Microbial biotransformation as a source of chemical diversity in cane toad steroid toxins R. Andrew Hayes, Andrew M. Piggott, Kristian Dalle, Robert J. Capon * Institute for Molecular Bioscience, The University of Queensland, Carmody Road, St. Lucia, Qld 4072, Australia article info Article history: Received 11 December 2008 Revised 20 January 2009 Accepted 21 January 2009 Available online 27 January 2009 Keywords: Biotransformation Bufo marinus Cane toad Bufadienolide Chemical ecology abstract The cane toad is an invasive pest that is rapidly colonising northern Australia. The cane toad parotoid gland secretes cardiotoxic steroids (bufadienolides) that are poisoning native predator species. This study reveals bufadienolide diversity within the secretions of Australian cane toads is different to cane toads from overseas, being far more structurally diverse than previously assumed. It is proposed that this var- iation is mediated by in situ bacterial biotransformation. Ó 2009 Elsevier Ltd. All rights reserved. The cane toad, Bufo marinus, is an invasive pest that was intro- duced to multiple locations around the world as an ultimately unsuccessful biocontrol agent for beetle pests of sugar cane. In sev- eral locations, including Australia, Fiji and Hawaii, the species has had a significant ecological impact on native animal populations. 1 The cane toad and other members of the genus Bufo are renowned for their ability to produce and deploy cardiotoxic steroids (bufadi- enolides) as a form of chemical defence. 1 Bufadienolides are antag- onists of Na + /K + -ATPase in much the same way as the plant derived cardenolides (such as digitalis) 2 and ingestion can lead to cardiac arrest and death. The prospect of fatal encounters with cane toads is enhanced by the presence of specialised parotoid glands that se- crete high concentrations of bufadienolides in response to preda- tory attack. In Australia, the cane toad is a threat to native predator species such as fresh water crocodiles, marsupials, snakes and lizards, which are highly vulnerable to cane toad poisoning. 3–5 Since its release in Australia in 1935, 6 the cane toad has ad- vanced south along the eastern seaboard from Queensland into New South Wales, and west through the Northern Territory to- wards Western Australia – colonising >1 million km 2 and seriously impacting native predator populations. 1 To date, cane toad control has been limited to local and short term techniques such as hand collection and trapping. 7 In an attempt to broaden the control agenda, and identify more permanent solutions effective on a lar- ger regional or national scale, we recently embarked on an analysis of cane toad chemical ecology. 8 This report describes one aspect of those investigations, namely an assessment of Australian cane toad bufadienolides, including the role played by microorganisms in diversifying and possibly enhancing bufadienolide toxicity. Of the order of 100 bufadienolides have been described from toads of the genus Bufo, of which only 30 have been attributed to B. marinus, 9 and only four, marinobufagin (1), telocinobufagin (2), bufalin (3) and resibufogenin (4), have been reported from the cane toad parotoid gland secretion. More significantly, none of these re- ported chemical analyses was performed on cane toads sampled from the resident Australian population. Our investigations into the parotoid gland chemistry of cane toads sampled from locations at the eastern and western extremes of the Australian colonisation range revealed a bufadienolide composition dominated by 1, with moderate levels of 2, 3, arenobufagin (5) and marinobufotoxin (6), lower levels of 4, hellebrigenin (7), marinobufagin-3-pimeloyl-L- arginine ester (8), bufalin-3-pimeloyl-L-arginine ester (9) and buf- alitoxin (10), and detectable levels of >30 minor bufadienolides (Fig. 1 and Scheme 1). 10 These studies reveal, for the first time, that Australian cane toad parotoid secretion chemistry is not identical to that reported from overseas cane toads, 9 and that the bufadien- olide chemical diversity is far greater than previously assumed. It has previously been demonstrated that bufadienolides can undergo biotransformation when exposed to cultures of bacte- ria 11–13 or plant cells. 14 Although these biotransformation studies lack ecological relevance, they do raise the possibility that in situ bacterial biotransformation could be a mechanism for chemical diversification within the parotoid gland. To test this hypothesis, we recovered bacterial isolates from swabs taken from the parotoid gland, ovary, tongue and stomach of a dissected mature 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.01.064 * Corresponding author. Tel.: +61 7 3346 2979; fax: +61 7 3346 2090. E-mail address: r.capon@imb.uq.edu.au (R.J. Capon). Bioorganic & Medicinal Chemistry Letters 19 (2009) 1790–1792 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl