High-Voltage Toxin'Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes.

The evolutionary interplay between predator and prey has significantly shaped the development of snake venom, a critical adaptation for subduing prey. This arms race has spurred the diversification of the components of venom and the corresponding emergence of resistance mechanisms in the prey and predators of venomous snakes. Our study investigates the molecular basis of venom resistance in pythons, focusing on electrostatic charge repulsion as a defense against α-neurotoxins binding to the alpha-1 subunit of the postsynaptic nicotinic acetylcholine receptor. Through phylogenetic and bioactivity analyses of orthosteric site sequences from various python species, we explore the prevalence and evolution of amino acid substitutions that confer resistance by electrostatic repulsion, which initially evolved in response to predatory pressure by Naja (cobra) species (which occurs across Africa and Asia). The small African species Python regius retains the two resistance-conferring lysines (positions 189 and 191) of the ancestral Python genus, conferring resistance to sympatric Naja venoms. This differed from the giant African species Python sebae, which has secondarily lost one of these lysines, potentially due to its rapid growth out of the prey size range of sympatric Naja species. In contrast, the two Asian species Python brongersmai (small) and Python bivittatus (giant) share an identical orthosteric site, which exhibits the highest degree of resistance, attributed to three lysine residues in the orthosteric sites. One of these lysines (at orthosteric position 195) evolved in the last common ancestor of these two species, which may reflect an adaptive response to increased predation pressures from the sympatric α-neurotoxic snake-eating genus Ophiophagus (King Cobras) in Asia. All these terrestrial Python species, however, were less neurotoxin-susceptible than pythons in other genera which have evolved under different predatory pressure as: the Asian species Malayopython reticulatus which is arboreal as neonates and juveniles before rapidly reaching sizes as terrestrial adults too large for sympatric Ophiophagus species to consider as prey; and the terrestrial Australian species Aspidites melanocephalus which occupies a niche, devoid of selection pressure from α-neurotoxic predatory snakes. Our findings underline the importance of positive selection in the evolution of venom resistance and suggest a complex evolutionary history involving both conserved traits and secondary evolution. This study enhances our understanding of the molecular adaptations that enable pythons to survive in environments laden with venomous threats and offers insights into the ongoing co-evolution between venomous snakes and their prey.

Sugar-coated survival: N-glycosylation as a unique bearded dragon venom resistance trait within Australian agamid lizards.

In the ongoing evolutionary arms race between predators and prey, adaptive innovations often trigger a reciprocal response. For instance, the emergence of α-neurotoxins in snake venom has driven prey species targeted by these snakes to evolve sophisticated defense mechanisms. This study zeroes in on the particular motifs within the orthosteric sites of post-synaptic nicotinic acetylcholine receptors (nAChR) that confer resistance to α-neurotoxins, often through structural alterations of nAChR. This research examined Australian agamid lizards, a primary prey group for Australian elapid snakes, which are subject to predatory selection pressures. We previously showed that Pogona vitticeps (Central bearded dragon) was resistant to α-neurotoxic snake venoms through a steric hindrance form resistance evolving within the nAChR orthosteric, specifically through the 187-189NVT motif resulting in the presence of N-glycosylation, with the branching carbohydrate chains impeding the binding by the neurotoxins. This adaptive trait is thought to be a compensatory mechanism for the lizard's limited escape capabilities. Despite the significance of this novel adaptation, the prevalence and evolutionary roots of such venom resistance in Australian agamids have not been thoroughly investigated. To fill this knowledge gap, we undertook a comprehensive sequencing analysis of the nAChR ligand-binding domain across the full taxonomical diversity of Australian agamid species. Our findings reveal that the N-glycosylation resistance mechanism is a trait unique to the Pogona genus and absent in other Australian agamids. This aligns with Pogona's distinctive morphology, which likely increases vulnerability to neurotoxic elapid snakes, thereby increasing selective pressures for resistance. In contrast, biolayer interferometry experiments with death adder (Acanthophis species) venoms did not indicate any resistance-related binding patterns in other agamids, suggesting a lack of similar resistance adaptations, consistent with these lineages either being fast-moving, covered with large defensive spines, or being arboreal. This research not only uncovers a novel α-neurotoxin resistance mechanism in Australian agamids but also highlights the complex dynamics of the predator-prey chemical arms race. It provides a deeper understanding of how evolutionary pressures shape the interactions between venomous snakes and their prey.

