Australian cane toad (Rhinella Marina) chemical ecology : the natural degradation and transformation of bufadienolies

Abstract

The cane toad (Rhinella marina) is a performant invasive species that has spread throughout sub-tropical and tropical Australia. Introduced in 1935 in a failed attempt to control agricultural pests, cane toads have since posed a threat to native ecosystems. By a chemical self-defence mechanism, they are poisonous to most Australian anurophagous at all life stages. The cane toad toxins are a complex mixture dominated by bufadienolides, a class of cardiotoxic steroids. The cane toad chemical ecology has become an important part of the fight against this invasive species because it has brought key knowledge that inspired practical control methods. Bufadienolides are principally stored in the form of stable bufotoxins in the adult cane toad parotoid glands. The secretion of parotoid toxins provokes the ex situ enzymatic hydrolysis of bufotoxins into more potent bufadienolides called bufagenins. In laboratory conditions, parotoid gland-associated bacteria were found to mediate either the degradation or transformation of bufagenins into oxidised or hydroxylated analogues respectively. However, road-killed cane toads were found to remain toxic to native predators in field studies. This research project intended to shed some light on the natural evolution of bufadienolides and evaluate the importance of microbiological degradation and transformation within the natural substrate. Firstly, investigations were conducted into the chemistry of parotoid glands of four road-killed cane toads in decomposition. They revealed that bufadienolide profiles first dominated by bufotoxins quickly evolved to the profit of bufagenins in a similar way than for parotoid secretion. The subsequent profiles remained unchanged and no evolution was observed. Only one hydroxylated analogue (11-hydroxymarinobufagenin) was detected but at a constantly low level. Then, parotoid gland-associated bacteria that were previously proved to degrade or transform bufagenins were challenged with marinobufagenin. The results helped describe and differentiate the different microbiological processes: the biodegradation of bufagenins is an oxidation mediated by gram negative bacteria that follows a sequence to produce oxidised scaffolds; the biotransformation of bufagenins is a hydroxylation mediated by gram positive bacteria independently yielding hydroxylated analogues. Finally, strains were isolated from the four road-killed cane toad parotoid glands and challenged with marinobufagenin. Each bufadienolide profile was compared with reference results as to determine if it was originated from microbial degradation or transformation. The comparison confirmed that parotoid glands are a source of degrading and transforming bacteria. Consequently, this research project demonstrated that bufagenins were the agent of the enduring toxicity of road-killed cane toads and remained stable despite the presence of biodegrading and biotransforming bacteria within the parotoid glands. This area of study has still got the potential to bring key knowledge in the general understanding of bufadienolide metamorphosis and inspire new ways to control the Australian cane toad.