Specialized acquisition behaviors maintain reliable environmental transmission in an insect-microbial mutualism

Understanding how horizontally transmitted mutualisms are maintained is a major focus of symbiosis research.1,2,3,4 Unlike vertical transmission, hosts that rely on horizontal transmission produce symbiont-free offspring that must find and acquire their beneficial microbes from the environment. This transmission strategy is inherently risky since hosts may not obtain the right symbiont every generation. Despite these potential costs, horizontal transmission underlies stable mutualisms involving a large diversity of both plants and animals.5,6,7,8,9 One largely unexplored way horizontal transmission is maintained is for hosts to evolve sophisticated mechanisms to consistently find and acquire specific symbionts from the environment. Here, we examine this possibility in the squash bug Anasa tristis, an insect pest that requires bacterial symbionts in the genus Caballeronia10 for survival and development.11 We conduct a series of behavioral and transmission experiments that track strain-level transmission in vivo among individuals in real-time. We demonstrate that nymphs can accurately find feces from adult bugs in both the presence and absence of those adults. Once nymphs locate the feces, they deploy feeding behavior that results in nearly perfect symbiont acquisition success. We further demonstrate that nymphs can locate and feed on isolated, cultured symbionts in the absence of feces. Finally, we show this acquisition behavior is highly host specific. Taken together, our data describe not only the evolution of a reliable horizontal transmission strategy, but also a potential mechanism that drives patterns of species-specific microbial communities among closely related, sympatric host species. •Choice assays show A. tristis nymphs are highly attracted to symbiont-rich feces•A. tristis nymphs use flush feeding to acquire symbionts from fecal matter•Nymphs are only attracted to conspecific feces, reinforcing host-microbe associations Villa et al. demonstrate how an agricultural pest reliably transmits their beneficial microbial symbionts despite having to acquire them from the environment every generation. Real-time tracking of fluorescently tagged symbionts shows that symbiont-free nymphs use species-specific homing and feeding behaviors to ingest symbionts from adult feces.