Transcriptomic identification and characterization of genes commonly responding to sublethal concentrations of six different insecticides in the common fruit fly, Drosophila melanogaster.

Pretreatment with sublethal concentrations (LC10) of six insecticides (chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb, ivermectin, and spinosad) significantly elevated tolerance of the common fruit fly Drosophila melanogaster to lethal concentration of the respective insecticide. Commonly responding genes to sublethal treatments of the six insecticides were identified by transcriptome analysis based on a fold change >1.5 or < -1.5, and p < 0.05 as selection criteria. Following treatment with all the six insecticides, 26 transcripts were commonly over-transcribed, whereas 30 transcripts were commonly under-transcribed. Reliability of the transcriptome data was confirmed by quantitative PCR. A majority of the over-transcribed genes included those related to olfactory behavior, such as odorant-binding proteins, as well as immune-related genes, including attacin, diptericin, and immune-induced molecule 18. In contrast, genes belonging to the mitochondrial respiratory chain, such as mitochondrial NADH-ubiquinone oxidoreductase chain 1/3/4/5 and mitochondrial cytochrome b/c, were commonly under-transcribed. Furthermore, genes related to eggshell formation and motion were also under-transcribed, which may indicate a possible energy trade-off for xenobiotic stress. In summary, most of the differentially expressed genes were not directly related to well-known detoxification genes, suggesting that the roles of commonly expressed tolerance-related genes are not likely related to direct metabolic detoxification, but rather are associated with restoration of homeostasis.

Transcriptomic identification and characterization of genes responding to sublethal doses of three different insecticides in the western flower thrips, Frankliniella occidentalis.

Pretreatment with sublethal concentrations (LC10) of three insecticides (chlorfenapyr, dinotefuran, and spinosad) enhanced tolerance to a lethal dose of the respective insecticide in the Western flower thrips, Frankliniella occidentalis. To identify genes responding to sublethal treatment with insecticides, transcriptome analysis was conducted for thrips treated with LC10 of the three insecticides. When based on a fold change >1.5 or < -1.5 as a selection criterion, 199 transcripts were commonly up-regulated, whereas 31 transcripts were commonly down-regulated following all three insecticide treatments. The differential expression levels of representative genes were validated by quantitative PCR. Most over-transcribed transcripts could be categorized as basic biological processes, such as proteolysis and lipid metabolism. Detoxification genes, such as one glutathione S transferase S1, three UDP-glucuronosyltransferases, four CYP450s, and one ABC transporter G family member 20, were commonly overexpressed in all three insecticide-treated groups. Knockdown of the five representative commonly overexpressed genes via ingestion RNA interference increased mortalities to all the three test insecticides, supporting their common role in tolerance induction. In contrast, three C2H2-type zinc finger-containing proteins were significantly down-regulated in all insecticide-treated thrip groups. Since the tested insecticides have distinct structures and modes of action, the roles of commonly expressed genes in tolerance were discussed.

Transcriptome-based identification and characterization of genes commonly responding to five different insecticides in the diamondback moth, Plutella xylostella.

When the 3rd instar larvae of the diamondback moth (DBM), Plutella xylostella, were pretreated with sublethal doses (LC10) and then subsequently exposed to lethal doses (LC50) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad via leaf dipping, their tolerance to insecticides was significantly enhanced. To identify genes that commonly respond to the treatment of different insecticides and are responsible for the tolerance enhancement, transcriptomic profiles of larvae treated with sublethal doses of the five insecticides were compared with that of untreated control. A total of 117,181 transcripts with a mean length of 662bp were generated by de novo assembly, of which 35,329 transcripts were annotated. Among them, 125, 143, 182, 215 and 149 transcripts were determined to be up-regulated whereas 67, 45, 60, 60 and 38 genes were down-regulated following treatments with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively. Gene ontology (GO) analysis of differentially expressed genes (DEGs) revealed little differences in their GO profiles between treatments with different insecticides except for spinosad. Finally, the DEGs commonly responding to all insecticides were selected for further characterization, and some of their over-transcription levels were confirmed by quantitative PCR. The most notable examples of commonly responding over-transcribed genes were two cytochrome P450 genes (Cyp301a1 and Cyp9e2) and nine cuticular protein genes. In contrast, several genes composing the mitochondrial energy generation system were significantly down-regulated in all treated larvae. Considering the distinct structure and mode of action of the five insecticides tested, the differentially expressed genes identified in this study appear to be involved in general chemical defense at the initial stage of intoxication. Their possible roles in the tolerance/resistance development were discussed.

