Essay 3. Are All Snakes Venomous? The Toxicofera Hypothesis And How We Define Venom.

One fine spring afternoon, I was bitten on the face by a nearly one meter long snake. Certainly a result of my own stupidity, this animal decided to clamp onto my left cheek. No permanent damage was done, save for my work shirt which suffered minor stains, and after a quick clean up we were back to work. Our job of course is to relocate snakes to more suitable habitats whenever they come across fearful humans, and we knew we were in no real danger. The animal in question was a common tree snake (Dendrelaphis punctulata), an effectively harmless, fangless colubrid snake which had been mistaken by warehouse staff members for a highly venomous eastern brown snake (Pseudonaja textilis). I humbly suggest, had it been P. textilis under the shelves, I would have been much more cautious, perhaps even restraining myself from holding it up near my face, allowing it to strike precisely at the moment I was explaining to watching staff members that these animals aren’t venomous (D. punctulata, that is!) and, while they do have teeth, they only very rarely bite even when handled (they truly are quite docile…most of the time). I was also defecated on and musked, a defensive tactic of releasing a foul smelling, pungent “musk” from glands in the cloaca, by the same individual. Ah, the infinite joys of working with native wildlife.

The common tree snake is a frequent visitor of people’s properties from Australia’s east coast and across the top end, avoiding the more arid inland and colder south (See Wilson & Swan, 2013 (1) for a field guide of Australian reptiles, Cogger, 2014 (1) as a comprehensive at-home companion). These laterally compressed, lightweight, elongate snakes are perfectly adapted for their non-venomous style of prey capture, climbing up to high into forest canopies and vine thickets where tree frogs hide, or slipping stealthily around aquatic and riparian vegetation. A particular fan of frogs, they simply latch on to prey animals small enough to overpower and swallow as soon as possible, often alive. This requires nothing more than small, sharp teeth, a contrast from the highly complicated venom systems of other snakes.

Why are we discussing this rather commonplace, non-venomous, seemingly unexceptional snake? Here we take a tentative step into a yet unsettled and at times especially heated debate in the world of reptile biology (please, nobody hit me!). For according to the captivatingly titled Toxicofera hypothesis, not only D. punctulata but all other snakes, including the various pythons which have also gifted me with unpleasant facial injuries, are venomous or share a venomous ancestor. No recent theory in the world of venomous reptiles has caused as much of a controversy as this idea. Take the non-venomous snakes I was so idly handling my whole life; were they actually in possession of a venom system, even if somewhat deteriorated over evolutionary time through non usage?

The Toxicofera Hypothesis daringly goes even further. The discovery of highly dangerous venom products in the mouths of theoretically non-venomous snakes poses the question; what else out there might be venomous? The venom research team of Fry et al. went looking for venom where there should be none. They found that many lizards, including the largest in the world, the Komodo Dragon (Varanus komodoensis), also possess venom and venom glands in their jaws. The Komodo in particular caught the fascination of the science media, as these gigantic monitor lizards were previously thought to capture their prey by delivering a septic bite full of bacteria, or relying on physical trauma caused by their serrated teeth, like well maintained steak-knives (see (3)). What a phenomenal discovery to further weaponize this already powerful and captivating beast!

Since the expansive work of Fry et al. 2006 (4), the Toxicofera hypothesis had been widely accepted. The Komodo Dragon’s newly acquired venomous status had even been cemented in culture by no lesser natural history orator than the celebrated Sir David Attenborough (stand and salute, or be shot) in the BBC Life documentary series.  Alas, certain aspects of their research and conclusions have come under fire in the following years from other toxinologists around the world, casting some shadow over this idea which had so captured the attention of both public and scientific audiences. What followed was a battle of words and reasoning between the two camps, taking stage on two of the grandest of battlefields known to modern scientists; the “letters to the editor” section of a specialist journal (Toxicon in this case), and a public scientific debate at Oxford. Perhaps we should closely examine the evidence for and against the Toxicofera hypothesis to better arm ourselves before we enter the fray in this venomous war of wits.

Let’s start with the origin of the Toxicofera and the work of Fry and colleagues. How did the authors determine that these animals all belong to the same clade, an evolutionary term for a grouping of related animals with a common ancestor? Traditionally, venom is thought to have arisen twice or even three times in the squamata; in the snakes (perhaps separately in the elapids/vipers and the colubrids) and in the heliodermatid lizards, the Gila Monster and the Beaded Lizard (3). After several years working on the evolution of venom systems in front fanged elapid snakes from the late ‘90s to the early 2000, Fry began investigating the venom systems of some less studied venomous snakes in the Colubridae family, an unresolved, numerous and diverse umbrella family which likely contains several separate families (see Lumsden et al. 2004 (5) for an example of some of their work). Not all colubrid snakes possess fangs or venoms, and where present they’re less sophisticated than in elapids and vipers. While the latter two families deliver a pressurized bolus of venom, passing from the temporally placed gland (venom reservoir inbuilt) through a venom-duct to the font maxilla where it is injected through modified hollow teeth or “fangs “, colubrids rely on simple enlarged teeth halfway down the back of the jaw (thus the term “rear-fanged” vs “front-fanged” for venomous colubrids and elapids/vipers respectively). The rear-fang is grooved at the back, trickling venom proteins down to the puncture site as they flood the mouth at the base of the fangs, extruded from more the simple glands referred to as Duvernoy’s Glands, often with small additional glands along the upper and lower jaw line under the labial scales (see 6, 7).

All this led to an intriguing question; what are the origins of this venom system in snakes? This issue has been debated within the herpetological community for some time, with many suggesting that the elapids and vipers evolved their venom system separately, with certain colubrids later developing their own similar system. This was cited as a case for convergent evolution, the process by which two species evolve similar adaptive traits due to similar selective pressures (7, 8). This is in contrast with shared ancestry, where homology (similarity) between features and species implies a common ancestor in possession of the shared trait, which is then inherited down many generations by separate lineages as they become separate species and groups.

Consider the evolution of flight in vertebrates. Only the birds and the bats are capable of powered flight, but we do not believe they evolved from a recent common ancestor. The birds are essentially a lineage of reptile, closely related to the dinosaurs, the bird line of crown-group Avemetatarsalia evolving within the Archosauria, trading scales for feathered plumage (although both are made of similar keratinized materials), while the mammals evolved much later in the history of life on Earth from smaller, ground-dwelling, lizard or mouse-like reptiles, perhaps similar to shrews or gerbils (9, 10). For these lightweight animals, in a world full of predators, the benefits of being able to glide, or perhaps simply control airtime and corner more precisely while bounding from log to rock to branch, are self evident. Any mutations for increased forearm feathers or perhaps thin membranes between elongate fingers might be quickly swept to ‘fixation’, that is, mutant offspring with these traits are so successful and breed so frequently that within a few generations the whole population is comprised of individuals in possession of their gene variant with the accompanying trait, and the mutant becomes the new norm. In this way, both bird-like and bat-like ancestors successively converged on the same solution independently, as opposed to sharing a flying ancestor like the modern descendents of these early birds or bats do. Flight, incidentally, seems to be a common point of convergence, first evolving in the Pterygota, the winged insects, 170 million years before any other life forms took flight (11).

