Knockdown-resistance (kdr) against pyrethroids in bed bugs (Cimex lectularis) is associated with the presence of several point mutations in the voltage-sensitive sodium channel alpha-subunit gene and/or an increased metabolic detoxification by cytochrome P450 monooxygenases (CYPs). In the present study, pyrosequencing assays were developed to quantify the presence of the kdr substitutions (V419L or L925I substitution) in bed bugs in Berlin, Germany. In 14 of 17 bed bug field strains, pymsequencing revealed the presence of the substitution L925I with allele frequencies between 30% and 100%. One field strain additionally carried the substitution V419L with allele frequencies of 40% in males and 96% in females. In seven of the 17 field strains, mRNA levels of four CYP genes were examined using RT-qPCR. Relative to a susceptible laboratory reference strain, five field strains showed significantly higher mRNA levels of cyp397a1 with 7.1 to 56-fold increases. One of these strains additionally showed a 4.9-fold higher mRNA level of cyp398a1 compared to the reference strain, while cyp4cm1 and cyp6dn1 showed no significant differences. Our findings indicate that multiple resistance mechanisms are present in German C. lectularius populations simultaneously.