Identification and interaction of multiple genes resulting in DDT resistance in the 91-R strain of Drosophila melanogaster by RNAi approaches.

4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will likely increase in terms of level and extent. Drosophila melanogaster is a model dipteran with a well annotated genome allowing both forward and reverse genetic manipulation, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold) and recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms. Their interactions, however, remain unclear. Use of Gal4/UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has identified the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in decreased sensitivity to DDT. These above findings have been further validated in 91-R flies using a nanoparticle-enhanced RNAi strategy, directly implication these genes in DDT resistance in 91-R flies.

Quantitative Sequencing for the Determination of Kdr-type Resistance Allele (V419L, L925I, I936F) Frequencies in Common Bed Bug (Hemiptera: Cimicidae) Populations Collected from Israel.

Human bed bug infestations have dramatically increased worldwide since the mid-1990s. A similar phenomenon was also observed in Israel since 2005, when infestations were reported from all over the country. Two single nucleotide polymorphisms (V419L and L925I) in the bed bug voltage-sensitive sodium channel confer kdr-type resistance to pyrethroids. Using quantitative sequencing (QS), the resistance allele frequencies of Israeli bed bug populations from across the country were determined. Genomic DNA was extracted from samples of 12 populations of bed bugs collected from Israel and DNA fragments containing the V419L or L925I and I936F mutations sites were PCR amplified. The PCR products were analyzed by QS and the nucleotide signal ratios calculated and used to predict the resistance allele frequencies of the unknown populations. Results of the genetic analysis show that resistant nucleotide signals are highly correlated to resistance allele frequencies for both mutations. Ten of the 12 tested populations had 100% of the L925I mutation and 0% of the V419L mutation. One population was heterogeneous for the L925I mutation and had 0% of the V419L mutation and another population was heterogeneous for the V419L mutation and had 100% of the L925I mutation. I936F occurred only at low levels. These results indicate that bed bugs in Israel are genetically resistant to pyrethroids. Thus, pyrethroids should only be used for bed bug management with caution using effective application and careful monitoring procedures. Additionally, new and novel-acting insecticides and nonchemical means of controlling bed bugs should be explored.

Toxicodynamic mechanisms and monitoring of acaricide resistance in the two-spotted spider mite.

The two-spotted spider (Tetranychus urticae) is one of the most serious pests world-wide and has developed resistance to many types of acaricides. Various mutations on acaricide target site genes have been determined to be responsible for toxicodynamic resistance, and the genotyping and frequency prediction of these mutations can be employed as an alternative resistance monitoring strategy. A quantitative sequencing (QS) protocol was reported as a population-based genotyping technique, and applied for the determination of resistance allele frequencies in T. urticae field populations. In addition, a modified glass vial bioassay method (residual contact vial bioassay, RCV) was implemented as a rapid on-site resistance monitoring tool. The QS protocol, together with the RCV, would greatly facilitate monitoring of T. urticae resistance. Recent completion of T. urticae genome analysis should facilitate the identification of additional resistance genetic markers that can be employed for molecular resistance monitoring.

RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster.

4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance.

Utilization of the human louse genome to study insecticide resistance and innate immune response.

