Abstract
Microbes associate with animal hosts, often providing shelter in a nutrient-rich environment. The gut, however, can be a harsh environment with members of the microbiome settling in distinct niches resulting in more stable, adherent biofilms. These diverse communities can provide orders of magnitude more gene products than the host genome; selection and maintenance of a functionally relevant and useful microbiome is now recognized to be an essential component of homeostasis. Germ-free (GF) model systems allow dissection of host-microbe interactions in a simple and direct way where each member of the symbiosis can be studied in isolation. In addition, because immune defenses in the gut are often naïve in GF animals, host immune recognition and responses during the process of colonization can be studied. Ciona intestinalis, a basal chordate, is a well-characterized developmental model system and holds promise for addressing some of these important questions. With transparent juveniles, Ciona can be exposed to distinct bacterial isolates by inoculating GF artificial seawater; concentrated bacteria can subsequently be visualized in vivo if fluorescent stains are utilized. Rearing GF Ciona is a first step in untangling the complex dialogue between bacteria and innate immunity during colonization.
Highlights
The ability to establish germ-free (GF) versions of both traditional and non-traditional model systems has become essential in studies that aim to develop and expand our understanding of the role of host-associated microbial communities in both health and disease
The embryo develops into a swimming tadpole larva that attaches to a substrate and Generating Germ-Free Ciona undergoes metamorphosis, losing its notochord and becoming a sessile adult (Chiba et al, 2004)
In the early stages of metamorphosis, the intestine disc begins to differentiate into its anatomical compartments and, at stage 4 of the 1st ascidian juvenile, the digestive tract opens to the external environment, initiating the process of feeding and microbial colonization
Summary
The ability to establish germ-free (GF) versions of both traditional and non-traditional model systems has become essential in studies that aim to develop and expand our understanding of the role of host-associated microbial communities in both health and disease. One week after fertilization, when the animals reach stage 4 of metamorphosis, the antibiotics and antifungal solution are removed, and the freshly changed water can be inoculated with one (monoassociation) or more (mixed community) microbes of choice for colonization experiments. (3) Dish preparation for larval development- timing 40 min To generate and maintain GF animals, sterilization steps are included that alter chorion integrity; embryos with disrupted chorions can stick to plastic dishes, a process that can significantly decrease viability.
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