May 2020, Leiden
In this visualization, you can see how dead and live microbes cover a zebrafish egg.
Already at the embryonic life stage, zebrafish acquire a consortium of microbes - their microbiota - that is important to their health and development. Just like our own microbiota, zebrafish microbiota contribute to processes such as nutrient uptake, metabolism, immune responses, and the bioactivation and detoxification of environmental pollutants.
In this movie you can see that zebrafish embryos develop in a sterile environment inside of the chorion. Only when the larvae hatch from their eggs, microbes from the water and chorion will colonize zebrafish larvae. This most likely starts with colonization of the skin and gills of the larvae, and will later on, when zebrafish larvae open their mouth and start feeding, be followed up by colonization of their gastrointestinal tracts.
Because zebrafish embryos initially develop inside of a sterile environment, it is relatively easy to raise the embryos under germ-free conditions. This can be achieved by rinsing the eggs with antimicrobial agents (iodine and bleach), and subsequently maintaining the eggs in sterile egg water.
Sterile zebrafish larvae are a very useful starting point for experiments that aim to increase our understanding of the role of host-associated microbiota. In a recent study, we for instance compared the sensitivity of germ-free and microbially-colonized larvae to different kinds of nanoparticles. In this way, we found that host-associated microbiota can protect zebrafish larvae against the toxicity of silver nanoparticles.
We labelled microbes with two fluorescent dyes: 1) a green dye - Syto9 - that labels the DNA of all microbes; combined with 2) a red dye - propidium iodide - that can only enter, and thus label, microbes with damaged membranes. As a result, live microbes appear green, and dead microbes will either appear red (if only the red dye entered the microbes) or yellow (if both the green and the red dyes labeled the microbes). We made pictures of these microbes using an inverted laser scanning microscope (LSM880, Zeiss). From pictures taken at different optical sections of the zebrafish egg, we could reconstruct the three-dimensional image that is shown in the visualization.
You can learn more about how we found that colonizing microbiota protect zebrafish larvae against silver nanoparticle toxicity in our recent publication in Nanotoxicology... or watch our project movie!