Since sequencing the human body louse genome, substantial advances have occurred in the utilization of the information gathered from louse genomes and transcriptomes. Comparatively, the body louse genome contains far fewer genes involved in environmental response, such as xenobiotic detoxification and innate immune response. Additionally, the body louse maintains a primary bacterial endosymbiont, Candidatus Riesia pediculicola, and a number of bacterial pathogens that it vectors, which have genomes that are also reduced in size. Thus, human louse genomes offer unique information and tools for use in advancing our understanding of coevolution among vectors, endosymbionts and pathogens. In this review, we summarize the current literature on the extent of pediculicide resistance, the availability of new pediculicides and information establishing this organism as an efficient model to study how xenobiotic metabolism, which is involved in insecticide resistance, is induced and how insects modify their innate immune response upon bacterial challenge resulting in enhanced vector competence.

Development of multifunctional metabolic synergists to suppress the evolution of resistance against pyrethroids in insects that blood feed on humans.

BACKGROUND: 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. RESULTS: 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). CONCLUSIONS: 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.

Identification and characterization of an esterase involved in malathion resistance in the head louse Pediculus humanus capitis.

Enhanced malathion carboxylesterase (MCE) activity was previously reported to be involved in malathion resistance in the head louse Pediculus humanus capitis (Gao et al., 2006 [8]). To identify MCE, the transcriptional profiles of all five esterases that had been annotated to be catalytically active were determined and compared between the malathion-resistant (BR-HL) and malathion-susceptible (KR-HL) strains of head lice. An esterase gene, designated HLCbE3, exhibited approximately 5.4-fold higher transcription levels, whereas remaining four esterases did not exhibit a significant increase in their transcription in BR-HL, indicating that HLCbE3 may be the putative MCE. Comparison of the entire cDNA sequences of HLCbE3 revealed no sequence differences between the BR-HL and KR-HL strains and suggested that no single nucleotide polymorphism is associated with enhanced MCE activity. Two copies of the HLCbE3 gene were observed in BR-HL, implying that the over-transcription of HLCbE3 is due to the combination of a gene duplication and up-regulated transcription. Knockdown of HLCbE3 expression by RNA interference in the BR-HL strain led to increases in malathion susceptibility, confirming the identity of HLCbE3 as a MCE responsible for malathion resistance in the head louse. Phylogenetic analysis suggested that HLCbE3 is a typical dietary esterase and belongs to a clade containing various MCEs involved in malathion resistance.

Permethrin alters adipogenesis in 3T3-L1 adipocytes and causes insulin resistance in C2C12 myotubes.

Pyrethroids are a class of insecticides structurally derived from the naturally occurring insecticides called pyrethrins. Along with emerging evidence that exposure to insecticides is linked to altered weight gain and glucose homeostasis, exposure to pyrethroids has been linked to altered blood glucose levels in humans. Thus, the purpose of this study was to determine the role of permethrin on lipid and glucose metabolisms. Permethrin was treated to 3T3-L1 adipocytes and C2C12 myoblasts to determine its role in lipid and glucose metabolisms, respectively. Permethrin treatment resulted in increased expression of key markers of adipogenesis and lipogenesis in adipocytes. Permethrin significantly reduced insulin-stimulated glucose uptake in myotubes. This is the first report on the role of permethrin in altered lipid metabolism in adipocytes and impaired glucose homeostasis in myotubes. These results may help elucidate fundamental underlying mechanisms between insecticide exposure, particularly permethrin, and potential risk of developing obesity and its comorbidities.

Knockdown resistance allele frequencies in North American head louse (Anoplura: Pediculidae) populations.

