To identify genes that commonly respond to the treatment of different insecticides and are responsible for the toleranceenhancement, transcriptomic profiles of larvae treated with sublethal doses of the five insecticides were compared withthat of untreated control. A total of 117,181 transcripts with a mean length of 662 bp were generated by de novo assembly,of which 35,329 transcripts were annotated. Among them, 125, 143, 182, 215 and 149 transcripts were determined tobe up-regulated whereas 67, 45, 60, 60 and 38 genes were down-regulated following treatments with these five insecticides.The most notable examples of commonly responding over-transcribed genes were two cytochrome P450 genes and ninecuticular protein genes. In contrast, several genes composing the mitochondrial energy generation system were significantlydown-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 initialstage of intoxication. Their possible roles in the tolerance/resistance development were discussed.

Human body and head lice are obligatory human ectoparasites. Although both body and head lice belong to a single species, Pediculus humanus, only body lice are known to be a vector of several bacterial diseases. The higher vector competence of body lice is assumed to be due to their weaker immune response than that of head lice. To test this hypothesis, immune reactions were compared between body and head lice following infections by two model bacteria, Staphylococcus aureus and Escherichia coli, and a human pathogen, Bartonella quintana. Following dermal or oral challenge, the number of these bacteria increased both in hemocoel and alimentary tract of body lice but not in head lice and the viability of the B. quintana was significantly higher in body louse feces, the major route of infection to human. In addition, body lice showed the lower basal/induced transcription level of major immune genes, cytotoxic reactive oxygen species and phagocytosis activity compared with head lice. These findings suggest that a reduced immune response may be responsible, in part, for the increased proliferation and excretion of viable bacteria which are associated with the high level of human infectivity seen in body versus head lice.

Both the body louse (Pediculus humanus humanus) and the head louse (P. humanus capitis) are obligatory human ectoparasites. The body louse is a serious public health threat because it transmits a variety of human diseases whereas the head lice causes one of the most prevalent human infestations, pediculosis. Recent genome analysis revealed that both body and head lice have one of the smallest insect genomes (~108 Mb). Comparison of transcriptional profiles uncovered that almost the same number of genes was annotated both in the head louse (10,770 genes) and the body louse (10,771 genes) among 10,775 protein-coding genes predicted from the body louse genome. Despite their similar genetic background, body and head lice have several differences in their biological features, such as choice of habitat on human host, body size and vector competence. Annotation of major detoxification genes revealed that they are dramatically reduced in human lice compared to other insects except for the honey bee and that, despite the overall reduction in number, human lice retain at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to insecticides, suggesting their high potential for resistance development. Comparison of insecticide target site gene sequences and transcription levels of detoxification genes enabled the identification of toxicodynamic and metabolic factors of insecticide resistance and further allowed the development of molecular markers for resistance detection. Transcriptional profiling during tolerance was used to identify ivermectinmetabolizing detoxification genes, indicating that such an approach may allow proactive resistance management. Comparison of genomes and transcriptomes between body and head lice suggested that vector competence difference is not attributed to the difference in the composition of immune related genes but rather to their transcriptional regulation and/or not-yet-identified epigenetic factors.

The molecular mechanisms and genetics of abamectin resistance mediated by target site insensitivity in the two-spotted spider mite, Tetranychus urticae, were investigated by comparing two isogenic AbaS and AbaR strains. Cloning and sequencing of full-length cDNA fragments of GABA-gated chloride channel genes revealed no polymorphisms between the two strains. However, sequence comparison of the full-length cDNA fragment of a T. urticae glutamate-gated chloride channel gene (TuGluCl) identified a G323D point mutation as being tentatively related with abamectin resistance. In individual F2 progenies obtained by backcrossing, the G323D genotype was confirmed to correlate with abamectin resistance. Bioassays using progeny from reciprocal crossings revealed that the abamectin resistance trait due to TuGluCl insensitivity is incompletely recessive.

Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel (VSSC) α-subunit gene have been identified in deltamethrin-resistant bedbugs. To predict resistance allele frequencies of sodium channel mutations (V419L and L925I) in bedbugs at a population level, we developed quantitative sequencing (QS) protocol. The signal ratios between resistant and susceptible nucleotides were generated from sequencing chromatogram and plotted against the corresponding resistance allele frequencies. Linear regression coefficients of the plots were close to 1 (r2 = 0.9928 and 0.9998), suggesting that the signal ratios are reliable correlated with the resistance allele frequencies. To enable on-site monitoring of pyrethroid resistance in bed bugs, residual contact vial (RCV) bioassay method was established and used to determine median lethal concentration (LC50) values to deltamethrin for various bed bug strains. Resistance allele frequencies in these bedbug strains predicted by QS were correlated well with the RCV bioassay results, confirming the roles of two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with RCV bioassay should greatly facilitate resistance monitoring of bedbugs in the field.

The present investigation establishes deltamethrin resistance in the common bed bug, Cimex lectularius, populationcollected from New York City (NY-BB). The mortality resistance ratio indicated that NY-BB population was 264-fold more resistant to 1% deltamethrin in contact bioassay compared to one insecticide- susceptible population collected in Florida (FL-BB). Specific enzyme activities (general esterase, glutathione S-transferase, and 7-ethoxycoumarin O-deethylase) of NY-BB were not statistically different from those of FL-BB, indicating that the metabolic factors are not associated with the deltamethrin resistance in NY-BB. Complementary DNA fragments that encoded the open reading frame of voltage-sensitive sodium channel α-subunit genes from the FL-BB and NY-BB were obtained by homology probing PCR and sequenced. Sequence alignment of the internal and 5’ and 3’ RACE fragments generated a 6500 bp cDNA sequence contig, which was composed of a 6084 bp open reading frame (ORF) encoding 2027 amino acid residues and 186 bp 5’ and 230 bp 3’ untranslated regions (5’ and 3’UTRs, respectively). Sequence comparisons of the complete open reading frames of the sodium channel genes identified two point mutations (V419L and L925I) that were presented only in the NY-BB population. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in the pyrethroid-resistant populations of whitefly (Bemisia tabaci) that was more than 100-fold resistant to fenpropathrin. V419L, located in the IS6 transmembrane segment, is a novel mutation. This evidence suggests that the two mutations are likely the major resistance-causing mutations in the deltamethrin-resistant NY-BB via a knockdown-type nerve insensitivity mechanism.

A quantitative sequencing (QS) protocol that detects the frequencies of sodium channel mutations (M815I, T917I and L920F) responsible for knockdown resistance in permethrin-resistant head lice was tested as a population genotyping method. Genomic DNA fragments of the sodium channel α-subunit gene that encompass the three mutation sites were PCR-amplified from individual head lice with either resistant or susceptible genotypes, and combined together in various ratios to generate standard DNA template mixtures for QS. Following sequencing, the signal ratios between resistant and susceptible nucleotides were calculated and plotted against the corresponding resistance allele frequencies. Quadratic regression coefficients of the plots were close to 1, demonstrating that QS is highly reliable for the prediction of resistance allele frequencies. Prediction of resistance allele frequencies by QS in several globally collected lice samples including 12 Korean lice populations suggested that permethrin resistance varied substantially amongst different geographical regions. Three local populations of Korean lice were determined to have 9.8-36.7% resistance allele frequencies, indicating that an urgent resistance management is needed. QS should serve as a preliminary resistance monitoring tool for proper management strategies by allowing early resistance detection.