“You spotted snakes with double tongue”- (William Shakespeare, A Midsummer Night’s Dream)
There comes a time in the life of a young man when he has to make a transition from childhood to adulthood. Most societies celebrate this rite of passage with a special ceremony of some kind. Quite often there is a task that needs to be successfully undertaken. In modern societies this might well be something intellectually or artistically challenging such as chanting from the Torah at a bar mitzvah. But that certainly isn’t the only way of doing it. Early explorers of the Amazonian jungles often reported that the tribes of indigenous peoples they encountered practiced rituals that had a much more physical aspect to them. In such cases the young man in question might have to overcome a confrontation with a fearful enemy; something so ghastly that it would forever leave its mark on him both physically and psychologically. There was one type of ceremony in particular, variants of which were practiced by tribes in both Brazil and Suriname. The same ceremonies are still carried out today. In this case the young man has to battle a monster capable of inflicting the most excruciating pain imaginable. And what kind of monster is this exactly? It has a sleek black skeleton with six articulated legs making it capable of traversing the ground very rapidly. Its armored helmeted head bears giant jaws, making it look like something that HR Giger, who designed the Alien movies, might have dreamed up. Protruding from the back of its thorax is a deadly weapon. This monster is something to be feared and respected. The monster is an ant. The name of the ant is Paraponera Clavata or the “Bullet Ant”. It’s quite a big ant, maybe up to an inch long; but still, it’s an ant; not Godzilla. Nevertheless, to the indigenous population of the Amazon rain forest this ant is one of the most feared of all creatures and to be avoided at all costs. The reason is its sting. To be stung by a Bullet Ant feels like being shot by a bullet. A single sting from a solitary ant can produce pain so intense that the victim is usually completely incapacitated, often just rolling around on the ground screaming. There do not seem to be words capable of adequately describing the experience; victims often attempt the poetic finding normal discourse falls very short of the mark.
The use of the bullet ant in tribal ritual requires a young man to survive not just one, but multiple ant stings and to remain as silent as possible. The procedure is as follows. Members of the tribe search the local jungle for a Bullet Ant nest. They then narcotize the ants using a drug prepared from the bark of a local tree. The ants are taken back to the tribal village where they are inserted into specially woven gloves or mats with their stingers protruding inwards. The young man now inserts his hands into the gloves or has the mat wrapped around him. Soon the ants wake from their slumber. They find themselves trapped and are not happy about it. So, they begin stinging. This is allowed to go on for a specified period of time during which the man is allowed to dance in silence. Eventually the ants are removed, and the tribe celebrate the youngster’s passage into manhood. The pain however lingers for many hours, it is not something that dissipates quickly. The man’s hands have become swollen and inflamed leading to a painful condition that lasts for days. Eventually the pain subsides but the memories echo on forever.
A Bullet Ant
Pain is always something to be overcome. There are many contexts for this. For the Amazonian Indians it may represent a passage to manhood. For a patient in hospital, it may be part of the pathway to recovery. Overcoming pain may even be recreational. Chili peppers are spicy but in excess may be really painful. The eating of such peppers can take on a competitive edge. Who can eat the hottest one?
In the case of the Bullet Ant, we may wonder where exactly the pain comes from? The jaws of the ant, though fearsome to look at, are surely not sufficient to produce such an excruciating and lingering experience. Indeed, the jaws are not the point. It is the stinger at the other end of the ant that is the key. This contraption is capable of injecting the venom of the ant into its victim. But here again we might wonder exactly how large a volume of its venom a single ant might inject? Surely this is a tiny amount? How could so small a quantity be responsible for such a devastating result?
The answer lies in the chemical composition of the venom. We know, of course, that the stings and bites of different animals can be extraordinarily painful. It doesn’t have to be a Bullet Ant, an ordinary bumble bee or wasp will certainly prove the point. This kind of pain does not result from an injury such as a burn or broken limb. It seems reasonable that such a thing would be painful. Rather we are faced with the question of how an extremely small quantity of venom can deliver pain with such intense qualities. There have been serious scientific investigations into the affective nature of different stings. Professor Justin O Schmidt, an eminent entomologist, has written eloquently on the topic. He has devised a scale rating sting related pain from zero to four accompanied by descriptions of the experience involved. In the world of ants, for example, the Delicate Trap Door ant is near the bottom end of the scale and ranks a score of just one, its sting described as “A tiny spark. Just enough to rouse you from a dreamy stroll in the woods A slight jolt as you return to reality”. Bullet ants on the other hand are the top of the scale, ranking four (plus) –“Pure intense brilliant pain. Like walking over flaming charcoal with a 3-inch nail imbedded in your heal”. It’s not only ants though. The Tarantula Hawk wasp also scores a 4. A bullet ant with wings-imagine that! Painful stings are not just produced by insects. You might also want to avoid, the Box Jellyfish, the Duck Billed platypus, the Gila monster, the stingray, the stonefish, diverse kinds of vipers and definitely scorpions. Some plants ranging from the ordinary stinging nettle to the fearsome dendocnide moroides, the Gympie-Gympie plant, are also things you might not want to plant in your garden. But why exactly are these plant and animal venoms painful? Where exactly does the pain come from?
The first thing to understand is that pain is good. I mean that seriously. Pain is essential for avoiding life threatening injury. Pain is a basic physiological necessity. If you put your hand on a hot grill or flame, you immediately feel intense pain and reflexively remove it. If you didn’t then you would suffer tissue damaging burns. If you tread on a nail or trap your finger in the door again the immediate result is pain which causes you to remove your limb immediately. In these cases, once the noxious stimulus is removed, the pain resolves. In other situations, pain is associated with inflammation and may linger, but here again it serves as a warning to avoid the use of infected or damaged tissue. Then there is pathological pain. This is pain that serves no useful function and can, in and of itself, be considered as a disease. Such chronic pain syndromes may follow damage to the nervous system, diseases such as diabetes, infection with a virus such as herpes virus or HIV-1or the use of some drugs like the anticancer medication cisplatinum.
