Many microorganisms engaged in host-microbe interactions pendulate between a free-living phase and a host-affiliated stage. How adaptation to stress during the free-living phase affects host-microbe associations is unclear and understudied. To explore this topic, the symbiosis between Hawaiian bobtail squid (Euprymna scolopes) and the luminous bacterium Vibrio fischeri was leveraged for a microbial experimental evolution study. V. fischeri experienced adaptation to extreme pH while apart from the squid host. V. fischeri was serially passaged for 2000 generations to the lower and upper pH growth limits for this microorganism, which were pH 6.0 and 10.0, respectively. V. fischeri was also serially passaged for 2000 generations to vacillating pH 6.0 and 10.0. Evolution to pH stress both facilitated and impaired symbiosis. Microbial evolution to acid stress promoted squid colonization and increased bioluminescence for V. fischeri, while symbiont adaptation to alkaline stress diminished these two traits. Oscillatory selection to acid and alkaline stress also improved symbiosis for V. fischeri, but the facilitating effects were less than that provided by microbial adaptation to acid stress. In summary, microbial adaptation to harsh environments amid the free-living phase may impact the evolution of host-microbe interactions in ways that were not formerly considered.

For microorganisms cycling between free‐living and host‐associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether adaptation to stress during the free‐living stage can impact microbial fitness in the host. To address this topic, the mutualism between the Hawaiian bobtail squid (Euprymna scolopes) and the marine bioluminescent bacterium Vibrio fischeri was utilized. Using microbial experimental evolution, V. fischeri was selected to low (8°C), high (34°C), and fluctuating temperature stress (8°C/34°C) for 2000 generations. The temperatures 8°C and 34°C were the lower and upper growth limits, respectively. V. fischeri was also selected to benign temperatures (21°C and 28°C) for 2000 generations, which served as controls. V. fischeri demonstrated significant adaptation to low, high, and fluctuating temperature stress. V. fischeri did not display significant adaptation to the benign temperatures. Adaptation to stressful temperatures facilitated V. fischeri’s ability to colonize the squid host relative to the ancestral lines. Bioluminescence levels also increased. Evolution to benign temperatures did not manifest these results. In summary, microbial adaptation to stress during the free‐living stage can promote coevolution between hosts and microorganisms.