Under the radar: differential responses of bed bugs to an entomopathogen, environmental bacteria, and a human pathogen

Background: Bed bugs (Hemiptera: Cimicidae) are a widely distributed, obligately blood-feeding insect, but they have never been linked to pathogen transmission in humans. Most other hematophagous insects that frequently bite humans transmit pathogens, and it is unclear why bed bugs do not. One hypothesis is that bed bugs have evolved a highly robust immune system because their mating system, traumatic insemination, exposes females to consistent wounding and bacterial infections. Although this has been proposed, very little is known about the bed bug immune system and how bed bugs respond to microbial challenges. Understanding the bed bug immune system could give insight to why bed bugs are not known to transmit disease and under what circumstances they could, while also facilitating biological control efforts involving microbes. Methods: To investigate the immune response of bed bugs to bacterial challenges, we exposed female bed bugs to three bacterial challenges. 1.) Pseudomonas fluorescens, an entomopathogen known to have harmful effects to bed bugs, 2.) bacteria cultured from a bed bug enclosure likely encountered during traumatic insemination, and 3.) Borrelia duttoni, a human vector-borne pathogen that causes relapsing fever. We compared the transcriptomes of infected bed bugs with uninfected bed bugs, focusing on immune-related genes. We also conducted phylogenetic analyses to understand patterns of gene duplication and function of potentially immune-related genes. Results: We found many known immune effector genes upregulated in response to P. fluorescens and traumatic insemination-associated bacteria, but interestingly, not in response to B. duttoni. Furthermore, we found significant overlap in the genes differentially expressed in response to P. fluorescens and the traumatic insemination associated bacteria, and between P. fluorescens and B. duttoni, but no significant overlap between traumatic insemination bacteria and B. duttoni. We also show that bed bug diptericin-like antimicrobial peptides underwent a lineage-specific gene duplication, and that they may have further functional specialization. Finally, we identify previously overlooked candidates for future study of immune function in bed bugs, including some putative cuticle-associated genes, a laccase-like gene, and a mucin-like gene. Conclusions: By taking comprehensive transcriptomic approach, our study is an important step in understanding how bed bugs respond to diverse immune challenges. ### Competing Interest Statement The authors have declared no competing interest.