The use of insecticides remains important in managing pest insects. Over the years, many insects manifested physiological and behavioral modifications resulting in reduced efficacy of insecticides targeted against them. Emerging evidence suggests that bacterial symbionts could modulate susceptibility of host insects against insecticides. Here, we explore the influence of host microbiota in affecting the susceptibility of insect host against different insecticides in the blood-sucking bed bug, Cimex hemipterus. Rifampicin antibiotic treatment resulted in increased susceptibility to fenitrothion and imidacloprid, but not against deltamethrin. Meanwhile, the host fitness parameters measured in the present study were not significantly affected by rifampicin treatment, suggesting the role of bacterial symbionts influencing susceptibility against the insecticides. 16S metagenomics sequencing revealed a drastic shift in the composition of several bacterial taxa following rifampicin treatment. The highly abundant Alphaproteobacteria (Wolbachia > 90%) and Gammaproteobacteria (Yersinia > 6%) in control bed bugs were significantly suppressed and replaced by Actinobacteria, Bacilli, and Betaproteobacteria in the rifampicin treated F1 bed bugs, suggesting possibilities of Wolbachia mediating insecticide susceptibility in C. hemipterus. However, no significant changes in the total esterase, GST, and P450 activities were observed following rifampicin treatment, indicating yet unknown bacterial mechanisms explaining the observed phenomena. Re-inoculation of microbial content from control individuals regained the tolerance of rifampicin treated bed bugs to imidacloprid and fenitrothion. This study provides a foundation for a symbiont-mediated mechanism in influencing insecticide susceptibility that was previously unknown to bed bugs.

Background The common bed bug, Cimex lectularius L., and the tropical bed bug, Cimex hemipterus (F.), are now widely regarded as important public health pests following their rapid global resurgence, largely due to insecticide resistance and an increased rate of global travel. The insecticide resistance mechanisms are well documented in C. lectularius, however, only one mechanism is validated in C. hemipterus thus far. This demands further understanding on the resistance mechanisms involved in C. hemipterus. Results Here, we identified differences in resistance to fenitrothion (organophosphate) and imidacloprid (neonicotinoid) related cuticle thickness in C. hemipterus. There is evidence of a possible association between cuticle thickness and resistance, but the association can be tenuous, likely because resistance is multifactorial in C. hemipterus. We also discovered a novel T1011 residue in domain IIS6 of the voltage‐gated sodium channel that likely enhanced susceptibility to deltamethrin (pyrethroid) despite the presence of a L1014F mutation known to confer pyrethroid resistance in C. hemipterus. Our findings also confirmed that the M918I mutation enhanced resistance to pyrethroid when present with the L1014F mutation, which was consistent with a super‐kdr phenotype, as reported previously. Multiple resistance mechanisms can be found within a single C. hemipterus population, and the presence of both M918I + L1014F mutations likely masked the influence of cuticle thickness in conferring resistance against deltamethrin. The elevated metabolic enzyme activities in some strains were not necessarily associated with increased insecticide resistance. Conclusion This study has enhanced our understanding on the penetration resistance mechanism and target site insensitivity of sodium channels in C. hemipterus.

The tropical bed bug, Cimex hemipterus (F.), has now emerged as an important public health pest in the tropics. Despite its alarming infestation rate, the information on its population genetics remains scarce. Here, we described the infestation structure and population dynamics of C. hemipterus in the tropics, especially Malaysia and Singapore, based on eight novel microsatellites and two mtDNA markers, including cytochrome c oxidase I (COI) and 16S rRNA genes. Across populations, microsatellite data revealed high genetic diversity with significant genetic differentiation and restricted gene flow. Analysis within populations revealed evidence of a recent bottleneck. Nonetheless, elevated genetic diversity in nearly all populations suggests that the propagule in C. hemipterus populations were much diverse, distantly related (mean r = 0.373), and not significantly inbred (mean FIS = 0.24) than that observed in Cimex lectularius from previous studies. We observed seven mtDNA haplotypes across the 18 populations studied (Hd = 0.593) and several populations displayed more than one matrilineal descent. The two markers were generally congruent in suggesting a common, genetically diverse (especially at the nuclear region) source population with possibilities of multiple introductions for the bed bug populations in the present study.