Proteomic Comparison Among New World Arboreal Pit-Viper Venoms
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toxins Article Canopy Venom: Proteomic Comparison among New World Arboreal Pit-Viper Venoms Jordan Debono 1,†, Chip Cochran 2,†, Sanjaya Kuruppu 3, Amanda Nouwens 4, Niwanthi W. Rajapakse 5,6, Minami Kawasaki 7, Kelly Wood 1, James Dobson 1, Kate Baumann 1, Mahdokht Jouiaei 1,8, Timothy N. W. Jackson 1,8, Ivan Koludarov 1,8, Dolyce Low 1, Syed A. Ali 1,8,9, A. Ian Smith 3, Andrew Barnes 7 and Bryan G. Fry 1,8,* 1 Venom Evolution Lab, School of Biological Sciences, University of Queensland, St Lucia, QLD 4072, Australia; [email protected] (J.D.); [email protected] (K.W.); [email protected] (J.D.); [email protected] (K.B.); [email protected] (M.J.); [email protected] (T.N.W.J.); [email protected] (I.K.); [email protected] (D.L.); [email protected] (S.A.A.) 2 Department of Earth and Biological Sciences, Loma Linda University, Loma Linda, CA 92350, USA; [email protected] 3 Department of Biochemistry & Molecular Biology, Biomedical Discovery Institute, Monash University, Clayton, VIC 3800, Australia; [email protected] (S.K.); [email protected] (A.I.S.) 4 School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia; [email protected] 5 Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria 3004, Australia; [email protected] 6 Department of Physiology, Biomedical Discovery Institute, Monash University, Clayton, VIC 3800, Australia 7 Aquatic Animal Health, School of Biological Sciences, University of Queensland, St Lucia, QLD 4072 Australia; [email protected] (M.K.); [email protected] (A.B.) 8 Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia 9 HEJ Research Institute of Chemistry, ICCBS, University of Karachi, Karachi-75270, Pakistan * Correspondence: [email protected]; Tel.: +61-400-193-182 † These authors contributed equally to this work. Academic Editor: Ronald A. Jenner Received: 22 August 2015; Accepted: 16 June 2016; Published: 8 July 2016 Abstract: Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries. Keywords: venom; evolution; pit-viper; enzyme 1. Introduction Snake venoms are complex secretions of various enzymes, peptides and small organic molecules that have diverse modes of action on both prey and accidental human bite victims [1–6]. Though targeting a wide array of physiological processes and targets, the proteins that make up snake venom come from relatively few protein families. This is due to core protein scaffolds being maintained with accelerated rates of evolution at the secondary through quaternary levels facilitating the evolution of multiple derived functions within each protein family [3,7–9]. The use of mass spectrometry Toxins 2016, 8, 210; doi:10.3390/toxins8070210 www.mdpi.com/journal/toxins Toxins 2016, 8, 210 2 of 19 Toxinsaccelerated2016, 8, 210rates of evolution at the secondary through quaternary levels facilitating the evolution2 of 19of multiple derived functions within each protein family [3,7–9]. The use of mass spectrometry in venom proteomics [7,10–19] and venom gland transcriptome analysis [9,20–28] has improved knowledge of in venom proteomics [7,10–19] and venom gland transcriptome analysis [9,20–28] has improved venom composition tremendously. Variation in venom profiles has been shown interspecifically knowledge of venom composition tremendously. Variation in venom profiles has been shown [7,13,18,19,29–34] and intraspecifically, with intraspecific differences found among geographic interspecifically [7,13,18,19,29–34] and intraspecifically, with intraspecific differences found among locales [12,35–43], between sexes [36,37,44,45], and between age classes [10,15,46–52]. Some authors geographic locales [12,35–43], between sexes [36,37,44,45], and between age classes [10,15,46–52]. Some have argued that diversity in venom composition is the result of neutral evolutionary processes and authors have argued that diversity in venom composition is the result of neutral evolutionary processes is not subject to natural selection [53,54], whereas others have argued that adaptation has been driven, and is not subject to natural selection [53,54], whereas others have argued that adaptation has been by strong natural selection, to specific prey species [19,29,36,37,55–60]. driven, by strong natural selection, to specific prey species [19,29,36,37,55–60]. New World pitvipers are believed to be descendants