Poison frogs, defensive alkaloids, and sleepless mice: critique of a toxicity bioassay Paul J. Weldon Chemoecology Evolution and mechanisms of the chemical base of ecological interactions ISSN 0937-7409 Volume 27 Number 4 Chemoecology (2017) 27:123-126 DOI 10.1007/s00049-017-0238-0 1 23 Your article is protected by copyright and all rights are held exclusively by Springer International Publishing AG. This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Chemoecology (2017) 27:123–126 DOI 10.1007/s00049-017-0238-0 CHEMOECOLOGY COMMENTARY Poison frogs, defensive alkaloids, and sleepless mice: critique of a toxicity bioassay Paul J. Weldon1 Received: 11 May 2017 / Accepted: 5 June 2017 / Published online: 16 June 2017 Ó Springer International Publishing AG 2017 Abstract In studies of defensive allomones, appropriate chemicals and quantified measures of responses by preda- methods of presenting chemicals and measuring their tors that detract from their success. deterrent effects on consumers are essential for under- standing the contributions that chemicals make to the Keywords Alkaloids Á Chemical defense Á Dendrobatidae Á survivorship of potential prey. However, unnatural chem- Poison frogs Á Toxicity bioassay ical presentations and/or ambiguous bioassay responses occasionally have left open questions on some allelo- chemical effects. This discussion critiques a toxicity Introduction bioassay of Neotropical poison frogs (Dendrobatidae), a group whose skins are known to possess a diverse array of Chemicals that deter consumers play pivotal roles in inter- bioactive alkaloids. The problematic bioassay entails actions throughout nature. Appropriate methods of injecting laboratory mice with the skin extracts of frogs and presenting chemicals and measuring their deterrent effects monitoring the time taken for mice to fall back to sleep to against predation are essential if the contributions that estimate extract toxicity, where longer latencies of sleep chemicals make to survivorship are to be properly evaluated onset were claimed to reflect greater toxicity. Dendrobatids and their ecological significance understood. Documentation do not invasively deliver skin alkaloids to offenders, hence throughout the literature of the repellence of predators or the method of injecting mice with skin extracts does not their post-attack rejections of prey and prey-derived chem- correspond to the frogs’ natural defense mechanisms. icals attests to the use of defensive allomones in many Neither does the injection of frog extracts permit gas- interactions. However, unnatural chemical presentations trointestinal deactivation or clearance mechanisms that and/or ambiguous bioassay responses occasionally have left may reduce the bioavailability of toxins. Whether or not open questions on some allelochemical effects. Here, I dis- increased sleep latencies induced by injected skin extracts cuss these problems arising in investigations of Neotropical reflect toxicity, irritability or other effects, the ultimate poison frogs (Dendrobatidae), a group widely cited in dis- protective value for frogs of prolonging the wakefulness of cussions of chemical defense and a quintessential exemplar mice (or relevant predators) is unclear. Defensible bioas- among vertebrates for the dietary sequestration of defensive says entail both modes of chemical delivery consistent with chemicals (Savitzky et al. 2012). those by which would-be predators normally encounter Handling Editor: Michael Heethoff. Dendrobatids and their alkaloid arsenal & Paul J. Weldon The Dendrobatidae contains ca. 180 species of small frogs [email protected] that occur in leaf litter to forest canopy habitats from Nicaragua south through northern South America to Boli- 1 Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA via (Frost 2017). Many dendrobatids are brightly colored, 22630, USA an aposematic feature related to the noxious skin chemicals 123 Author's personal copy 124 P. J. Weldon that they primarily sequester from their diet of ants, mites, dendrobatids. One concern pertains to predator proxies and other arthropods. More than 550 alkaloids representing being injected with skin extracts; although some amphib- over 20 compound classes have been isolated from the skin ians, including anurans, possess skeletal elements that of dendrobatids (Saporito et al. 2012). Some of these protrude through their integument and subcutaneously compounds, tested singly, exhibit