrin, cypermethrin, esfenvalerate, etofenprox) were ineffective. Aerosols containing various pyrethroid insecticides with two to four different active ingredients were effective against the bedbugs. The results obtained from this study suggested that both species of bedbugs in Thailand have developed marked resistance to various groups of insecticides, especially those in the pyrethroid group, which are the most common insecticides used for pest control. Therefore, an integrated pest management should be implemented for managing bedbugs in Thailand.”
Tomita, T. 2010. Insecticide resistance of bedbugs. Medical Entomology and Zoology. 61: 223–229. doi: 10.7601/mez.61.223
A literature review (in Japanese) focusing on the mechanisms for pyrethroid resistance in bed bugs. Lowered sensitivity of the voltage gated sodium channel and increased metabolism by cytochrome P450s are involved in major pyrethroid resistance.
Romero, A., M. F. Potter, D. A. Potter, and K. F. Haynes. 2009. Evaluation of piperonyl butoxide as a deltamethrin synergist for pyrethroid-resistant bed bugs. Journal of Economic Entomology. 102: 2310–2315. doi: 10.1603/029.102.0637
“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 andWOR-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.”
Steelman, C. D., A. L. Szalanski, R. Trout, J. A. McKern, C. Solorzano, and J. W. Austin. 2008. Susceptibility of the bed bug Cimex lectularius L. (Heteroptera: Cimicidae) collected in poultry production facilities to selected insecticides. Journal of Agricultural and Urban Entomology. 25: 41–51. doi: 10.3954/1523-5475-25.1.41
“Cimex lectularius L. is a widespread hematophagus insect pest around the world and is currently experiencing a reemergence as a public health pest of concern. One possible source of bed bugs to the human environment is the movement of bed bugs from poultry facilities to human structures by poultry workers. No recent studies have been conducted on the susceptibility of this insect to a wide range of insecticides. In addition, populations of bed bugs from poultry facilities have not been screened against insecticides for over 15 yr. Adult bed bugs collected from three poultry facilities in northwest Arkansas were exposed for 24 or 48 h (25°C) to glass vials treated with various dilutions of 12 insecticides dissolved in acetone to determine the concentration–response relationship. The order of toxicity, from most to least based on the LC50s was: λ-cyhalothrin, bifenthrin, carbaryl, imidacloprid, fipronil, permethrin, diazinon, spinosyn, dichlorvos, chlorfenapyr, and DDT. Significant differences in LC50 and LC90 values for diazinon was observed among the three populations due to the previous history of repeated exposure to a mixture of tetrachlorvinphos and dichlorvos over a 10 yr period when compared to the LC50s of two populations that had been exposed to the tetrachlorvinphos and dichlorvos mixture during 2–3 flock cycles. Bed bugs in each of the three populations exhibited high levels of DDT resistance, LC50 > 100,000 ppm, which confirms that resistance to this insecticide continues in bed bug populations. This study documents baseline toxicological data for 12 insecticides in three populations of bed bugs and provides the first data on bed bug susceptibility to fipronil, spinosyn, and imidacloprid.”
Romero, A., M. F. Potter, D. A. Potter, and K. F. Haynes. 2007. Insecticide resistance in the bed bug: a factor in the pest’s sudden resurgence? Journal of Medical Entomology. 44: 175–178. doi: 10.1093/jmedent/44.2.175
“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 lambda -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.”