UV-irradiated rotifers for the maintenance of gnotobiotic zebrafish larvae.

Host-associated microbial communities profoundly impact the health of humans and other animals. Zebrafish have proven to be a useful model for uncovering mechanisms of host-microbe interactions, but the difficulty of maintaining germ-free or gnotobiotic zebrafish beyond 1 week post-fertilization has limited their utility. To address this, we have developed a simple protocol using UV irradiation of rotifers, a common and nutrient-rich prey species for larval zebrafish, to reduce the bacterial load associated with the rotifers by several orders of magnitude while maintaining their motility and viability. We find that though feeding with UV-treated rotifers does not preserve the sterility of germ-free fish, it enables the maintenance of pre-existing bacterial communities. Normal feeding, in striking contrast, leads to the near-total depletion of these prior populations. We measure the abundance of single- and three-species consortia of zebrafish-commensal bacteria inoculated into initially germ-free larvae in a series of experiments extending to 8 days of feeding, or 13 days post-fertilization. We find, in fish-fed UV-treated rotifers, the persistence of bacterial populations on timescales of days, together with strong species-specific variation. In addition, re-inoculation of differently labeled strains of the same zebrafish-commensal species alongside feeding leads to colonization by the new bacteria without displacement of earlier microbes. Our method will facilitate the use of gnotobiotic zebrafish for investigations of phenomena that emerge later in animal development and for studies that probe microbiome composition fluctuations and stability over extended timescales.IMPORTANCEAll animals, including humans, are host to vast microbial communities that contribute to health and disease through mechanisms that remain largely mysterious. These microbiomes are challenging to study, spurring the use of various model organisms, including zebrafish. Zebrafish, however, are difficult to raise beyond 1 week post-fertilization under gnotobiotic conditions, in other words, germ free or with known microbial constituents, a consequence of normally feeding on live prey that brings their own, generally unknown, microbes. Therefore, we developed a simple protocol in which UV irradiation of rotifers, a widely used small-animal food for larval zebrafish, facilitates the maintenance of gnotobiotic larvae. We show that pre-existing bacterial communities in larvae are minimally affected by feeding on UV-treated rotifers, in strong contrast to feeding on untreated rotifers. We demonstrate that this feeding method allows investigations of zebrafish-associated bacterial community stability over several days, allowing investigation of previously intractable questions about microbiome stability.