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MASTERS OF PHYSIOLOGY
Venoms are not accidents, poisons may be.
If you decided to create a list of the most improbable animals on the planet, the platypus is an easy first pick. The platypus is so peculiar that even the great naturalist George Shaw, who provided the first scientific description of the animal in 1799, could hardly believe it was real. “A degree of skepticism is not only pardonable, but laudable,” he wrote in the tenth volume of his Naturalist’s Miscellany, “and I ought perhaps to acknowledge that I almost doubt the testimony of my own eyes.” It is a sentiment I understand. As I sat staring at a large male platypus at the Lone Pine Koala Sanctuary in Melbourne, Australia, I could hardly believe the creature in front of me was real. Even up close, it looked like some kind of masterful puppet, Jim Henson’s greatest feat.
Rebecca Bain, known as Beck, the head mammal keeper and one of the people responsible for Lone Pine’s two male platypuses, was kind enough to let me in behind the scenes to indulge my interest in the animal. As Beck wrestled the older male from his nest box, I was surprised by his beaveresque tail, duck-like bill, and ottery feet. But while these traits are all fantastically unthinkable, there is one feature of the platypus that stands out among these oddities. It was the feature that drew me to Australia, the reason I came to see the bizarre creatures in person. Beware the male platypus: of the 5,416 currently recognized species of mammals, he alone possesses a venomous sting, using toxic ankle spurs to fight over females.
We know of twelve venomous mammals; all except for the platypus deliver a venomous bite. There are four species of shrew, three vampire bats, two solenodons (long-snouted, rodent-like burrowing mammals), one mole, the slow loris, and the platypus. There’s some evidence that the slow loris may actually be four species of slow lorises, which would bump the total to fifteen, but even so, that’s still just three handfuls of venomous mammals.
Of the animal lineages, there are venomous representatives in the phyla Cnidaria, Echinodermata, Annelida, Arthropoda, Mollusca, and Chordata—the phylum that includes humans. Compared with other groups of animals, the mammals boast very few venomous members; the Cnidaria, including jellyfishes, anemones, and corals, are an entire phylum—more than nine thousand species—of venomous animals, though if we want to talk sheer numbers, the venomous arthropods, including spiders, bees and wasps, centipedes, and scorpions, undoubtedly reign supreme. There are venomous snails, venomous worms, and venomous urchins. And that’s not even including the rest of the venomous vertebrates in the Chordata. There are venomous fishes, frogs, snakes, and lizards.
The term venomous carries with it an explicit set of requirements. Many species are toxic: they possess substances that cause a substantial degree of harm in small doses (a toxin). We used to think of the terms toxic, poisonous, and venomous as interchangeable; now modern scientists distinguish between them. Both poisonous and venomous species are indeed toxic, for they produce or store toxins in their tissues. You may have heard that everything is a toxin in the right dose, but that’s not quite true. A large enough dose can make something toxic, but if it takes a lot to kill you, then a substance isn’t a toxin. Sure, you can drink enough cans of Coke for it to be fatal, but sodas are not considered toxins because the amount it takes for them to be toxic is huge (you’d have to chug liters at a time). The secretion of the anthrax bacterium, on the other hand, is a toxin because even a teeny bit can be deadly.
We can furthµer classify species that are toxic based on how those toxins arrive in a victim. Any toxin that causes harm through ingestion, inhalation, or absorption is considered a poison. Poisonous species, like dart frogs or pufferfishes, must wait for other species to make a mistake before inflicting their toxins. Some scientists would argue there is a third subcategory of toxic, in addition to poisonous and venomous—the toxungenous animals—which are essentially poisonous with purpose: toxungenous animals are equipped with poisons, but they’re more impatient. Animals like the poison-squirting cane toads or the spitting cobras actively aim their poisons at offenders when they’re annoyed, refusing to wait to be touched or bitten, like other poisonous animals, to transmit their toxins.
To earn the prestigious descriptor of “venomous,” an organism must be more than just toxic; it must also have a specific means of delivering its dangerous goods into another animal. It has to be proactive about its toxicity. Snakes have fangs. Lionfish have spines. Jellyfish have stinging cells. Male platypuses have spurs.
The venomous spurs on the platypus aren’t hard to spot. As Beck described the animals and their care at Lone Pine, I stared at the yellow toothlike points jutting from the hind legs. At about an inch long, they are much larger than I had expected. There’s no doubt that any wound created by such impressive spurs would be terribly painful even without the venom. As I placed my hands within inches of the spurs to get a close-up photograph, I shuddered at the thought of how much it would hurt to be stung by the animal in front of me.
Platypuses are really awfully, terribly venomous. From what I’ve heard, being stung by a platypus is a life-changing experience, as any deeply traumatic event shapes who you are. Their venom causes excruciating pain for several hours, even days. In one recorded case, a fifty-seven-year-old war veteran was stung in his right hand when he stumbled on what seemed like a wounded or sick platypus while he was out hunting and, concerned for the little guy, picked it up. For his kindness, he was hospitalized for six days in excruciating agony. Over the first half hour of his treatment, doctors administered a total of 30 milligrams of morphine (the standard for patients in pain is usually 1 milligram per hour), but it had almost no effect. The veteran said the pain was far worse than the pain from the shrapnel wounds he’d gotten as a soldier. Only when the medics numbed all feeling in his hand with a nerve-blocking agent did he finally feel relief.
Even more bizarre is that the venom the platypus delivers is very different from the venoms of its mammalian relatives. Similar to the animal’s outward appearance, with its collection of body parts seemingly taken from other species, it is as if the platypus’s venom is composed of a random spattering of proteins stolen from other animals. There are eighty-three different toxin genes expressed in the platypus venom gland, some of whose products closely resemble proteins from spiders, sea stars, anemones, snakes, fish, and lizards, as if someone cut and pasted genes from the entire diversity of venomous life into the platypus’s genome. Both externally and internally, the platypus is a testament to the power of convergent evolution, the phenomenon in which similar selective pressures can lead to strikingly similar results in very different lineages. Yet they are also wonderfully unique animals, the only ones we know of that use venom primarily for masculine combat rather than for feeding or defense.
Before she placed him back in his nest box, Beck allowed the platypus to release his rage. She pulled out a towel and dangled it behind him. The animal quickly and gleefully grabbed the towel with his hind legs and began writhing vigorously. The fervor with which he envenomated the cloth was adorable and terrifying. I silently thanked the awkward animal for accommodating my presence, however unwillingly. I’m pretty sure he imagined it was my arm and not the towel he clung to.
Unlike the platypus, many species use needle-like teeth to deliver potent toxins from modified salivary glands, the method preferred by the snakes and most of the mammals. The slow loris, however, has its own way of delivering a venomous bite. The small nocturnal primate, a contender with the platypus for most bizarre venomous animal on the planet, uses grooved teeth called tooth combs to deliver its painful venom. But before it can do so, it has to collect venom from glands on its elbows. Spiders, centipedes, and many other arthropods also inflict a venomous bite with the aid of fangs or other modified mouthparts. You could even say that some snails deliver a venomous “bite”: they strike their food with a harpoon-like structure called a radula, which I think of as a kind of hardened tongue.
Then there are the other stingers. Bees, wasps, ants, and scorpions are the most well-known for their stings, as are the venomous rays (or stingrays). A wide array of spiky armaments are employed by caterpillars, urchins, and plants to deliver a potent sting. The Cnidaria possess a unique mechanism—the “stinging cells” (or cnidocytes) exclusive to the phylum. They are found along the tentacles of these jellies, corals, and anemones, and can be readily triggered to launch a tube-tethered microscopic needle into whatever comes too close. While we tend to think of them as a venom delivery system, cnidocytes are diverse in form and function, with only some serving to deliver venom. Others discharge glue-like substances or simple hooks to ensnare potential prey.
The two main categories of wounding implements reflect the two main uses for venom: to aid in acquiring or eating prey, or to defend oneself against potential predators. The different uses lead to different selective pressures, and, often, to different venom activities. Those that bite generally use venom predominantly for offense. The stingers, instead, are defensive adaptations. Of course, there are exceptions to each. The scorpion and jellyfish sting to kill prey, and the slow loris bites in defense. And often, species will use their venoms for both, switching from offense to defense as necessary.
Offensive venoms tend to be more physically disastrous. They’re often packed with potent neurotoxins to paralyze the intended food or awful cytotoxins that help digest the meal. But they can also be the mildest venoms as far as humans are concerned: if the venoms are intended for an insect or some other species dissimilar to our own, the venom components might not cause the same effects in our tissues as they do in the animal’s prey. Or the delivery system may not be tough enough to get through our skin: many species of anemones, for example, are harmless to us because their nematocysts—the most common type of cnida, the “firing” organ inside each cnidocyte—can’t penetrate our dermal layers. Meanwhile, the defensive venoms generally contain different neurotoxins—ones meant to induce horrific, inescapable pain, a warning to select a different dinner. Because they’re meant as a warning, most defensive venoms aren’t lethal.
One thing is true of all venoms: they’re expensive. I don’t mean they cost a lot of money on the black market (though some do fetch a pretty penny)—I mean they cost a lot of energy to produce. An animal has to devote hard-earned calories to producing and maintaining its toxic weaponry rather than to other important uses, such as growth or reproduction.
Scientists know that venoms are costly from several kinds of evidence. Perhaps the simplest clue is that even within venomous branches (what scientists refer to as clades) of an evolutionary tree, there are often species that have lost their toxic touch. If venoms are so damned useful evolutionarily, why would any species give up the advantage unless it cost more than it was worth? A shift in diet from active to passive prey, for example, might make a predatory venom far less useful to possess. That’s why scientists believe that when the marbled sea snakes switched to eating eggs, they lost their potent venom.
In many venomous groups, there are similar key examples of reduced or lost toxicity. The constrictor snakes could be one such example: some scientists believe that the origins of venomous reptiles date back to before snakes split from their lizard relatives, but the ones that could catch enough prey with constriction had no further need of their venomous bite. The venomous fish lineages are scattered among nonvenomous groups, suggesting that the gain and loss of venom is frequent in fishes. Being toxic just wasn’t worth it from an evolutionary perspective.
Something else is true of all venomous animals: we’re fascinated by them. Detailed descriptions of venomous animals and how their bites and stings torment our bodies can be found in some of the earliest medical texts known, and have been pondered at length by the likes of Aristotle and Cleopatra. Mithridates VI of Pontus, a formidable enemy of Rome, was so obsessed with venoms and poisons that he became known as “the poison king.” His father was murdered by poison when he was only twelve, so from a young age, Mithridates sought a universal cure to any toxin. He began ingesting small amounts of toxins on a daily basis, believing that he could build an immunity to all poisons over time.
Following on the heels of the poison king were the physicians Nicander (roughly 185–135 B.C.) and Galen (A.D. 131–201), both of whom wrote extensively about venomous animals and treatments for injuries inflicted by their diverse toxins. These physicians were considered some of the best authorities on venom and medicine in general, and well into the fifteenth and sixteenth centuries, their writings were still read and translated into Latin and other languages.
Though many physicians and writers would talk about venomous animals, it would take until the seventeenth century for scientists to begin systematic studies of these dangerous creatures. Francesco Redi (1621?–1697) was among the first to compile what was known about snake venoms at the time, and to demonstrate that they were in fact venoms and not poisons—that many were harmless if they were ingested, but deadly if injected under the skin. In the nineteenth century, taxonomy as we know it emerged, and scientists began classifying and sorting venomous animals.
Strangely enough, though the platypus’s spurs were noted in some of the first specimens—indeed, the first documented sting was in 1816—scientists would debate for decades whether the animals were actually venomous. Henri de Blainville (1777–1850), chair of anatomy and zoology at the University of Paris, created one of the first detailed descriptions of the spur and its associated glands, concluding that the spurs were venom organs, intended to inject toxins “comme cela a lieu dans les serpens venimeux” (“as occurs in the venomous snakes”). Yet in 1823, an anonymous medical commenter assured The Sydney Gazette that “I have dissected this animal particularly, to ascertain this much controverted point, and have not been able to trace, either in the living or dead animal, the virus supposed to be contained in the sac; and I am not solitary in my opinion, that there is no poison; nor is it, properly speaking, a gland which the spur is conjoined to.”