The experience of pain has several components including the rapid reflex that ensures immediate removal a limb from potentially damaging circumstances, to the conscious appreciation of the affective qualities of pain. The rapid reflex is basically mediated by nerves in the spinal cord whereas the affective aspects of the pain experience require the participation of the brain. All of these things need to be coordinated so that they work together, and this is achieved by the integrating properties of the nervous system. Nerve pathways run from the skin or our internal organs into the spinal cord and then up to cerebral cortex of the brain, the highest region of the neuraxis which is responsible for producing our conscious experience. The information that travels along these nerve pathways takes the form of electrical impulses called action potentials. These action potentials trigger the release of chemical neurotransmitters at synaptic contacts between nerve cells. Drugs used for treating pain, including opioid drugs such as morphine, produce their effects by reducing the flow of information along these pain conducting nerve pathways and reducing the release of neurotransmitters. How then can the venom of an insect produce pain?
The secret lies in the molecular mechanisms that underlie the generation of pain. How does a painful stimulus actually engage the nerve cells that initiate the pain response? Somehow the terminals of the nerve fibers that innervate skin or organs must possess molecular mechanisms that can detect painful stimuli and turn these into action potentials that then travel into the central nervous system and indicate pain. Indeed, we know that there are proteins that act as molecular detectors that transduce pain signals into electrical responses. First there are molecules that respond to painfully hot or cold temperatures. These are known as Transient Receptor Potential or TRP channels. Then there are molecules that respond to acids. These are Acid Sensing Ion Channels or ASICs. Other types of molecules known as PIEZO channels respond to painful mechanical pressures. All of these “receptor” molecules act as ion channels. When they are activated, they allow the flow of positively charged sodium and calcium ions into the nerve cell (or potentially the flow of negatively charged chloride ions out of the cell.) This influx of positive charge produces a depolarization of the nerve membrane potential which acts as a stimulus initiating the firing of action potential by the nerve. These action potentials now carry information into the central nervous system. If an insect sting is painful, it must have a method through which it can access some component of the pain signaling system-and that is exactly what happens.
The venoms of insects, snakes and other animals are not simple. Most of them are mixtures of hundreds, if not thousands, of different chemicals that have evolved to work together to produce a particular effect. There are really two major kinds of effects produced by venoms. The first of these is pain. If you can inject something into a predator that is extremely aversive, they are likely to go away and attempt to eat something else that is a little more forgiving. Another strategy is for the venom to paralyze or kill its victim. If you are a predator and you can paralyze your prey, then you can do what you like with it. Drag it back to your lair and snack on it at your leisure. Paralysis rather than death also has the advantage that the meat will stay fresher. Sometimes a venom that produces paralysis in one species will produce pain in another such as a human.
Venoms that produce paralysis contain substances that block the ability of nerves to regulate the contraction of muscles. On the other hand, venoms that produce pain usually contain substances that interact with one of the ion channels on nerves that are responsible for the initiation of pain. By recruiting these mechanisms, they enhance the firing of action potentials by these nerves and consequently produce a pain response. What is really marvelous is the number of ways which Nature has devised for producing painful venoms. In order to illustrate this point, it is an interesting exercise to select several members from the list of the “World’s Most Painful Stings” and examine exactly how they work.
Let us start out at the very top. The number one most painful sting (at least as far as we know), is indeed the Bullet Ant, Paraponera Clavata. Ants are an incredibly diverse group of animals, including around 13,000 species, the vast majority of which use venoms in a predatory or defensive manner. It’s true that ants aren’t very big, but they make up for their lack of size by their enormous numbers. It has been estimated that approximately 20% of the biomass of tropical rainforests consists of ants –and that’s a lot of ants. Many ants act as solitary hunters straying from their nests and feeding on things like earthworms or other insects like spiders, centipedes, and millipedes. On the other hand, some species of ants are highly social and form well organized armies of predators. Some ants, such as the Bullet Ant, are mainly vegetarians, but need meat in order to feed their developing offspring.
Considering the number of ants in existence and the complexity of all their venoms, we know very little about what they consist of and how they work. The venom of the Bullet Ant is one of the more thoroughly investigated at this point. Although it contains many components, it is believed that the major pain producing molecule is called Poneratoxin. Poneratoxin (PTx) is what is known as a peptide, a relatively short string of amino acids linked together in a specific sequence. In the case of PTx, there are 25 amino acids in the sequence. PTx is rather unique, and its sequence bears little resemblance to other peptides that have been discovered even in the venoms of other ants. The question is why does PTx produce such painful effects?
The nerves that initially detect painful stimuli are mostly located in the peripheral nervous system in groups known as dorsal root or trigeminal ganglia. Generally speaking, nerves that are specialized for transmitting noxious pain signals from the periphery into the central nervous system are known as nociceptors. We know that the information which nerves carry consists of action potentials; action potentials are basically formed by ions flowing in and out of nerve cells through protein channels selective for sodium ions and potassium ions. The initial excitatory phase of the action potential is due to the opening of sodium channels allowing sodium ions to pour into the nerve cell. To this knowledge we can add another interesting fact. Although the opening of sodium channels is responsible for triggering the firing of an action potential, the exact type of sodium channel in a particular type of nerve cell may differ slightly one from another. Sodium channels make up a small family of molecules that are all very similar in their structure and function but exhibit some subtle differences. One result of this is that drugs that affect one type of sodium channel may not affect another. One major type of sodium channel that exists in nociceptors is called Nav 1.7 and it has some unique properties.
One way we can assess the role of this sodium channel in human physiology is to observe the effects of mutations that sometimes appear in the human population. These are very rare and may have devastating effects. Typically, people who carry mutations that cause the Nav1.7 channel not to work properly, present with a history of not experiencing any form of pain anywhere on their bodies. Truly, they are incapable of feeling pain, even after burns, bone fractures or severe injuries to their lips and tongues. They also do not experience visceral pain, that is pain derived from internal organs. These rare individuals cannot protect themselves from things that cause serious injuries to normal people. In countries like India, they have been found working in carnivals in isolated villages where they would deliberately injure themselves with knives and exhibit no adverse effects. Such observations clearly demonstrate that this sodium channel plays a vital role in the processing of pain information by the nervous system, presumably because nociceptors are not able to generate action potentials under these circumstances. On the other hand, there are people who harbor gene mutations which make the Nav1.7 channel function more effectively than normal. In this case the mutation doesn’t allow the channel to go through its normal course of activation and inactivation. Rather the channel tends to stay open rather than inactivate during the normal course of an action potential. The result of this is that the nerve is excited for long periods of time and so fires an abnormal number of action potentials, even under nonthreatening circumstances. These numerous action potentials are interpreted as pain responses even though the individual is not really responding to anything dangerous in the environment. The result of this is that patients carrying these mutations exhibit a variety of disorders where they are constantly in pain. This is the case for patients with “primary erythromelalgia” or “paroxysmal extreme pain disorder”. Such patients have constant pain accompanied by extreme redness and swelling of their limbs. Some relief can be obtained by keeping their hands and feet in buckets of ice water, but it is a very miserable situation. Nevertheless, these naturally occurring human genetic “experiments” clearly indicate that changes to the normal activity of sodium channels found in nociceptors can radically alter our pain responses.
