Background: Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained\r\nhigh impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has\r\nbeen attributed to numerous factors including frequent international travel, narrower pest management practices,\r\nand insecticide resistance.\r\nResults: We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed\r\ngenes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference\r\ntranscriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived\r\nfrom de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs).\r\nThe two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and\r\nPS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins,\r\ncytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl\r\ncholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and\r\ndevelopment-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian\r\ntubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a\r\ncandidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad\r\nrange of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid.\r\nConclusions: We developed significant molecular resources for C. lectularius putatively involved in metabolic\r\nresistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains\r\ncombined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C.\r\nlectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets\r\nsuch as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the\r\ngenetic basis of insecticide resistance in C. lectularius.
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