Microbes ubiquitously inhabit animals and plants, often affecting their host's phenotype. As a result, even in a constant genetic background, the host's phenotype may evolve through indirect selection on the microbiome. 'Microbiome engineering' offers a promising novel approach for attaining desired host traits but has been attempted only a few times. Building on the known role of the microbiome on development in fruit flies, we attempted to evolve earlier eclosing flies by selecting on microbes in the growth media. We carried out parallel evolution experiments in no- and high-sugar diets by transferring media associated with fast-developing fly lines over the course of four rounds of selection. In each round, we used sterile eggs from the same inbred population, and assayed fly mean eclosion times. Ultimately, flies eclosed seven to twelve hours earlier, depending on the diet, but selection had no effect. 16S sequencing showed that the microbiome did evolve, particularly in the no sugar diet, with an increase in alpha diversity over time. Thus, while microbiome evolution did affect host eclosion times, these effects were incidental. Instead, any experimentally enforced selection effects were swamped by independent microbial evolution. These results imply that selection on host phenotypes must be strong enough to overcome other selection pressures simultaneously operating on the microbiome. The independent evolutionary trajectories of the host and the microbiome may limit the extent to which indirect selection on the microbiome can ultimately affect host phenotype. Random-selection lines accounting for independent microbial evolution are essential for experimental microbiome engineering studies.

The microbiome wants what it wants: microbial evolution overtakes experimental host-mediated indirect selection