A Russian Doll of Resistance: Nested Gains and Losses of Venom Immunity in Varanid Lizards.

The interplay between predator and prey has catalyzed the evolution of venom systems, with predators honing their venoms in response to the evolving resistance of prey. A previous study showed that the African varanid species Varanus exanthematicus has heightened resistance to snake venoms compared to the Australian species V. giganteus, V. komodoensis, and V. mertensi, likely due to increased predation by sympatric venomous snakes on V. exanthematicus. To understand venom resistance among varanid lizards, we analyzed the receptor site targeted by venoms in 27 varanid lizards, including 25 Australian varanids. The results indicate an active evolutionary arms race between Australian varanid lizards and sympatric neurotoxic elapid snakes. Large species preying on venomous snakes exhibit inherited neurotoxin resistance, a trait potentially linked to their predatory habits. Consistent with the 'use it or lose it' aspect of venom resistance, this trait was secondarily reduced in two lineages that had convergently evolved gigantism (V. giganteus and the V. komodoensis/V. varius clade), suggestive of increased predatory success accompanying extreme size and also increased mechanical protection against envenomation due to larger scale osteoderms. Resistance was completely lost in the mangrove monitor V. indicus, consistent with venomous snakes not being common in their arboreal and aquatic niche. Conversely, dwarf varanids demonstrate a secondary loss at the base of the clade, with resistance subsequently re-evolving in the burrowing V. acanthurus/V. storri clade, suggesting an ongoing battle with neurotoxic predators. Intriguingly, within the V. acanthurus/V. storri clade, resistance was lost again in V. kingorum, which is morphologically and ecologically distinct from other members of this clade. Resistance was also re-evolved in V. glebopalma which is terrestrial in contrast to the arboreal/cliff dwelling niches occupied by the other members of its clade (V. glebopalma, V. mitchelli, V. scalaris, V. tristis). This 'Russian doll' pattern of venom resistance underscores the dynamic interaction between dwarf varanids and Australian neurotoxic elapid snakes. Our research, which included testing Acanthophis (death adder) venoms against varanid receptors as models for alpha-neurotoxic interactions, uncovered a fascinating instance of the Red Queen Hypothesis: some death adders have developed more potent toxins specifically targeting resistant varanids, a clear sign of the relentless predator-prey arms race. These results offer new insight into the complex dynamics of venom resistance and highlight the intricate ecological interactions that shape the natural world.

The Target Selects the Toxin: Specific Amino Acids in Snake-Prey Nicotinic Acetylcholine Receptors That Are Selectively Bound by King Cobra Venoms.

Snake venom is an adaptive ecological trait that has evolved primarily as a form of prey subjugation. Thus, the selection pressure for toxin diversification is exerted by the prey's physiological targets, with this pressure being particularly acute for specialist feeders, such as the King Cobra species, all of which are snake-prey specialists. However, while extensive research has been undertaken to elucidate key amino acids that guide toxin structure-activity relationships, reciprocal investigations into the specific sites guiding prey-lineage selective effects have been lacking. This has largely been due to the lack of assay systems amenable to systematic amino acid replacements of targeted proteins in the prey's physiological pathways. To fill this knowledge gap, we used a recently described approach based upon mimotope peptides corresponding to the orthosteric site of nicotinic acetylcholine receptor alpha-1 subunits, a major binding site for snake venom neurotoxins that cause flaccid paralysis. We investigated the venoms of four different types of King Cobra (Cambodian, Javan, Malaysian, and Thai). This approach allowed for the determination of the key amino acid positions in King Cobra snake prey that are selectively bound by the toxins, whereby replacing these amino acids in the snake-prey orthosteric site with those from lizards or rats resulted in a significantly lower level of binding by the venoms, while conversely replacing the lizard or rat amino acids with those from the snake at that position increased the binding. By doing such, we identified three negatively charged amino acids in the snake orthosteric site that are strongly bound by the positively charged neurotoxic three-finger toxins found in King Cobra venom. This study, thus, sheds light on the selection pressures exerted by a specialist prey item for the evolution of lineage-selective toxins.