Several studies have now revealed that this is exactly how PTx works. If you measure the properties of sodium channels in nociceptors (Nav 1.7 in this instance) and add some PTx two powerful effects are rapidly observed. One of these is that it becomes much easier to activate the sodium channels. Normally you have to give cell a bit of a push represented by an electrical “depolarization” of the membrane potential to persuade it to fire an action potential. This is what painful stimuli normally do in order to trigger a pain response. But after adding PTx this becomes unnecessary, and the nerve cell just begins to fire action potentials spontaneously. Then there is a second effect. Normally after a sodium channel opens and participates in an action potential it closes again and is inactive for a while before it can be called upon again. But in the presence of PTx, sodium channels don’t close again after they have opened, and they do not spend any time in their normally inactive state. The result of these two effects –opening very easily and not closing again, means that the nerve cell is prompted to fire a huge number of action potentials signaling intense pain. When the stinger of the bullet ant enters the skin a small amount of PTx is rapidly introduced. Many pain nerves innervate the skin, and they will be exposed to this small amount of the toxin. But don’t forget, although the amount of toxin in small, its effects are extremely potent and long lasting. Moreover, when pain nerves in the skin are excited in this way, they also release numerous other chemical factors that produce an inflammatory response which also adds to the painful experience and ensures that it lasts for a long time. Hence, in addition to feeling intense pain, the victim’s limbs will become swollen and inflamed.
The effects of Bullet Ant venom raise the question as to whether the painful stings of other animals are produced by using the same strategy, that is by increasing the sodium channel activity in pain nerves. The answer is that it is a very widely used strategy indeed. Several kinds of animals including some spiders, wasps, snakes, scorpions, cone snails and sea anemones have venoms that contain peptide toxins that produce similar kinds of effects as PTx. Some of these animals are famous stingers. Consider scorpions, for example, who generally speaking have a reputation for having some of the nastiest stings known to man. Even among scorpions however, the Arizona Bark Scorpion (Cenruroides Sculpturatus) is notorious for its particularly painful sting. This type of scorpion lives in the Sonoran Desert in the southwest USA, a very dry and unhospitable environment.
Arizona Bark Scorpion
Not too many creatures live there but there is a type of mouse called a Grasshopper mouse. Dining options for the mouse are not too extensive but it will eat Arizona Bark scorpions. This is a very unusual behavior. If you put any other kind of mouse, or for that matter any creature, together with an Arizona Bark scorpion, it will run a mile in the opposite direction, which seems like reasonable behavior. Injection of a tiny amount of Arizona Bark scorpion venom into the paw of a normal mouse causes to exhibit extreme discomfort and intense licking behavior of the paw. This is more or less what happens if a human sits or treads on one. However, if you put a Grasshopper mouse in a box with some Arizona Bark scorpions it will just gobble them up like candy. How is such a thing possible? Even if you take some of the painful Arizona Bark scorpion venom and inject it into a Grasshopper mouse paw it will just shrug it off. Apparently, it does not produce pain. If you put the venom on pain nerves from a normal mouse, they fire action potentials like crazy because it has effects on sodium channels similar to those described for PTx from the Bullet ant. If you do the same thing to the Grasshopper mouse, there is no response at all. Scientists have figured out what is going on. They isolated the sodium channels from the pain neurons of normal and Grasshopper mice. These are large proteins consisting of many amino acids linked together in a very long chain. It is possible to figure out exactly where in this sequence the scorpion toxin interacts with the sodium channel in order to alter its properties. However, when you do this with the channel protein from the Grasshopper mouse you find that a few amino acids are different. This change in sequence results in a sodium channel which cannot interact with the toxin any longer and is therefore not affected by it. It’s an amazing result of evolution. The mouse that lives in proximity to the scorpion has evolved to become resistant to it and so it can act as a predator, and it has achieved this by changing the properties of the sodium channel that is normally the target of the scorpion’s venom. This type of toxicological arms race is often observed in nature, where species become resistant to the venoms of other animals resulting in the development of novel venomous strategies, further resistance and so on.
We can quickly survey some of the other types of animals whose venoms contain sodium channel activating toxins. Spiders can give a pretty painful sting. As we shall see there are a variety of ways that spiders can inflict pain. They certainly don’t all do it the same way, but some of them use venoms that affect sodium channels. One real monster is The Brazilian “Armed” spider Phoneutria nigriventer.
Brazilian Armed Spider
This is an extremely aggressive spider which hunts and eats a vast variety of animals including many species of insects, other spiders, and even small rodents. Phoneutria nigriventer does not construct webs and its success as a predator is in part explained by the diversity of potent toxins present in its venom, including sodium channel activating toxins.
You are also not safe if you decide to leave land and go for a swim in the sea. There are plenty of painful creatures waiting for you there and some of them use toxins that activate sodium channels. The best studied of these are Cone snails, a carnivorous type of snail found in tropical waters. Treading on a sea anemone can also be painful because they also produce venoms that contain sodium channel toxins. Of course, being bitten by a snake must also rank as a very nasty experience and snake venoms are certainly chock full of interesting toxins, although they don’t usually produce pain by activating sodium channels. One exception to this rule however is the gorgeous blue coral snake Calliophis bivirgata which is native to Malaysia and other parts of Southeast Asia. This snake, sometimes known as “the snake with a scorpions bite” has a very potent venom that can sometimes be fatal to humans and an extremely long venom gland that can extend to about a quarter of the length of its body. One component of the venom is d-calliotoxin which has been shown to be a strong sodium channel activator. So, activating sodium channels in nociceptors is certainly a widely used method for producing a painful sting. It’s an interesting example of convergent evolution.
