CHAMPAIGN, Ill. — A new advance in animal husbandry involving a popular aquarium fish should speed the pace of discovery in laboratory studies of host-microbe interactions, researchers report.
The new findings are detailed in the journal mSystems, a publication of the American Society for Microbiology.
Zebrafish are a desirable study animal because one pair can produce hundreds of offspring in a day at a cost that is roughly 70 times lower than the cost of doing the same work in mice. It also helps that zebrafish larvae are transparent, allowing scientists to peer into their insides to see how things develop under different conditions.
So far, the findings from microbiome studies in zebrafish parallel those conducted in mice, suggesting that the mechanisms that drive host-microbe interactions are conserved across species, said Christopher Gaulke , a professor of pathobiology at the University of Illinois Urbana-Champaign who led the new study with graduate student Lydia Okyere .
A problem arises, however, from the use of "germ-free" animals, which lack the microbes that normally colonize the body. Raising germ-free lab animals is essential to understanding how various species grow, develop and function — with and without their microbiota. Studies of germ-free animals also let scientists see how the animals respond to various chemicals or drugs and test whether host microbes help shape responses to such compounds.
Raising germ-free animals means providing them with germ-free food, Gaulke said. This is problematic, as current methods for raising zebrafish involve feeding them live food, which cannot be easily sterilized. This has limited studies of germ-free zebrafish to their larval phase.
The two most common methods for developing sterile animal chow — by exposing the food to extreme heat in an autoclave or subjecting it to ultraviolet radiation — sometimes come with undesirable limitations or side effects, Gaulke said. UV radiation sterilizes only the food surface, and heat from an autoclave can produce toxic compounds in the chow.
A third option, the use of gamma irradiation, has been used to sterilize chow fed to mammals with success. But this approach had not been perfected in fish chow.
Gaulke and Okyere decided to try using gamma irradiation to sterilize zebrafish chow. If successful, this approach would support the germ-free fish as they develop, extending studies well beyond the larval stage.
"We began by investigating how increasing doses of gamma-irradiated fish chow affected both normal and germ-free zebrafish," Gaulke said. "We then developed a long-term protocol for raising germ-free zebrafish."
The team found that the irradiated chow had no harmful effects on normal or germ-free zebrafish and sustained the fish well into the juvenile stages of development, overcoming a major hurdle.
Further studies revealed significant differences between the germ-free zebrafish and those with intact microbiomes. The germ-free zebrafish had delayed development and experienced other changes, Okyere said.
"Our germ-free animals had very distinct gene-expression profile," she said. Pathways related to metabolizing foreign agents, like agricultural chemicals or pharmaceutical drugs, were downregulated in the germ-free zebrafish, as were genes related to lipid metabolism and immune function. These findings parallel findings in mice and suggest that host microbes play similar roles in immunity and metabolism across species.
"The most interesting part about this work for me is that now we've been able to develop a new husbandry protocol for raising germ-free zebrafish beyond the larval stages that we've been limited to for a very long time," Okyere said. "This is an advance that the field can build upon and use to answer a lot of questions that we've been wanting to ask about how the microbiome influences host responses to toxicants like pesticides."
Such questions include studies that Gaulke is currently undertaking, with funding from the National Institutes of Health, exploring whether the microbiome affects how animals metabolize agricultural chemicals like atrazine and glyphosate.
"We'll also be looking at how individual microbes and chemicals may interact to predispose individuals to metabolic disorders," Gaulke said.
Other avenues of research include studies of how host microbiota influence the onset or pathology of behavioral disorders or the development of a healthy immune system, he said.
"Developing a low-cost, high-throughput animal model like the germ-free zebrafish will accelerate the pace of discovery in microbiome research and allow us to more quickly and more rigorously test our hypotheses in the lab," Gaulke said. "This opens the doors to tens of thousands of studies we couldn't do before."
The National Institute of Environmental Health Sciences supported this research through grant R01ES036174.