toxicity in investigations deliver skin toxins to offenders (Jared et al. 2015), den- of their pharmacological mode of action, e.g., binding to drobatids do not. Hence, the method of injecting mice with types of neurotransmitter receptors (Santos et al. 2016). skin extracts does not correspond to the frogs’ natural Several compounds, notably pumiliotoxins, are known to defense mechanisms. act as contact toxins against insects, penetrating their Neither does the injection of frog extracts permit gas- cuticle and inducing convulsions, leg autotomy, immobil- trointestinal deactivation nor clearance mechanisms that ity, and death (Weldon et al. 2006, 2013). may reduce the bioavailability of toxins (e.g., Gavhane and Yadav 2012). As Williams et al. (2002) stated, under- standing the relationship between the effects of a toxin The bioassay and its purported significance administered orally and through injection is critical if an oral dose is the exclusive natural mode of exposure. The In a series of studies of anti-predator defense and sexual skin and other organs of newts (Taricha spp.), for example, signaling of dendrobatids, Cummings and colleagues com- contain tetrodotoxin (TTX), an alkaloid that blocks volt- pared the skin toxicities of different frog species or morphs age-gated sodium channels and inhibits the propagation of (Darst and Cummings 2006; Darst et al. 2006; Maan and action potentials in muscles and nerves. North American Cummings 2012; Cummings and Crothers 2013). To obtain deer mice (Peromyscus maniculatus) treated with newt skin extracts, frogs were euthanized by topically applying ben- extracts (Brodie 1968), and laboratory mice and garter zocaine to their head and venter, their skin was removed, and snakes (Thamnophis sirtalis) treated with measured doses skin chemicals were extracted with methanol. The extracts of TTX (Williams et al. 2002), required significantly were evaporated to dryness and the residues were re-dis- greater oral doses of these substances to induce toxicosis solved in saline solution. The extracts from (usually five) than when they were administered by intraperitoneal individual frogs of each species or morph were then injected injections. Williams et al. (2002) hypothesized that TTX subcutaneously into (usually five) sleeping female mice administered orally may be degraded by stomach acidity. (Harlan Laboratories, outbred strain CD-1), one frog extract Darst et al. (2006) cited Daly and Myers (1967) for per mouse, awakened for treatment. Saline solution and, in establishing the skin extract injection of laboratory mice as some experiments (Maan and Cummings 2012), the skin ‘‘a standard protocol’’ for assessing frog toxicity. However, extracts of the Talamanca rocket frog (Allobates talaman- Daly and Myers (1967) injected mice to assess the lethality cae), an alkaloid-free dendrobatid, served as controls. The of different frogs’ extracts, an outcome obviously more time taken for mice to fall back to sleep was used to estimate pertinent both to toxicity and to frog defense (mode of extract toxicity, where longer latencies of sleep onset were chemical delivery notwithstanding) than is latency of sleep claimed to reflect greater toxicity. On the basis of this onset. Maan and Cummings (2012) asserted that their bioassay, referred to here as the sleepless mouse bioassay method represents a ‘‘more sensitive toxicity assay’’ than (SMB), the relative toxicities of frogs were assigned in that used by Daly and Myers (1967). Darst et al. (2006: purported demonstrations of (1) the correlation between frog 5856) had clarified that they actually ‘‘use the term ‘toxi- toxicity and skin color brightness (Darst et al. 2006; Maan city’ to refer to relative irritant effect of frog skin and Cummings 2012; cf. Daly and Myers 1967), (2) the alkaloids’’. establishment of learned avoidances by predators of Batesian Apart from toxicity and/or irritancy, the potential mimic and model frogs (Darst and Cummings 2006; Darst insomnolent effects of dendrobatid skin alkaloids cannot be et al. 2006), and (3) the interaction of natural and sexual dismissed as confounding the SMB. Many plant alkaloids, selection in the evolution of aposematism and signaling e.g., caffeine and nicotine, are documented stimulants, related to female mate choice and male–male competition increasing the latency of sleep onset in mammals (Sh- (Cummings and Crothers 2013). neerson 2005). Nicotine also occurs