Blue Coral snake
But it’s not the only method through which animal venoms can produce intense pain. As we have discussed, the Bullet Ant is generally accepted as being the animal with the most painful sting. It’s top of the list. What then is second on the list? The answer is the Tarantula Hawk wasp (Hemipepsis ustulata) commonly found in the southeast of the United States. (In fact, it was voted in as the “state insect” for New Mexico.) Whereas the discovery that the most painful animal in the world is an ant might be somewhat surprising, one might not be at all surprised to find out that the Tarantula Hawk wasp was close to the top of the list. It’s consistent with our everyday experience. We all know that bees and wasps have painful stings and this one is an absolute whopper. A huge menacing looking wasp about the size of a human hand that lives a solitary life killing tarantulas. The Tarantula Hawk normally eats plants such as milkweed. The tarantula killing behavior only occurs when it needs to feed its young. A Tarantula Hawk will first find a tarantula hiding in its burrow. The wasp will then partly enter the burrow and evict the spider. Then they fight. When the spider raises its legs to take on an aggressive posture, the wasp quickly stings it in its soft underbelly exactly at the position of its major nerve plexus, thus paralyzing it-think Sam Gamgee killing Shelob. Now the wasp drags the comatose spider back to its lair and lays an egg on it and then covers the hole up. When the egg hatches the wasp larva burrows into the spider, who is still alive at this point, and so its meat is fresh. Gradually the spider is consumed and finally the wasp forms a pupa and eventually matures into an adult which exits the lair leaving the remains of the spider behind. It isn’t clear whether the venomous components responsible for spider paralysis are the same as those that cause pain when stinging an animal such as a human, after all the goals of these two things are different-food and deterrence. Spider wasps are members of the Pompilidae family, and some members of this family make toxins, known as pompilidotoxins that activate sodium channels and so may be partly responsible for their painful sting. However, as we shall see wasp venoms contain other toxins as well that may be primarily responsible for the pain in this case.
Tarantula Hawk wasp
Clearly, then, a venomous animal can induce pain by activating pain nerves and making them fire action potentials; many animal toxins can target the sodium channels that participate in action potentials directly. But there are other targets as well. Anything that produces a depolarization of the nociceptor will provide a kick that will cause the nerve to fire an action potential and so we should think about whether there are any other ways this can be achieved. As we discussed above pain nerves express a variety of molecules whose raison d’etre is to detect potentially damaging stimuli and turn these into a pain warning by inducing pain nerves to fire action potentials. These would include things like TRP (Transient Receptor Potential) and ASIC (Acid Sensing Ion Channel) channels that normally detect damaging damaging temperatures or acids. The effect of activating TRPV1 channels that are found in many pain nerves will be well known to countless people because it is this channel that is activated by hot peppers. These peppers contain a substance called capsaicin which can bind to TRPV1 channels and activate them. Because the channel is normally activated by hot temperatures, its activation by capsaicin fools us into thinking that is what has happened. This is quite acceptable if we restrict ourselves to eating spicy food. But get speck of habanera extract on a cut or in your eye by mistake and the pain will be excruciating.
The TRPV1 channel would obviously make an excellent target for a venomous animal wanting to inflict pain and there is ever increasing evidence that this is the case. One thing that has been well known since antiquity is that it is very painful to be bitten by a “tarantula”, a name that applies to a number of types of spiders that exist around the Mediterranean and other parts of the world. This is particularly the case if you happen to sit on one. The victim of a tarantula bite is likely to get up and jump around as if performing a mad dance in order to find relief. The ever-creative Italians based a dance form on these movements. It’s called the Tarantella-basically a dance that is meant to look like you have been bitten by a tarantula. It’s a very lively dance indeed not only in its original folk form but as appropriated by many classical composers. Scientists have now shown that several types of tarantulas produce venoms that contain toxins that are activators of TRPV1 channels. Interestingly, these venoms are not known to be fatal to humans, just to cause intense pain. For example, three similar toxins have been identified from the venom of the Trinidad Chevron tarantula Psalmopoeus cambridgei, a spider from the West Indies whose sting produces intense pain through activation of TRPV1 channels. Tarantulas from other countries such as Ornithoctonus huwena, a Chinese tarantula, produce similar toxins that share certain key structural features and also activate TRPV1. The fact that these toxins work this way is interesting but also useful because scientists can use them as specific probes for finding out exactly how TRPV1 works at a molecular level.
Another type of receptors found in pain neurons are the Acid Sensing Ion Channels (ASICs) which produce pain in response to an acidic environment. These would also seem be good targets for pain producing toxins. Again, nature has obliged. Some snakes, such as Coral snakes, produce very painful bites. Coral snakes are a large group of snakes that fall into two categories , “Old” and “New” world snakes. We have already met the Blue Coral snake that is found in Southeast Asia. The Old-world Texas coral snake (Micrurus tener tener) produces a toxin called MitTx which produces pain by activating ASIC channels in pain neurons. Interestingly, the way the toxin works is to activate ASIC channels unrelentingly as opposed to acid that only produces its effect transiently. This means that the pain from the bite of the snake just keeps on going. It’s as if you were being constantly burned by concentrated acid, an excellent strategy if you just want to be as painful as possible
