Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans

Abstract

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species.

Fig 1. Life cycle of chytrid fungi.

As illustrated here with images of Bsal, chytrid fungi have a biphasic life cycle characterized by a stationary growth phase called a sporangium (top) and a motile dispersal phase called a zoospore (bottom). Images taken at 100X using differential interference contrast (DIC) microscopy.

Fig 2. Inhibition of Bd growth in liquid media.

Percent of Bd growth in liquid media supplemented with (A) Hygromycin, (B), Zeocin, (C) Blasticidin, (D) Puromycin, and (E) Neomycin as compared to an antibiotic free control for three temporally isolated replicates (circle, square, and triangle, shades of blue). Orange symbols indicate concentrations at which no growth occurred after three days in all three replicates.

Fig 3. Inhibition of Bsal growth in liquid media.

Percent of Bsal growth in liquid media supplemented with (A) Hygromycin, (B), Zeocin, (C) Blasticidin, (D) Puromycin, and (E) Neomycin as compared to an antibiotic free control for three temporally isolated replicates (circle, square, and triangle, shades of blue). Orange symbols indicate concentrations at which no growth occurred after four days in all three replicates.

Fig 4. Inhibition of Bd growth on solid media.

(A) Examples of Bd growth after three days on antibiotic selection plates. The ‘+’ demonstrates the relative zoospore activity of each plate compared to an antibiotic-free control plate. The box highlights zoospores, which appear as small dots while the bracket highlights sporangia. The zoospores in the ‘0’ image are immotile (see S1 Video). Scale bar 50 μm. (B) Bd growth on antibiotic selection plates. Concentrations highlighted in bold and orange are the lowest concentrations that prevent growth for at least 14 days post zoospore plating.

Fig 5. Inhibition of Bsal growth on solid media.

(A) Examples of Bsal growth after four days on antibiotic selection plates. The ‘+’ demonstrates the relative zoospore activity of each plate compared to a no antibiotic control plate. The box highlights zoospores, which appear as small dots while the bracket highlights sporangia. The zoospores in the ‘0’ image are immotile (see S1 Video). Scale bar 50 μm. (B) Bsal growth on antibiotic selection plates. Concentrations highlighted in bold and orange are the lowest concentrations that prevent growth for at least 14 days post zoospore plating.