Chemotaxis promotes early colonization and microbiogeography of Caballeronia insecticola in the gut symbiotic organ of Riptortus pedestris

ABSTRACT Many plants and animals form specific symbioses with microorganisms, relying on bidirectional host–bacteria interactions. However, knowledge about the evolution of symbiont traits enabling such specificity remains limited. The bean bug Riptortus pedestris acquires Caballeronia from environmental soil and harbors it in its gut symbiotic organ. This bug- Caballeronia symbiosis is an ideal model to clarify the evolutionary process of symbiotic bacteria because members of outgroups, such as Paraburkholderia and Pandoraea , can also colonize the host symbiotic organ but are outcompeted when co-inoculated with the native symbiont, Caballeronia . Here, we investigated mechanisms underlying the competitive performance of Caballeronia within the insect gut. Spatial analyses revealed that wild-type Caballeronia showed directional localization toward the symbiotic organ and colonized the M4 region faster than related bacteria. A cheA insertion mutant exhibited delayed early colonization and reduced competitive performance in co-infection assays although both strains ultimately reached similar levels of crypt colonization. In addition, microscopic observations revealed strain-specific aggregation patterns within the symbiotic organ, providing additional insight into bacterial spatial organization during host colonization. Together, these findings suggest that chemotaxis accelerates early symbiotic organ colonization and enhances competitive performance during the initial stages of symbiont establishment. More broadly, our results highlight the importance of spatial and temporal dynamics in shaping host–microbe associations and provide a framework for understanding how bacterial traits contribute to the evolution of symbiotic specificity. IMPORTANCE Riptortus pedestris , a major soybean pest in East Asia, acquires symbiotic bacteria from the environment every generation, yet its gut is consistently and specifically colonized by Caballeronia species. The evolutionary traits that underlie this strong symbiotic specificity remain poorly understood. Here, we demonstrate that chemotaxis accelerates early colonization of the symbiotic organ by Caballeronia and enhances its competitive performance relative to closely related bacteria. Using comparative colonization assays with wild-type, chemotaxis-deficient mutants, closely related bacteria, and an out-group species, we show that chemotaxis contributes to competitive performance during the initial stages of symbiotic organ colonization. Our findings indicate that ecological and behavioral traits, including chemotaxis, contribute to the establishment of exclusive symbiotic associations, providing new insight into how symbiotic specificity may evolve in horizontally transmitted systems.