Carbon dioxide (CO 2 ) plays a crucial role in the host search/localization process of bed bugs, Cimex lectularius L., and Cimex hemipterus (F.). Current research on the CO 2 perception by bed bugs mainly focuses on their behavioral responses, and the molecular mechanisms are still unclear. In addition, existing research has mainly been conducted on C. lectularius with very little research on C. hemipterus . In this study, we investigated the behavioral response of C. hemipterus to CO 2 and analyzed the role of antennae in the CO 2 sensing process. Then, potential CO 2 receptor genes were screened through antennal RNA sequencing and tissue-specific expression profiling analysis. Finally, the function of CO 2 receptors was further validated using RNA interference. Results indicated that increasing the CO 2 concentration in the air not only stimulated the activity of C. hemipterus , but also presented a directional attraction effect on them. CO 2 , with a concentration of 0.06%–1.8%, had a significant attraction effect on C. hemipterus . Removal of the antennae led to the loss of bed bugs’ preference for CO 2 . Four candidate CO 2 receptor genes ( ChGR1 , ChGR2 , ChGR3 , and ChGR4 ) were identified through transcriptomic analysis of the bed bug antennae, and 3 of them ( ChGR2 , ChGR3 , and ChGR4 ) were highly expressed in the antennae. Silencing ChGR2 or ChGR4 individually led to a significant reduction or even loss of CO 2 sensing ability in C. hemipterus . In conclusion, CO 2 can induce the host searching behavior of C. hemipterus ; moreover, ChGR2 and ChGR4 are crucial for detecting CO 2 in C. hemipterus .
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