It will be clear then that some venomous animals can produce pain by directly activating TRPV1 or ASIC channels. However, there is also a second possible mechanism through which venoms can activate these channels indirectly. The fact is that pain nerves are covered with numerous types of receptors that mediate the effects of many different substances that act on these nerves. Not all of these receptors are ion channels like the sodium channels, TRPV1 or ASICs which can directly alter the permeability of the nerve cell membrane to ions and so directly affect the membrane potential. Many of these other receptors are G protein coupled receptors (GPCRs), similar to the muscarinic acetylcholine receptors, the site of action for the effects of Belladonna related drugs. When activated these receptors can produce a very large number of effects. Many of these involve the initiation of biochemical pathways within the target cell. Some of these biochemical reactions can modify the properties of other molecules like TRPV1 or ASIC channels expressed in exactly the same cell, so that now these channels become much easier to activate. As these channels mediate pain, the result is that activation of a GPCR in this case will also produce pain through the ultimate activation of TRPV1 or ASICs. Many of the substances normally associated with the phenomenon of inflammation act in this way and that is one reason why inflammation is painful. It therefore isn’t surprising that some venomous animals have developed a strategy by which they have evolved painful toxins that act upon the same pain producing pathways as inflammatory mediators. Of particular importance in this regard are a group of peptide mediators known as kinins. Bradykinin is the archetypal member of this family found in humans. Kinins are produced in response to different types of tissue injury and act upon on variety of tissues including the smooth muscles of blood vessels, the kidneys and the sensory nerves that transmit pain, thereby coordinating the overall response of the body to tissue injury. The site of action of bradykinin in all these tissues is a GPCR known reasonably enough as the bradykinin receptor. When this receptor is activated on pain neurons, it produces the release of biochemical mediators that sensitize the activity of TRPV1 and so produce a pain response. As it turns out bradykinin like molecules are widely distributed in the venoms of many animals that have painful stings. Wasps are a good example of this phenomenon. Wasps are members of the Hymenoptera ,the third-largest order of insects, which also includes ants and bees. Over 150,000 living species of Hymenoptera have been described and it appears likely that the venoms of many of these contain kinins. We have already encountered the Tarantula Hawk Wasp as an example of an animal with an uber-painful sting and many other varieties of wasps have stings that are also fairly high up the pain scale. Kinins are likely to be major contributors to the majority of these. This strategy is not restricted to the stings of insects. For example, the Black Mamba (Dendroaspis polylepis) is considered to be the most dangerous of all African snakes. Black Mambas have an average length of about 3 meters and can move along at quite a rapid rate of up to 7km/hr. When confronted with an animal that they consider threatening they will rear up, spread their hoods somewhat like a cobra and make a horrible hissing noise. Unfortunately, they can also leap into the air allowing them to bite their victims on the upper parts of their bodies. If all of this isn’t bad enough their venom is extremely toxic and can lead to death within an hour or so if the victim is not treated properly. Apparently, it is also horribly painful. Black Mamba venom, as well as the venom of the related Green Mamba, is chock full of nasty toxins. Among these is something called Mamba Intestinal Toxin 1, a long peptide consisting of 81 amino acids. Interestingly, a peptide with a similar sequence has also been isolated from the Yellow-Bellied frog (Bombina Variegata). It was observed that these toxins were able to induce pain in conventional tests where they are injected into the paw of a mouse or under the skin of a human and this eventually led to the identification of an entirely new receptor system in animals. These receptors, which are expressed in pain nerves as well as other parts of the body, are known as prokineticin receptors and the mammalian proteins that normally activate them are known as prokineticins. There are two prokineticin receptors and they are both GPCRs. These proteins and receptors seem to have a normal role in pain physiology as well as other important physiological processes such as control of the cardiovascular system. MIT1 is a very good activator of prokinecitin receptors and so, like the kinins, can indirectly activate TRPV1 like channels and therefore produce pain.
It is clear then that the venoms of many animals contain a witches’ brew of toxins designed to produce phenomena such as pain and paralysis in their victims. Common targets for these toxins are receptors that exist in nociceptors including sodium channels, TRP channels and ASIC channels, as well as GPCRs which may activate these targets indirectly. It is an interesting exercise to consider some of the world’s most famously painful animals and seeing how they stack up in this regard. High up in most lists is a truly nightmarish creature, the Box jellyfish (Chironex fleckeri).
Box Jellyfish
This jolly little fellow has tentacles that are up to 10 feet long that are covered with stinging organs known as nematocysts. Upon contact with its victim the nematocyst discharges explosively, expelling the organ at high speed and releasing its contents into its victim’s the blood stream.
A “Boxie”, as they are affectionately known “down under”, can wrap its tentacles around its unfortunate pray delivering hundreds of simultaneous stings. The result is not only horribly painful but can also lead to cardiac arrest and death within a few minutes if not treated properly. The other thing to note is that Boxies are very intelligent (at least by jellyfish standards) and can swim rapidly through the waters hunting down their pray in a purposeful manner. Like other venomous animals the composition of box jellyfish venom is complex, containing proteins that can activate pain nerves, lyse cells, kill cells or produce inflammation. Recently, a venom component was identified that could activate TRPV1 possibly explaining the intense pain associated with a Boxie sting. Although it seems reasonable that being wrapped in the arms of a giant jellyfish should elicit such a painful response, it is interesting to note that there are jellyfish that are much worse than the Boxie .Indeed, the absolute worst jellyfish experience you can have results from the sting of the tiny Irukandji jellyfish that is only about 1cm long .You probably wouldn’t even notice it and its initial sting is just a tiny pinprick. However, within minutes the effects of its venom kicks in resulting in extensive pain, vomiting, headache, anxiety, cramping, and — most distinctively — a state that scientists have described as “a feeling of impending doom.” A course of intravenous opioids plus benzodiazepines is used to treat victims of Irukandji syndrome and, if you are lucky, may take the edge off things a bit. Yet like many venoms we still don’t really know how it works but presume that it must represent a combination of the effects of a complex brew of different venom constituents.
Looking down the list of animals with the most painful bites there are a few other creatures that deserve at least a brief mention. Curiously, one of the animals that regularly features in lists of this sort is Ornithorhynchus anatinus, the Duck Billed Platypus, an animal that has excited human curiosity ever since it was first discovered.