Back to venom in snakes, did the colubrid and elapid/viper venom systems evolve separately in convergence? Or is the front-fanged condition a derived, more complicated and effective version of the one original rear-fanged condition? Of course, proponents exist for both ideas. Some suggest that it could be naught else but a venom system in infancy, others say that even if so, a venom is defined by its use in prey capture or defense, and can be only defined by its biological function, which has not been demonstrated yet. For example, humans also possess toxic oral compounds in our saliva, known for pre-digestion etc. rather than hunting, but we would hardly be considered venomous (7). Still, whether venom or some other protein product, the shared point of agreement was that the oral products of non-venomous colubrids, even those without fangs or Duvernoy’s Glands, needed more investigation.
Fry was in the process of studying the glands of these less dangerous colubrids when the first suggestions of the Toxicofera came to light. Using LC-MS (Liquid Chromatography–Mass Spectrometry), as well as protein isolation and sequencing from the Asian Ratsnake (12), the team could investigate what proteins were being produced and what genes may be involved. In the mouths of some putatively non-venomous colubrids, highly toxic venomous protein compounds continued to be located, albeit in small quantities. These included three-finger proteins which the team suspects may be the ancestors of the three-finger neurotoxins (3FTXs) of highly venomous elapid snakes. Where does the story of squamate venom end? In following this, Fry and colleagues spend much field time with various Australian lizards. By creating libraries of the DNA expression in oral tissues, they found similar toxic venom products in an agamid and a varanid lizard (3). These proteins closely matched those found in the venom glands of serpents.

Around the same time, another type of study was making waves. Morphological evidence from precise physical measurement of bones and fossils, and the mathematical groupings of these measurements to delineate species and higher orders of taxonomic groups, has a centuries long standing as the standard method for evolutionary research and the study of natural history. It might’ve come as a rude shock to proponents of the traditional schools when a newer style of evolutionary data, molecular evidence from examining sequence variation and expression patterns of DNA, RNA and proteins (the information storage, transcript papers, and physical matter of biology itself), began placing doubts on some of these morphological conclusions. Most notably for us, squamate phylogenies built from multiple gene sequences placed the evolution of iguanid reptiles much later history (13). In fact, they have buddied up right next to the Serpentes lineage, the latest major evolutionary and morphological radiation in the squamate reptile group, a radical difference from the basal placement suggested by morphology.

Both the molecular phylogenetic data and the protein sequence data seemed to agree upon this rearrangement of Iguania. Furthermore, according to the findings of Fry and colleagues, these lizards share the similar venom products in their mouths to all snakes. The blow to our pillar of understanding resounded throughout biology; all snakes are venomous, and so are, most probably, many lizards (excluding several lineages like the geckoes and skinks which diverged before the development of the “venom genes” typifying the Toxicofera). All share a venomous common ancestor some 170 million years ago, lizard-like in form, with some subsequent lineages losing the apparatus through lack of usage. So sayeth the Toxicofera hypothesis. Further support came in the following years with more so called “incipient venom systems” found, including that of the famous Komodo Dragon. Glands and venom proteins were discovered in the mouth tissues of various iguanid and varanid lizards, including several commonly kept commercial pet species, such as the central bearded dragon (Pogona spp). The Toxicofera hypothesis was picking up steam. That’s when someone threw a chair, and a fight broke out.

Not everyone was on board with team Toxicofera. It is easy to view such dissent for new idea as nothing more than the Old Guard complaining that the new kids are being too noisy (with their gosh darn rap music and DNA technologies), but this would dismiss two major tenants of science: that no idea is beyond review, and that an exceptional statement or revision requires exceptional supporting evidence. Scientists complain, particularly about other scientists, and this is how we improve, usually in a polite and courteous manner. While one might publically comment online in various forums, this means little in the world of peer review. What to do? Like any polite but disagreeable reader, one might feel compelled to write a strongly worded, thoroughly research letter to the editor, expressing your discontent and reasoning, for the target audience to see in the next issue of the journal.

Such was the response of Weinstein et al. 2012 (14) to the several years of Toxicofera papers that had been published since 2006. This group of toxinologists and evolutionary biologists had decided to challenge certain aspects of the Toxicofera. Namely they were targeting the definition of these animals as venomous. Team Toxicofera hit back (15), writing a letter to the editor themselves to dispute the points brought up by Weinstein et al., which was naturally replied to in kind (16). If ever the opportunity arises, I thoroughly recommend these letters not just for their scientific content but for entertainment. I truly enjoyed these papers not just for the well formed arguments, but for the salvos of criticism and rebuttal peppered with as much passive aggression as any journal can let past their editorial desk. While Fry and colleagues argued from the functional lab tests on blood pressure, homology of proteins, DNA/RNA sequences and phylogenies, as well as their mere presence in the oral tissues, the detractors maintain it is premature to call this “venom” as its ecological role has yet to be demonstrated.

Fry and colleagues do not accept this and argue instead for a revision in how we define venoms and venom systems. I personally find this hard to agree with as the already-in-use term “oral product/secretion” would suffice for any toxic compound without such a biological function (See 6). The various snakes which have bitten my face were not, in my preferential use of the term, “venomous”, despite perhaps possessing small amounts of toxic compounds in their saliva. The many, many bearded dragon (P. barbata and vitticeps) bites I have sustained on my fingers also never caused excessive bleeding, swelling, or other illness. Even if the toxic compounds in their tissues eventually were recruited to give rise to true venoms through splice variants, gene duplications, and adaptive selection, as may indeed be the case, these pre-venom products still have no natural use in prey capture in bearded dragons, which are primarily vegetarian.

While we of course do not fall within the Toxicofera phylogenetically, humans also possess some of these putative venoms in our buccal cavity (17). These include toxins with similar LD50s (a measure of toxicity) to those being used to attribute venomous status to the Toxicofera. Should we suggest humans are venomous since an intravenous injection of our saliva would disrupt the body’s delicate homeostasis? Does it not seem more consistent to continue using “toxic oral products” or a similar term to refer to any compounds produced in the oral cavity that are toxic, until the ecological function has been ascertained? Or do all snakes, many lizards, and probably a host of other species, including humans, now have the honour of being venomous fauna? In response to the need to ascertain the ecological role of these proteins, Fry has been quoted as saying, somewhat dismissively, “just give us a couple more centuries, an army of research students, unlimited funding, and we will be with you on that” (See this link (18) in the Atlantic), implying perhaps the task of confirming the ecological role of all of these proteins in every species is too arduous, an impossible and preposterous standard to set. This seems like a somewhat facetious argument as a response to genuine criticism. To suggest that every species’ oral products must be tested for function would of course be foolish. However, if it could be demonstrated in but a handful of species across the Toxicofera lineages that these proteins have an envenomation function, the venomous status of these creatures would be gloriously and righteously vindicated. Strong support indeed and hard for anyone to argue against!

Alas, there are no such examples in any taxa thus far. Even the Komodo Dragons venomous status is under debate, with detractors suggesting the amount of trauma generated by the serrated, saw-like teeth seems more than enough to cause bloodloss and shock rather quickly. Ecological function of all of these putative venom compounds thus remains a mystery. Should we perhaps withhold upgrading them from “oral products” to “venoms” until we decide whether ecological function or simple toxicity will be the standard-barer for biology as a whole, us included, from here on? Evolutionary definition can be a bumpy road, but such standards and definitions are set exactly to moderate the kind of confusion currently being generated.

