Since their introduction to the toad-free Australian continent cane toads (Bufo marinus) have caused a dramatic increase in naïve varanid mortality when these large lizards attempt to feed on this toxic amphibian. In contrast Asian–African varanids, which have coevolved with toads, are resistant to toad toxin. Toad toxins, such as Bufalin target the H1-H2 domain of the α1 subunit of the sodium-potassium-ATPase enzyme. Sequencing of this domain revealed identical nucleotide sequences in four Asian as well as in three African varanids, and identical sequences in all 11 Australian varanids. However, compared to the Asian–African varanids, the Australian varanids showed four-base-pair substitutions, resulting in the alteration in three of the 12 amino acids representing the H1-H2 domain. The phenotypic effect of the substitutions was investigated in human embryonic kidney (HEK) 293 cells stably transfected with the Australian and the Asian–African H1-H2 domains. The transfections resulted in an approximate 3000-fold reduction in resistance to Bufalin in the Australian HEK293 cells compared to the Asian–African HEK293 cells, demonstrating the critical role of this minor mutation in providing Bufalin resistance. Our study hence presents a clear link between genotype and phenotype, a critical step in understanding the evolution of phenotypic diversity.

Among-species interactions form the foundation of adaptive evolution and the diversification of species. Such coevolutionary interactions are, hence, one of the major processes responsible for global biodiversity (Thompson 1999). Animals migrating to islands, however, often experience only a subset, or even a total lack of the selective regimes to which they have been subjected to on the mainland. Thus, island living may result in migrants acquiring significant novel physiological, behavioral, as well as morphological adaptations (Blumstein and Daniel 2005; Aubret et al. 2006; Whittaker and Fernández-Palacios 2007; Calsbeek et al. 2009; Losos and Ricklefs 2009). Moreover, such novel adaptations may make island biotas highly susceptible to anthropogenic introduction of invasive species (Pimm et al. 1994; Primack 1995; Grant 1998). For example, the accidental introduction of a novel predator, the brown tree snake (Bioga irregularis), to the island of Guam has caused the extirpation of majority of the island's 25 resident bird species (Wiles et al. 2003). Following similar anthropogenic introductions of invasive species, Australia, like smaller islands, has also suffered from the extinctions of numerous native species (Flannery 1994; Low 2001). Although the impacts of introduced species often vary among ecosystems, the introduction of toxic prey into naïve predator faunas may become particularly destructive (Phillips et al. 2003). An example of such an introduction was the release of the highly toxic South American cane toad (Bufo marinus) to the toad-free Australian continent in 1935. Since their introduction to sugar cane fields in Queensland, cane toads have rapidly spread over large areas of tropical Australia, causing a dramatic increase in mortality of naïve predators, such as large varanids lizards, which rapidly succumb when attempting to feed on this highly toxic amphibian (Doody et al. 2006; Ujvari and Madsen 2009). African and Asian varanids that coexist with toads (family Bufonidae), however, often include these toxic amphibians in their diet (Losos and Greene 1988), demonstrating that they are resistant to toad toxin.

By targeting the 36 nucleotides of the extracellular H1-H2 domain of the α1 subunit of the sodium-potassium-ATPase enzyme, toad toxins such as bufogenines and bufotoxins, as well as other cardiac steroids, for example, ouabain and digoxin, inhibit the enzyme's ability to maintain cellular homeostasis due to intracellular sodium accumulation, and hence affect a wide range of essential cellular functions and crucial physiological processes (Price et al. 1990; Jaisser et al. 1992; Lingrel 2010; Hasenfuss and Teerlink 2011). In the present study, we demonstrate that minor mutations of the H1-H2 domain of the α1 subunit sodium-potassium-ATPase enzyme have made Australian varanids extremely susceptible to toad toxin.

Materials and Methods


Genomic varanid DNA of four Asian and three African varanids, as well as 11 Australian varanid taxa was isolated by phenol–chloroform extraction (Table 1 lists the 18 species used in the study, the three groups of varanids are henceforth referred to Asian–African and Australian). Using the primers described in Holzinger et al (1992), DNA corresponding to the extracellular loop, the H1-H2 domain, of the α1 subunit of the sodium-potassium-ATPase gene in the 18 varanid taxa was subsequently amplified and sequenced on ABI 3130xl Genetic Analyzer using BigDye Terminator Kit V.3.1 (Applied Biosystems, Foster City, CA).

