Venom in Non-Serpent Squamates
Venom in Non-Serpent Squamates
Introduction
Venom has captivated inquisitive people ever since its discovery in the fangs of snakes and stinging appendages on insects, but it has been largely unknown in the squamates that have not lost their legs over the eons. The accepted assumptions have traditionally been that, in squamates, venom was exclusively restricted to snakes and members of the genus Heloderma after evolving multiple times (Arbuckle, 2009, Casewell et al., 2012), but there has been new research emerging since the early 2000’s that challenges these assumptions. This research has presented new opportunities for scientific exploration in both the field of taxonomy and evolutionary history (Fry, Vidal, Norman et al., 2006) and medical research (Dobson et al., 2019). These recent studies, however, are not without criticism from other researchers that are skeptical of the conclusions drawn and methods used by Fry and those expanding on his work. The research being done on both sides of the scientific conversation pertaining to venom in non-serpent squamates emphasizes how little we know about the evolutionary history of these animals that warrants a continuation of scientific exploration and a possibility of filling some gaps in medical venom research.
Heloderma in History
Genus Heloderma is represented by the beaded lizards, which have historically been classified as the only venomous lizards (Arbuckle, 2009, Casewell et al., 2012), however this classification was only granted after much controversy in the early twentieth century (Woodson, 1947). Heloderma’s status as a genus of venomous lizards came after the meticulous documentation of the symptoms that arose in those unfortunate enough to end up on the wrong end of a bite from members of the genus mirrored symptoms of envenomation from other known venomous species. These symptoms include extreme pain, nausea, paralysis, edema, increased perspiration, and convulsions and were noted to vary in severity depending upon the type of bite in question (Strimple et al., 1997). In these early studies it was noted that two distinct types of bites were documented, those where the animal latched on and “chewed” venom into the wounds with their lower teeth, and “slashing” with little resultant contact with the skin of victims, the former consistently resulted in more intense symptoms of envenomation.
At this point it was hypothesized that the venom apparatus in these lizards could be found in the lower jaw, however this was still awaiting definitive confirmation, and little was known about the molecular makeup of the venom produced by members helodermatids. In the early twenty-first century, it was confirmed that helodermatids house their venom glands in their lower jaws, and the proteins that make their venom toxic were identified. Of the proteins found in the venom, some are unique to the genus Heloderma, specifically helokinestatin peptides, and have been highly conserved since their evolution approximately 30 million years ago (Dobson et al., 2019, Fry et al., 2010, Fry et al., 2010). Neurotoxins known to cause almost immediate pain have also been discovered which may indicate that the venom in genus Heloderma has persisted primarily as a defense mechanism as opposed to an adaptation to aid in prey capture. Helodermatid venom function will be discussed in a later section.
The Creation of Toxicofera
Until the early twenty-first century, it was widely assumed that venom in squamates was unique to snakes and the genus Heloderma, and had evolved separately in both groups, however, in 2006 evidence for venom in other lizard species was presented resulting in the proposal for a new clade of squamates, Toxicofera (Fry, Vidal, Norman et al., 2006) which includes iguanians, anguimorphs, and snakes. The discovery of lobular non-compound venom-secreting glands on both the upper and lower jaws of the iguanian lizard Pogona barbata, as well as the identification of nine shared toxins in the venom of lizards and snakes suggest that orally secreted venom is the basal characteristic of the Toxicofera clade that may have arisen around 200 million years ago as the result of gene duplication. The iguanian condition of venom secreting glands on both the upper and lower jaws is likely the specific basal characteristic of the clade while the snakes and anguimorphs lost either the lower or upper venom glands with specialization. Despite the morphological differences that are reported among the venomous species, molecular study does support Toxicofera as a monophyletic clade where speciation and specialization of venom has been influenced by diet and other ecological pressures (Arbuckle, 2009, Fry et al, 2010, Koludarov et al, 2017, Vidal and Hedges, 2009) potentially resulting in the loss of venom entirely in some extant species.
The Komodo Dragon (Varanus komodoensis) and Varanid Venom Research
Historically, it was thought that the Komodo dragon (Varanus komodoensis) employed weaponized bacteria cultivated in their oral flora to aid in the subdual of large prey items (Arbuckle, 2009, Dobson et al, 2019, Fry et al., 2009, Goldstein et al., 2013), and the possibility of the presence of venom was largely ignored. After magnetic resonance imaging (MRI) scans of a preserved V. komodoensis head revealed a large mandibular venom gland with six total compartments and ducts leading up to serrated teeth (Fry et al., 2009) the weaponized bacteria hypothesis was called into question. Future studies found that the oral flora of captive V. komodoensis individuals were identical to the surface and gut flora of their food items and nearly the same as other carnivorous species with none of the bacteria strains present in the samples known to cause rapid death in any possible prey species (Goldstein et al., 2013). This implied that any bacteria in the flora of wild counterparts would be incidental and unreliable as a method of prey capture in an environment where finding prey is not guaranteed.
