Variation in Chemical Defense Among Natural Populations of Common Toad, Bufo bufo, Tadpoles: the Role of Environmental Factors

Abstract

Defensive toxins are widespread in nature, yet we know little about how various environmental factors shape the evolution of chemical defense, especially in vertebrates. In this study we investigated the natural variation in the amount and composition of bufadienolide toxins, and the relative importance of ecological factors in predicting that variation, in larvae of the common toad, Bufo bufo, an amphibian that produces toxins de novo. We found that tadpoles' toxin content varied markedly among populations, and the number of compounds per tadpole also differed between two geographical regions. The most consistent predictor of toxicity was the strength of competition, indicating that tadpoles produced more compounds and larger amounts of toxins when coexisting with more competitors. Additionally, tadpoles tended to contain larger concentrations of bufadienolides in ponds that were less prone to desiccation, suggesting that the costs of toxin production can only be afforded by tadpoles that do not need to drastically speed up their development. Interestingly, this trade-off was not alleviated by higher food abundance, as periphyton biomass had negligible effect on chemical defense. Even more surprisingly, we found no evidence that higher predation risk enhances chemical defenses, suggesting that low predictability of predation risk and high mortality cost of low toxicity might select for constitutive expression of chemical defense irrespective of the actual level of predation risk. Our findings highlight that the variation in chemical defense may be influenced by environmental heterogeneity in both the need for, and constraints on, toxicity as predicted by optimal defense theory.

A matter of proportion? Associational effects in larval anuran communities under fish predation

“…The total initial number of tadpoles was 40 tadpoles m −2 , which is within the natural densities of R. temporaria and B. bufo (Van Buskirk 2005; Gazzola and Van Buskirk 2015; Bókony et al 2016). In the results section, treatments are expressed as the proportion of B. bufo of the total initial number of tadpoles in a cage (Table 1).…”

“…Thus, B. bufo ’s tendency to avoid heterospecific tadpoles and form single-species aggregations (Griffiths and Denton 1992; Bardsley and Beebee 1998) could be viewed as an adaptation counteracting associational susceptibility. Elevated toxin levels may act similarly, but recent findings show that B. bufo toxicity increases in the presence of both conspecific and heterospecific tadpoles (Bókony et al 2016, 2017). …”

Age- and environment-dependent changes in chemical defences of larval and post-metamorphic toads

“…We filtered the samples using nylon syringe filters (FilterBio, pore size = 0.22 μm). We identified compounds as bufadienolides by inspecting the UV spectrum of peaks [27, 33, 45] and by using commercially acquired bufalin, bufotalin, resibufogenin, gamabufotalin, areno- and telocinobufagin (Biopurify Phytochemicals, Chengdu, China), cinobufagin (Chembest, Shanghai, China), cinobufotalin (Quality Phytochemicals, New Jersey, USA) and digitoxigenin (Santa Cruz Biotechnology, Dallas, TX, USA) as standards (Fig. 1).…”

“…213 [47]), as well as the MS ( c ) and UV ( d ) spectra of bufotalin. Further examples of representative chromatograms of the common toad can be accessed in the online appendix of [45] …”

“…We chose the common toad as the study species, because it displays relatively weak inducible defences during the larval stage in terms of morphology and behaviour [36–38] and appears to be unpalatable to several predator species [39, 40], suggesting heavy reliance on chemical defence. Also, the chemical composition of Bufo skin secretions is relatively well known, their main defensive chemicals being bufadienolides and biogenic amines [41–44], and B. bufo are known to contain toxins already in the larval stages [41, 45]. …”