X. laevis bone marrow-derived mast cells possess classical mast cell cytology and transcriptional profiles.

Neutrophils (A, C, E, G) and mast cells (B, D, F, H) were stained with Giemsa (A, B) and Leder to visualize specific esterase activity (SE) (C, D) or imaged with scanning and transmission electron microscopy (SEM: E, F and TEM: G, H). (I) Heat map of the top 30 differentially expressed genes (DEGs) identified with RNA sequencing analyses of X. laevis mast cell (N=4) and neutrophil (N=4) cultures. Log2fold change in expression represented as color scale.

Frog mast cells and neutrophils possess gene profiles similar to their mammalian counterparts.

The differentially expressed genes from the RNA sequencing analyses of X. laevis mast cells and neutrophil cultures were profiled for those encoding (A) transcription factors associated with mast cell- or neutrophil-specific lineages and (B) granulocyte antimicrobial components and growth factor receptor genes. All depicted genes were significantly differentially expressed between the two populations, N=4 per group.

Enriching frog cutaneous mast cells lowers Bd loads.

Representative images of SE stained (A) control and (B) mast cell-enriched skin 12 hpi. (C) We confirmed the enriched population was composed of mono-morphonuclear cells. (D) Mast cell enrichment was optimized across several time points by quantifying SE-positive cells per field of view under 40x magnification. Results represent means ± SEM from 3 animals per time point (2 experimental repeats). (E) Mast cell-enriched and control dorsal skins were collected from X. laevis 7- and 21-dpi. Bd loads are represented as the number of zoospore genomic equivalents (GE) x 104 per μl of total input DNA. Time points were analyzed independently. Results represent means ± SEM from 7 animals per experimental group (N=7). Asterisks indicate significance: p < 0.05 by (D) one-way ANOVA with Tukey post-hoc analysis or (E) Student’s t-test.

Consequences of cutaneous mast cell and neutrophil enrichment.

(A) RNAseq analysis of skin tissue from control (r-ctrl) or mast cell-enriched (rSCF) Bd-infected X. laevis at 21 dpi. Heat map of the top 30 DEGs, numbers matched to colors represent log2 fold change in expression. (B & C) Representative images of control and mast cell-enriched, Bd-infected skins, 21 dpi, demonstrating differences in epidermal thickening. (D & E) Representative images of control (r-ctrl) and neutrophil-enriched (rCSF3) skins collected 12 hpi demonstrating differences in epidermal thickening. (F) Kinetics of neutrophil enrichment following subcutaneous rCSF3 administration. Results are means + SEM of SE-positive cells per field of view from 4 animals per time point (N=4). (G) Bd loads from control and neutrophil-enriched skin tissue 7 dpi, (N=6). Asterisks indicate significance: p < 0.05 by (F) one-way ANOVA with Tukey post-hoc analysis or (G) Student’s t-test.

Cutaneous mast cells promote filled mucus glands.

Control (r-ctrl-injected) or mast cell-enriched (rSCF-injected) X. laevis were mock-infected or challenged with Bd for 21 days. Mucin content was examined in cutaneous mucus glands with Alcian Blue/PAS stain. Representative images of Bd-infected (A) control and (B) mast cell-enriched animals. Mucus glands are denoted by ‘m’, and epithelia are denoted by ‘Ep’. (C) ImageJ software was used to determine the proportion of positive mucin staining within each mucus gland. Results are mean + SEM percent positive mucin staining per mucus gland. Letters indicate group mean significance: p < 0.05 for treatment effects and interaction by two-way ANOVA. Skin tissue pieces from six individual frogs were used per treatment group (N=6). (D) Microbial phyla distribution across groups. Low abundance phyla (< 5% relative abundance are not shown). 10 dpi (E) community composition (Jaccard distances shown with 80% confidence ellipses) differed among all treatments. (F) Relative abundance of Bd-inhibitory bacteria and (G) bacterial richness were examined in control and mast cell enriched frogs, 10 days post Bd or mock challenge. Letters above bars in C, F and G indicate statistically different groups.

List of primer sequences

In vitro analyses of frog mast cells and neutrophils challenged with Bd (A) Mast cells and neutrophils derived from bone marrow of 6 individual frogs (N=6) were co- cultured with Bd (5 fungal cells per granulocyte) for 6 hrs prior to gene expression analyses of the antimicrobial peptide genes PGLa (pgla) and magainin (mag) or interleukin-4 (il4). (B) Changes in gene expression of selected genes in skin following subcutaneous injection of rIL4 (N=7). (C) Frogs were exposed to Bd and 24 hrs later injected subcutaneously with rIL4 or r-ctrl. Skin Bd loads were assessed 9 days following injection (N=8, per group). For B, asterisks denote statistically significant differences in expression following Bd-challenge compared to medium controls in mast cell cultures, one- way ANOVA with Tukey post-hoc analysis p < 0.05. For C, asterisk indicates significantly increased expression of cd36 by Student’s t test, p < 0.05.

Mucosomes from mast cell-enriched frogs do not confer Bd killing.

Differences in musocome-killing capacities were determined by incubating zoospores with total mucosome contents for 16 hrs. Mucosomes were acquired from 10- or 21-day mock- or Bd-infected X. laevis that were injected with rSCF (mast cell-enriched) or r-ctrl (control). Results are mean + SEM and were analyzed via a two-way ANOVA; 10- and 21-day experimental groups were analyzed independently; alpha set at 0.05. N = 5 experimental animals per treatment group for 10 dpi analyses and N= 8 experimental animals per treatment group for 21 dpi analyses.

Mast cell enrichment protects frogs from Bd-mediated changes to skin microbiomes.

21 days post mock or Bd-challenge, we examined (A) community composition (Jaccard distances shown with 80% confidence ellipses), (F) relative abundance of Bd-inhibitory bacteria and (G) bacterial richness. Letters above bars indicate statistically distinct groups.