Further exploration into the venom of lizards in the genus Varanus has revealed that varanid venom varies greatly from species to species with it being more pronounced in the larger varanids with diets consisting of more vertebrates (Arbuckle, 2009, Dobson et al., 2019). While the venom in varanid species varies, the commonality in the genus is the presence of coagulotoxins that prevent clotting and increase blood loss from bite wounds that might otherwise not prove to be fatal in vertebrate prey. The specific fribrinogenolytic mechanism that these toxins use is not known to stimulate pain reactions which suggests these venoms are used as an adaptation for subduing relatively large prey compared to the size of the lizard. The anticoagulative properties of varanid venom may potentially have medical applications, specifically in research for new stroke and clotting disorder medications. Varanid venom research, however, is still a fledgling field that needs more time for scientific understanding to develop and questions to be answered.
The Function of Non-Serpent Venom
Venom function is a debated topic regardless of the animals being studied, but four potential functions have been proposed for non-serpent venom: prey capture, defense, digestive aid, and maintenance of oral hygiene, the former two being the ones with the most compelling evidence (Arbuckle, 2009). Venom that evolved primarily for defensive purposes causes some sort of near immediate pain following envenomation allowing the animal to escape (Ward-Smith et al., 2020), the benefit from the defense then outweighs the energetic consequence of producing venom that may not aid in the capture of prey. This is observed in the genus Heloderma, the lizards in this genus have diets consisting primarily of eggs and other items that need little to no subduing implying that their venom noted to cause extreme negative symptoms in bite victims is likely a defensive adaptation in species that are otherwise vulnerable to predation (Dobson et al., 2019). To date, the genus Heloderma is the only anguimorph genus that produces venom known to stimulate an immediate pain response, which is directly contrasted by varanid lizard venom.
As noted in the previous section, varanid venoms contain toxins that prevent blood clotting and promote excess bleeding from bite wounds inflicted in prey animals. The presence of venom in varanid lizards is most frequently observed in the larger species with a primary diet of vertebrates and inhabit environments where the loss of prey could potentially have deadly consequences (Dobson et al., 2019). In these species, the venom allows the lizard to bite and then follow their prey until they eventually succumb to or are weakened by blood loos, guaranteeing the varanid a meal. These venoms also do not contain pain inducing toxins, therefore venom as a defense adaptation is unlikely in varanid lizards. The specific toxins found in the varanid venoms vary from species to species and have likely been influenced by diet and other selective pressures that arose during their evolution (Fry et al., 2010). This phenomenon explains the specializations in the various lizard venoms and may be better understood with future research.
Scientific Criticism of Toxicofera and Denial of Venom in Varanid Lizards
Although the proposed clade Toxicofera neatly organizes the snakes, iguanians, and anguimorphs into a well resolved clade (Fry, Vidal, and Normal et al., 2006), there have been some studies challenging the validity of the proposed clade. It is suggested that it is highly unlikely for a gene to be duplicated, expressed in the venom gland, then revert back to nontoxic tissues when venom is no longer an advantageous adaptation, and that the protein families that the shared toxins are from likely originate from general “housekeeping” genes that are expressed in multiple places in the body (Casewell et al., 2012, Hargreaves et al., 2014). The studies criticize the earlier work for failing to sample tissues in the non-venom secreting portions of the body, concluding that when these tissues are included in samples it is impossible to conclude that primitive venom mechanisms developed in a single ancestor then was refined or lost as different species evolved. Instead it is suggested the original model is more accurate and venom evolved at least three separate times, once in snakes, once in genus Heloderma, and considering the current research, once in genus Varanus. However, it is important to note that there is a minority arguing that there is little to no evidence for venom in varanid lizards, and the weaponized bacteria hypothesis is the only valid conclusion for venom-like symptoms in prey of those species (Sweet, 2016).
The most recent molecular research, however, does support the Toxicofera clade and it is noted that the controversy is to be expected and likely stems from ambiguity in the definition of venom and relative toxcicity (Koludarov et al., 2017). The current way venom is discussed often relates defines the toxicity by its effect on humans and overlooks the effect on the prey species it was evolved to subdue indicating that more research is necessary to reach a definitive conclusion about the evolution of venom in squamates.