Duck Billed Platypus
18th and 19th century zoologists really didn’t know what to make of it . Was it a mammal, a reptile or perhaps something else? In 1800 the governor of New South Wales, John Hunter, considered this ‘‘amphibious animal’’ a tripartite creation merging the characters of fishes, birds, and quadrupeds. But if it was a mammal, why did it lay eggs? Moreover, as humans began to study it intensively it also became apparent that the platypus was venomous. Unlike a reptile however platypus venom was released from a hollow spur situated on the back of its claws. Only the male platypus is venomous and the degree to which this occurs seems to be related to several different factors such as the time of the year, whether the animal is involved in mating and so on. Observations of platypus behavior in the wild suggest that the venom may be used by males when they fight over females during the mating season. There are also reports of humans being stabbed by a platypus spur and experiencing immediate excruciating pain followed by long lasting inflammation and swelling of the affected limb. Nowadays the rapid advances in molecular genetics have allowed the characterization of the complete “venome”, that is the complete genetic makeup of the venom of the platypus and many other venomous animals. Scientists carrying out this exercise with the platypus identified 83 novel putative platypus venom genes from 13 toxin families, which are homologous to known toxins from a wide range of vertebrates (fish, reptiles, insectivores) and invertebrates (spiders, sea anemones, starfish). So, clearly, as with other animals, the overall effects of the venom are due to a combination of multiple different factors. Interestingly, one protein found in the platypus venom exhibits a homology with some of the sodium channel toxins found in other venomous animals but fails to show any effects on sodium channels indicating that it has now evolved to carry out distinct and, as yet, unknown, functions. Another protein associated with the venom is nerve growth factor (NGF). As can be seen from its name this molecule was originally discovered to have an important role in the growth and development of the nervous system. This particular function involves activating a program of gene transcription in nerves that regulates their development and survival. Such effects take many hours to become apparent. A more recent observation, however, has been the role the NGF protein has in the physiology of pain. Receptors for NGF are localized on pain neurons and, like GPCRs, they are linked to TRPV1. So, one of the first things that happens when NGF activates its receptor is that it triggers TRPV1 mediated pain. NGF induced pain can be quite intense and so it is a good candidate for the agent from platypus venom that produces immediate pain In fact, it is now thought that NGF is a pain mediator in several human diseases such as osteoarthritis (OA) Antibodies against NGF have proved to be highly effective in treating pain associated with OA and have been considered a potential therapeutic breakthrough for a disease which causes unrelenting pain in millions of patients throughout the world. Initial efforts to test this hypothesis in human OA patients confirmed that the antibodies could greatly ameliorate pain but, unfortunately, produced important side effects that limited their ultimate use as therapeutics.
Another exotic creature from the “Most Painful List” with a really painful bite is the Gila monster, clearly the inspiration of its fictitious alter ego Godzilla. Gila monsters inhabit the Southwestern deserts of the US and a very closely related lizard, the Mexican Beaded lizard, is found to the south in Central America. Gila monsters are quite large, ranging up to 2 feet long and weighing up to six pounds. They generally spend most of their time in underground burrows and don’t move around very quickly. They are not known to be very aggressive and only bite humans in extremis, if you try to pick one up or threaten it in some way. If it does bite you, the results are extremely unpleasant. For one thing rather than a quick strike such as a snake would make, the Gila monster is likely to remain clamped to your hand or other limb with its teeth deeply inserted into it for up to 15 minutes during which time it is almost impossible to remove. While attached to your limb the creature’s venom passes through special grooves in its teeth into your bloodstream where its many components can flow around your body wreaking havoc. Here is a brief description of the result of being bitten by a Guatemalan Beaded Lizard (Heloderma horridum charlesbogerti),a close relative of the Gila monster. “After disengaging the lizard, the patient experienced severe local pain, dizziness and diaphoresis. Approximately one minute later the patient experienced paresthesia in his left hand and arm, and it became difficult to move the fingers of the bitten hand. Around 3 minutes later, it became difficult for the patient to move the fingers of the right hand, and he experienced paralysis of the left hand. There was considerable edema of the left hand, and pain extended from the tip of fingers to the shoulder. Swelling of the mid-left tongue made speech difficult. At this time the patient experienced shortness of breath and was transported to a hospital”. One can easily see how the combination of different venom components spreads around the victim’s bloodstream recruiting a variety of symptoms. Exactly what is responsible for the “severe local pain” isn’t clear. However, the best bet is that it is due to kallkrein, an enzyme that can release bradykinin and similar peptides that produce pain through the indirect activation of TRPV1, as described above. Moreover, kinins also have potent effects on the vascular system which might help to explain some of the other symptoms observed.
Let us now return briefly to the oceans which, aside from the jellyfish, harbor a huge number of painfully venomous creatures. Some of these are fish. One of the most commonly encountered of these, and indeed one of the most toxic of all marine animals, is the stonefish. There are several types of stonefish in the family of fish known as the Synanceiidae .The stonefish is a rather an ugly, grumpy looking fish that is found in shallow tropical waters. It is a master of disguise and that makes it very hard to detect, as it camouflages itself very expertly blending in with coral reefs or the sandy sea bottom. Generally speaking, you might just think it is a piece of gravel and so you might easily step on it by mistake. If you do step on it however you will become aware of its presence very rapidly. The fish protects itself using 13 razor sharp venom filled spines capable of slicing through your shoes and injecting its venom deep into your foot. The resulting pain is crippling, can last for days and may result in amputation of a limb or death. It is not that unusual for a human to die following stonefish venom poisoning. A recent report featured an Australian man who had this to say about his experience. "I guess I could best describe the pain as holding an oxy-acetylene torch on your foot, and then working its way up your whole leg over an hour or so, then smashing your leg with a sledgehammer every 10 seconds, not to mention the associated nausea, the fever, the hyperventilation, you know trying to breathe. My eyes were rolling in the back of my head, I was clenching a towel with my teeth, just going blue in the face. I really was in a bad way. They got me on a drip and injected me with morphine — five shots of morphine over about an hour until the breathing came under control — and then they administered antibiotics, anti-inflammatories, tetanus shots and the rest. It took about two hours for the pain to come down below 10 out of 10." The victim survived but it took nearly six months before he could walk properly once again without the aid of crutches.
Can you spot the stonefish?
Analysis of the venom of the stonefish Synanceia horrida has revealed a complex mixture of factors as is the case for most other venoms. It isn’t clear which of these components are responsible for the excruciating pain. However, to date the best characterized toxins are stonustoxin and verrucotoxin. These toxins act differently to those we have already discussed. Basically, they act as “perforins’, that is proteins that can perforate or punch holes in cells. As can be imagined punching holes in cells may have all kinds of deleterious consequences. Cells killed by this process will trigger an inflammatory response as observed following stonefish envenomation and that is certainly painful. However, it may well be that these toxins can also punch holes in pain neurons, causing a collapse in their membrane potential and leading to aberrant pain signaling.
