Serum-based inhibition of pitviper Venom by Eastern Indigo Snakes (Drymarchon couperi)

Abstract

When organisms possess chemical defenses, their predators may eventually evolve resistance to their toxins. Eastern indigo snakes ( Drymarchon couperi ; EIS) prey on pitvipers and are suspected to possess physiological resistance to their venom. In this study, we formally investigated this hypothesis using microassays that measured the ability of EIS blood sera to inhibit (A) hemolytic and (B) snake venom metalloproteinase (SVMP) activity of copperhead ( Agkistrodon contortrix ) venom. To serve as controls, we also tested the inhibitory ability of sera from house mice ( Mus musculus ) and checkered gartersnakes ( Thamnophis marcianus ), a snake that does not feed on pitvipers. Sera from both EIS and gartersnakes inhibited over 60% of SVMP activity, while only EIS sera also inhibited venom hemolytic activity (78%). Our results demonstrate that EIS serum is indeed capable of inhibiting two of the primary classes of toxins found in copperhead venom, providing the first empirical evidence suggesting that EIS possess physiological resistance to venom upon injection. Because we documented resistance to hemolytic components of pitviper venom within EIS but not gartersnakes, we speculate this resistance may be driven by selection from feeding on pitvipers while resistance to SVMP may be relatively widespread among snakes.

Phenotypic and functional variation in venom and venom resistance of two sympatric rattlesnakes and their prey

“…We used rattlesnake venom and mammal blood serum samples to conduct in vitro bioassays following the method of Biardi, Nguyen, et al. (2011) to quantify both venom activity and mammal resistance (Finger et al., 2020; Goetz et al., 2019; Holding, Biardi, et al., 2016; Pomento et al., 2016). First, we measured protein concentration in serum and venom using a Quick Start Bradford Protein Assay (Bio‐Rad Laboratories).…”

“…Although pooling the venom samples reduces statistical power by precluding the ability to estimate venom‐related variance in SVMP from individual snakes, we chose to pool venom to expedite population‐level comparisons between sites. We then used the EnzChek Gelatinase/Collagenase Assay Kit (Life Technologies) to measure the collagenase activity of each venom pool, which has been previously linked specifically to SVMP activity in rattlesnakes (Biardi, Nguyen, et al., 2011; Finger et al., 2020; Goetz et al., 2019; Holding, Biardi, et al., 2016; Pomento et al., 2016) and is a proxy for the tissue‐destroying haemorrhagic activities of these SVMPs (henceforth ‘SVMP activity’). We measured fluorescence intensity in relative fluorescence units (RFU) every 1.5 min for an hour using a Tecan Infinite M200 fluorescence microplate reader (Tecan Group Ltd).…”

Preying dangerously: black widow spider venom resistance in sympatric lizards

“…From a mechanistic perspective, the absence of organ or cellular damage in E. multicarinata may provide clues about the molecular or physiological basis of resistance. In similar systems where the mode of venom resistance is at least partially understood, venoms are often prevented from attacking target tissues because they are bound by macromolecules [8,[77][78][79]. These 'toxin-scavenging' molecules are usually inhibitor proteins that actively circulate in the blood stream (e.g.…”

Snakebite Therapeutics Based on Endogenous Inhibitors from Vipers

“…Natural inhibitors isolated from resistant animals are detailed in reviews by Domont et al [34,68] and Bastos et al [37], which summarized toxin resistance corresponding to several snake species from the plasma, serum, and muscle of mammals. There are a few reports of natural inhibitors isolated from nonvenomous snakes, such as NtAH from Natrix tessellate [69] and a 59 kDa protein from Dinodon semicarinatus [70]. Recently, the whole serum of Drymarchon couperi [71] has also shown antihemorrhagic activity to venom, but the protein responsible for this activity has yet to be determined.…”