Molecular physiology of pumiliotoxin sequestration in a poison frog

Abstract

Poison frogs bioaccumulate alkaloids for chemical defense from their arthropod diet. Although many alkaloids are accumulated without modification, some poison frog species can metabolize pumiliotoxin (PTX 251D) into the more potent allopumiliotoxin (aPTX 267A). Despite extensive research characterizing the chemical arsenal of poison frogs, the physiological mechanisms involved in the sequestration and metabolism of individual alkaloids remain unclear. We first performed a feeding experiment with the Dyeing poison frog (Dendrobates tinctorius) to ask if this species can metabolize PTX 251D into aPTX 267A and what gene expression changes are associated with PTX 251D exposure in the intestines, liver, and skin. We found that D. tinctorius can metabolize PTX 251D into aPTX 267A, and that PTX 251D exposure changed the expression level of genes involved in immune system function and small molecule metabolism and transport. To better understand the functional significance of these changes in gene expression, we then conducted a series of high-throughput screens to determine the molecular targets of PTX 251D and identify potential proteins responsible for metabolism of PTX 251D into aPTX 267A. Although screens of PTX 251D binding human voltage-gated ion channels and G-protein coupled receptors were inconclusive, we identified human CYP2D6 as a rapid metabolizer of PTX 251D in a cytochrome P450 screen. Furthermore, a CYP2D6-like gene had increased expression in the intestines of animals fed PTX, suggesting this protein may be involved in PTX metabolism. These results show that individual alkaloids can modify gene expression across tissues, including genes involved in alkaloid metabolism. More broadly, this work suggests that specific alkaloid classes in wild diets may induce physiological changes for targeted accumulation and metabolism.

Noninvasive Detection of Chemical Defenses in Poison Frogs Using the MasSpec Pen

“…The concentration of DHQ for all alkaloid-fed samples was determined using the linear fit from the response curve, accounting for the mass of the dorsal skin used in the homogenate, and was determined to be 13 ± 7 ng DHQ/μg skin. Note that while alkaloids have been detected in frog skin as soon as 4–5 days after initial administration by LC-MS, , we did not observe an increase in normalized DHQ signal among alkaloid-fed individuals versus vehicle-fed individuals until day 10 during the feeding study. As alkaloid detection depends on the sensitivity of the mode of analysis and total quantities of accumulated DHQ in skin have been shown to increase with higher doses of the alkaloid, we expect that administration of a higher dose of DHQ would result in earlier detection of the alkaloid in vivo using the MasSpec Pen.…”

“…The name “poison frog” refers to several genera of frogs from anuran families including dendrobatidae, bufonidae, mantellidae, and myobatrachidae, all of which sequester alkaloids from their diet of arthropod prey. − Chemists and biologists have great interest in studying poison frogs for the medicinal implications of alkaloids , and better understanding how the physiological mechanisms allowing alkaloid sequestration evolved. − Alkaloids can be detected on the mucous membrane of the mouth, stomach, intestine, and liver of the frog, prior to storage in granular glands in the skin and excretion upon threat of predation. The mechanism of this chemical sequestration is under investigation but has been found to occur rapidly within 4 days of alkaloid ingestion and may involve plasma carrier proteins and proteins involved in small molecule transport and metabolism. , Over 800 different alkaloid compounds have been described in poisonous amphibians, and alkaloid profiles vary between species, sexes, populations, habitats, and seasons, processes that are largely related to arthropod species availability and diversity. − …”

A toxic environment selects for specialist microbiome in poison frogs

“…Since the frogs do not synthesize their own alkaloids, but instead obtain them from their diet, frogs are non-toxic in the laboratory. Laboratory reared frogs can also be fed alkaloids experimentally to recapitulate the toxic phenotype [21][22][23] . Our ability to rear poison frogs in the lab enables comparisons across lab and field studies to infer generalizable principles of chemical defense and microbial community dynamics.…”

Binding and sequestration of poison frog alkaloids by a plasma globulin

“…Epibatidine was first identified in the genus Epipedobates and specifically binds certain nicotinic receptors, leading it to be proposed as an analgesic alternative to morphine ( Spande et al, 1992 ). Although there is limited research into the mechanisms of sequestration and autoresistance of alkaloids in poison frogs ( Abderemane-Ali et al, 2021 ; Caty et al, 2019 ; Alvarez-Buylla et al, 2022 ; O’Connell et al, 2021 ; Tarvin et al, 2017 ), it is likely this process involves alkaloid transport through circulation for these dietary compounds to end up in skin storage glands. Based on the extensive work on plasma small molecule transport in mammals, one might expect that proteins like albumin, which is an abundant and promiscuous small molecule binder in the blood ( Peters, 1995 ; Baker, 2002 ; Czub et al, 2020 ), or vitamin transporters ( Haddad et al, 1993 ; Hall, 1975 ; Kanai et al, 1968 ), which are able to interact with diet derived molecules, might be involved in alkaloid sequestration in poison frogs.…”