What then are the roles of these oral proteins if they’re not venoms? Many functions have been suggested, for example, lubricating prey to aid swallowing, pre-digestion (just as the enzyme amylase starts digestion of starch compounds in our mouths before food even reaches the stomach), anti-putrefaction since large prey may take days to digest inside a snake, detoxifying/disinfecting prey surfaces, or general oral health and hygiene (Kardong, 1982). However, just as suggestions for venom function in lizards, the above are nothing more than suggestions. Proof requires evidence, which has yet to be gathered, thus judgments should be withheld until we know for sure.

More recent evidence may suggest that these “venom” proteins are not venom proteins at all. A 2014 publication by Hargreaves et al., using emerging technologies, surveyed DNA expression in multiple body tissues of Toxicoferan animals, including the Burmese python and the leopard gecko (19). Comparison of the genes expressed in these tissues with online databanks of gene sequences found matches where one would not expect. These “venom” genes were being expressed throughout multiple parts of the body, and in comparable levels to those found in the oral tissues. How could this happen? The authors suggest that these genes are in fact nothing more than “housekeeping” genes, used for general maintenance purposes rather than venom. Of 74 putative venom genes studies, only two (Laminoacid oxidase b2 and PLA2 IIA-c for those playing at home) showed significant expression of gland-specific splice variants as expected of true venom proteins. This was considered a heavy blow to the Toxicofera hypothesis, although Fry and colleagues maintained that true venom genes had to come from somewhere, and these housekeeping genes were recruited for their toxicity, a by-product of their original function, at some point in the early Toxicofera history. If you suspect that this was far too ambiguous an answer to satisfy everyone, particularly those opposing team Toxicofera, you would be correct.

Obviously the matter needed to be settled before the scientific community came to blows (scientists are after all known for their violent temper and rage issues). The battle in the editorial section had garnered quite an audience, but as yet seemed unresolved. The challenge was set; a public debate at Oxford University. Who wouldn’t appreciate the Schadenfreude of beating one’s opponent in debate at such a renowned establishment, in the grand public arena no less? Both parties naturally agreed and brought their best case to the fore, however the result was an almost unanimous vote against the singular evolution of venom in reptiles (for a brief rundown, albeit from only Hargreaves pen since history is always written by the victorious, see link 20).

Credit must be given here, for as arguing against the establishment is difficult, the house almost always wins, and team Toxicofera obviously stuck to its convictions. The conclusions of Hargreaves and others on the other hand are increasingly hard to deny, as demonstrated by the thoroughly one sided final score. While a convincing victory, the Toxicofera proponents are by no means licked, and not yet abandoning their hypothesis due to the opinion of the masses, be they scientists or otherwise and further interesting research is undeniably in the works from both camps. For example, a recent combined molecular morphological study claims to have resolved the squamate phylogeny once and for all, coming down on the side of the Toxicofera, however this paper’s methods (including removing what they call “rogue” taxa which don’t conform in their phylogeny, a curiously convenient way to prune a tree) have been criticized (21). Why can’t it ever go smoothly?

So, the number of times venom has arisen in the reptiles remains unsettled. But where does this leave us with the unresolved squamate phylogeny? Here I believe, or at least hope that future evidence shows, that we can have our cake and eat it too, while not being venomous people. The Toxicofera clade seems to be consistently recovered by many molecular investigations, and the homology of some oral tissues and protein products seem to suggest shared ancestry. What is the problem with assuming a non-venomous role for these oral products, particularly in light of their low expression in glands and expression throughout the body? Could not such oral products still confer some selective advantage and lead to a successful clade of animals? If, for example, the common oral products of the Toxicofera are simple anti-microbial tools to protect the delicate mouth of the snake, which gave them an immune advantage that was inherited throughout the clade, then while the clade Toxicofera may truly define an evolutionary lineage, then only the implication in the name would be misleading. While a clade of successful reptiles with an improved ability to resists oral infections (I dub this hypothetical clade “Antimicrobifera”) may be a less captivating idea than the highly toxic or bacteria laden bite of the Komodo dragon, weirder things have happened.

But this is all conjecture. For now, we must satisfy ourselves with what we know; current molecular data indicates the Toxicofera may in fact be a real evolutionary grouping sharing a common ancestor, but whether they’re truly “venomous” as the namesake suggests remains to be proven. Until the function of these products is proven, I personally shall simply say they appear to be common in much of the putative Toxicofera group, for some reason or other. Vague I know, for vagueness is our refuge in the face of uncertainty, until sufficient evidence draws us out into the light. Such caution keeps theories alive in the face of predators, for the tastier the theory, the more they shall try to tear it to shreds.

A final thought on complexity. It is tempting to view venom systems as on an upward climb in complexity through the squamata (disregarding examples of degeneration). This view is in agreement with the parsimony principle, referring to finding the simplest solution, the smallest amount of evolutionary branches on the tree, as morphological and evolutionary changes do not happen lightly. However while organs can increase in complexity, it is more important to remember that evolution does not strive for progress, only survival and reproduction. Any “progress” we perceive in biological systems is merely a by-product of life’s branching nature and the great spread of variety and form, rather than a march towards some perfect animal or system. What intricacy life generates is not simply to increase in complexity towards a particular ideal; there is no ideal, or if there is it exists as a broad, fluctuating spectrum built atop a sliding scale, and even we humans can make no claim for superiority or perfection. This, at least, we share with all snakes, lizards, and the rest of life on Earth.

