U. of I. pathobiology professor Christopher Gaulke, right, graduate student Lydia Okyere and their colleagues overcame a major hurdle to raising “germ-free” zebrafish beyond the larval stage. Their advance will speed the pace of research into host-microbe interactions.
Photo Credit: Craig Pessman
Scientific Frontline: Extended "At a Glance" Summary: Germ-Free Zebrafish Microbiome Modeling
The Core Concept: A novel husbandry protocol utilizes gamma-irradiated feed to rear germ-free zebrafish beyond their larval phase, establishing the species as a viable, long-term animal model for studying host-microbe interactions.
Key Distinction/Mechanism: Traditional feed sterilization methods, such as autoclaving or ultraviolet radiation, introduce toxic compounds or fail to penetrate the feed surface. Gamma irradiation fully sterilizes the feed without degrading its nutritional profile or introducing harmful byproducts, allowing germ-free zebrafish to survive into juvenile developmental stages.
Major Frameworks/Components:
- Gnotobiotic Animal Models: The use of organisms completely devoid of an endogenous microbiome to isolate and study physiological baselines.
- Gamma Sterilization: The application of ionizing radiation to eliminate microbial life from complex biological matrices, like animal feed, without thermal degradation.
- Transcriptomics and Host Response: Analyzing distinct gene-expression profiles in germ-free subjects, specifically noting the downregulation of pathways related to lipid metabolism, immune function, and xenobiotic metabolism.
Branch of Science: Microbiology, Toxicology, Genetics, and Immunology.
Future Application: This model enables high-throughput testing of how the microbiome influences the metabolism of agrochemicals (such as atrazine and glyphosate) and pharmaceuticals. It also provides a platform to study the microbiome's role in metabolic disorders, behavioral conditions, and immune system development.
Why It Matters: Zebrafish share conserved host-microbe interaction mechanisms with mammals but reproduce at a vastly higher rate and at roughly 1/70th the cost of murine models. Overcoming the larval-stage developmental barrier enables large-scale, long-term host-microbe studies that were previously economically unfeasible.
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.
Zebrafish are desirable animal models because one breeding pair can produce hundreds of offspring in a single day at a cost roughly 70 times lower than equivalent studies using mice. Additionally, zebrafish larvae are transparent, allowing scientists to observe internal anatomical development under varying conditions.
So far, findings from microbiome studies in zebrafish parallel those conducted in mice, suggesting that the mechanisms driving host-microbe interactions are conserved across species, according to 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, regarding the use of "germ-free" animals, which lack the microbes that normally colonize the body. Raising germ-free laboratory animals is essential to understanding how various species grow, develop, and function—with and without their microbiota. Studies involving germ-free animals also allow scientists to observe how the subjects respond to various chemicals or drugs, and to test whether host microbes help shape responses to such compounds.
Getting to "Germ-Free"
Raising germ-free animals requires providing them with germ-free food, Gaulke noted. This requirement is problematic; current methods for rearing zebrafish involve feeding them live food, which cannot be easily sterilized. Consequently, studies of germ-free zebrafish have been limited to their larval phase.
The two most common methods for developing sterile animal chow—exposing the food to extreme heat in an autoclave or subjecting it to ultraviolet (UV) radiation—sometimes present undesirable limitations or side effects, Gaulke said. UV radiation sterilizes only the surface of the food, and heat from an autoclave can produce toxic compounds within the chow.
A third option, gamma irradiation, has been used successfully to sterilize mammalian chow. However, this approach had not yet been optimized for fish chow.
Gamma Irradiation to Sterilize Zebrafish Chow
Gaulke and Okyere tested the use of gamma irradiation to sterilize zebrafish chow. If successful, this approach would support 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 it sustained the fish well into the juvenile stages of development, overcoming a major methodological hurdle.
Further studies revealed significant physiological differences between the germ-free zebrafish and those with intact microbiomes. The germ-free zebrafish exhibited delayed development and experienced other phenotypic changes, Okyere noted.
Zebrafish Model Comparable to Mice
"Our germ-free animals had a very distinct gene-expression profile," she said. Pathways related to the metabolism of foreign agents, such as agricultural chemicals or pharmaceutical drugs, were downregulated in the germ-free zebrafish, as were genes related to lipid metabolism and immune function. These results parallel findings in mice, suggesting 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."
New Zebrafish Model "Opens the Doors" for Microbiome Research
Such questions include studies that Gaulke is currently undertaking, with funding from the National Institutes of Health, to explore whether the microbiome affects how animals metabolize agricultural chemicals, such as 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, as well as 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 rigorously test our hypotheses in the lab," Gaulke said. "This opens the doors to tens of thousands of studies we couldn't do before."
Published in journal: mSystems
Title: Long-term culture of germ-free zebrafish using gamma-irradiated feeds
Authors: Lydia Okyere, Angela Di Fulvio, and Christopher A. Gaulke
Source/Credit: University of Illinois Urbana-Champaign | Diana Yates
Edited by: Scientific Frontline
Reference Number: mcb060426_01