Bed bugs (Heteroptera: Cimicidae) harbor obligate bacterial symbionts that supplement their blood diet with missing nutrients, especially B vitamins. The primary symbiont, transovarially transmitted Wolbachia, is notable for a horizontally acquired biotin operon. Additional maternally inherited bacteria, including Symbiopectobacterium and Tisiphia, have been detected but are considered facultative and nonessential. However, nearly all current knowledge is derived from the human-associated Cimex lectularius, leaving symbiont diversity across more than 100 bed bug species largely unknown. Using amplicon and metagenomic data, we identified Wolbachia, Symbiopectobacterium, Sodalis, Serratia, and Tisiphia as candidate symbionts, with at least 16 independent acquisition events across the cimicid species, sometimes involving multiple strains per host. Phylogenetic comparisons indicated that some of these origins were followed by cospeciation. Wolbachia was present in most hosts except Cacodminae, where Symbiopectobacterium occurred as the sole symbiont, suggesting its obligate role. Analysis of 23 draft genomes revealed heterogeneity in size and gene content, consistent with varying stages of symbiotic reduction. Most lineages lost many biosynthetic pathways; only riboflavin and lipoic acid synthesis remained universally conserved. Our survey reveals a dynamic evolution of bed bug symbioses, with repeated symbiont acquisitions, cospeciation, and frequent coinfections. Despite independent origins, most symbionts belong to Wolbachia, Symbiopectobacterium, or Sodalis, implying unknown mechanisms shaping host specificity. Two points merit further study. First, Symbiopectobacterium as the sole obligate symbiont in Cacodminae suggests broader sampling may uncover greater symbiotic diversity. Second, uncertainties in biotin synthesis function call for deeper investigation into the evolution of this pathway in symbiotic bacteria.IMPORTANCEBed bugs are obligate blood-feeding insects that depend on bacterial partners to supply nutrients missing from their diet. Most previous research has focused on the human-associated species Cimex lectularius, leaving little known about symbiont diversity across other species. By surveying a broad phylogenetic range, we found that bed bugs have repeatedly acquired different bacteria as symbionts, including lineages not previously recognized as essential. Notably, finding Symbiopectobacterium as the sole symbiont in one subfamily shows that the nutritional partnerships in bed bugs are more dynamic than previously thought. At the same time, the majority of the 16 independent acquisitions involve only four bacterial genera, suggesting efficient mechanisms that constrain and shape bed bug-symbiont specificity.

Department of Ecology Faculty of Environmental Sciences Czech University of Life Sciences Prague Prague Czech Republic

Department of Parasitology Faculty of Science University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre ASCR v v i České Budějovice Czech Republic

University Museum of Bergen Bergen Norway

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