References

1. Wilson, S. & Swan, G. (2013) A Complete Guide to Reptiles of Australia; Fourth Edition. New Holland Publishing.
2. Cogger, H. (2014) Reptiles And Amphibians Of Australia; Seventh Edition. CSIRO Publishing.
3. Fry, B.G., Wroe, S., Teeuwisse, W., van Osch, M.J., Moreno, K., Ingle, J., McHenry, C., Ferrara, T., Clausen, P., Scheib, H., Winter, K.L., Greisman, L., Roelants, K., van der Weerd, L., Clemente, C.J., Giannakis, E., Hodgson, W.C., Luz, S., Martelli, P., Krishnasamy, K., Kochva, E., Kwok, H.F., Scanlon, D., Karas, J., Citron, D.M., Goldstein, E.J.,McNaughtan, J.E., Norman, J.A.. (2009) A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus(Megalania) priscus. Proc Natl Acad Sci U S A.106:22
4. Fry, B.G., Vidal, N., Norman, J.A., Vonk, F.J., Scheib, H., Ramjan, S.F.R., Kuruppu, S., Fung, K., Hedges, S.B., Richardson, M.K., Hodgson, W.C., Ignjatovic, V., Summerhayes, R., Kochva, E. (2006) Early evolution of the venom system in lizards and snakes. Nature. 439, 7076.
5. Lumsden, N.G., Fry, B.G., Kini, R.M., Hodgson, W.C. (2004) In vitro neuromuscular activity of ‘colubrid’ venoms: clinical and evolutionary implications. Toxicon. 43
6. Kardong, K.V. (1982) The evolution of the venom apparatus in snakes from colubrids to viperids and elapids. Memoirs of the Institute of Butanan 46: 105–118.
7. Kardong, K.V. (1996) Snake toxins and venom evolution. Herpetologica 52.1
8. Jackson, K (2003) Evolution Of Venom-Delivery Systems in Snakes. Zoological Journal of the Linnean Society The Linnean Society of London, 137
9. Brusatte, S.L. , Benton, M.J. , Desojo, J.B., Langer, M.C. (2010) The higher-level phylogeny of Archosauria (Tetrapoda: Diapsida), Journal of Systematic Palaeontology, 8:1
10. Close, R.A., Friedman, M., Lloyd, G.T., Benson, R.B.J  (2015) Evidence for a Mid-Jurassic Adaptive Radiation in Mammals. Current Biology Volume 25, Issue 16.
11. Engel, M. (2015) Insect Evolution. Current Biololgy. 25
12. Fry, B.G., Wuster, W., Ramjan, S.F.R., Jackson, T., Martelli, P.,  Kini, R.M. (2003). Analysis of Colubroidea snake venoms by liquid chromatography with mass spectrometry: evolutionaryand toxinological implications. Rapid Commun. Mass Spectrom. 17: 2047–2062
13. Vidal, N., Hedges, S.B. (2005). The phylogeny of squamate reptiles (lizards, snakes, and amphisbaenians) inferred from nine nuclear protein-coding genes. C R Biol. 328(10-11)
14. Weinstein, S., Keyler, D.E., White, J. (2012) Replies to Fry et al. (Toxicon 2012, 60/4, 434–448). Part A. Analyses of squamate reptile oral glands and their products: A call for caution in formal assignment of terminology designating biological function. Toxicon. 60(4)
15. Jackson, T.N.W., Caswell, N.R., Fry, B.G. (2012) Response to “Replies to Fry et al. (Toxicon 2012, 60/4, 434–448). Part A. Analyses of squamate reptile oral glands and their products: A call for caution in formal assignment of terminology designating biological function”. Toxicon 2012. 1-17
16. Weinstein, S.A., White, J., Keyler, D.E. (2013) Response to Jackson et al. (2012), Toxicon. 64.
17. Bonilla, C.A., Fiero, M.K., Seifert, W. (1971) Comparative biochemistry and pharmacology of salivary gland secretions. I. Electrophoretic analysis of the proteins in the secretions from human parotid and reptilian parotid (Duvernoy’s) glands. J Chromatogr.  56(2)
18. http://www.theatlantic.com/science/archive/2015/11/reptile-scientists-bear-their-fangs-in-debate-over-venom/413485/
19. Hargreaves, A.D., Swain M.T., Logan, D.W., Mulley, J.F. (2014) Testing the Toxicofera: Comparative transcriptomics casts doubt on the single, early evolution of the reptile venom system. Toxicon. 92
20. http://www.ox.ac.uk/news/science-blog/amicable-venomous-debate
21. Reeder, T.W., Townsend, T.M., Mulcahy, D.G., Noonan, B.P., Wood, P.L. Jr, Sites, J.W. Jr, Wiens, J.J. (2015) Integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected placements for fossil taxa.PLoS One.10(3)

Essay 2. Hide and Sea Snakes: The Rediscovery of Two Potentially Extinct Species in WA

“No one knows the diversity in the world, not even to the nearest order of magnitude…We don’t know for sure how many species there are, where they can be found or how fast they’re disappearing.” — Edward O. Wilson

Extinction is a bitch. Unless humanity has some serious new biological technologies which I’m yet to hear about, there is no return. The loss of a species is truly a terrible thing, an artwork of nature lost to the ages, leaving nothing but memories and, if we’re very lucky, some physical remains from which we may interpret the mode of their life and demise. In these marvelous times, the early 21st century, the rates of species extinction are unfathomable. I mean quite literally, it’s impossible to count exactly how many species at any given moment we are relegating to history’s dark corners through habitat destruction, overexploitation, and…well, let’s face it, I could list the ways humanity is contributing to this decline for the next the several pages (1). Furthermore, we don’t even know how many species of plants and animals currently exist, not to mention the unfathomable numbers of fungi, bacteria, archaea and viruses, all constantly mutating and evolving. With around 1.5 million species currently described, it’s astonishing that biologists predict at least one-third of all species remain to be discovered (2) (incidentally, we don’t even have a great idea how to define a species! See (3) for a review of this issue in the Bacteria and Archaea). Human nature, it seems, is often to simply proceed at will using ignorance as a map, until the stacking negative consequences force us to reign in our “progress” and examine our actions more objectively, a skill we are apparently developing when it comes to balancing our population requirements with those of other organisms and the planet’s natural resources.

What a depressing way to start! Kill me now, or let’s have some good news! Luckily, while we will discuss population declines and extinction, I truly do intend to discuss a rather more cheerful topic; the recent rediscovery of potentially extinct species. While tempting to view these situations as a somewhat miraculous, phoenix-like revival, they often share more in common with finding a mystery five-note in the back pocket…Score! Still, for conservationists who frequently are in the painful position to observe and record the decline of their favourite study species as they attempt recovery efforts (or at least learn something about how to prevent future declines in conspecifics), these discoveries are occasions for tremendous joy, even shock, like a long deceased friend contacting you out of the blue for a meal and a catch up. Even so, the joy is short-lived, for a species so rare it was thought extinct must be either especially cryptic and/or nearly impossible to find, that or it must truly be in critically low population numbers right on the brink of collapse, and extinction is still a real threat.

It is with thus with much joy and some trepidation that I write about the findings of D’Anastasi et al. (2016) (4) describing the discovery of not one, but two sea snake species, the Leaf Scaled Sea Snake (Aipysusrus folliosquama) and the Short Nosed Sea Snake (A. apraefrontalis), which recently disappeared from their only known geographic range. Both species were known only from the Ashmore and Hibernia Reefs, an isolated area of the Timor Sea, home to an incredible diversity of sea snakes. A study in 1973, for example, collected more than 350 snakes from 9 species and observed many more, while estimates of almost 40,000 snakes were made for Ashmore Reef in 1994 (5). However, populations of all sea snakes in the region began a steep decline for completely unknown reasons in the early 1990s, and continued to do so until several species disappeared completely. Neither of these species, known only from this tiny area of the Timor Sea, had been recorded in surveys since 2002.

The severe declines in these and other sea snake species on the reefs came along quickly. Further frustration and despair would’ve been in ample supply for the conservationists working on these isolated reefs, with no explanation for the declines, and no option but to observe and record the losses while searching for answers. Extinction seemed likely for these rare, open-ocean reef dwelling snakes (both were in fact listed as “Critically Endangered” on the IUCN Red List and Australia’s EPBC Act, one step shy of “Extinct in the Wild”, or when no individuals are maintained in captivity, “Extinct”). That is until the authors of (4) discovered both species not far off the northwest coast of Australia, around Western Australia’s Ningaloo Reef and further south at Shark Bay.

The paper by D’Anastasi et al. (2016) (4) from James Cook University’s ARC Centre of Excellence for Coral Reef Studies entails the search for and successful field observations of these species. After their disappearance and listing as Critically Endangered in 2002, no more sightings occurred in their known home range, though older anecdotal evidence suggested they may occur in coastal Western Australia (5). The most interesting evidence for their presence was from just one or two specimens of each species, deposited in the WA museum. A single A. foliosquama was found washed up on Barrow Island in 2010, with two A. apraefrontalis caught in 2012, one as prawn trawling by-catch and one washing up on a beach near Broome. These were mostly dismissed as probable vagrants, washed out of their natural geographic range by powerful ocean currents. However to others, this all suggested the possibility of undiscovered populations. Why not survey the coastline in the hope of discovering the hidden outposts of these rare sea snakes?

