Comment on Amézquita et al. (2017) “Conspicuousness, color resemblance, and toxicity in geographically diverging mimicry: The pan-Amazonian frogAllobates femoralis”

Abstract

Amézquita et al. (2017) recently concluded that species of the Allobates femoralis group are toxic to mice at levels equivalent to syntopic alkaloid-containing poison frogs, which they attributed to the presence of alkaloids in skin secretions. However, the chemical composition of skin secretions was not analyzed, and here we present additional data supporting the absence of alkaloids in skin secretions of the Allobates femoralis group. Instead, we suggest the observed toxicity was caused by the anesthetic benzocaine, which was applied to the buccal cavity to euthanize frogs prior to skin removal. We show that orally administered benzocaine is rapidly incorporated into the skin of species that sequester and do not sequester alkaloids, which casts doubt on the conclusion that Allobates femoralis group skin secretions are toxic and makes the results of experiments with alkaloid-containing species of Adelphobates and Ameerega uninterpretable. To prevent experimental errors and misinterpretations in studies of amphibian chemical defense, we encourage researchers to test the chemical composition of samples prior to experimentation, include all necessary controls to detect false positives, conduct small pilot studies for new methods, and consider the limitations of particular methods and their ability to address the intended research questions.

Transcriptomic Signatures of Experimental Alkaloid Consumption in a Poison Frog

“…[32]. Because epibatidine is a highly toxic alkaloid (LD50 ~0.2 mg/kg; [43]), and A. femoralis is considered a non-toxic dendrobatid species ([44], but see [45] and [46]), we judged using the less toxic alkaloid sparteine (LD50 ~200 mg/kg; [47]) for this species. Sparteine is an alkaloid from plants of the genus Lupinus , which has a similar structure to epibatidine but belongs to the quinolizidine class (Figure 1B).…”

Weak warning signals can persist in the absence of gene flow

“…Notably, we initially collected 2 frogs from the Kaw Mountains in 2013, which were used for molecular analysis, but to increase sample size for alkaloid variability, we collected 6 additional frogs in 2014. Frogs were killed by deep-freezing to avoid possible contamination from killing agents (58). Livers were preserved in 95% ethanol, and whole skins were preserved in 100% methanol for subsequent alkaloid analysis.…”

Evaluation of benzocaine-based anesthetic gel in anuran skins extracts: A case study using the frog Lithodytes lineatus (Anura: Leptodactylidae)

“…The use as an anuran death protocol is highly effective concerning lethality even when compared to other anesthetics PLOS ONE [20,21,31,32] besides to being considered a low-cost product and without restrictions on the purchase, facilitating access [12,18]. On the other hand, the use of anesthetics to kill frogs that will subsequently have their skins removed for the preparation of extracts used in biological tests or investigation of the chemical composition present in the integument can be a problematic factor due to the potential contamination [22].…”

“…Benzocainebased anesthetics are highly effective for both, anesthesia and death in amphibians, not requiring a large amount of the product to be able to kill them [12,[18][19][20][21]. However, recently, Saporito and Grant [22] found traces of the benzocaine substance coming from the Orajel © (liquid) product in skin extracts made from individuals of the species Melanophryniscus moreirae and Lithobates clamitans killed with the oral application of this anesthetic and concluded that the application directly in the mouth in certain species of anurans, may invalidate potential studies on the chemical composition of the extracts of these animals, signalling false positives, such as inaccurate detection of substances and/or incorrect information about potential biological activities. The authors gave as an example the study by Amézquita et al [23] who, through experiments, suggested that some populations of the frog Allobates femoralis showed higher toxicity in the extracts when used in mice and was considered by the authors to be from alkaloids present in the integument of this species.…”

“…The authors gave as an example the study by Amézquita et al [23] who, through experiments, suggested that some populations of the frog Allobates femoralis showed higher toxicity in the extracts when used in mice and was considered by the authors to be from alkaloids present in the integument of this species. As A. femoralis belongs to a frog family that has no representative known for producing or sequestering diet alkaloids [24][25][26][27][28], this result was contested by Saporito and Grant [22] who described that the possible toxicity in A. femoralis is due to the presence of benzocaine substance that was converted to the animals' skin, detecting the presence of this substance experimentally using mass spectrometry (MS).…”