Table 1.  List of the 18 varanid species from which the H1-H2 domain of the sodium-potassium-ATPase gene was sequenced.
Asian taxa Varanus bengalensis, V. dumerili, V. rudicollis, V. salvator
African taxa V. albigularis, V. exanthematicus, V. niloticus
Australian taxa V. acanthurus, V. eremius, V. giganteus, V. gouldi, V. mertensi, V. mitchelli, V. panoptes, V. scalaris, V. storri, V. tristis. V. varius


Site-directed mutagenesis was performed using the Stratagene (La Jolla, CA) Quik-ChangeTM kit according to the manufacturer's instructions. Briefly, complementary primers of 25–35 bases were designed for each mutagenesis reaction with the mutations placed in the centers of the primers. The α1 subunit of the human Na+/K+-ATPase (GenBank accession number NM_00070101.6) was used as the template and was obtained in the plasmid vector, pCMV6-XL5 (OriGene, Rockville, MD). Mutagenesis products were amplified using Pfu DNA polymerase (Stratagene) using 12–16 cycles in a DNA thermal cycler (Perkin Elmer, Sydney, NSW, Australia). After digestion of the template DNA with Dpn I (New England Biolabs), the amplified mutant DNA was transformed into Escherichia coli (DH5α) as described by Mun et al. (2005). Incorporation of the desired mutations was confirmed by automated DNA sequencing (Australian Genome Research Facility, Brisbane, Australia).

With respect to the human H1-H2 domain of the α1 subunit amino acid sequence (residues 512–547), two varanid sequence modifications were obtained corresponding to QAGTEDDPAGDN (Australian varanids) and LAGTEDDPSR-DN (Asian–African varanids).


Human embryonic kidney 293 cells (HEK 293 cells) were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum and 1/200 diluted streptomycin and penicillin (0.5%; v/v) under standard conditions (5% CO2, 37°C).

HEK293 cells were maintained in 25 cm2 culture flasks and transfected with 8 μg of the α1 subunits of the Asian–African and Australian varanid-modified versions of the human Na+-K+-ATPase gene (3.5 kb) in pCMV-XL5 using LipofectAMINE 2000TM according to the manufacturer's instructions (Invitrogen).

After 24 h transfected cells were transferred to 24-well plates and cultured for a further 24 h. Selection of stable transformants was performed by exposing transfected HEK293 cells to 100 μg/mL G418 (Invitrogen). Individual resistant clones were isolated 3 weeks later and exposed to ouabain (10 mM, Sigma, St. Louis, MO) for a further 3 weeks. The success of the stable transfections of the α1 subunits of the sodium-potassium-ATPase gene containing the Asian–African or Australian varanid H1-H2 domains was subsequently verified by sequencing genomic DNA extracted from the two HEK293 cell lines.


The transfected Asian–African and Australian HEK 293 cells were seeded in 96-well plates at 37°C in a humidified 5% CO2 atmosphere (approximately 5000 cells/well). When reaching a confluence of 50% the cells were subjected to Bufalin (MW 386.52, Sigma) concentrations ranging from 0.0005 to 2000 μM for 24 h (see Table 2 for Bufalin concentrations and sample sizes used).

Table 2.  Bufalin concentrations (μM) and sample sizes (number of wells) of the two cell lines exposed to Bufalin toxin.
Bufalin concentration (μM)Australian varanidsAsian–African varanids
0.001 30  
0.0075 30  
0.025 30  
0.1 30  
5 30 15
25   15
50 30
75   30
150   30
200 30
300   30
1000 15
2000   15

The effects of Bufalin on the Asian–African and Australian variant α1 subunits expressed in HEK 293 cells were quantified using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI). This assay is based on a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The assays were performed by adding 20 μl of the CellTiter 96® AQueous One Solution Reagent directly to each culture well. The cells and reagent were then incubated at 37°C for 2 h. The absorbance in each well was subsequently recorded at 490 nm using a 96-well plate reader.