“It is my firm conviction that the animal has not the power of instilling poison by its spur,” wrote the lawyer Thomas Axford in 1829. He even went so far as to say, “I am so convinced that the spur is harmless, that I should not fear a scratch from one.”
The view that the platypus was harmless would hold through the nineteenth century despite reliable reports of envenomations. Even in 1883, the English naturalist Arthur Nicols scoffed at the idea that the platypus was venomous, condescendingly dismissing those wary of the animals: “On seeing me handle my specimen with perfect indifference to the supposed weapon, the black fellow expressed very decided apprehension, and pointed to the spur with gestures of alarm. Here, then, was another example of the ignorance of practical natural history among the Australian natives.” The platypus was considered remarkable for its placement as an evolutionary bridge between mammals and reptiles—a mammal that lays eggs!—not because of its potent venom. Scientists were far more focused on its reproduction than its toxins. But, as the nineteenth century came to a close, a growing group of scientists would take interest in venoms, spurring advances in technology that became the foundation of modern studies. The science of venoms was about to take off, just in time to end the debate about the venomousness of the platypus.
The early studies of venoms were largely generated by professionals interested in the clinical implications of dangerous animals. The medical literature is littered with experiments to determine potency, physiological responses, and effectiveness of treatments. These scientists developed and mastered tests for specific activities of different venoms—what we now call functional assays or bioassays. This meant that for the first time, scientists could reliably study the various effects of venoms, often referred to as “activities,” such as whether a venom kills cells or stimulates muscle contraction. By comparing the results of such assays with studies in living organisms, researchers were able to gain a better understanding of which venoms attack which systems, and thus develop emergency treatments. They could also begin to compare similar venoms from different species. For example, scientists could determine how effectively the venom from one snake kills red blood cells (a common activity of necrotic venoms) compared to another species in the same genus, allowing them to better understand why certain animals are more dangerous than others.
Scientists had also discovered the secret to treating the worst bites and stings; by 1896, Albert Calmette (a protégé of Louis Pasteur) had created the first antivenom. He was in what is now Vietnam when a flood forced monocled cobras into the village he was staying in, and the sudden increase in bites prompted him to search for a way to treat the deadly envenomations. His solution was to inject a horse with cobra venom, then use its blood serum to treat the bitten, creating the first antivenom. Antivenoms take advantage of the adaptive immune system to create targeted antibodies that bind venom toxins, rendering them harmless. Antivenoms save countless lives every single year, but there is still much room for improvement. Modern scientists are on the hunt for a universal antivenom to streamline treatments and ensure that inaccurate identification of venomous perpetrators doesn’t lead to improper dosing. How successful they will be remains unclear.
So while venom science had changed much over the nineteenth century, beliefs about the platypus were still stuck in the past, until the question of the purpose of the platypus’s spurs resurfaced in the late 1890s. In 1894, The British Medical Journal dared to challenge the conventional view that had prevailed since the 1830s, asking “Is the platypus venomous?” as reliable reports of stings were starting to accumulate. In 1895, the first experiment on a live animal was finally conducted—this one on rabbits—injecting platypus venom milked from the spurs to see how it would act. The results of that test were clear: there is a “remarkable analogy between the venom of Australian snakes and the poison of the Platypus.” Chemical analysis of the venom showed that it contained enzymes (known as proteases) that could cut proteins, and the study also explained why accounts of envenomation were inconsistent: toxicity was seasonal, with the most toxic venoms produced during the reproductive season, solidifying the argument that the venom was used by males in combat over access to females.
In 1935, the venom scientists Charles Kellaway and D. H. Le Messurier stated unequivocally that the venom was similar to “a very feebly toxic viperine venom.” Exactly what was in that venom, though, went unstudied for another thirty years. That’s because starting around the 1930s, the study of venoms began to shift away from the almost purely clinical work of early researchers, which had sought to connect venom doses to envenomations in people and how to treat them. While there are still many scientists who investigate venoms and antivenoms from a medical standpoint, a new wave of researchers emerged during the 1940s and ’50s who studied venoms by conducting basic research into their molecular mechanisms of action. Technological advances also have allowed researchers to begin to understand the evolution of venoms and their components, which has led to novel insights into their pharmaceutical potential.
