Molecular physiology of chemical defenses in a poison frog

Abstract

Poison frogs sequester small molecule lipophilic alkaloids from their diet of leaf litter arthropods for use as chemical defenses against predation. Although the dietary acquisition of chemical defenses in poison frogs is well documented, the physiological mechanisms of alkaloid sequestration has not been investigated. Here, we used RNA sequencing and proteomics to determine how alkaloids impact mRNA or protein abundance in the little devil frog (Oophaga sylvatica), and compared wild-caught chemically defended frogs with laboratory frogs raised on an alkaloid-free diet. To understand how poison frogs move alkaloids from their diet to their skin granular glands, we focused on measuring gene expression in the intestines, skin and liver. Across these tissues, we found many differentially expressed transcripts involved in small molecule transport and metabolism, as well as sodium channels and other ion pumps. We then used proteomic approaches to quantify plasma proteins, where we found several protein abundance differences between wild and laboratory frogs, including the amphibian neurotoxin binding protein saxiphilin. Finally, because many blood proteins are synthesized in the liver, we used thermal proteome profiling as an untargeted screen for soluble proteins that bind the alkaloid decahydroquinoline. Using this approach, we identified several candidate proteins that interact with this alkaloid, including saxiphilin. These transcript and protein abundance patterns suggest that the presence of alkaloids influences frog physiology and that small molecule transport proteins may be involved in toxin bioaccumulation in dendrobatid poison frogs.

Rapid toxin sequestration modifies poison frog physiology

“…The amphibian protein saxiphilin, which binds the lethal marine alkaloid saxitoxin (Mahar et al, 1991;Yen et al, 2019), increased in abundance in the intestines and skin of toxic frogs (Figure 2A, Table 1). We have previously shown that saxiphilin is more abundant in the plasma of non-toxic frogs and that saxiphilin may bind DHQ using thermal shift assays (Caty et al, 2019). These data support the hypothesis that saxiphilin may carry alkaloid toxins in poison frogs.…”

“…The three experimental groups received fruit flies dusted with a 1% decahydroquinoline (DHQ; Sigma-Aldrich, St. Louis, MO, USA) in a vitamin mix (Dendrocare vitamin/mineral powder, Black Jungle, Turners Falls, MA, USA) for either 1, 4, or 18 days ( Figure 1A). DHQ-like alkaloids are sequestered by O. sylvatica in the wild (Caty et al, 2019) and represent an ecologically relevant treatment. Control frogs (N=3) received flies dusted with vitamin mix without DHQ.…”

“…We confirmed this transcriptome-based method was the best approach by comparing the number of unique peptides that matched the O. sylvatica transcriptome-derived proteome compared to Xenopus and human reference proteomes ( Figure S2). All analyses were performed with a database generated from a O. sylvatica transcriptome (Caty et al, 2019) using the PHROG workflow (proteomic reference with heterogeneous RNA omitting the genome; (Wühr et al, 2014)), which was concatenated with their reversed sequences as decoys for FDR determination. Common contaminant sequences were similarly included.…”

“…We conducted an experiment to determine how alkaloid sequestration alters frog physiology and tested the general hypothesis that alkaloid bioaccumulation alters the abundance of proteins involved in small molecule transport and metabolism. We used the Diablito frog (Oophaga sylvatica) to address this question, which complements our previous studies examining the chemical ecology of this species (Caty et al, 2019;McGugan et al, 2016;Roland et al, 2016).…”

“…Frogs were fed the alkaloid decahydroquinoline (DHQ), a commercially available alkaloid they naturally sequester in the wild (Caty et al, 2019), and we examined how alkaloid sequestration influences frog physiology using quantitative proteomics. Our experiments show that poison frogs sequester alkaloids within four days of exposure, shifting protein abundance across tissues, including plasma glycoproteins and cytochrome P450s.…”