Comparative Biochemistry and Physiology, Part C 211 (2018) 7–14 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc Does size matter? Venom proteomic and functional comparison between T night adder species (Viperidae: Causus) with short and long venom glands Francisco C.P. Coimbraa, James Dobsona, Christina N. Zdeneka, Bianca op den Brouwa, Brett Hamiltonb, Jordan Debonoa, Paul Mascic, Nathaniel Frankd, Lilin Gee,f, Hang Fai Kwoke, ⁎ Bryan G. Frya, a Venom Evolution Lab, School of Biological Sciences, University of Queensland, St Lucia, QLD 4072, Australia b Centre for Advanced Imaging & Centre for Microscopy and Microanalysis, University of Queensland, St Lucia, QLD 4072, Australia c Princess Alexandra Hospital, Translational Research Institute, University of Queensland, St Lucia, QLD 4072, Australia d Mtoxins, 1111 Washington Ave, Oshkosh, WI 54901, USA e Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, China f Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, School of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Qixia District, Nanjing 215400, China ABSTRACT Night adders (Causus species within the Viperidae family) are amphibian specialists and a common source of snakebite in Africa. Some species are unique in that they have the longest venom glands of any viper, extending approximately 10% of the body length. Despite their potential medical importance and evolutionary novelty, their venom has received almost no research attention. In this study, venoms from a short-glanded species (C. lichtensteinii) and from a long-glanded species (C. rhombeatus) were compared using a series of proteomic and bioactivity testing techniques to investigate and compare the toxin composition and functioning of the venoms of these two species. Both C. rhombeatus and C. lichtensteinii were similar in overall venom composition and in- hibition of blood coagulation through non-clotting proteolytic cleavage of fibrinogen. While the 1D gel profiles were very similar to each other in the toxin types present, 2D gel analyses revealed isoformic differences within each toxin classes. This variation was congruent with differential efficacy of South African Institute for Medical Research snake polyvalent antivenom, with C. lichtensteinii unaffected at the dose tested while C. rhombeatus was moderately but significantly neutralized. Despite the variation within toxin classes, the similarity in overall venom biochemistry suggests that the selection pressure for the evolution of long glands served to increase venom yield in order to subjugate proportionally large anurans as a unique form of niche partitioning, and is not linked to significant changes in venom function. These results not only contribute to the body of venom evo- lution knowledge but also highlight the limited clinical management outcomes for Causus envenomations. 1. Introduction due to their ambush predatory strategy and associated low rates of food intake (Ineich et al., 2006). However, night adders stray from this Night Adders (Family Viperidae, genus Causus) are a clade of small classic viperid profile, as they have slender heads and gracile bodies African sub-Saharan batrachophagous (anuran specialist) vipers which that are morphologically convergent with many non-venomous snakes contains six well-recognized species, that, as the common name in- or elapids (Mallow et al., 2003; Ineich et al., 2006). Furthermore, as dicates, are primarily nocturnal (Luiselli et al., 2001; Ineich et al., 2006; opposed to other vipers, night adders are active pursuit predators that Toledo et al., 2007; Akani et al., 2012). C. lichtensteinii is the slenderest engage in fossorial to semi-fossorial life styles (Mallow et al., 2003; (Ineich et al., 2006) and one of the smallest species in the genus Ineich et al., 2006). They are also reported to prey upon multiple frogs (averaging 30 cm in length), while C. rhombeatus is the largest species or toads in succession and particularly large ones, digesting meals (95 cm) (Ineich et al., 2006; Mallow et al., 2003). within 7–10 days (Mallow et al., 2003), which is a relatively short time Viperid snakes typically possess short, robust bodies with a broad compared to other vipers, which is indicative of a higher metabolic rate head and thin neck, and they are thought to have low metabolic rates (Ineich et al., 2006). Because of their morphological and ecological ⁎ Corresponding author. E-mail address: [email protected] (B.G. Fry). https://doi.org/10.1016/j.cbpc.2018.05.003 Received 1 January 2018; Received in revised form 3 May 2018; Accepted 8 May 2018 1532-0456/ © 2018 Elsevier Inc. All rights reserved. F.C.P. Coimbra et al. Comparative Biochemistry and Physiology, Part