J Chem Ecol (2009) 35:391–399 DOI 10.1007/s10886-009-9608-6
Ontogenetic Variation in the Chemical Defenses of Cane Toads (Bufo marinus): Toxin Profiles and Effects on Predators R. Andrew Hayes & Michael R. Crossland & Mattias Hagman & Robert J. Capon & Richard Shine
Received: 30 October 2008 / Accepted: 11 February 2009 / Published online: 5 March 2009 # Springer Science + Business Media, LLC 2009
Abstract We conducted a quantitative and qualitative chemical analysis of cane toad bufadienolides—the cardioactive steroids that are believed to be the principal cane toad toxins. We found complex shifts in toxin composition through toad ontogeny: (1) eggs contain at least 28 dominant bufadienolides, 17 of which are not detected in any other ontogenetic stage; (2) tadpoles present a simpler chemical profile with two to eight dominant bufadienolides; and (3) toxin diversity decreases during tadpole life but increases again after metamorphosis (larger metamorph/juvenile toads display five major bufadienolides). Total bufadienolide concentrations are highest in eggs (2.64±0.56 μmol/mg), decreasing during tadpole life stages (0.084±0.060 μmol/mg) before rising again after metamorphosis (2.35±0.45 μmol/mg). These variations in total bufadienolide levels correlate with toxicity to Australian frog species. For example, consumption of cane toad eggs killed tadpoles of two Australian frog species (Limnodynastes convexiusculus and Litoria rothii), whereas no tadpoles died after consuming late-stage cane toad tadpoles or small metamorphs. The high toxicity of toad eggs reflects components in the egg itself, not the surrounding jelly coat. Our results suggest a dramatic ontogenetic shift in the danger that toads pose to native predators, reflecting rapid changes in the types and amounts of toxins during toad development. Electronic supplementary material The online version of this article (doi:10.1007/s10886-009-9608-6) contains supplementary material, which is available to authorized users. R. A. Hayes : R. J. Capon Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia M. R. Crossland : M. Hagman : R. Shine (*) School of Biological Sciences A08, University of Sydney, Sydney, New South Wales 2006, Australia e-mail:
[email protected]
Keywords Anuran . bufadienolides . Bufo marinus . toxicity . ontogeny . bufadienolides . cardiac steroids
Introduction Understanding the pathways by which introduced species modify the ecosystems they invade is a central challenge for conservation biology; unless we understand those effects, we are unlikely to mitigate them. However, the challenge is a daunting one because of the diverse array of mechanisms involved in the ecological impact of invasive species. Some introduced species act as predators, some as competitors, and others as parasites (Sandlund et al. 1999; Mack et al. 2000; Mooney and Cleland 2001; Schlaepfer et al. 2002). Another category of impact involves toxic invaders, species that imperil native species via exposure to toxins lethal to the native taxa. If an invading species belongs to a phylogenetic lineage not represented in the fauna of the invaded area, it may possess toxins to which native predators are naïve and, hence, are unable to tolerate. For example, Australia has no endemic bufonids (“toads”), and the invasion of South American cane toads (Bufo marinus; see Frost et al. 2006; Pramuk 2006; Pramuk et al. 2008 for alternative generic allocations) has killed a diverse array of Australian predators (including crocodiles, marsupials, snakes, lizards, and anurans) through poisoning when they attempt to eat the toxic invader (Burnett 1997; Doody et al. 2006; Griffiths and McKay 2007; Crossland et al. 2008; Letnic et al. 2008). Although conventional wisdom (including publications for the general public from government departments and museums) asserts that all life history stages of cane toads are highly toxic to predators, this generalization is not supported by empirical evidence. Although it is more than a
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century since Phisalix (1903, 1922) reported that toxicity of bufonid larvae (Bufo bufo) decreased with increasing tadpole stage, few empirical data are available on the toxicity of bufadienolides from cane toads (Lever 2001). This lack of empirical support for the conventional wisdom motivated the present study. To our knowledge, there have been no previous analyses of the chemical composition of bufonid toxins as a function of ontogeny. The most direct evidence of shifts in toxicity comes from studying the effects of toad eggs and larvae on predators. Crossland and Alford (1998) reported no apparent ontogenetic change in toxicity of cane toads (for example, both eggs and hatchlings of toads were toxic to frog tadpoles but not to crayfish), but their experiments were not designed to focus specifically on ontogenetic shifts. In a more detailed study, Crossland (1998) reported that belostomatid water bugs experienced higher mortality rates from consuming late-stage toad tadpoles than from eating earlier-stage conspecifics. Ontogenetic changes in palatability of toad tadpoles also hint at underlying toxicity (presumably, predators detect and avoid toxins). Several authors have reported that the palatability of toad tadpoles differs among developmental stages (see review by Gunzburger and Travis 2005). For example, Heyer et al. (1975) personally tasted small and large tadpoles of B. marinus and reported that larger tadpoles were less palatable. Similarly, Lawler and Hero (1997) found that palatability of cane toad tadpoles to fish (barramundi, Lates calcarifer) decreased during ontogeny. In adult toads, toxins are localized largely in parotid glands, where they can be expressed as a secretion during a predatory event. At the molecular level, the toxic chemicals in cane toad parotid secretions are attributed to steroids known as bufadienolides (Lever 2001). Bufadienolides are not unique to toads and are known to exert a cardiotoxic effect by inhibiting membrane-bound heart tissue Na+/K+ adenosine triphosphatase (ATPase; Steyn and van Heerden 1998). The potency and selectivity of toxicity vary among different bufadienolides and with different isoforms of Na+/ K+ ATPase in different tissues and animal species (e.g., Akimova et al. 2005; Keenan et al. 2005). Given this variability in sensitivity to cane toad bufadienolides, it is impossible to establish a single definitive bioassay— biochemical-, cell-, or animal-based—that measures absolute toxicity. To generate ecologically relevant measures of “overall toxicity,” we need detailed analysis not only of the toxin components within each ontogenetic stage but also of their relative toxicity to predators. The latter measures could be obtained in a standard Na+/K+ ATPase bioassay, but this is not definitive. A more ecologically relevant assay would be based on actual measures on the predator species of concern (in the case of cane toads in Australia, this would involve taxa such as varanid lizards, crocodiles, and
J Chem Ecol (2009) 35:391–399
quolls), but this becomes prohibitive (on both logistical and ethical grounds) when each species requires its own optimized assay. Rather than this, we used a chemical approach to assess likely levels of toxicity by measuring the presence and composition of the different bufadienolides. Australian cane toad parotid secretions are dominated by several major bufadienolides (some of which have not been reported previously from parotid secretions), with >90 additional, as yet unidentified, bufadienolides occurring at low concentrations (Hayes and Capon, unpublished). Although many of these minor cane toad bufadienolides are present at concentrations
