Figure 4: Intracellular colonization by Bd in zebrafish larvae.
(a) Zebrafish larvae bath water was inoculated with high (>200 zsp per
μl) dose Bd zoospores and incubated for 72 h.p.i., then fixed and labelled with
Hoechst (for DNA; blue) and mAb 5C4 (for Bd; green) for visualization by
confocal microscopy. Images taken at 63 X, maximum intensity projection of
Z-stack shown here. Cartoon depicts imaged region. Representative images with
arrows highlight colocalization between Bd and blisters on larvae skin. Scale
bar, 50 μm. (b) Zebrafish larvae bath water was inoculated with high dose Bd
zoospores and incubated for 72 h.p.i., then fixed and labelled with Hoechst (for
DNA; blue), mAb 5C4 (for Bd; green) and phalloidin (for F-Actin; red) for
visualization by confocal microscopy. Images taken at × 63, maximum intensity
projection of Z-stack shown here. Cartoon depicts imaged region. Representative
images with insets highlight Bd adjacent to host cell actin rearrangements.
Scale bar, 50 μm. See also Supplementary Fig. 4c for host cell actin in control
treated larvae. (c) Zebrafish larvae bath water was treated as in a. Images
taken at × 40 or × 63, maximum intensity projection of Z-stack shown here.
Images showing different stages of Bd invasion and infection on larvae, also
depicted using cartoons in the right column. 1, rhizoid-like germ tube attached
to encysting zoospore, 2, chytrid thallus growth on zebrafish larvae skin, 3,
encysted sporangium amongst hyperplasic epithelial cells. Scale bars,
Full size image
Our study shows that Bd is able to
infect and multiply on zebrafish larvae treated with antibiotics in a dose
dependent manner that mimics the process of infection seen in amphibians. This
demonstration of a non-amphibian vertebrate host being infected by Bd widens the
host range previously known to be exploited by this hypervirulent chytrid
lineage. Using a Bd monoclonal antibody (mAb 5C4), we were able to image the
different stages of Bd infection with unprecedented resolution in vivo.
Collectively, these results validate zebrafish larvae as a powerful aquatic
model system within which these host-Bd interactions can be more fully explored.
Furthermore, our observations that treating zebrafish with antibiotics results
in higher cable accessories burdens of
infection highlight the use of probiotic bacteria to combat Bd infection34.
Although the specific host factors necessary for Bd infection remain to be
discovered, Bd is commonly found to parasitize the keratinized tissue of both
amphibian and non-amphibian hosts8,9,35,36. Consistent with these observations,
we found Bd parasitizing zebrafish larvae structures known to express high
levels of keratin, such as the edges of the caudal fin32, where we observed fin
erosion, tissue damage and apoptotic cells. These observations are in agreement
with studies showing widespread apoptosis of amphibian skin cells in response to
Bd infection6,31. However, in common with many macroparasite disease systems37,
not all larvae became infected and mortality is heterogeneous among experiments
(Supplementary Table 1), suggesting that there are unknown factors underlying
the susceptibility of zebrafish larvae to Bd.