Fig 1.
Alkaloid sequestration in different tissue types.
Boxplots showing abundance of different compounds in different tissues and treatment types, with DHQ-fed frogs in yellow and DHQ+PTX-fed frogs in blue. (A) Frogs were orally administered either DHQ or DHQ+PTX once a day for five days. (B) DHQ abundance differed by tissue but not treatment group and was highest in the liver and skin (GLMM tissue, X2 (3) = 203.642, p < 2e-16). (C) PTX levels differed by tissue and treatment, and were higher in the liver and skin of the DHQ+PTX fed group (GLMM tissue:treatment, X2 (3) = 57.265, p < 2e-12). (D) The hydroxylated metabolite aPTX was found in the DHQ+PTX fed frogs (Wilcoxon test, W = 0, p-value = 0.012, n = 5). (E) aPTX abundance differed across tissues within the DHQ-PTX group (Kruskal-Wallis, X2 (3) = 13.727, p = 0.003), and was found primarily in the skin, with some in the liver.
Fig 2.
Differentially expressed genes in different tissues.
Boxplots show TMM normalized expression levels in the DHQ-fed group (yellow) and DHQ+PTX fed group (blue) for a subset of differentially expressed genes. (A) Differentially expressed genes in the intestines include Cytochrome P450 Family 3 Protein 29 (CYP3A29) and MHC Class I alpha (MHCIα). (B) Differentially expressed genes in the liver include vitellogenin 2 (VTG2) and MHCIα. (C) Differentially expressed genes in the skin include syntaxin 1A (STX1A) and solute carrier family 2 (SLC2). (FC indicates log2 fold change values, * indicates adjusted p-value < 0.05, *** indicates adjusted p-value < 0.005; y-axes of individual plots have different scales).
Fig 3.
Human CYP2D6 PTX 251D metabolism.
(A) Human CYP2D6 rapidly clears PTX 251D in-vitro compared to other human CYPs. (B) Human CYP2D6 creates two hydroxylation products from PTX 251D corresponding to a single hydroxylation event (m/z = 266), suggestive of aPTX 267A, and two hydroxylation events (m/z = 282). Mass-to-charge ratio of the peak is indicated with “m/z”, and “peak area” indicates the area under the curve of each peak corresponding to the m/z indicated.
Fig 4.
CYP2D6 expression and sequence conservation.
(A) A CYP2D6-like protein was identified in the D. tinctorius transcriptome that was upregulated with PTX feeding. (B) Alignment of CYP2D6-like proteins in poison frogs and other amphibians show that important human CYP2D6 binding residue Asp301 is conserved, however other active site residues are changed in frogs. Blank spots indicate residues not present in the alignment of those species due to shorter protein sequences. The orange lines on the dendrobatid phylogeny indicate independent origins of chemical defense. Species in black have been tested for ability to metabolize PTX 251D into aPTX 267A, whereas species names in gray have not been tested. Asterisks (*) indicate species that sequester aPTX 267A when fed PTX 251D. Amino acid residues are colored using the RasMol scheme, which corresponds to amino acid properties.