Search for: All records

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. 

Intestinal microbes, whether resident or transient, influence the physiology of their hosts, altering both the chemical and the physical characteristics of the gut. An example of the latter is the human pathogenVibrio cholerae’sability to induce strong mechanical contractions, discovered in zebrafish. The underlying mechanism has remained unknown, but the phenomenon requires the actin crosslinking domain (ACD) ofVibrio’s type VI secretion system (T6SS), a multicomponent protein syringe that pierces adjacent cells and delivers toxins. By using a zebrafish-nativeVibrioand imaging-based assays of host intestinal mechanics and immune responses, we find evidence that macrophages mediate the connection between the T6SS ACD and intestinal activity. Inoculation withVibriogives rise to strong, ACD-dependent, gut contractions whose magnitude resembles those resulting from genetic depletion of macrophages.Vibrioalso induces tissue damage and macrophage activation, both ACD-dependent, recruiting macrophages to the site of tissue damage and away from their unperturbed positions near enteric neurons that line the midgut and regulate intestinal motility. Given known crosstalk between macrophages and enteric neurons, our observations suggest that macrophage redistribution forms a key link betweenVibrioactivity and intestinal motility. In addition to illuminating host-directed actions of the widespread T6SS protein apparatus, our findings highlight how localized bacteria-induced injury can reshape neuro-immune cellular dynamics to impact whole-organ physiology. IMPORTANCEGut microbes, whether beneficial, harmful, or neutral, can have dramatic effects on host activities. The human pathogenVibrio choleraecan induce strong intestinal contractions, though how this is achieved has remained a mystery. Using a zebrafish-nativeVibrioand live imaging of larval fish, we find evidence that immune cells mediate the connection between bacteria and host mechanics. A piece ofVibrio’s type VI secretion system, a syringe-like apparatus that stabs cellular targets, induces localized tissue damage, activating macrophages and drawing them from their normal residence near neurons, whose stimulation of gut contractions they dampen, to the damage site. Our observations reveal a mechanism in which cellular rearrangements, rather than bespoke biochemical signaling, drives a dynamic neuro-immune response to bacterial activity.