We used the DeltaGraph 5 software (Red Rock Software, Salt Lake City, UT) to generate the cell proliferation curves of the Asian–African and the Australian cell lines exposed to increasing Bufalin concentrations (the two curves were based on the mean cell proliferation rates recorded at each of the Bufalin concentrations used in the study). The comparison in Bufalin sensitivities of the Asian–African and Australian transfected HEK 293 cells was based on the Bufalin concentration resulting in a 50% reduction in proliferation absorbance between the control untransfected HEK 293 cells (n= 60) not exposed to Bufalin and the absorbance recorded of methanol untransfected HEK 293 cells (n= 60; [2.632 nm + 0.277 nm, respectively]/2 = 1.450 nm). The latter treatment completely inhibits cell proliferation. The “50% absorbance value” was subsequently entered in the DeltaGraph software to generate the corresponding Bufalin concentration in the Asian–African and the Australian varanid HEK 293 cell lines.


The nucleotide sequences of the H1-H2 domains in all seven toad toxin resistant varanids (four Asian and three African taxa) were identical. Moreover, the sequences of the H1-H2 domains in all 11 toad toxin-sensitive Australian varanid taxa were also identical. However, compared to the Asian–African varanids, the Australian varanids showed four-base-pair substitutions, resulting in the alteration of three amino acids at positions 111, 119, and 120 (Table 3).

Table 3.  Amino acid and nucleotide sequence of the H1-H2 domain of the alpha-1 subunit of the Na+/K+-ATPase gene in Asian–African and Australian varanid lizards. Amino acid substitutions in the Australian compared to that of the Asian–African varanids are highlighted in bold (positions 111, 119, and 120).
Asian– African varanids
Position 111 112113114115116117118 119 120 121122
Amino Leucine Alanine Glycine Threonine Glutamic Aspartic Aspartic Proline Serine Arginine Aspartic Aspargine
 acids      acid acid acid     acid  
Australian varanids
AminoGlutamineAlanineGlycineThreonineGlutamicAsparticAsparticProline Alanine Glycine AsparticAspargine
 acids    acidacidacid   acid 

We failed to observe any significant difference in mean cell proliferation rates of Bufalin concentrations up to 0.01 μM of the Australian varanid HEK 293 cells and of up to 25 μM of the Asian–African HEK 293 cells compared to control untransfected HEK 293 cells not exposed to Bufalin (t88= 0.94, P= 0.35, t73= 0.69, P= 0.49, respectively, Figure 1). We also failed to record any significant difference in mean cell proliferation rates of Australian HEK 293 cells at Bufalin concentrations equal and above 100 μM (t43= 0.08, P= 0.91, Figure 1) and that of control methanol treated untransfected HEK 293 cells, whereas in the Asian–African HEK 293 cells such a lack of difference was only observed at the highest Bufalin concentration (2000 μM, t73= 0.45, P= 0.65; Figure 1).

Figure 1.

Cell-proliferation rates (absorbance at 490 nm, with associated standard deviations) of control untransfected HEK 293 cells (no Bufalin added, filled quadrate), control untransfected HEK 293 treated with 70% methanol (open quadrate) and cell-proliferations rates of the Australian varanids (filled black circles) and Asian–African varanids (open gray circles) subjected to increasing levels of Bufalin. The horizontal stippled line denotes 50% reduction in cell proliferation rate between control (no Bufalin added) and methanol treated untransfected HEK 293 cells. The curves were fitted to mean absorbance of the depicted values obtained from the Asian–African and Australian HEK 293 cells using the DeltaGraph 5 software.

In the Australian HEK 293 cells a 50% reduction in cell proliferation was observed at a Bufalin concentration of 0.058 μM, whereas in the Asian–African HEK 293 cells a 50% reduction in cell proliferation occurred at a concentration of 170 μM, approximately 3000 times higher than that recorded in the Australian HEK 293 cells.


As in all members of the family Bufonidae, the parotoid glands of the cane toad contain potent bufogenines as well as bufotoxins (Zug and Zug 1979; Clark 1997). Both of these toxic compounds are steroids having a nucleus of three 6- and one 5-membered rings (Clark 1997). This basic chemical structure of toad toxins has been observed in all members of the family Bufonidae so far investigated (Pettit and Kamano 1972; Flier et al. 1980; Shimada and Nambara 1980; Shimada et al. 1984; Shimada et al. 1985a,b; Tashmukhamedov et al. 1995). Bufalin shares the same basic chemical steroid structure with other bufogenines and bufotoxins (Rohrer et al. 1982), and, like bufogenines and bufotoxins, Bufalin has been shown to inhibit the activity of sodium-potassium-ATPase gene (Pamnani et al. 1994; McGowan et al. 1999). Moreover, except for one H+, Bufalin has the identical structure as resibufogenin found in cane toad toxin (B. marinus; Pamnani et al. 1994). Thus, the results obtained in the present study based on only using one of toxic compounds (Bufalin) strongly suggest an overall significant difference in general toad toxin resistance between Asian–African and Australian varanid lizards.