As a final example of things with very painful stings, we might look beyond the animal kingdom. Plants, of course, are also capable of delivering stings –the common stinging nettle being something that many readers will have encountered. However, there are plants out there which can deliver far nastier stings than the humble nettle. Perhaps the most notorious of all stinging plants would be the fictitious Triffid. As described by John Wyndham in his classic “The Day of the Triffids”, these mobile plants can kill a man instantly with a lash from their ten-foot-long stingers. Triffids aside, however, one might do well to avoid the Gympie-Gympie plant (Dendrocnide moroides), which is most commonly found in Northeastern Australia and is a good example of the family of Australian “stinging trees”. The Gympie-Gympie can grow to a height of several feet, probably as large as a decent sized Triffid, but unlike the latter they cannot walk around. Nevertheless, their sting, delivered by small hollow spines that cover the stalks and leaves, is reported to be extremely painful. Indeed, the Gympie-Gympie is also known as the ‘suicide plant” which should tell you something. A scientist who has spent a considerable time studying plants like the Gympie-Gympie described it as, “the worst kind of pain you can imagine - like being burnt with hot acid and electrocuted at the same time”. It seems that once the spines have pierced your skin, they are sometimes difficult to remove and can engender a severe allergic reaction which naturally adds to the long-term unpleasantness of the entire experience. Really serious cases have led to the death of animals and occasionally even humans. It is clear that the hypodermic needle like structures that surround the plant inject some type of toxin into its victims. One might imagine that the type of toxins used by a plant would be extremely different from those used by animals. Until very recently the chemical nature of Gympie-Gympie venom was incompletely characterized. It was shown to contain a molecule called moroidin which consists of eight amino acids arranged in a ring like structure. Injection of moroidin under the skin has been reported to be painful and so it was assumed that it contributes to the overall effects of Gympie-Gympie venom. However, very recently the situation has been clarified. Scientists in Australia isolated a 36 amino acid peptide from the Gympie-Gympie which was extremely painful when injected under the skin. It was the real deal! Amazingly, the mechanism of action of what have been called “Gympietides” is very similar to that of painful toxins from the Bullet Ant and other animals that work by activating sodium channels in pain neurons. A fascinating example of convergent evolution. Although the amino acid sequences of gympietide and something like poneratoxin are completely different, their three-dimensional shape is likely to be similar meaning that they can produce the same biological effects when they interact with proteins like sodium channels.
It’s clearly a dangerous world out there. Pain is just about everywhere. Just walking around in the countryside may bring you in contact with any number of pain inducing animals or plants and paddling in the sea at the beach is just as dangerous. But the news is not all bad. In spite of the fact that animal and plant venoms have developed over the course of evolution for purposes such as defense or the provision of food, human ingenuity coupled with modern day sophisticated biochemical and genetic techniques has come up with numerous ways of using these things to our advantage.
There have also been attempts at developing the components of some animal venoms for the treatment of pain, in fact some of these are already in use for this purpose. The best example of this is provided by the venom of an extremely interesting creature known as a Cone snail. Cone snails are marine predatory carnivorous snails, many of which are piscivorous, that is to say they eat fish. If you think about it this seems very odd. After all fish are rather fast swimmers and snails are rather…well they’re snails, enough said. So, how exactly can a snail survive by hunting fish? To understand this, you have to look carefully at how a cone snail is constructed and also watch it in action. It’s certainly one of the most surprising and terrifying things you will ever see. Consider the following. If a snail is going to kill a fish and eat it, it needs a method for doing this that will incapacitate the fish immediately so it can access its prey easily. Making it feel a little woozy won’t do the job because by the time the fish dies it may well have swum a hundred feet away and that is just too far for a snail to journey for its dinner. So, this is what happens. First of all, the snail hides in the mud or gravel at the bottom of the sea. It has an organ that allows it to sense a suitable fish swimming in the vicinity. Within the snail is a venom sack containing one of the most potent venoms known to man. Indeed, the sting of a large Cone snail can easily kill a human. The venom is packaged into small structures derived from teeth-they look like small, barbed harpoons. The snail now extends a long proboscis with a harpoon attached to the end. Once the fish is close the harpoon is fired into its side. It is absolutely worth watching this take place on a YouTube video. As soon as the fish is speared by a harpoon the toxins go to work. Some components of the venom are vasodilators that open up the fish’s blood stream allowing other venom components to reach all parts of the fish without delay. A mixture of potent neurotoxins targets different ion channels and receptors that produce immediate paralysis, and, with scarcely a flap of its fins, the fish just drops to the sea floor. Now the snail comes out of hiding and open its enormous maw into which the paralyzed fish is maneuvered by the snail’s proboscis. Once engulfed, the fish is digested at leisure until after several hours, with an aquatic belch, the residue of bones and scales are released back into the sea. It may come as a surprise to know that one of the components of cone snail venom is now used to treat serious pain. The venoms contain proteins called w-conotoxins. These toxins inhibit the release of neurotransmitters from the terminals of nerves. Normally, when an action potential reaches a nerve terminal the depolarization it produces opens up a type of channel called a calcium channel. This allows positively charged calcium ions to enter the nerve terminal and trigger the release of neurotransmitters. w-conotoxins block these Ca channels which inhibits Ca entry into the nerve and the release of neurotransmitters. In the case of the cone snail this action helps to paralyze the fish by blocking the release of neurotransmitters that are essential if its muscles are to work properly. If the toxin was injected into the blood stream of a human ,then it would be extremely poisonous. But consider this possibility. What if one just applied the toxin to the terminals of pain nerves? If one just blocked the release of neurotransmitter from these nerves in particular, then the result would be inhibition of pain and nothing else. The terminals of pain neurons end up in the spinal cord. Some patients have particularly intractable pain symptoms that are difficult to control even with powerful drugs like morphine. In some of these cases it has been shown that if one takes the w-conotoxin from the cone snail Conus Magnus and carefully injects it into the ventricular space of the spinal cord it acts as a powerful analgesic agent, just as one would predict. This preparation, called ziconotide (Prialt), is now widely used for the treatment of intractable pain. Unlike morphine it isn’t addictive but, of course, it is difficult to administer.