The Next Logical Step
Figure 1. If the hypothesis that Toxicofera is a monophyletic clade sharing nine specific toxins in the venom of members of the clade, it is expected that observations of each toxin will be relatively similar with the dissimilarities explained by specification and specialization of sampled venoms.
Venom research in non-serpent squamates is a relatively new scientific endeavor, and as such there are more questions than available answers. Considering the age of this field, the next logical step is to continue with the work currently being done do identify venom in species where it is currently unknown, determine the molecular makeup of those venoms, and determine the presence or lack thereof of the proposed shared toxins that support the proposed monophyletic Toxicofera clade. If the monophyletic clade is supported, then I would expect to see a relatively even distribution when one graphs the number of observations (see Figure 1) of each shared toxin in analyzed venom samples. It is, however, reasonable to see some differences in the frequency of observations from toxin to toxin when specialization of venom as the result of different selective evolutionary pressures is considered. I would also suggest that while more lizard venom is identified and classified that it may be used to fill in any gaps in medical venom research.
Conclusion
The discovery of venom in non-serpent squamates has offered new insights into the evolutionary history of squamate reptiles, specifically those that fit into the proposed Toxicofera clade. Research surrounding the iguanian and anguimorphs is still a new field that is facing scientific criticism despite the evidence presented, and there will need to be much more work done to answer the questions that have been asked in the years to come. Future research will offer new understanding of the evolution of the venomous squamates and introduce new possibilities for medical research by analyzing and identifying the toxins and proteins found in non-serpent squamate venom.
References
Arbuckle, K (2009). Ecological Function of Venom in Varanus, with a Compilation of Dietary Records from the Literature. Biawak 3(2), 46-56.
Casewell, N. R., et al. (2012). Dynamic evolution of venom proteins in squamate reptiles. Nat. Commun. 3, 1066. doi:10.1038/ncomms2065.
Dobson, J. S. et al. (2019). Varanid lizard venoms disrupt the clotting ability of human fibrinogen through destructive cleavage. Toxins 11, 255. doi:10.3390/toxins11050255.
Fry, B., Vidal, N., Norman, J., et al. (2006). Early evolution of the venom system in lizards and snakes. Nature 439, 584-588. doi: 10.1038/nature04328
Fry, B. G., et al. (2009). A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Megalania) priscus. PNAS 106, 8969-8974. doi:10.1073/pnas.0810883106.
Fry, B. G., et al. (2010). Novel venom proteins produced by differential domain-expression strategies in beaded lizards and gila monsters (genus Heloderma). Mol. Biol. Evol. 27, 395-407. doi:10.1093/molbev/msp251.
Fry, B. G., et al. (2010) Functional and structural diversification of the anguimorpha lizard venom system. Molecular & Cellular Proteomics 9, 2369-2390. doi:10.1074/mcp.M110.001370.
Goldstein, E. J. C., et al. (2013) Anaerobic and aerobic bacteriology of the saliva and gingiva from 16 captive komodo dragons (Varanus komodoensis): new implications for the ‘bacteria as venom’ model. Journal of Zoo and Wildlife Medicine 44, 262–72.
Hargreaves, A. D., et al. (2014) Testing the toxicofera: comparative transcriptomics casts doubt on the single, early evolution of the reptile venom system. Toxicon 92, 140–56. doi:10.1016/j.toxicon.2014.10.004.
Koludarov, I., et al. (2017) Enter the dragon: the dynamic and multifunctional evolution of anguimorpha lizard venoms. Toxins 9, 242. doi:10.3390/toxins9080242.
Strimple, P. D., et al. (1997) Report on envenomation by a gila monster (Heloderma suspectum) with a discussion of venom apparatus, clinical findings, and treatment. Wilderness & Environmental Medicine 8, 111–16. DOI.org (Crossref), doi:10.1580/1080-6032(1997)008[0111:ROEBAG]2.3.CO;2.
Sweet, S.S. (2016) Chasing flamingos: Toxicofera and the misinterpretation of venom in varanid lizards. In Proceedings of the 2015 Interdisciplinary World Conference on Monitor Lizards, 123–149.
Vidal, N., and Hedges, S. B. (2009) The molecular evolutionary tree of lizards, snakes, and amphisbaenians. Comptes Rendus Biologies 332, 129–139. doi:10.1016/j.crvi.2008.07.010.
Ward-Smith, H., et al. (2020) Fangs for the memories? a survey of pain in snakebite patients does not support a strong role for defense in the evolution of snake venom composition. Toxins 12,201. doi:10.3390/toxins12030201.
Woodson, W. D. (1947) Toxicity of Heloderma venom.” Herpetologica 4, 31–33.
