Nutrient requirements for cell differentiation progression in Batrachochytrium dendrobatidis

Abstract

Batrachochytrium dendrobatidis (Bd) is the causative agent of the deadly amphibian disease chytridiomycosis, which has decimated amphibian populations across the globe. Bd reproduces via a multistage asexual pathway. The Bd life cycle begins with a motile zoospore. Upon contact with a surface, the zoospore anchors itself and forms a cyst. The cyst then develops into a germling that produces rhizoid structures. Finally, the germling matures into a zoosporangium which then releases multiple new zoospores. In this study, we investigated the effects of nutrients on the growth and differentiation of Bd. The presence of carbohydrates, vitamins, and trace minerals had a negligible effect on Bds ability to progress through its life cycle. In contrast, the amino acids L-leucine and L-isoleucine (and to a lesser extent L-arginine) were required for cysts to germinate into germlings. However, none of these amino acids were able to sustain further cellular development. Furthermore, the presence of nitrate or ammonium aided progression from germlings into immature zoosporangia that were not able to produce zoospores. The progression to mature zoosporangia was blocked by nitrite or the combined action of calcium ions and bicarbonate. The addition of soluble and insoluble protein sources alone also allowed Bd to develop into immature zoosporangia and affected the rate of development. Finally, protein sources in combination with L-amino acids allowed the completion of the Bd cycle by allowing development from zoospores to mature zoosporangia that were able to release new zoospores. ImportanceEven though the cell differentiation cycle of Bd has been microscopically described, not much is known about the requirements for Bd to progress throughout its life stages. The identification of the molecular cues that allow Bd cell cycle progression may be crucial to disease mitigation efforts. As a corollary, we have identified conditions that allow for the arrest of Bd at various stages of its life cycle. By changing the metabolite availability, we can efficiently obtain cultures enriched for zoospores, cysts, germlings, immature zoosporangia, or spore-releasing zoosporangia. The ability to temporarily freeze and then restart Bd at different points of its life cycle, can allow for determining the mechanisms behind individual transitions between cell types.