How does one go about mounting such a survey? With sweat, for one. Let it not be said that field biologists are lazy, particularly those who are fit enough to make the marine environment their office. Aside from the technicalities of mapping out and planning a thorough but efficient search, to cover maximum territory for optimal time/cost, the physical effort was incredible. Over 200 hours of survey were conducted at Shark Bay, Ningaloo Reef and other northwest coastal regions, as well as at the Scott Reef in the Timor Sea.  The team performed more than 30 hours of both snorkel and scuba surveys, as well as over 25 hours of so called “Manta Tow” survey to cover a greater area. Developed in 1969 to assess crown-of-thorns starfish densities, this method involves towing a buoyant ‘manta board’, complete with waterproof recordkeeping sheets, behind a boat constant speed while a harnessed in observer clings to the board, using a snorkel and goggles to observe underwater as they pass. Around 40 hours by-catch prawn trawling surveys were also conducted around Shark Bay and Exmouth, and opportunistic sightings were also recorded wherever possible. As said by more than one field biologist in their time, the only way to find something is to look for it.

This mammoth effort was rewarded with gusto. Several individuals of the species Aipysurus apraefrontalis, the short nosed sea snake, were spotted around the Ningaloo Reef. Imagine the elation of finding this little known species of sea snake, 15 years after the last one was seen, 1700km south of its known distribution, and when all but a few were holding out hope for its continued existence. Not only that, two individuals observed appeared to be in the middle of courtship, hopefully a sign indicating a healthy breeding population. How very sweet for this stunning, golden yellow and cream banded sea serpent which had seemed destined to join the museum cupboards of the Dodo and the Dinosaurs. Further specimens were collected near Exmouth in WA Department of Fisheries prawn trawl by-catch surveys, another good indicator of a viable breeding population in the area.

But the story continues. The second discovery came only a few hours boating further south of Ningaloo. Another of Ashmore Reef’s lost sea snakes, the Leaf Scaled Sea Snake (Aipysusrus folliosquama), was found happily drifting among the sea grass flats of Shark Bay, a significant change from their typical setting of isolated coral atolls and reefs in the Timor Sea. To find this coral reef specialist in sea grass beds is very odd indeed, and the description as a shallow water tropical reef specialist requiring significant cover and structure will need some review. As with A. apraefrontalis, can we be sure this an established breeding population? While certainty is a rare commodity, a total of 16 A. folliosquama were caught in by-catch surveys between 2013 and 2015, all from Shark Bay and surrounding areas. It seems they hit the jackpot!

All’s well that ends well, no? In fact, not, and at this point some interesting questions start to arise. Ashmore and Hibernia Reefs supported diverse, abundant populations of sea snakes. For such seemingly successful animals in what appeared a very suitable habitat, how could such rapid population declines occur? What impacts are they under threat from in the waters off northwest Australia? Furthermore, now that we know they’re here just off the WA coastline, what can we do to minimize the stresses on these potentially new populations and increase their chances of survival? Can we prevent them from slipping away again?

In Australian waters, sea snakes themselves are not harvested for any commercial purposes, however through parts of southeast Asia there continues to be a small local market for sea snakes, usually for the colourful skins of sea kraits (sea snakes of the Laticaudidae subfamily, less common in Australia than in southeast Asia, all which lay eggs on land rather than on reef/rocky substrates like the Hydrophiinae subfamily, the true sea snakes), as an imported menu item, or for traditional medicine (6). Being in Australian waters, and since 1983 within the Ashmore Reef National Nature Reserve, direct harvesting is not likely to have much impact on these isolated populations, which are themselves classified as protected marine fauna under the Nature Conservation Act of 1992.

Trawl fisheries generally pose a much greater threat. Sea snakes need to surface to breath, using lungs which, aside from a few notable adaptations (extreme elongation, one dominant lung, a devascularised posterior “scuba tank” unique to sea snakes), are rather like ours. While they obviously possess a phenomenal breath holding ability, around 4 hours under optimal conditions, they can actually drown just like a person, dolphin, emu, or a crocodile. Add to this damage from being net-dragged across the benthos or crushed in a turbulent net by hundreds of kilos of rocks, crustaceans, and fish including sharks and rays, all struggling and making repeated attempts to escape, and the impacts of these fisheries are obviously a potentially serious threat (7).

Luckily the Ashmore and Hibernia Reefs have some reprieve from our constant plundering of the ocean. At least locally, the depth just off the edge of the coral platforms and the coral itself makes for difficult-to-impossible trawling conditions. Trawling is more difficult at depths and around structure, the potential for snagging and damage to the net being an important financial consideration. Furthermore the trawling rates and rates of decline do not correlate; while rates of trawling have remained relatively steady, sea snake numbers began to decline dramatically in 1994. Trawl fisheries, an initially likely culprit, seem to be in the clear for the moment.

So, direct human harvesting and the local trawl fisheries both seem to have little impact on these disappearing sea snake populations on the reefs. What then could have caused such rapid declines? Several possible causes, at very different spatial scales, are currently under suspicion; firstly, on the small scale of cells, tissues and organisms themselves, the potential for disease or demographic reasons for these declines has to be considered; secondly, on the middle scale, habitat loss might be to blame, with coral bleaching events occurring in 2003, though these could hardly cause the declines from 1994; on a large scale, environmental changes, such as shifts in temperature and/or prey abundance due to human induced climate change, are also on the suspect list (5). Investigations into these areas will hopefully provide much needed insight in the near future, maybe even an answer as to why these populations collapsed.

As for the newly discovered coastal populations, what can the observations tell us? One thing we can infer with some confidence, from the aforementioned courting/breeding pair of A. apreafrontalis spotted off Ningaloo Reef, and the substantial number of A. folliosquama found during the rather short survey, is that these are most likely true local breeding populations, rather than ephemeral waifs floating adrift on the currents, being fed into the survey areas from some outside source population. This is further supported by the apparent strong population structure of A. folliosquama, Shark Bay populations being genetically distinct from the washed up specimen from Barrow Island, itself distinct from historical Ashmore Reef samples. This all suggests that the Barrow Island specimen may be of a separate coastal breeding population, yet undiscovered. Along with the habitat extension, it seems that populations  of A. folliosquama may really be out there in hidden in the WA sea grass beds, and A. apraefrontalis may be casually courting one another of Ningaloo and other reefs without our knowing! Unfortunately, while this is all rather hopeful conjecture, we can just as easily assume that whatever afflicted the Ashmore Reef populations can also affect these “unknown” populations, and they may disappear before we ever know they existed. One can rarely ever know what diversity and complexity has been lost, or how it was lost, without the benefit of painful hindsight.