The study examines the extent and frequency of a knockdown-type resistance allele (kdr type) in North American populations of human head lice. Lice were collected from 32 locations in Canada and the United States. DNA was extracted from individual lice and used to determine their zygosity using the serial invasive signal amplification technique to detect the kdr-type T917I (TI) mutation, which is most responsible for nerve insensitivity that results in the kdr phenotype and permethrin resistance. Previously sampled sites were resampled to determine if the frequency of the TI mutation was changing. The TI frequency was also reevaluated using a quantitative sequencing method on pooled DNA samples from selected sites to validate this population genotyping method. Genotyping substantiated that TI occurs at high levels in North American lice (88.4%). Overall, the TI frequency in U.S. lice was 84.4% from 1999 to 2009, increased to 99.6% from 2007 to 2009, and was 97.1% in Canadian lice in 2008. Genotyping results using the serial invasive signal amplification reaction (99.54%) and quantitative sequencing (99.45%) techniques were highly correlated. Thus, the frequencies of TI in North American head louse populations were found to be uniformly high, which may be due to the high selection pressure from the intensive and widespread use of the pyrethrins- or pyrethroid-based pediculicides over many years, and is likely a main cause of increased pediculosis and failure of pyrethrins- or permethrin-based products in Canada and the United States. Alternative approaches to treatment of head lice infestations are critically needed.

Imidacloprid, a neonicotinoid insecticide, induces insulin resistance.

Recently, scientific evidence supports a connection between environmental chemical exposures, which includes insecticides, and development of type 2 diabetes. However, there is limited information about the link between influences of neonicotinoid insecticides and incidence of type 2 diabetes. Thus, the purpose of the study was to determine effects of imidacloprid, a neonicotinoid insecticide, on glucose metabolism. Three different cell models were used; adipocytes (3T3-L1), hepatocytes (HepG2), and myotubes (C2C12). These cells were treated with imidacloprid (0, 10, and 20 µM) for 4-6 days followed by treatment with insulin for 15 min to determine responses. Insulin stimulated glucose uptake was reduced by imidacloprid in all three cell culture models. Treatment with imidacloprid reduced phosphorylation of protein kinase B (AKT), one of the major regulators of insulin signaling, without changing overall AKT expression. Subsequently, imidacloprid reduced phosphorylation of ribosomal S6 kinase (S6K), which is a downstream target of AKT and also a feed-back inhibitor of insulin signaling. These results suggest that imidacloprid could induce insulin resistance by affecting the insulin signaling cascade, particularly up-stream of AKT, in adipocytes, liver, and muscle.

Bartonella quintana deploys host and vector temperature-specific transcriptomes.

The bacterial pathogen Bartonella quintana is passed between humans by body lice. B. quintana has adapted to both the human host and body louse vector niches, producing persistent infection with high titer bacterial loads in both the host (up to 10(5) colony-forming units [CFU]/ml) and vector (more than 10(8) CFU/ml). Using a novel custom microarray platform, we analyzed bacterial transcription at temperatures corresponding to the host (37°C) and vector (28°C), to probe for temperature-specific and growth phase-specific transcriptomes. We observed that transcription of 7% (93 genes) of the B. quintana genome is modified in response to change in growth phase, and that 5% (68 genes) of the genome is temperature-responsive. Among these transcriptional changes in response to temperature shift and growth phase was the induction of known B. quintana virulence genes and several previously unannotated genes. Hemin binding proteins, secretion systems, response regulators, and genes for invasion and cell attachment were prominent among the differentially-regulated B. quintana genes. This study represents the first analysis of global transcriptional responses by B. quintana. In addition, the in vivo experiments provide novel insight into the B. quintana transcriptional program within the body louse environment. These data and approaches will facilitate study of the adaptation mechanisms employed by Bartonella during the transition between human host and arthropod vector.

Ovicidal response of NYDA formulations on the human head louse (Anoplura: Pediculidae) using a hair tuft bioassay.