of an ancestral Asian pitviper species, by New World pitvipers are believed to be descendants of an ancestral Asian pitviper species, by way of a single New World colonization event, via the Bering land bridge [61–64]. In regards to the way of a single New World colonization event, via the Bering land bridge [61–64]. In regards to the current study particular interest is given to the members of the genera Bothriechis and Bothrops that current study particular interest is given to the members of the genera Bothriechis and Bothrops that have independently evolved to occupy arboreal niches. That Bothriechis is a distinctive genus from have independently evolved to occupy arboreal niches. That Bothriechis is a distinctive genus from the “bothropoid” genera [61] has been asserted by multiple authors [61–67]; however, phylogenetic the “bothropoid” genera [61] has been asserted by multiple authors [61–67]; however, phylogenetic relationships within the bothropoids, in particular the validity of Bothriopsis, have proven more relationships within the bothropoids, in particular the validity of Bothriopsis, have proven more difficult difficult due to limited sampling of taxa. Numerous studies have presented evidence of the paraphyly due to limited sampling of taxa. Numerous studies have presented evidence of the paraphyly of of Bothrops with respect to Bothriopsis [61–72] leading some authors to advocate for the synonymizing Bothrops with respect to Bothriopsis [61–72] leading some authors to advocate for the synonymizing of Bothriopsis and Bothrocophias with Bothrops [70,72] or Bothriechis [73], while others have maintained of Bothriopsis and Bothrocophias with Bothrops [70,72] or Bothriechis [73], while others have maintained that Bothriopsis and Bothrocophias should remain distinct [61,62,65,74,75] (Figure 1). Regardless of that Bothriopsis and Bothrocophias should remain distinct [61,62,65,74,75] (Figure1). Regardless of taxonomical divisions preferred, it is clear that the arboreal specialization has occurred upon two taxonomical divisions preferred, it is clear that the arboreal specialization has occurred upon two separate occasions. separate occasions. Figure 1. Taxonomical relationships of American pitvipers, based on Castoe 2006 [65]. Figure 1. Taxonomical relationships of American pitvipers, based on Castoe 2006 [65]. Recent studiesstudies utilizingutilizing aa combinedcombined morphological/molecularmorphological/molecular approach and increased sampling of taxa have disagreed on on generic generic assignment assignment as as well. well. Fenwick Fenwick et etal. al.(2009),(2009), sampling sampling 90% 90% of the of theknown known “bothropoid” “bothropoid” taxa taxa and andutilizing utilizing both both morphological morphological characters characters and mtDNA and mtDNA sequence sequence data datafound found support support for several for several monophyletic monophyletic clades within clades Bothrops within (Bothropssensu lato()sensu and proposed lato) and taxonomic proposed taxonomicrevisions, including revisions, recognition including of recognition Bothriopsis of, basedBothriopsis on the, baseddistinct on species the distinct groups speciesrecovered groups [76]. recoveredCarrasco et [76 al.]. (2012)Carrasco [77]et incorporated al. (2012) [77 ]additionalincorporated morphological additional morphological characters not characters utilized by not Fenwick utilized byet al.Fenwick (2009) [76]et al. in(2009) addition [76] into additionpreviously to previouslypublished molecular published sequences molecular and sequences ecological and characters. ecological characters.Citing possible Citing paraphyly possible paraphylyof Bothrops ofwithBothrops respectwith to respectBothriopsis to Bothriopsis (with the (withinclusion the inclusion of Bothrops of Bothropssanctaecrucis sanctaecrucis), they proposed), they proposed following following the recommendation the recommendation of Salomão of et Salomão al. (1997)et [70] al. and(1997) Wüster [70] andet al. Wüster (2002) [72]et al. and(2002) synonymized [72] and synonymized Bothriopsis withBothriopsis Bothropswith [77].Bothrops We have[77 followed]. We have the followedproposals the of proposalsSalomão et of al. Salomão (1997) [70]et al. and(1997) Wüster [70] et and al. (2002) Wüster [72]et al.in recognizing(2002) [72] in the recognizing bothropoids the as bothropoids a single genus, as a singleBothrops genus,,