One of the problems with studying venoms until very recently was that we didn’t have very good ways of teasing apart what was present in the crude substances milked from animals. Chemists had nearly mastered the art of separating vastly different types of compounds, such as lipids and proteins, but such methods didn’t finely separate venom components. It was like sorting laundry: they could pull shirts from socks, but couldn’t separate based on fabric color, or distinguish long sleeves from short ones. Some venoms have hundreds of different peptides (small proteins), all of which might be soluble in water, for example. That means that an “aqueous fraction,” or subsection of a venom separated using water, might contain hundreds of different venom compounds, making it impossible to determine if one or many are responsible for any activity seen when that fraction is injected into a mouse.
Luckily, back in the early twentieth century, the Russian scientist Mikhail Tsvet invented a method to separate pigments from plants that came to be known as chromatography, which, with many later variations and refinements, has helped current scientists to isolate and identify venom components. In chromatography, mixtures are dissolved in a fluid (referred to as the mobile phase) which is then passed through a structure (the stationary phase) with certain properties. This structure can be simply a column of material through which the solution passes, drawn by gravity; or it can have specific chemical properties that make it “sticky” to particular types of molecules. When the mixture is run through the stationary phase, even small variations in the compounds’ size, 3-D structure, or chemical properties cause molecules to travel at different speeds, allowing scientists to separate venom components on a much finer scale.
Throughout the 1940s and ’50s, new kinds of chromatography were invented, and what is now known as high performance liquid chromatography (HPLC) entered the scene. HPLC, which uses high air pressure instead of gravity to move the solution through a finer-textured column, is now one of the most important techniques in the study of venoms, as it allows scientists to separate venom samples into individual components. And conveniently, during the mid-twentieth century, scientists also invented gel electrophoresis for separating molecules of protein, DNA, or RNA. Gel electrophoresis uses an electric field to pull compounds through a gel by attracting negatively charged molecules to one end, while the gel’s properties affect which things will move through it more easily, traveling farther in a given amount of time. You can imagine how much faster a needle can be pushed into molasses than a finger can, for example, if they were both pressed with the same amount of force. When it comes to proteins, electrophoresis is mostly used to separate based on size, giving scientists a rough idea of the number of different proteins present in a venom. It also has become an invaluable method for determining whether genetic extractions or amplifications were successful, and is an absolute requirement in just about every lab that studies venom today.
The modern era of venom research followed on the heels of these two major advances in separation technology. By the 1970s, labs worldwide could examine different components of a venom and their individual activities rather than the crude venom as a whole, and they began separating out the ones responsible for the most noticeable venom actions. Captopril—one of the best-selling drugs of all time, used to treat high blood pressure and heart failure—was isolated (from the venom of a Brazilian pit viper, Bothrops jararaca) during this time, as were many other venom compounds.
As a part of his Ph.D. thesis published in 1973, Peter Temple-Smith took advantage of the new battery of techniques to determine the contents and activities in platypus venom. He found at least ten different proteins through electrophoresis and chromatography, and isolated the components that were lethal in mice from ones that caused convulsions. However, the scope of his research was limited, as the separation methods and bioassays still required relatively large amounts of venom (Temple-Smith couldn’t complete lethality tests, for example, because he didn’t have enough venom to work with). Snakes are easy, as they can be milked repeatedly and produce milliliters, and even liters, of venom fairly readily, but many of the other groups of venomous animals provide only 1/1000th or less of the volume required to run such tests. Though the platypus is capable of delivering upwards of 4 milliliters of venom with each spur, actually getting that much raw material is exceptionally difficult. On average, Temple-Smith and others found they could extract only 100 microliters at a time—too little, in those days, for detailed analyses.