Using the in vitro rearing system in conjunction with the hair tuft bioassay, NYDA and NYDA without fragrances formulations (92% wt:wt dimeticones) were 100% ovicidal (0% of treated eggs hatched) after an 8-h exposure of the eggs of the human head louse (Pediculus humanus capitis De Geer) following the manufacturer's instructions. Comparatively, 78 and 66% of eggs similarly exposed hatched after distilled deionized water or Nix (1% permethrin) treatments, respectively. NYDA and NYDA without fragrances formulations were also statistically and substantially more ovicidal than either distilled deionized water or Nix treatments after 10, 30 min, and 1 h exposures. Only the 10 min exposure of eggs to NYDA and NYDA without fragrances formulations resulted in hatched lice that survived to adulthood (5-8% survival). Of the lice that hatched from eggs exposed to NYDA formulations for 10 min, there were no significant differences in the time it took them to become adults, female fecundity or the viability of eggs laid by surviving females. The longevity of adults, however, was reduced after the 10 min treatments of eggs with NYDA and NYDA without fragrances formulations compared with either the distilled deionized water or Nix treatments.

Advances in the mode of action of pyrethroids.

The ability to clone, express, and electrophysiologically measure currents carried by voltage-gated ion channels has allowed a detailed assessment of the action of pyrethroids on various target proteins.Recently, the heterologous expression of various rat brain voltage-gated sodium channel isoforms in Xenopus laevis oocytes has determined a wide range of sensitivities to the pyrethroids, with some channels virtually insensitive and others highly sensitive. Furthermore, some isoforms show selective sensitivity to certain pyrethroids and this selectivity can be altered in a state-dependent manner. Additionally, some rat brain isoforms are apparently more sensitive to pyrethroids than the corresponding human isoform. These finding may have significant relevance in judging the merit and value of assessing the risk of pyrethroid exposures to humans using toxicological studies done in rat.Other target sites for certain pyrethroids include the voltage-gated calcium and chloride channels. Of particular interest is the increased effect of Type II pyrethroids on certain phosphoforms of the N-type Ca(v)2.2 calcium channel following post-translational modification and its relationship to enhanced neurotransmitter release seen in vivo.Lastly, parallel neurobehavioral and mechanistic studies on three target sites suggest that a fundamental difference exists between the action of Types I and II pyrethroids, both on a functional and molecular level. These differences should be considered in any future risk evaluation of the pyrethroids.

Enhanced repellency of binary mixtures of Zanthoxylum armatum seed oil, vanillin, and their aerosols to mosquitoes under laboratory and field conditions.

The repellency of Zanthoxylum armatum seed oil (ZA-SO), alone or in combination with vanillin (VA), its six major constituents, and another four major previously known Zanthoxylum piperitum fruit oil constituents, as well as aerosol products containing 5 or 10% ZA-SO and 5% VA, was evaluated against female Aedes aegypti in laboratory and field studies. Results were then compared with those of N,N-diethyl-3-methylbenzamide (DEET) as a standard. Hand in cage laboratory tests showed that 0.2, 0.1, and 0.05 mg/cm2 ZA-SO resulted in > 92% protection through 30-min postexposure and was not significantly different than 0.05 mg/cm2 DEET. Skin treated with linalool and limonene (from Z. armatum) provided > 80% repellency to female Ae. aegypti at 10-min exposure, whereas cuminaldehyde, citronellal, geranyl acetate, and cuminyl alcohol (from Zanthoxylum piperitum) provided > 90% protection during this same time period. Only cuminaldehyde and citronellal provided complete protection comparable to DEET at 10-min postexposure. After that time, repellency of all plant constituents to mosquitoes was considerably decreased (< approximately 65%). An increase in repellency and duration of effectiveness was produced by a binary 1:4 mixture of ZA-SO and VA (0.05:0.2 mg/cm2) that was significantly more effective than 0.05 mg/cm2 DEET through 90 min. In field tests, an aerosol formulation containing 5 or 10% ZA-SO plus 5% VA gave 100% repellency at 60-min postexposure. Although these formulations were equal to the level of protection afforded by 10% DEET, repellency to the binary ZA-SO aerosol formulations at 90 min was significantly less effective than DEET. However, mixtures formulated from ZA-SO and VA merit further study as potential repellents for protection of humans and domestic animals from biting and nuisance caused by mosquitoes.