If these populations are true, established breeding populations as we suspect, one troubling thought comes to mind. Despite the relative safety of the Ashmore and Hibernia Reefs from trawling impacts, the now known extension of these two species’ geographic ranges and habitat to coastal areas including sea grass beds, and the fact that the vast majority of specimens were caught in prawn trawl by-catch surveys, suggests the impact of trawling on coastal populations may be much higher. In fact, these coastal populations might naturally be subjected to all manner of man-made coastal disturbances, such as industrial/agricultural run-off, seismic activity from mineral and gas exploration, increased recreational activities such as boating and fishing to compete with, and much more. Being so isolated, it is easy to assume that the reef populations were somewhat protected from many of these activities, and yet they’re gone. What does this mean for our chances to protect the newly discovered coastal populations? With all the increased human impacts are we doomed to fail? Or, does their mere continued presence indicate some resilience, perhaps due to the amount of structural variation in coastal environments, or their sheer size and continuity around the coastline, which the outposts in the Timor Sea did not have?

In summary, we know little, as is most often the case. What we know is that we don’t know the true population size, distribution, or spatial ecology of these rediscovered sea snakes. More field observations are necessary to understand both the ecology of these populations and the threats they face, as well as surveys of other coastal areas for more unknown populations if we are to have a thorough inventory to base conservation efforts on. Such are the difficulties involved in working with rare animals and small population sizes. For now though, let us take five to give the fine people behind these discoveries, and to all who work with endangered species, fighting for tiny populations who persevere at the edge of oblivion, a seriously deserved round of applause. It is very close to the least we could do. We can, and should, do much more.

PS On further rediscovered reptile news…

As I write this essay another small step was made in restoring our reptile biodiversity! This time in the spotlight is a lizard species which, throughout my childhood, had been presumed extinct. The Pygmy Blue Tongue Lizard (Tiliqua adelaidensis) is a true pygmy of the robust and moderate sized blue tongue lizards. Within the Tiliqua genus most species reach over 40cm in length, while the T. adelaidensis tops out at a tiny 18 cm. After an absence from their native habitats for 30 years, the future seemed uncertain for these little gems. As a kid I knew them only from pictures of deceased museum specimens in my reptile encyclopedia, the status next to them reading “Presumed Extinct”. While well preserved, they were somewhat less inspiring than the rest of the creatures in the book, perhaps due to signs of dessication and rigor, and the final shame of a finely engraved voucher label attached to the hid legs, as if just another artifact of history. Then, while driving along a country road one day in 1992, a herpetologist outside of Adelaide accidentally ran over an eastern brown snake sunning itself on the road. Upon examination, he found two “extinct” T. adelaidensis inside its stomach. Not only that, surveys found local populations living in spider holes in the surrounding native grasslands, and study taught us much about their ecology (8).

As of January 2016, Zoos South Australia confirmed the successful captive breeding of the Pygmy Blue Tongue Lizard (9). Twenty years after first being fascinated and saddened by pictures of these creatures which I thought I would never see, I am currently sipping a cup of tea and looking at images online of newly born babies. The tiny, tiny offspring of this diminutive species of blue tongue are coloured more like some of our speckled Ctenotusspecies, but the stocky little body, small tail, and big head are characteristic Tiliqua. They’re damned cute. My first pets were also in the genus (mine were Southern Blue Tongued Lizards,T. nigrolutea), and I have an inordinate fondness for the Tiliqua, from the common garden visitors and the desert dwellers to this spider-specialist dwarf. To find both of these articles, the lizards and the sea snakes, making the rounds and gaining interest on social media networks, all in one week, caught me by surprise. Perhaps there is some hope for humanity after all.

References

1. Proença, V. &  Pereira, H. M. (2013) Comparing Extinction Rates: Past, Present, and Future. Encyclopedia of Biodiversity (Second Edition). P167–176
2. Costelo, M. J. (2015) Biodiversity: The Known, Unknown, and Rates of Extinction. Current Biology 25, R362–R383
3. Rosselló-Móra, R. & Amann, R (2015) Past and future species definitions for Bacteria and Archaea. Systematic and Applied Microbiology. Volume 38, Issue 4, 209–216
4. D’Anastasi, B.R., Van Herwerden, L., Hobbs, J.A, Simpfendorfe, C.A., Lukoschek, V. (2016) New range and habitat records for threatened Australian sea snakes raise challenges for conservation. Biological Conservation. 194. 66–70
5. Lukoschek, V., Beger, M., Ceccarelli, D., Richards, Z., Pratchett, M. (2013) Enigmatic declines of Australia’s sea snakes from a biodiversity hotspot Biological Conservation 166. 191–202
6. Van Cao N, Thien Tao N, Moore A, Montoya A, Redsted Rasmussen A, Broad K, Voris HK, Takacs Z.(2014) Sea snake harvest in the gulf of Thailand. Conserv Biol. 28(6):1677-87
7. Wa ssenberg, T.J., Milton, D.A., Burridge, C.Y. (2001) Survival rates of sea snakes caught by demersal trawlers in northern and eastern Australia. Biol. Conserv. 100, 271–280.
8. Armstrong, G. & J. Reid (1992). The rediscovery of the Adelaide Pygmy Bluetongue Tiliqua adelaidensis (Peters, 1863). Herpetofauna. 22 (2):3-6.
9. http://www.abc.net.au/news/2016-02-24/monarto-zoos-pygmy-blue-tongue-lizard-breeding-success/7195136

Essay 1. The Brown Snake, The Taipan, and The Paradox

“I love fools’ experiments. I am always making them.” – Charles Darwin

As a zoology student in Brisbane, Australia, whenever classmates would ask me for study tips, they were often disappointed to find I rarely start at the beginning of anything. My ideal study method is to dive into the middle of a subject at an interesting juncture and read widely around this nugget of intrigue, often at the temporary expense of the subject at large. As my own interest in a subject blossoms, so does my curiosity for the overall details of whatever system I am learning about, then perhaps I am ready to tackle the early introductory subject matter (not to mention lecture slides on general housekeeping and assessment dates, the horrors of which no undergraduate need reminisce on). Without further delay, let us then dive headlong into the world of snakes, and perhaps throughout come to a greater understanding of this bizarre collection of animals. We should I believe, for interest’s sake, start with a discussion on a peculiar aspect of the venoms of two of Australia’s deadliest snakes; one the most common venomous snake we have, the other the longest and arguably most formidable.

When it comes to the venoms of various animals, neurotoxins are typically the most complex and the most lethal. While a typical “neurotoxin” is simply any substance harmful to nerve tissues, the protein based neurotoxins developed for hunting, defense, or competition, in various groups of organisms are among the most dangerous substances on the planet. In fact, the most toxic substance currently known to humanity is a neurotoxic excretion from the Clostridium botulinum bacteria and its relatives, known as Botulinum Toxin (BTX), or Botox to those in the cosmetic industry, blocking cholinergic neuromuscular junctions, which prevents motor fiber stimulation and can cause death by paralysis of the respiratory system (1). If it seems ludicrous to be injecting this bacterial neurotoxic protein, the most dangerous substance known to humanity, into one’s cheeks to lift and tighten the skin, well, I may agree with you however we must both somewhat acknowledge that the science of using sub-lethal doses of toxic substances for therapeutic purposes is well established. As for the various neurotoxins of the animal world, how very few, relative to the expansive available variety, have been recklessly injected into people’s cheeks for vanity and/or science? Think of what we might learn with but a little bravery!

