Attempts to characterize the mechanisms of resistance in these resurgent bed bug populations have followed. Yoon et al. found that resistance to deltamethrin in a bed bug strain from New York City was the result of two point mutations in the alpha-subunit of the voltage-gated sodium channel, which is the target of pyrethroid insecticides. Subsequently, Zhu et al. screened 110 bed bug strains collected from different regions of the U.S. and found that 88% of those populations had either one or both of these target site mutations, indicating that this target site resistance is widespread in reemerging bed bug populations. While a target site mutation, or kdr resistance, was identified as the primary mechanism of resistance in the previously noted studies, other mechanisms of resistance such as enhanced detoxification enzyme activity have not been as well documented in modern bed bug strains. The New York strain analyzed by Yoon et al showed no elevated activity in any of the major insecticide-metabolizing enzyme systems; glutathione-S-transferases, carboxylesterases, and cytochrome P450 monooxygenases using model substrates. However, Romero et al. observed a decrease in resistance to pyrethroid insecticides using piperonyl butoxide, an inhibitor of P450 activity, in bed bugs collected in Cincinnati, OH and Worcester, MA. This suggests that detoxification is an important mechanism of resistance in these strains, though other resistance mechanisms must also be present. Likewise, Bai et al found increased transcript levels for a cytochrome P450 in bed bugs collected in Columbus, OH, compared with those of susceptible strains. Biochemical analyses of resistance phenotypes are laborintensive and costly. As such, PCR-based methods are preferred to screen large numbers of individuals/populations for a defined trait such as a BAY-60-7550 distributor nucleotide polymorphism or for differences in gene expression levels, which are strongly associated with a resistance phenotype. While such a diagnostic PCR method has been developed for surveying target site resistance, no such assays have been associated with metabolic resistance in bed bugs. In this paper, we present both biochemical and genetic evidence that bed bug populations collected in Richmond, VA carry both target site and metabolic resistance traits, and these correlate with,5200- fold resistance to the pyrethroid deltamethrin. Further, we have identified through deep sequencing a large cohort of full-length P450, GST and CE-encoding sequences, several of which are significantly upregulated in resistant bed bugs. These sequences will aid in surveillance efforts to detect and monitor metabolic resistance in resurgent bed bug populations and enable studies involving the biochemical characterization of precisely how bed bugs metabolize and inactivate insecticides. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type and metabolic resistance to pyrethroid nsecticides. The resistance ratios we quantified for this strain suggest that pyrethroid insecticides would be largely ineffective in controlling this multi-resistant population. Pyrethroid insecticides target the voltage-gated sodium channel, with the sequence of the bed bug homolog reported in just 2008.