Toxins 2014, 6, 1979-1995; doi:10.3390/toxins6071979 OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Article Comparative Studies of the Venom of a New Taipan Species, Oxyuranus temporalis, with Other Members of Its Genus Carmel M. Barber 1, Frank Madaras 2, Richard K. Turnbull 3, Terry Morley 4, Nathan Dunstan 5, Luke Allen 5, Tim Kuchel 3, Peter Mirtschin 2 and Wayne C. Hodgson 1,* 1 Monash Venom Group, Department of Pharmacology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria 3168, Australia; E-Mail: [email protected] 2 Venom Science Pty Ltd, Tanunda, South Australia 5352, Australia; E-Mails: [email protected] (F.M.); [email protected] (P.M.) 3 SA Pathology, IMVS Veterinary Services, Gilles Plains, South Australia 5086, Australia; E-Mails: [email protected] (R.K.T.); [email protected] (T.K.) 4 Adelaide Zoo, Adelaide, South Australia 5000, Australia; E-Mail: [email protected] 5 Venom Supplies, Tanunda, South Australia, South Australia 5352, Australia; E-Mails: [email protected] (N.D.); [email protected] (L.A.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +61-3-9905-4861; Fax: +61-3-9905-2547. Received: 5 May 2014; in revised form: 11 June 2014 / Accepted: 16 June 2014 / Published: 2 July 2014 Abstract: Taipans are highly venomous Australo-Papuan elapids. A new species of taipan, the Western Desert Taipan (Oxyuranus temporalis), has been discovered with two specimens housed in captivity at the Adelaide Zoo. This study is the first investigation of O. temporalis venom and seeks to characterise and compare the neurotoxicity, lethality and biochemical properties of O. temporalis venom with other taipan venoms. Analysis of O. temporalis venom using size-exclusion and reverse-phase HPLC indicated a markedly simplified “profile” compared to other taipan venoms. SDS-PAGE and agarose gel electrophoresis analysis also indicated a relatively simple composition. Murine LD50 studies showed that O. temporalis venom is less lethal than O. microlepidotus venom. Venoms were tested in vitro, using the chick biventer cervicis nerve-muscle preparation. Based on t90 values, O. temporalis venom is highly neurotoxic abolishing indirect twitches far more rapidly than other taipan venoms. O. temporalis venom also abolished responses to exogenous acetylcholine and carbachol, indicating the presence of postsynaptic Toxins 2014, 6 1980 neurotoxins. Prior administration of CSL Taipan antivenom (CSL Limited) neutralised the inhibitory effects of all taipan venoms. The results of this study suggest that the venom of the O. temporalis is highly neurotoxic in vitro and may contain procoagulant toxins, making this snake potentially dangerous to humans. Keywords: Oxyuranus temporalis; taipan; antivenom; neurotoxicity; snake; venom 1. Introduction The taipans (Oxyuranus genus) are highly venomous Australo-Papuan elapids consisting of the Inland Taipan (O. microlepidotus), Coastal Taipan (O. scutellatus) and Papuan Taipan (previously O. s. canni; now O. scutellatus). However, phylogenetic studies have shown that even though the Papuan Taipan has been considered a distinct subspecies to the Coastal Taipan, there are no significant differences between the populations [1,2]. More recently, a third distinct species of taipan (O. temporalis, Western Desert Taipan) has been discovered [3]. Due to the apparent remote geographical distribution of this species only a handful of specimens have been collected and studied. Therefore, limited data exists about the distribution, appearance, diet and genetic variation of this species (see [2–4] for further details). Currently there are two wild-caught specimens housed at the Adelaide Zoo in South Australia. The venom of O. temporalis has not been studied. Taipan venoms contain a variety of components including pre- and post-synaptic neurotoxins. Paradoxin (O. microlepidotus, [5]), taipoxin (O. scutellatus, [6]) and cannitoxin (previously O. s. canni, [7]) are presynaptic neurotoxins isolated from taipan venoms consisting of three subunits (α, β and γ) and molecular masses of approximately 45–47 kDa [5–8]. Postsynaptic neurotoxins including oxylepitoxin-1 (O. microlepidotus, [9]), α-scutoxin 1 (O. scutellatus, [10]), α-oxytoxin 1 (previously O. s. canni, [10]), taipan toxin 1 (O. scutellatus, [11]) and taipan toxin 2 (O. scutellatus, [11]) have also been isolated. These short-chain postsynaptic neurotoxins consist of a single subunit, with molecular masses ranging from 6726 Da to 6789 Da, are similar to those isolated from other elapid venoms [12]. Taipan venoms also contain other components including natriuretic-like peptides [13,14], prothrombin activators [14–17], toxins that reversibly block calcium channels (i.e., taicatoxin, [14,18]), Kunitz-type plasma kallikrein inhibitors [14,19] and cysteine-rich secretory proteins (CRISP) [14]. Studies using the chick biventer cervicis nerve-muscle preparation (i.e., a skeletal muscle preparation) have shown that all taipan venoms have in vitro neurotoxic activity [7,8,20,21]. CSL taipan antivenom has