Comparison of the humoral and cellular immune responses between body and head lice following bacterial challenge.

The differences in the immune response between body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, were investigated initially by measuring the proliferation rates of two model bacteria, a Gram-positive Staphylococcus aureus and a Gram-negative Escherichia coli, following challenge by injection. Body lice showed a significantly reduced immune response compared to head lice particularly to E. coli at the early stage of the immune challenge. Annotation of the body louse genome identified substantially fewer immune-related genes compared with other insects. Nevertheless, all required genetic components of the major immune pathways, except for the immune deficiency (Imd) pathway, are still retained in the body louse genome. Transcriptional profiling of representative genes involved in the humoral immune response, following bacterial challenge, revealed that both body and head lice, regardless of their developmental stages, exhibited an increased immune response to S. aureus but little to E. coli. Head lice, however, exhibited a significantly higher phagocytotic activity against E. coli than body lice, whereas the phagocytosis against S. aureus differed only slightly between body and head lice. These findings suggest that the greater immune response in head lice against E. coli is largely due to enhanced phagocytosis and not due to differences in the humoral immune response. The reduced phagocytotic activity in body lice could be responsible, in part, for their increased vector competence.

Ivermectin acts as a posteclosion nymphicide by reducing blood feeding of human head lice (Anoplura: Pediculidae) that hatched from treated eggs.

The 0.5% ivermectin topical cream formulation was not directly ovicidal to treated eggs of head lice, as hatchability was not decreased. Nevertheless, the percent of hatched lice from treated eggs that took a blood meal significantly decreased (80-95%) compared with lice that hatched from untreated eggs and all treated lice died within 48 h of hatching, including those that fed. Dilutions of ivermectin formulation of 0.15 and 0.2 microg/ml, which were topically applied to 0-8 d old eggs, were not lethal to lice at 24 h posteclosion. However, 9 and 16% less lice fed when hatched from these treated eggs, respectively. Total [3H] inulin ingested by untreated first instars significantly increased over a 48 h feeding interval but was significantly less in instars that hatched from eggs receiving the 0.15 (36% less) and 0.2 (55% less) microg/ml ivermectin treatments compared with placebo. The reduced feeding that occurred after the 0.15 and 0.2 microg/ml ivermectin treatments occurred in the absence of mortality and suggests a unique mode of action of ivermectin on feeding that is separate from the mode of action of ivermectin leading to mortality. Failure of hatched instars to take a blood meal after egg treatments with formulated ivermectin is likely responsible for its action as a posteclosion nymphicide.

Establishment of quantitative sequencing and filter contact vial bioassay for monitoring pyrethroid resistance in the common bed bug, Cimex lectularius.

Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel alpha-subunit gene have been identified in deltamethrin-resistant bed bugs. A quantitative sequencing (QS) protocol was developed to establish a population-based genotyping method as a molecular resistance-monitoring tool based on the frequency of the two mutations. The nucleotide signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were shown to highly correlate with resistance allele frequencies (r2 > 0.9928). As determined by QS, neither mutation was found in a bed bug population collected in 1993. Populations collected in recent years (2007-2009), however, exhibited completely or nearly saturating L925I mutation frequencies and highly variable frequencies of the V419L mutation. In addition to QS, the filter contact vial bioassay (FCVB) method was established and used to determine the baseline susceptibility and resistance of bed bugs to deltamethrin and lambda-cyhalothrin. A pyrethroid-resistant strain showed >9,375- and 6,990-fold resistance to deltamethrin and lambda-cyhalothrin, respectively. Resistance allele frequencies in different bed bug populations predicted by QS correlated well with the FCVB results, confirming the roles of the two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with FCVB should greatly facilitate the detection and monitoring of pyrethroid-resistant bed bugs in the field. The advantages of FCVB as an on-site resistance-monitoring tool are discussed.