Conus Magnus
Ziconotide is certainly not the only venom component that might be useful for the treatment of pain or indeed for other medical purposes. As we have discussed above one of the major receptors that detects potentially damaging processes by inducing a pain response is the ASIC family of channels which detect acids. A few years ago, scientists discovered that the venom of the Black Mamba contained a group of substances that were capable of blocking these ASIC receptors. These molecules named mambalgins are 57 amino acid long peptides that were found to produce powerful analgesia in a number of pain models in animals. Levels of analgesia that rivalled those of morphine were reported and so naturally there is great interest in pursuing the potential use of these molecules in the clinic. Observations like these further highlight the role of ASIC receptors in pain transduction, as not only do blockers of ASIC channels such as the mambalgins block pain but other molecules, such as toxins derived from the venom of the Texas Coral snake which activate ASIC channels produce intense pain. However, such molecules eventually fare in the clinic, they are extremely useful tools that can be used experimentally by scientists who are interested in studying the physiological basis of pain. Moreover, and of great significance, is the idea that the identification of toxins that can produce or inhibit pain may provide us with ideas about how to develop new kinds of pain killing drugs, something that is considered to be a very important goal in the light of the current opioid epidemic.
And it’s not just in the field of pain that animal venoms are providing clues for the development of new therapeutic agents. Pride of place here goes to agents that act as inhibitors of the Angiotensin Converting Enzyme-the well-known “ACE” inhibitors, which are widely used for the treatment of hypertension. The development of these extremely useful drugs followed the discovery of a nine amino acid peptide in the venom of the Brazilian Pit Viper that acted as a specific ACE inhibitor and enabled researchers to understand the central role of this enzyme in the development of hypertension. Because the original venom peptide was not effective when given orally screening programs were developed which led to the discovery of captopril the first ACE inhibitor drug. In quite another area of medicine, discoveries using the venom of the Gila monster have turned out to be of considerable importance for the treatment of diabetes. In 1992 a protein called exenatide was isolated from Gila monster venom. Exenatide turned out to look a lot like a human hormone called “Glucagon like peptide-1”. The human hormone is potentially useful to diabetics helping them to control their aberrant metabolism and has now been further developed in drugs like Ozempic. The venom protein also proved to have similar effects with the added advantage that they lasted longer. A synthetic version of exenatide is now marketed under the name Byetta for the treatment of diabetes.
Overall, it seems that a drug discovery strategy based on the effects of the components of stings and venoms may be a very fruitful one. Nevertheless, because venoms are certainly “natural products” their use sometimes falls into an area of therapeutics which is somewhat controversial. Take bee venoms for example. Just as all the other venoms discussed above, bee venom is a complex mixture of hundreds of different components. Of course, being stung by a bee is painful; many of us can attest to that. The major component of bee venom is a 26 amino acid peptide called melittin. It is highly likely that melittin is important in the production of bee sting pain. Injection of small amounts of pure melittin, much less than found in a bee sting, subcutaneously in a human produces a strong pain response. There are several ways in which melittin might produce pain. First of all, melittin can probably produce biochemical mediators that can activate TRPV1 channels, which, as we have discussed, is a good way of producing pain. Secondly, mellitin can activate cells of the immune system which causes them to release more factors that can also activate pain neurons. Thirdly, melittin can produce factors that increase the activity of the sodium channel NaV1.7, the same channel that can be activated by the toxin found in bullet ant venom. Put these effects together and it is going to hurt. Moreover, there are also minor components of bee venom that can produce pain. Of course, as we have also seen, mankind has also tried, often successfully, to take Sir Robert Boyle’s advice and “turn poisons into medicines” and this is certainly true of bee stings. For example, a very ancient literature, has attested to the fact that bee venom might actually be “good for you”. Although the initial sting of the bee may well hurt, the longer-term effects may be beneficial. There is a lot of folklore about this attesting to the fact, for example, that beekeepers never get arthritis. In more modern times this line of reasoning has been revived. In 1888 a book entitled ‘‘Report about a Peculiar Connection Between Bee Stings and Rheumatism’’ was published by the Austrian, Philip Terc, followed in 1935 when Dr. Bodog F. Beck published another book on the use of bee venoms in the treatment of rheumatic arthritis. In order to produce such effects patients can either allow themselves to be stung by bees or have the venom injected into them hypodermically. It is said that bee venom can reduce the inflammation that is the cause of arthritic pain. There is some evidence that this is the case but not enough to satisfy the majority of medical professionals working under the umbrella of conventional medicine. For practitioners of alternative medicine, it’s another matter. Moreover, the therapeutic use of bee venom appears to be increasing these days and moving into a variety of other areas including things like the treatment of Parkinson’s disease and Multiple Sclerosis. Part of the reason for this is that holistic medicine is currently very popular and so anything that seems “natural” gets a lot of attention, and also because of the fact that bee venom treatment or “apitherapy” has been talked up by a lot of A-list celebrities like Gwenyth Paltrow. One popular New Age therapy is bee acupuncture. In this approach, rather than applying needles to acupoints, bees are applied instead. The bees normally just sit there. Now you have to annoy the bee so that it applies its stinger to the acupoint and stimulates it, thereby combining the effect of acupuncture with the effects of bee venom injection. This may sound a little like something from Monty Python’s Flying Circus (“bamboozle a bee”), but really it is taken very seriously. And maybe there are some beneficial effects of procedures like this. However, there are at least a couple of problems with it all. The first is that modern medicine has specific standards that need to be met in order to say that some new therapy really works or not. These standards are usually met through a series of clinical trials, and these have generally not been applied to the field of apitherapy. The second thing is that it is quite possible for people to become allergic to components of bee venom or the venoms of other animals. If a person is sensitized in this way, then an injection of venom can result in a degree of anaphylactic shock which can be fatal. Indeed, there has been a recent highly publicized report of a woman in Spain who died in this way during a course of bee therapy. As the clinics that administer these treatments are not normal medical facilities, they may not have the resources to deal with somebody suffering from intense anaphylactic shock. It is unlikely that the public’s infatuation with “natural” products will go away anytime soon and in many respects it’s a very laudable thing to want to live responsibly in harmony with Nature and with our environment. However, it is important to try to distinguish between doing this and the allure of pseudoscience that appears to be able to cure any complaint even if there is a lot of buzz about the therapeutic uses of bee venom
The above discussion highlights the incredible complexity of animal venoms. They are evolutionary marvels. Given the fact that many animals, aside from mammals, are venomous, humans have really only explored a tiny section of their chemical richness. Of around 50,000 species of spiders, for example, the venoms of only about 50 have been characterized. Not only is a knowledge of venoms of great importance for understanding how animals live their lives but the molecular components of venoms provide scientists with extraordinarily important tools for carrying out their research. There are clearly many more venomous secrets for us to uncover. In the meantime, wear long sleeves and watch out where you tread!