Best practices for germ-free derivation and gnotobiotic zebrafish husbandry

Abstract

All animals are ecosystems with resident microbial communities, referred to as microbiota, which play profound roles in host development, physiology, and evolution. Enabled by new DNA sequencing technologies, there is a burgeoning interest in animal–microbiota interactions, but dissecting the specific impacts of microbes on their hosts is experimentally challenging. Gnotobiology, the study of biological systems in which all members are known, enables precise experimental analysis of the necessity and sufficiency of microbes in animal biology by deriving animals germ-free (GF) and inoculating them with defined microbial lineages. Mammalian host models have long dominated gnotobiology, but we have recently adapted gnotobiotic approaches to the zebrafish (Danio rerio), an important aquatic model. Zebrafish offer several experimental attributes that enable rapid, large-scale gnotobiotic experimentation with high replication rates and exquisite optical resolution. Here we describe detailed protocols for three procedures that form the foundation of zebrafish gnotobiology: derivation of GF embryos, microbial association of GF animals, and long-term, GF husbandry. Our aim is to provide sufficient guidance in zebrafish gnotobiotic methodology to expand and enrich this exciting field of research.

Low-dose antibiotics can collapse gut bacterial populations via a gelation transition

“…Wild-type (AB × TU strain) zebrafish were derived germfree (GF) and colonized with bacterial strains as previously described [21] with slight modifications. Briefly, fertilized eggs from adult mating pairs were harvested and incubated in sterile embryo media (EM) containing ampicillin (100 μ g/ml), gentamicin (10 μ g/ml), amphotericin B (250 ng/ml), tetracycline (1 μ g/ml), and chloramphenicol (1 μ g/ml) for 6 h. Embryos were then washed in EM containing 0.1% polyvinylpyrrolidone-iodine followed by EM containing 0.003% sodium hypochlorite.…”

Evolutionary “Experiments” in Symbiosis: The Study of Model Animals Provides Insights into the Mechanisms Underlying the Diversity of Host–Microbe Interactions

“…[56] The small size and optical transparency of the embryos and larvae have made this animal a premiere model for developmental biologists. The ex utero embryonic development allows for surface sterilization of the chorion and easy derivation of hundreds of axenic or "germ-free" larval animals at a time, [58] enabling gnotobiotic experiments on a scale that would be impossible to achieve with mammalian systems. [57] All of these features of accessible embryology, transparency during development, easy husbandry with high fecundity, and available genetic tools have made the zebrafish an excellent model for studying vertebrate interactions with microorganisms.…”

Comparative models for human nasal infections and immunity

“…Complex hostmicrobe interactions can be investigated in real time using life imaging and fluorescently labeled pathogens. Finally, germ-free (GF) zebrafish provide a platform for the identification of microbial contributions to disease onset and progression (Kostic et al, 2013;Melancon et al, 2017;Rawls et al, 2004).…”