Molecular genotyping of pyrethroid resistant mutations and their haplotypes in bed bug populations from Greece

The resurgence of bed bugs poses significant risks to public health and tourism-driven economies in Southern Europe, including Greece. Control efforts largely rely on pyrethroids; however, the widespread selection of knockdown resistance (kdr) mutations has compromised their effectiveness. Molecular monitoring is therefore essential for accurate species identification and resistance surveillance to support evidence-based pest management strategies. In this study, we analyzed bed bug populations collected from Athens, Thessaloniki, and Heraklion between 2021 and 2024. Species identification was performed via DNA barcoding of the cytochrome oxidase I (COI) gene, while kdr mutations in the voltage-gated sodium channel (VGSC) gene-specifically V419L, L925I, and I936F-were assessed to determine their frequencies and haplotype distributions. All specimens were identified as Cimex lectularius. The L925I mutation reached fixation (100 %) in Thessaloniki and Heraklion, while V419L was detected at frequencies of 30.00 % and 50.00 %, respectively; I936F was not detected in these populations. In Athens, L925I was highly prevalent (98.40 %), while V419L (5.27 %) and I936F (0.60 %) were detected at lower frequencies. Haplotype analysis revealed Haplotype B (L925I only) as the most common in Athens (91.20 %) and Thessaloniki (60.0 %), while Haplotype C (L925I + V419L) predominated in Heraklion (76.92 %). Additional haplotypes were identified in Athens, including Haplotype B^b (L925I + I936F), marking its first detection in Europe. These findings highlight the widespread presence of kdr mutations and underscore the urgent need for integrated pest management (IPM) strategies, incorporating resistance monitoring, alternative insecticides, and non-chemical control methods to mitigate the growing challenge of pyrethroid resistance in bed bugs.