Assessment of virally vectored autoimmunity as a biocontrol strategy for cane toads.

BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach.

Positive Darwinian selection results in resistance to cardioactive toxins in true toads (Anura: Bufonidae).

Members of the Family Bufonidae, true toads, are famous for their endogenously synthesized cardioactive steroids that serve as defensive toxins. Evolution of resistance to these toxins is not understood. We sequenced a key region of the toxin's binding site in the Na(+)/K(+) ATPase for relevant taxa representing Hyloidea (including bufonids), Ranoidea and Archaeobatrachia and tested for positive selection in a phylogenetic context. Bufonidae were distinct from other Hyloidea at 4-6 of 12 sites and, with one exception, had a homologous amino acid sequence. Melanophryniscus stelzneri had a distinct sequence, consistent with other independent evidence for a differentiated toxin. Tests within Bufonidae detected positive selection within the binding region, providing, to our knowledge, the first evidence of this type for positive selection within Amphibia. There was no evidence for positive selection on Bufonidae or M. stelzneri lineages. Sequence change in Leptodactylus ocellatus, a leptodactylid predator of Bufonidae, provides a molecular basis for predator resistance possibly associated with gene duplication.

Cane toad toxicity: an assessment of extracts from early developmental stages and adult tissues using MDCK cell culture.

Extracts of the cane toad (Bufo [Chaunus] marinus) adversely affected the growth of Mardin-Darby canine kidney (MDCK) cells during culture. In a similar manner to ouabain treatment, application of toad extracts over a 24 h period resulted in high levels of cytotoxicity, as indicated by cell detachment, increased membrane permeability and loss of mitochondrial function. Cell viability and growth were unchanged for controls (PBS) and increased with the application of Limnodynastes peronii tadpole and adult frog extracts. We investigated the general cytotoxicity of cane toad developmental stages (e.g., eggs, embryonic hatchlings, tadpoles and post-metamorphic toadlets) as well as selected adult tissues (e.g. skin, gut, liver). Our results showed that pre-metamorphic cane toad aqueous extracts used at 1 mg/ml on MDCK cells generated cytotoxicity levels comparable to ouabain treatment (3 microM). After normalisation, extracts from 2-3-month-old toadlets appeared less toxic than pre- and early metamorphic stages. Adult tissues revealed a gradient of cytotoxicity levels ranging from non-toxic brain to highly toxic dorsal skin extracts.

Genes induced during the early developmental stages of the Cane Toad, Bufo (Chaunus) marinus.

Metamorphosis, a critical stage in the development of toads and frogs, involves rapid levels of morphological change. In the current study, we have used microarray analysis to identify shifts in gene expression between tadpole and toadlet stages of the cane toad, Bufo (Chaunus) marinus. Here, we report on nine genes that show the greatest induction during metamorphosis; the gut-associated gastrokine and trefoil factor, blood components haemoglobins alpha/beta, apolipoprotein and serum albumin, a nasal gene olfactomedin, a lens gene gamma-crystallin, and a novel gene with low homology to frog harderin. We present both temporal and spatial expression patterns of these genes identified in developing and adult cane toads. This study extends our knowledge of the molecular basis of toad metamorphosis, and not only offers insights to the genes induced during the general remodelling that occurs but also reveals possible targets for control and manipulation of amphibian pest species, for example, the cane toad in Australia.