MixedWolbachiainfections resolve rapidly duringin vitroevolution
Authors: Mirchandani Cade , Wang Pingting , Jacobs Jodie , Genetti Maximilian , Pepper-Tunick Evan , Sullivan William T , Corbett-Detig Russ , Russell Shelbi L 
Published: March 2024

AbstractThe intracellular symbiontWolbachia pipientisevolved after the divergence of arthropods and nematodes, but it reached high prevalence in many of these taxa through its abilities to infect new hosts and their germlines. Some strains exhibit long-term patterns of co-evolution with their hosts, while other strains are capable of switching hosts. This makes strain selection an important factor in symbiont-based biological control. However, little is known about the ecological and evolutionary interactions that occur when a promiscuous strain colonizes an infected host. Here, we study what occurs when two strains come into contact in host cells following horizontal transmission and infection. We focus on the faithfulwMel strain fromDrosophila melanogasterand the promiscuouswRi strain fromDrosophila simulansusing anin vitrocell culture system with multiple host cell types and combinatorial infection states. MixingD. melanogastercell lines stably infected withwMel andwRi revealed that wMel outcompeteswRi quickly and reproducibly. Furthermore,wMel was able to competitively excludewRi even from minuscule starting quantities, indicating that this is a nearly deterministic outcome, independent of the starting infection frequency. This competitive advantage was not exclusive towMel’s nativeD. melanogastercell background, aswMel also outgrewwRi inD. simulanscells.Overall,wRi is less adept at in vitrogrowth and survival thanwMel and itsin vivostate, revealing differences between cellular and humoral regulation. These attributes may underlie the observed low rate of mixed infections in nature and the relatively rare rate of host-switching in most strains. Ourin vitroexperimental framework for estimating cellular growth dynamics ofWolbachiastrains in different host species, tissues, and cell types provides the first strategy for parameterizing endosymbiont and host cell biology at high resolution. This toolset will be crucial to our application of these bacteria as biological control agents in novel hosts and ecosystems.Author SummaryWolbachia pipientisis one of the most common bacterial endosymbionts due to its ability to manipulate host reproduction, and it has become a useful biological control tool for mosquito populations.Wolbachiais passed from mother to offspring, however the bacterium can also “jump” to new hosts via horizontal transmission.When aWolbachiastrain successfully infects a new host, it often encounters a resident strain that it must either replace or co-exist with as a superinfection. Here, we use aDrosophila melanogastercell culture system to study the dynamics of mixedWolbachiainfections consisting of the high-fidelitywMel and promiscuouswRi strains. ThewMel strain consistently outcompetes thewRi strain, regardless ofwMel’s initial frequency inD. melanogastercells. This competitive advantage is independent of host species. While both strains significantly impede host cell division, only thewMel strain is able to rapidly expand into uninfected cells. Our results suggest that thewRi strain is pathogenic in nature and a poor cellular symbiont, and it is retained in natural infections because cell lineages are not expendable or replaceable in development. These findings provide insights into mixed infection outcomes, which are crucial for the use of the bacteria in biological control.