Sequestered Alkaloid Defenses in the Dendrobatid Poison Frog Oophaga pumilio Provide Variable Protection from Microbial Pathogens

Abstract

Most amphibians produce their own defensive chemicals; however, poison frogs sequester their alkaloid-based defenses from dietary arthropods. Alkaloids function as a defense against predators, and certain types appear to inhibit microbial growth. Alkaloid defenses vary considerably among populations of poison frogs, reflecting geographic differences in availability of dietary arthropods. Consequently, environmentally driven differences in frog defenses may have significant implications regarding their protection against pathogens. While natural alkaloid mixtures in dendrobatid poison frogs have recently been shown to inhibit growth of non-pathogenic microbes, no studies have examined the effectiveness of alkaloids against microbes that infect these frogs. Herein, we examined how alkaloid defenses in the dendrobatid poison frog, Oophaga pumilio, affect growth of the known anuran pathogens Aeromonas hydrophila and Klebsiella pneumoniae. Frogs were collected from five locations throughout Costa Rica that are known to vary in their alkaloid profiles. Alkaloids were isolated from individual skins, and extracts were assayed against both pathogens. Microbe subcultures were inoculated with extracted alkaloids to create dose-response curves. Subsequent spectrophotometry and cell counting assays were used to assess growth inhibition. GC-MS was used to characterize and quantify alkaloids in frog extracts, and our results suggest that variation in alkaloid defenses lead to differences in inhibition of these pathogens. The present study provides the first evidence that alkaloid variation in a dendrobatid poison frog is associated with differences in inhibition of anuran pathogens, and offers further support that alkaloid defenses in poison frogs confer protection against both pathogens and predators.

Variable Alkaloid Defenses in the Dendrobatid Poison Frog Oophaga pumilio are Perceived as Differences in Palatability to Arthropods

“…Arthropod predators come into direct contact with chemically defended frogs using their antenniform, pedipalps, etc. and sample or taste the prey before making decisions to consume them (Gray et al 2010;Hantak et al 2016;Hovey et al 2016;Isman 1992;Murray et al 2016;Weldon et al 2013). Therefore, the palatability assays utilized in the present study may represent a more biologically relevant measure of alkaloid defenses against arthropod predators that use chemoreception.…”

“…Alternatively, arthropods primarily use contact chemoreception to assess prey (Fritz 1981;Hantak et al 2016;Hovey et al 2016;Isman 1992;Murray et al 2016;Szelistowski 1985;Weldon et al 2013), and have a diversity of chemoreceptors that are located on structures such as antenniform, maxillae, labium, pedipalps, etc. (Isman 1992).…”

“…Alkaloids isolated from whole skin fractionations were manually injected into the instrument using 1 μl of the final 100 μl alkaloid extract, whereas alkaloids isolated from TAS samples were analyzed by injecting 2 μl of the 100 μl final alkaloid extract using the auto-sampler function on the GC-MS. Each individual frog extract (n = 15 for whole frog skin fractionations; n = 66 for TAS fractionations) was run in triplicate on EI-MS for identification and quantification purposes (n = 45 for whole skin fractionations; n = 198 for TAS fractionations) and once on CI-MS for alkaloid identification, resulting in a total of 324 individual runs on the GC-MS. Individual alkaloids for each run on the GC-MS were identified by comparing retention times and mass spectral data to known alkaloids found in dendrobatids (see Daly et al 2005;Saporito et al 2006;Hovey 2016). Dendrobatid alkaloids have been assigned a coding system with boldface numbers and letters that distinguish different alkaloids by molecular weight.…”

“…More than 232 different alkaloids have been identified in O. pumilio (categorized into 24 structural classes), and individual frogs possess between 4 and 44 different alkaloids (Saporito et al 2007a). Ants and spiders are important natural predators of this species (Hovey et al 2016;Murray et al 2016;Santos and Cannatella 2011), both of which use chemoreception to detect prey and therefore may be particularly sensitive to differences in alkaloid defenses. Recently, Murray et al (2016) investigated differences in predation by bullet ants (Paraponera clavata) and red-legged banana spiders (Cupiennius coccineus) on different life stages of O. pumilio, and provided evidence that ant predators are sensitive to differences in alkaloid defenses; however, it is not known how alkaloid variation among populations of O. pumilio translates to predator avoidance.…”

Land use impacts poison frog chemical defenses through changes in leaf litter ant communities

“…Central and South American poison frogs (Family Dendrobatidae) have evolved unique physiological traits that are tightly linked to the trophic structure of rainforest arthropod communities [12]. Poison frogs acquire alkaloid-based chemical defenses from their diet for defense against predators and pathogens [13,14]. Chemical defenses have independently evolved four times within the Dendrobatidae family, coinciding with a dietary specialization on ants and mites in some species [15].…”