also been shown to either delay or prevent the neurotoxicity of these venoms in vitro [20]. Taipan envenoming in humans results in a range of common clinical effects including neurotoxicity, venom-induced consumption coagulopathy and mild rhabdomyolysis [22,23]. Other effects, though rarely reported, include renal failure, thrombotic microangiopathy and haemolytic anaemia [22]. The clinical symptoms of O. temporalis envenoming remain unknown as there have been no documented bites. Toxins 2014, 6 1981 This study, being the first investigation of O. temporalis venom, examined the neurotoxic effects, lethality, and biochemical properties of the venom in comparison to the more well studied taipan venoms. This study provides valuable insight into the venom components and the likely effects of human envenoming. 2. Results 2.1. Size-Exclusion Chromatography Size-exclusion HPLC profiles of all venoms were obtained using a Superdex G-75 column (Figure 1). The profile of O. temporalis venom was simplistic when compared to the other taipan venoms with only one major peak and three minor peaks being apparent (Figure 1A). The major peak, which eluted at approximately 32 min, constitutes a substantial proportion of the venom. From the O. temporalis venom profile, there appears to be no peak eluting over the period of 20–24 min, which is a time period where a peak is observed in the profiles of the other venoms (Figure 1B–E) and has been previously shown to include the presynaptic neurotoxin in these venoms [7,8]. Figure 1. Size exclusion HPLC chromatograph of (A) O. temporalis; (B) O. scutellatus; (C) O. microlepidotus; (D) O. scutellatus (Saibai Island); and (E) O. scutellatus (Merauke) venoms run on a Superdex G-75 column equilibrated with ammonium acetate buffer (0.1 M, pH 6.8) at a flow rate of 0.5 mL/min. Toxins 2014, 6 1982 2.2. Reverse-Phase Chromatography Reverse-phase HPLC profiles of the taipan venoms were obtained using the Jupiter analytical C18 column (Figure 2). As per size-exclusion profiling, reverse-phase HPLC analysis indicated that O. temporalis venom is not a complex venom with a smaller number of peaks compared to the other taipan venoms (Figure 2A). The peak eluting between 15 min and 17 min represents a large proportion of the venom and is likely to contain short-chain postsynaptic neurotoxins. Figure 2. Reverse-phase HPLC chromatograph of (A) O. temporalis; (B) O. scutellatus; (C) O. microlepidotus; (D) O. scutellatus (Saibai Island); and (E) O. scutellatus (Merauke) venoms run on a Jupiter analytical C18 column equilibrated with 0.1% trifluoroacetic acid and gradient conditions of solvent B buffer (90% acetonitrile in 0.09% trifluoroacetic acid) at a flow rate of 0.2 mL/min. 2.3. SDS-PAGE Gel Experiments: Protein Molecular Size All venoms were analysed using SDS-PAGE under non-reducing conditions (Figure 3A). Scans of the gel and computer estimations of the protein band molecular weights of the gel plate showed again that O. temporalis venom is less complex than other taipan venoms with only light bands appearing in the 100–120 kDa and 50–53 kDa with the vast majority of the proteins found below 27 kDa (Figure 3B). Toxins 2014, 6 1983 Figure 3. (A) SDS-PAGE analysis under non-reducing conditions (using a 4%–12.5% acrylamide gel plate); and (B) scan and computer estimations of the protein band molecular weights of the SDS-PAGE gel plate of taipan venoms (1) O. temporalis; (2) O. scutellatus; (3) O. scutellatus (Saibai Island); (4) O. scutellatus (Merauke); and (5) O. microlepidotus. MW indicates molecular standard markers. Note: The MW markers used in this electrophoresis are not reliable below 17 kDa molecular mass. The major taipan venom toxins have been highlighted in their approximate molecular weight position. (A) (B) Figure 4. Protein mobility of (A) taipan venoms (1) O. temporalis; (2) O. scutellatus; (3) O. scutellatus (Saibai Island); (4) O. scutellatus (Merauke); and (5) O. microlepidotus; and (B) venom components (A) O. scutellatus venom; (B) Oscutarin; (C) Taicatoxin; (D) Taipoxin; (E) Postsynaptic taipan venom neurotoxins separated on agarose gel electrophoresis. Toxins 2014, 6 1984 2.4. Agarose Gel Electrophoresis: Protein Mobility From the protein mobility assay, it is clear that O. temporalis venom is comprised of proteins that mainly migrate towards the negative electrode at pH 8.6 indicating that they have positively charged proteins (Figure 4A). The O. temporalis venom appears to contain greatly reduced concentrations of several major toxins that are present in the other taipan venoms examined. This is evident by the light protein bands in positions where procoagulant toxins and presynaptic neurotoxins migrate (Figure 4B). 2.5. Lethality (LD50 Assay) in Mice For O. temporalis venom, all mice in dose level groups 1.0× anticipated LD50 and lower survived. All mice in dose level groups 1.5× anticipated LD50 and higher did not survive. Therefore, the measured LD50 was calculated to be 0.075 mg/kg (i.e., 1.25× anticipated LD50 of 0.06 mg/kg).