Of course, this is foolishness. I simply hope to impress upon the reader two things; first, the efficacy and usefulness of biological neurotoxins for human medical science, and second, the potential untapped resources for biodiscovery in the diversity of our planet’s venomous fauna (2, interested readers would do well to check out this 2014 review by Alan L Harvey for the journal Toxicon). The study of toxic substances, venoms included, is called toxicology. The integration of chemistry, biology, and medicine is what makes toxicology so fascinating, with many toxicologists working broadly in all three fields. The field is not restricted to  just fauna; environmental toxicology, for example, is an extremely useful tool for societies who wish to avoid the pitfalls of environmental contaminants, poisons and catastrophes. Toxinology is the more specific study of animal, plant, and microbial toxins, & the Toxicologists who study animal venoms are a dedicated bunch, often travelling far and wide to find some elusive subject to “milk” for venom, which is stored on ice and rushed back to the laboratory as fast as the local means of transport will allow.

While this sounds all rather adventurous, we will instead focus on the recent work of toxicologists on something more familiar. Here on Australia’s east coast, you don’t have to go far to find our first subject. The Eastern Brown Snake (Pseudonaja textilis), also rightfully known as the Common Brown Snake (hereon referred to as “EB”), is a relatively large, alert, fast-moving snake of the family Elapidae, the front fanged snakes including cobras and coral snakes (3). While often regarded as dangerous or “aggressive”, in reality it is quick to avoid people and will flee rather than fight. When cornered or agitated, however, the EB’s defensive display is formidable. The fore-body is raised in an S-shape, displaying the often vibrant reddish-brown “belly spots” on the ventral surface, with the bright pink mouth open. They are capable of moving in this position using the posterior part of the body, keeping the S-shape raised as they approach a would-be attacker (4). This display is so effective that anecdotal reports of being “chased by a brown snake” often arise from those fleeing in terror. Needless to say, once you’re outside the snakes perceived threat perimeter, the defensive posture collapses and they resume looking for shelter, but I’m certain the story of the Epic Chase continues over cold beverages.

It is only the truly unlucky or foolhardy who get bitten by Australian snakes, with most bites caused by accidentally treading on snakes, followed by those actively trying to harm or capture them, and the EB is no different in this regard (5,6, with more on snake bite fatalities in Australia). However its venom contains potent neurotoxins, procoagulents, and more nasty surprises for any biological system it encounters. Based on the LD50 (volume needed to kill, on average, 50% of a population of lab mice within a given time period) it is the second most venomous snake on land, though it is important to remember that this value is not based on human physiology, something to bear in mind as we introduce another formidable serpent (7).

Our next subject, the Coastal Taipan (Oxyuranus scutellatus, hereon referred to as “CT”), has possibly the most feared reputation of all Australian snakes. Found from east to north Australia and across to Papua New Guinea, these large, fast moving elapids are highly venomous, perhaps a consequence of their strict diet of warm blooded mammals, with the rather challenging prey of bandicoots as a favourite (8). In fact, there was a time not long ago, the 1960s I believe, when a confirmed Taipan bite came with a 100% mortality rate (9). It is also the longest venomous snake in Australia, beat out in weight only by the Mulga or King Brown Snake (a member of the genus Pseudechis rather than Pseudonaja, thus not a true “Brown” snake, but the name King Brown now has a mythical quality to some Australians, and if a name makes the foe that much more menacing at the retelling…), and arguably the fastest and most agile of our large elapids (8). While in possession of a similar cocktail of chemical weaponry, which I’m delighted to say we shall soon discuss in length, the venom delivery system in the CT is phenomenal, with 13mm long fangs on a large, wide hinged jaw for consuming prey as large as bandicoots (I suppose analogous in size to a small rabbit). The CT is also known to flee readily, in fact they can be darned hard to find, I can count on one hand the occasions I’ve seen them in the wild myself, even as a reptile obsessed geek who searched for wildlife almost constantly (thankfully with little or no supervision or guidance, and thus my numerous injuries and near misses). Unlike the EB however, the defensive posture is much lower and less static, lashing out in multiple strikes rather unpredictably, often inflicting multiple bites (10).

Beautiful creatures, no? I suppose some would disagree, however the aesthetic appreciation of snakes can wait for another time. Why are we discussing these two snakes, other than for their apparent badassitude in terms of death and venom? The reason I bring up these two snakes, in relation to toxicology specifically, is due to a rather interesting medical mystery in the patient outcomes of human victims of EB and CT bites. The so called “Brown Snake Paradox”, coined in 2002, refers to the puzzling phenomenon that an EB bite rarely results in systemic neurotoxicity, while for a CT bite neurotoxicity is the major feature, manifesting as loss of muscle control, including the muscles controlling the respiratory and circulatory system (11). While both snakes have a variety of procoagulent toxins which interfere with natural clotting processes, causing internal bleeding and hemorrhages, this seems to be the main effect of EB venom. The main clinical effect of brown snake bites is VICC (Venom Induced Consumption Coagulopathy, say it with me now…) where the powerful procoagulent venom molecules consume all the clotting factors available in the blood (fibrinogens etc.). This inhibits clotting ability, causes microangiomas, and a host of other complications. It kind of like Stigmata on your everything.

The surprising part comes when you compare the LD50 of these snakes. When comparing the whole venom of these snakes, containing both pre- and post-synaptic neurotoxins, as well as myotoxins and procoagulants, the CT comes in as the 3rd most venomous snake in Australia, but it is beaten out by the humble, “common brown”! In fact, the EB ranks as the second most venomous land snake in the world, beat out only by the Inland Taipan (Oxyuranus microlepidotus) (9). Given this information, shouldn’t we suspect that an EB bite would result in severe neuropathy?

The Brown Snake Paradox was a medically significant conundrum for years. When it comes to treating a snakebite victim, the variety of complications is wide and varied, but the possible complications are still somewhat defined by the type of venom and its activity in the body (12). Neurotoxins often cause respiratory distress and require assisted breathing, while procoagulents may necessitate clotting factor replacement, or dialysis in cases of renal failure. Early treatment is of course the best course for survival and recovery, so we would prefer to understand the biological activity of these venoms and treat them as necessary, rather than inferring from symptoms what treatments are required. Whether this supposedly powerful textilotoxin causes human neurotoxicity is important for the whole snakebite scenario “Chain-Of-Survival”. While CPR and assisted breathing may certainly be necessary, if the main systematic threat is loss of clotting factors, then, brothers and sisters, you better hope there are some clotting factors on the way for you! Anti-venom therapy can only do so much, and the damage done by the venom on the way to hospital is still a health hazard which may require treatment. Knowing which treatments work for which species of snake, from which regions of Australia (enjoyably, snake venoms can vary in their bioactivity regionally, see 13), and early treatment are key.

So we have a paradox; the highly “neurotoxic” EB which causes little to no neurotoxicity. Could perhaps its lethality be limited to other venom functions? How actively neurotoxic are they? These questions and more are usually where the Toxicologists step in. To be fair, they’ve been here the whole time and are the only reason any of us actually know what an LD50 is! Thanks to some phenomenal work by Carmel M. Barber et al (2012) the Brown Snake Paradox is effectively solved, if only in how and perhaps not the why. Lets us first consider this newly illuminated how, and perhaps we might speculate on the unknown why.