But soon enough, tests miniaturized, and better technologies emerged to determine the shape and structure of different molecules, removing the large-volume requirements that had hindered progress. Scientists who made advances in mass spectrometry (MS) and nuclear magnetic resonance (NMR) won Nobel Prizes in chemistry, and for the first time, these advances allowed them to deduce the chemical composition of larger, more complex compounds like those found in venoms. Even small volumes of crude venom could be evaluated to find compounds that are responsible for key activities such as reducing blood pressure, shutting down nerve impulses, or destroying red blood cells.
In the 1990s, several studies picked up where Temple-Smith left off. Scientists taking a closer look at platypus venom isolated active peptides, two different proteases, and a hyaluronidase (enzymes also referred to as venom “spreading factors” because they cut hyaluronic acid, a major component of skin and the connective “goo” between cells). They could even obtain short sequences from some of these components, and determine that they are similar to snake venom constituents.
Then a new technology completely changed the way in which scientists study venomous animals and their toxins: genomics. Watson, Crick, and Franklin had deduced the structure of DNA in 1953, and thirty years later, scientists invented a method for amplifying fragments of DNA based on their sequence. Polymerase chain reaction (PCR) formed the basis for the first sequencing technology, Sanger sequencing, which is still in use today. The first full gene was sequenced in 1989, and the first full non-viral genome (a bacterium) in 1995. In the twenty or so years since, genetics and genomics have proved to be among the most rapidly changing fields in science. High-throughput technologies can now sequence entire genomes in a matter of hours, and new methods are regularly introduced that produce more information in less time for a lower price. It took years and cost millions to sequence the first human genome, which was finished in 2003—and it’s possible that within the next five to ten years, sequencing an entire human genome will cost less than $1,000.
When it came to studying venoms, the genetics revolution opened up avenues that had never been imagined. Scientists could use genes to look at evolutionary relationships, and determine which species were closely related. They could compare the sequences of toxins to other proteins, and begin to understand how venoms evolve. And it wasn’t just DNA—scientists have developed methods of sequencing ribonucleic acid (RNA), the step in between DNA and proteins, and can determine which genes are being expressed. Genomics meant that they could sequence every protein expressed in a venom gland to look at the composition of a venom even without a single drop of it. Drug companies can build libraries of venom toxins and search them for ones that might act as enzymes, or have the potential to interact with a “target” such as an ion channel (I discuss these later). By combining venom separation and component isolation with genomics, researchers have shifted from the study of venoms to venomics. Through such integrated research, we have come to know venomous animals far more intimately than at any point in history, and we have learned that their biochemical prowess is far more impressive than we ever imagined.
Without genomics, we wouldn’t be able to compare the dozens of different venom components, and acknowledge how strange it is that a mammal’s venom contains toxins that look like those of stonefish, snakes, sea stars, and spiders. We wouldn’t know just how bizarre the platypus really is.
Scientists are excited about the potential applications of this burst of platy-gene discovery. “The unusual symptoms of platypus envenomation suggest that platypus venom contains many unique substances which may also be clinically useful,” wrote the Sydney-based venom scientist Camilla Whittington and her colleagues. But the platypus still guards some secrets. No one really knows, for example, what part of the venom is responsible for the excruciating pain that accompanies stings. When we learn that, it will tell us even more about the animals, and it may help us understand ourselves better, too. “It is possible,” according to Whittington et al., “that this could lead to the discovery of new human pain receptors and thus targets for painkillers.”
Back at Lone Pine, after Beck has allowed the shy mammal to return to its home, I stand in front of his aquarium, watching as he swims in search of his shrimpy breakfast. He tumbles and twists, moving through the water with the grace of a fish. When he finds his treasure, he instantly consumes it, his cute little butt waggling side to side with his head as he swallows his meal. The sanctuary won’t open for another fifteen minutes, so for now, I have the area to myself. I try to imagine what it must have been like for those early explorers to encounter this weird furball for the first time. If it were me, I would have been entranced. I wouldn’t have even considered the notion that platypuses could be dangerous—I would have felt compelled to catch one to get a closer look. Even now, knowing what they’re capable of, I feel drawn to the mammal. Only a glass wall keeps me from his serpentine venom. When I meet some of the other infamous venomous animals in the world, there will be no such barrier.
Copyright © 2016 by Christie Wilcox