Substitution at position 120 of the H1-H2 domain by positively charged arginine residue in the Asian–African varanid sequence to a neutral glycine residue in the Australian varanid sequence causes a local change in net charge in the sodium-potassium-ATPase enzyme. Such alterations have been shown to have a significant impact on the enzyme's response to cardiac steroids (Price et al. 1990; Jaisser et al. 1992), and most likely constitute a major factor in the significant decrease in the Australian varanids resistance to Bufalin. However, changes in amino acid hydrophobicity and other structural properties can also affect cardiac steroid resistance (Hasenfuss and Teerlink 2011). Consequently, the substitutions at positions 111 and 119 may also have contributed to the substantial difference in Bufalin resistance between Asian–African and Australian varanid HEK 293 cell lines.

Asian varanids are estimated to have colonized Australia 10–15 million years ago (Baverstock et al. 1993; Pianka 1995; Fuller et al. 1998). As all Asian varanids examined in the present study exhibited the Bufalin resistant H1-H2 domain of α1 subunit of sodium-potassium-ATPase enzyme, we suggest that the first ancestral Asian migrants to Australia were most likely also resistant to Bufalin. As mentioned above, Asian–African varanids that coexist with toads often include toads in their diet (Losos and Greene 1988). Moreover, in the Philippines Varanus salvator has been observed feeding on introduced cane toads without suffering any effects of the toad's toxins (C. Wanger, pers. comm.), clearly demonstrating that Asian–African varanids are resistant to the toxins produced by these amphibians. Had similar H1-H2 domain mutations, as observed in the Australian varanids, occurred in Asian–African varanids such mutations would have resulted in a dramatic decrease in toad toxin resistance, and prevented Asian–African varanids to use this, sometimes abundant, prey resource. Members of the family Bufonidae are, however, not native to Australia (Cogger 2000), and after ancestral Asian varanids arrived on the toad-free Australian continent the four-base-pair mutations recorded in the Australian varanids would most likely not have resulted in any significant negative fitness consequences. We therefore suggest that relaxed selection among the ancestral Australian varanids to maintain a toad-toxin resistant H1-H2 domain of α1 subunit of sodium-potassium-ATPase enzyme most likely explains their present susceptibility to Bufalin. This is supported by examples in other taxa where among-population variation in predator exposure to a toxic prey has been shown to result in concomitant genetic adaptations in the predator (Brodie et al. 2002; Geffeney et al. 2005).

Until the recent development of novel genetic techniques, adaptive evolution was believed to involve mutations affecting large parts of the genome (Lande 1983; Falconer and Mackay 1996). However, the results from the present study clearly show that complex phenotypic traits can be controlled by minor mutations. Indeed, minor mutations have been demonstrated to have significant phenotypic effects in other organisms such as the single gene controlling body size in dogs (Sutter et al. 2007) and adaptations to high altitude in deer mouse (Peromyscus maniculatus; Storz et al. 2009). Thus, the results of our study provide a clear link between genotype and phenotype, a critical step in understanding the evolution of phenotypic diversity.

As mentioned above, our demographic studies of one of Australia's large (up to 6 kg) carnivorous varanids, the yellow-spotted goanna (V. panoptes), conducted on the Adelaide River floodplain in the Northern Territory of Australia, show that since the arrival of cane toads in October 2005, more than 95% of the yellow-spotted goannas have died while attempting to feed on toads (Ujvari and Madsen 2009). Similar cane-toad-related declines of this taxon have been recorded along the Daly River (Doody et al 2006; approximately 150 km southwest of our study site), clearly demonstrating the devastating effect of an exotic and invasive toxic prey on naïve predator populations. The dramatic population declines recorded may imperil the long-term survival of this large predator on the floodplains of tropical Australia.

Associate Editor: J. Fordyce


We are grateful to P. Baverstock and J. Vindum for supplying us with varanid lizard samples and to G. Torr, R. Shine, G. Brown, and M. Crossland for comments on the manuscript.