The neurotoxins of elapid snakes can be classified into two groups major: postsynaptic neurotoxins and presynaptic neurotoxins (14). In 1979, the presynaptic neurotoxins of our stories two protagonists were identified by separate research groups, taipotoxin from Taipans (15), and textilotoxin from the EB (16) (again, the Latin name is P. textilis, thus the name of the venom).  When tested on mice, textilotoxin has a much lower LD50 than taipotoxin, meaning it requires significantly less of the venom to kill the same amount of a test population. It was thus determined that textilotoxin was the largest and most potent neurotoxin isolated. How then did the team of Carmel M. Barber and co. defy this claim in 2012? As many venomous snake fanatics will tell you, LD50s alone are not to be taken as the Gospel of Snake Venom Toxicity (thou shalt not assume LD50s apply to all fauna?). What works in mice may not work in humans, but more significantly, the LD50 test itself is based on a whole organism test within a certain time limit, and “death within 12 hours or it’s all gravy” fails to take into account the amount of internal damage done to tissues and organs within those 12 hours. Damage which may suddenly result in further complications after the stopwatch for the LD50 test has finished. If you want to compare the neurotoxicity of two venoms, a more precise, direct method is needed.

This is precisely what Barber et al. achieved a few years ago (11). When new investigative methods arise, old prejudices must die like so many lab mice. Or in this case, lab chickens. Aside from extracting and comparing the amounts of the toxins, the two were compared on a Chick biventer cervicis nerve-muscle (CBCNM) preparation, a technique from the sixties that can help determine whether a toxin in pre- or post-synaptic in action, and help quantify it’s direct affect on nerve signal transduction from a motor neuron to a muscle fibre. From a euthanized chicken, a motor neuron and connected muscle tissue are delicately dissected and, in a medium, hooked up to a small electrical generator. Electrical signals are then used to simulate signals from the brain, causing the muscle to twitch at a defined interval. To compare potencies of the venoms, they compared the time taken to block the twitches from nerve stimulation by 90%. This value, t90, was much, much lower in taipotoxin, indicating it actually has a much greater effect on the nervous system then textilotoxin. This finding, along with the lower concentrations of textilotoxin found in EB venom suggests a resolution to the paradox; the more potent Taipotoxin makes a greater proportion of CT venom, thus imbuing their bite with a highly neurotoxic effect, while the textilotoxin on the EB has a comparatively lower effect on the nervous  system, and is present in lower concentrations. Other venom components, such as the prothrombin activator (a procoagulent which makes up over a third of EB venom) frequently cause EB victims to suffer from VICC and other complications, rather than paralysis. The paradox is, for now, fairly well dead.

So, we now accept that textilotoxin has a reduced activity on nerve function. Where does this leave us? Is there a ready explanation for this reduced neurotoxic activity? After all, we can assume from the wide variety of neurotoxic snakes that such venoms are historically very effective and a successful survival tool. Why abandon neurotoxicity?

While no solid hypotheses have yet been subjected to experiment, several potential theories come to mind. First, despite the lower neurotoxicity to the CBCNM preparation, rodents still seem rather susceptible to EB neurotoxins, and perhaps some accumulated differences in our receptor sites renders humans, and perhaps chickens, with some immunity. Second, textilotoxin is but one (albeit large and complex) neurotoxin in the EB cocktail, in fact we have completely ignored the post-synaptic neurotoxins, which act at the neuromuscular junction and more rapidly than our current focus, the pre-synaptic neurotoxins. The cocktail of post-synaptic neurotoxins may hide their own secrets! (For example, see this investigation of post synaptic neurotoxins between putative subspecies of CT, 17)

Thirdly, perhaps this reduction is a result of selective pressures acting on the venom composition of EB populations. It has already been shown that there is some variation in venom between geographically distant populations due to, it is thought, differences in dietary preference and availability (13). Venom genes evolve rapidly (18, 19), as do their upstream control genes, and perhaps with this rapid mutation rate the genes for non-neurotoxic venom components are, for some reason, achieving greater success. I can imagine a scenario where prey populations are under such strong pressure to evolve immune defenses to the newly evolved neurotoxins, and neurotoxins in turn evolve greater potency, and so on until building powerful, large, complex neurotoxins is the norm, and native prey items require strong immunity. Such evolutionary arms races are commonplace, and venoms are the result of this chemical arms race over thousands of generations.

But during such an arms race for neurotoxicity specifically, perhaps the selection pressures to evolve immunity to other newly evolved proteins, such as the prothrombin activators in EB venom, are relaxed somewhat in comparison (though not enough to disappear completely as they are still a minor component of many venoms) (20). If then, perhaps, a mutation in the control regions for a gene which is responsible for producing such procoagulents etc. causes an increase in production to well above what the prey’s immune system has historically encountered, the owner of said mutation might have greater success and leave more offspring. This of course increases the frequency of this successful mutation in the population. Given time, with snakes now relying on procoagulent effects rather than neurotoxicity, the gene for textilotoxin might be allowed to accumulate mutations at a near-neutral rate, with no functional purging of less effective variants since it is of little significance to catching dinner. The rifle gets relegated to the back of the attic to collect dust among the rest of the now retired hunting tools, replaced by the compound carbon-fiber hunting bow.

Speculation can be dangerous. The human mind’s ability to project ourselves into a virtual future have been proven flawed repeatedly, and we are prone to false conclusions, unconscious biases, and personal preferences which make fools of us all. As such, the above “whys” are to be taken with much more salt than the “hows” of the Brown Snake Paradox. Nonetheless, without imagination, we might never conceive the true nature of complex, time dependent processes, like poisoning for example. Imagination, conceptualization, and my-best-educated-guess are fundamental tools of science. After such fanciful thought wanderings, however, science puts its imagination on a scale. One consistently incredible facet of science to me is how scientists can imagine new ways to verify previously imagined realities. Perhaps, thanks to imaginative and dedicated toxicologists, we will one day know why the Brown Snake Paradox exists.

New methods lead to new discovery. Very few of us would consider that in our lifetime medically significant new information on the venom of the common, humble brown snake would come to light. Those deep in the field however know differently, and my admiration for those that continue to explore how the chemistry of life interacts with itself is limitless. Taking a deeper look into the neurotoxicity of these deadly and beautiful snakes, the Eastern Brown Snake and the Coastal Taipan, is a challenge. They are agile snakes, alert and fast, very capable of defending themselves if push comes to shove. Still, I like to imagine the smiles on the faces of some snake handlers and toxicologists in the field, trying to explain to baffled onlookers or locals how the venom from the gloriously deadly animal in his catch bag had a date with destiny, to be applied to electrically stimulated chicken flesh in order to save human lives.

Sometimes it’s a lot of fun, and very weird, to dig a little deeper.

References:

1.      Neghina A.M., Marincu I., Moldovan R., Iacobiciu 1., Neghina R., (2010) Foodborne botulism in southwest Romania during the post-communism period 1990—2007. International Journal of Infectious Diseases. 14, e96—e101
2.      Havey A.L., (2014) Review: Toxins and drug discovery. Toxicon, 92, e193-200
3.      Shine R., (1989) Constraints, Allometry, and Adaptation: Food Habits and Reproductive Biology of Australian Brownsnakes (Pseudonaja: Elapidae). Herpetologica. Vol. 45, 2,195-207
4.      P. B. Whitaker P.B., Ellis K, Shine R., (2000) The Defensive Strike of the Eastern Brownsnake, Pseudonaja textilis (Elapidae). Functional Ecology. Vol. 14, 1, pp. 25-31
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