Rapid toxin sequestration modifies poison frog physiology

Abstract

Poison frogs sequester their chemical defenses from their diet of leaf litter arthropods for defense against predation. Little is known about poison frog physiological adaptations that confer this unusual ability to bioaccumulate dietary alkaloids from the intestines to skin glands for storage. We conducted an alkaloid-feeding experiment with the Diablito poison frog (Oophaga sylvatica) to determine how quickly toxins are accumulated and how toxins impact frog physiology using quantitative proteomics. Diablito frogs were able to rapidly accumulate the alkaloid decahydroquinoline to the skin storage glands within four days of dietary exposure, with decahydroquinoline also detected in the intestines and liver. Alkaloid exposure impacted tissue physiology, with protein abundance shifting in the intestines, liver, and skin. Many proteins that increased in abundance with toxin accumulation are plasma glycoproteins, including components of the complement system and the toxin-binding protein saxiphilin. Other protein classes that change in abundance with toxin accumulation are membrane proteins involved in small molecule transport and metabolism, including ABC transporters, solute carrier proteins, and cytochrome P450s. Overall, this work shows that poison frogs can rapidly accumulate alkaloid toxins, which alter carrier protein abundance, initiate an immune response, and alter small molecule transport and metabolism dynamics across tissues. More broadly, this study suggests that acute changes in diet may quickly change the chemical arsenal and physiology of poison frogs, which may have important consequences in their defense against predation.

Evaluating False Transfer Rates from the Match-between-Runs Algorithm with a Two-Proteome Model

“…Human cell lysate and yeast cell lysate were prepared for label-free LC-MS/MS analysis following protocols previously described. 2 Briefly, a protein level bicinchoninic acid protein assay (Pierce) was performed on the clarified lysates to determine protein concentration. Lysates were then reduced at room temperature in the dark with incubation of 5 mM tris(2carboxyethly)-phosphine (TCEP), followed by alkylation with 10 mM iodoacetamide to covalently block reactive cysteine groups.…”

“…Both unfractionated samples were each analyzed 20 times consecutively on an Orbitrap Fusion Lumos mass spectrometer operated in positive-mode with a Proxeon EASY-nLC 1200 liquid chromatograph (Thermo Fisher Scientific) as described previously. 2 Peptide fractionation was performed on a 100 μm inner diameter microcapillary column packed with 35 cm of Accucore C18 resin (2.6 μm, 150 Å, Thermo Fisher Scientific). Approximately 1 μg of peptide was loaded onto the column for LC-MS/MS analysis.…”

“…1 Attempts to address this issue have been both chemical and computational. [2][3][4] Chemically, various labeling methods have been developed to analyze multiple MS experiments simultaneously and address missing values. 5 Isobaric labeling techniques like tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ) provide peptide level modification and simplify downstream analysis while increasing sample throughput.…”

“…8,9 Label-free quantitation inherently does not require chemical or metabolic labeling, but is susceptible to stochasticity. 2,10 As such, attempting to quantify across independent, labelfree LC-MS/MS analyses can result in inaccuracies and missing data. To combat this, several computational methods have been applied.…”

Sodium channel toxin-resistance mutations do not govern batrachotoxin (BTX) autoresistance in poison birds and frogs

“…Together these observations suggest that poison frogs have a means to prevent BTX and STX from reaching their NaVs. It is notable that other frogs resist STX poisoning (31,63,64) and it is thought that the soluble STX binding protein saxiphilin (13,31,65) acts as a 'toxin sponge' to sequester and neutralize the lethal effects of this and possibly other neurotoxins (13,14,30,31,66,67).…”