Published On: Tue, Mar 26th, 2019

Serum-based inhibition of pitviper venom by eastern indigo snakes (Drymarchon couperi) [RESEARCH ARTICLE]


Physiological resistance to toxins may evolve in predators that eat chemically-defended prey (Brodie, 1990; Rowe and Rowe, 2008) and selection for greater resistance is predicted to be stronger in predators that exhibit greater diet specialization (Arbuckle et al., 2017). Animal poisons and especially venoms are complex mixtures of toxins (Casewell et al., 2013; Fox and Serrano, 2008; Fry et al., 2009, 2012). The amount of damage caused by specific toxins, however, varies greatly and resistance may be achieved by inhibition of relatively few toxins (Arbuckle et al., 2017).

Blood sera components are one potential mechanism facilitating venom resistance, as they may bind to venom toxins and neutralize them, thereby inhibiting venom activity and minimizing damage. This serum-based toxin resistance appears to have independently evolved in a taxonomically-diverse suite of organisms in response to different ecological pressures (Arbuckle et al., 2017; Holding et al., 2016a; Perez et al., 1978). For example, resistance to pitviper venom has been documented in both snake prey and predators (de Wit, 1982; Perez et al., 1979; Pomento et al., 2016; Poran et al., 1987; Voss and Jansa, 2012), including three snake species that eat venomous prey (Lomonte et al., 1982; Tomihara et al., 1988; Weinstein et al., 1992) as well as pitvipers themselves, presumably as a form of autoresistance (Clark and Voris, 1969; Weinstein et al., 1991). Perhaps the best studied example of serum-based resistance to pitviper venom involves California ground squirrels (Spermophilus beecheyi) inhibiting the activity of rattlesnake (Crotalus spp.) venom. Ground squirrels are a major dietary component of many co-occurring rattlesnakes and ground squirrel sera contains factors that neutralize the digestive and hemostatic effects of pitviper venom (Biardi et al., 2006). Furthermore, detailed investigations have revealed among-population variation in both snake venom activity and squirrel resistance that suggests a co-evolutionary relationship (Biardi et al., 2006; Holding et al., 2016b) and supports the idea that prey capture, not antipredator defense, is likely the primary selective factor acting on snake venom evolution (Fry et al., 2008; Li et al., 2005; Richards et al., 2012).

Eastern indigo snakes (Drymarchon couperi; EIS) are predators of a variety of venomous snakes, and thus provide an appropriate model organism to explore ideas related to the evolution of venom resistance. Historically restricted to southern portions of the southeastern coastal plain of USA, EIS are associated with open-canopy pine savannahs and are considered dietary generalists preying on a variety of mammals, birds, reptiles, and amphibians. Prey records indicate that snakes, including a number of pitviper species, are the most commonly consumed food item (Steen et al., 2016; Stevenson et al., 2010). An experimental investigation of EIS response to prey odors revealed a preference for pitvipers over all other prey scents tested (Goetz et al., 2018). Together, qualitative and quantitative evidence indicates that EIS and pitvipers likely share a co-evolutionary history shaped by predator/prey dynamics.

Long-standing suggestions that snakes in the genus Drymarchon are resistant to the effects of pitviper venom (Boos, 2001; Keegan and Andrews, 1942; Mole, 1924) primarily stem from observations of successful predation of pitvipers by indigo snakes. Survival following possible envenomation, however, serves as a poor test of resistance (Arbuckle et al., 2017). For example, pitvipers can meter the quantity of venom injected during bites (Hayes, 2008; Hayes et al., 2002) thus it is not possible to estimate the amount of venom, if any, delivered during an observed bite. Moreover, envenomation by pitvipers requires penetration of outer epithelial layers (i.e. wound formation) and the large, thick scalation of EIS likely serves as formidable barrier to penetration of snake fangs. Finally, the predatory sequence of EIS typically begins by grasping and crushing the head of snake prey; therefore, toxic defenses may be bypassed altogether by subduing pitvipers before they can strike (Keegan and Andrews, 1942; Moulis, 1976). A more direct, experimental approach is necessary to determine if EIS possess physiological mechanisms to inhibit venom protein activity.

Here, we used a pair of venom activity assays to formally evaluate the ability of EIS blood sera to inhibit two of the primary groups of toxins in copperhead venom (Agkistrodon contortrix; Linnaeus, 1766). We first assessed serum inhibition of hemolytic factors, including toxins that damage erythrocytes and disrupt hemostasis (Biardi and Coss, 2011). We also investigated the inhibition of snake venom metalloproteinases (SVMPs) that damage proteins in the extracellular matrix and hydrolyze collagen (Biardi et al., 2011; Holding et al., 2016b; Pomento et al., 2016). Because collagen is found in both the lining of blood vessels and muscle tissue, SVMPs can cause hemorrhagic effects.

Our objective was to evaluate the hypothesis that EIS possess serum-based resistance to pitviper venom; we assumed a strong signal of venom inhibition would suggest the presence of circulating inhibitor molecules in the blood. We predicted EIS serum would exhibit greater toxin inhibition compared with house mouse (Mus musculus) serum, which lacks venom protein inhibitors and served as our experimental control. To add additional ecological context to our investigation, we also evaluated the inhibitory ability of serum from checkered gartersnakes (Thamnophis marcianus; Baird and Girard, 1853) that do not prey on pitvipers (Ernst and Ernst, 2003). We predicted greater inhibition by EIS compared with gartersnakes because the former is more likely to participate in antagonistic interactions with pitvipers.

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