C 211 (2018) 7–14 distinction among vipers, Causus was thought to be phylogenetically particular focus of the study was to investigate if the substantial dif- basal and plesiomorphic relative to all other genera within the Viperidae ferences in venom gland size and prey size are related to substantial family (Viperinae subfamily [vipers] and Crotalinae subfamily [pit-vi- variation of the venom composition or functional activity. pers]) (Janssen et al., 1990; Ineich et al., 2006). However, genetic re- search revealed strong support for their phylogenetic placement within 2. Materials and methods Viperinae subfamily (Wüster et al., 2008; Pook et al., 2009; Pyron et al., 2013; Alencar et al., 2016), although the precise intra-subfamily pla- 2.1. Venoms cement is still unresolved. The morpho-ecological characteristics of Causus are therefore derived traits (i.e. apomorphic) rather than re- Pooled, lyophilized C. lichtensteinii (from multiple adults, geo- presenting an ancestral viperid condition (plesiomorphic) (Wüster graphical original unknown) venom was supplied by MToxins (http:// et al., 2008). www.mtoxins.com/home.html). Pooled, lyophilized C. rhombeatus C. rhombeatus (and the recently split sister species C. maculatus) are (from multiple adults, geographical origin unknown) venom was pur- the only representatives of this genus that have elongated venom glands chased from the Miami Serpentarium Laboratories (Punta Gorda, extending beyond the base of the skull and are unique to all Viperidae in Florida, USA). Lyophilized venoms were resuspended in deionized H2O this regard (Underwood, 1967; Parker, 1977; Mallow et al., 2003; Fry before being centrifuged at 14,000 relative centrifugal force (RCF) at et al., 2015). This confers the ability of delivering very large venom 2 °C, for 15 min. The supernatants were collected and protein con- yields (up to 300 mg) relative to their narrow heads (Spawls and centrations (mg/ml) determined using a ThermoFisher Scientific Branch, 1995). Proportional to the size of the snakes, these two species Nanodrop™ 2000c Spectrophotometer. 50% deionized water/50% gly- also consume much larger anurans than all the others, with prey weight cerol (> 99%, Sigma-Alrich) working stocks of both venoms were sometimes equaling the snake's own body weight, along with prey prepared at 10 mg/ml and 1 mg/ml and stored at −20 °C to preserve diameter dramatically exceeding body diameter and jaw length (Ineich enzymatic activity and reduce enzyme degradation. et al., 2006; Mallow et al., 2003). All other Causus species prey upon much smaller prey proportional to snake size, with C. lichtensteinii 2.2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS having one of the smallest prey to body diameter ratio, where prey PAGE) diameter rarely exceeds body diameter and never exceeds jaw length (Ineich et al., 2006). These snakes also possess venom glands that, like 1 dimensional (1D) and 2 dimensional (2D) SDS-PAGE gels were all other vipers, are confined to the skull and do not extent beyond the prepared and MS/MS analysed as previously described by us base of the neck (Mallow et al., 2003; Ineich et al., 2006). C. rhombeatus (Koludarov et al., 2017). MSMS raw results are included in Supple- is known to preferentially feed on robust toads from the genus Bufo, mentary File 1. while C. lichtensteinii preferentially feeds on more gracile frogs from the Ptychadaena, Chiromantis, Cardioglossa and Leptodactylodon genera 2.3. Fibrinogen proteolysis (Channing, 2001; Ineich et al., 2006; Mallow et al., 2003; Minter et al., 2004). While the venom appears exceptionally potent to amphibians To quantify the proteolytic activity upon human fibrinogen chains (with prey items succumbing rapidly – Broadley, 1990)(Broadley, we used a modification of our previous protocol (Dobson et al., 2017; 1990; Mallow et al., 2003) the venom composition and bioactivity Koludarov et al., 2017). This methodology was expanded not only to characterization remain virtually unknown. include a greater number of time periods within a 60-min frame but Although not usually considered life threatening to humans, night also to include different venom concentrations and tests for antivenom adders should be considered potentially dangerous. They have pro- efficacy. The reaction concentration of fibrinogen was set to a 1 mg/ml duced serious symptoms in human bite victims and have killed do- constant and a series of experiments were run with venom at con- mestic animals (De Cramer et al., 2012). Documented human en- centrations of 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and venomations by C. rhombeatus report extreme swelling, drowsiness, 800 μg/ml. The designated