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Ossip 1 Cnidarian Toxin Production and the Function of Nematocysts in Box Jellyfish Rachel Ossip [email protected] BSPM570 Abstract The box jellyfish, or the “sea wasp,” is one of the most deadly animals on earth. The venom from the tentacle of one of these creatures can have catastrophic effects on the cardiovascular system of the victim, resulting in a quick and painful death. But how do these unsubstantial animals produce such a potent toxin? This article will explore the chemical process that allows the box jellyfish to manufacture one of the deadliest substances on earth, and how that toxin works inside prey’s body to shut down the heart. Box jellyfish is a common way of speaking of 36 species of cnidarian invertebrates, and three of these species are best known for their deadly sting – Chironex fleckeri, Carukia barnesi, and Malo kingi. These animals’ tentacles are full of highly complex intracellular structures called nematocysts, which are activated chemically and are capable of becoming embedded in the skin, releasing tiny tubules that eject complex protein compounds directly into the jelly’s prey. There are at least 61 known proteins involved in the process of nematocyst chemical triggering and envenomation, and it is thought that at least one of the key proteins responsible for the extreme deadliness of these animals – glycosylation – is a post-translational modification of the cnidarian toxin family. Other lipolytic and proteolytic proteins from the nematocyst catabolize prey tissues, and eventually, the porins in the toxin will cause cardiovascular cells to leak potassium out of them, a condition called hyperkalemia, which leads to catastrophic cardiovascular failure and death can occur within minutes. As if the heart-stopping venom and searing pain weren’t enough, the box jellyfish sting Ossip 2 also releases a myriad of amines and hormones such as serotonin, caissarone, and histamine that flood the vascular tissue and accelerate the pathogenic effects of the deadly toxins. Although these creatures are made of about 95 percent water, they have evolved to produce some of the most exquisitely complex and potent venoms found on earth. Introduction: Box jellyfish – named for the cube-like shape of their bells – manufacture and deliver a multitude of proteins and toxins directly into their prey through stinging barbs called nematocysts (Endean, Monks, and Cameron, 1993). The chemical make-up of these toxins, their effects on prey, and measures to counteract these effects are the subject of great scientific scrutiny, as over 40 people a year fall victim to stings in the Philippines alone (“Jellyfish by the Numbers”). This paper will discuss the chemical formation of toxins within these animals’ tentacles and the mechanisms of envenomation. There are 36 species of jellyfish with this box-shaped bell, but not all are carriers of such deadly neurotoxins (Bentlage et al., 2009). Each species produces their own special venomous compound, but some have immediate and identical effects on prey – an immediate and severe burning pain, muscle spasms, cardiac failure, and death (Brinkman, Mulvenna et al., 2012). These compounds work by attaching highly specialized proteins to the receptors for hormones in nerve cells and disrupting their ability to receive signals from other cells, resulting in a cascading chain of chemical reactions that shut down body functions (Jouiaei et al., 2015). At the same time, specialty hemolytic toxins and myotoxins cause widespread cardiac failure, and hormones flood the vascular system creating a massive anaphylactic response (Bentlage et al., 2009; Bloom et al., 1998). An unfortunate swimmer can be killed by this deadly cocktail in a matter of minutes Ossip 3 (“Jellyfish by the Numbers”), while others may only be effected hours after envenomation – a rare delayed response reaction called Irukandji Syndrome, caused by excess catecholamine production in the areas affected (Bloom et al., 1998). It has long been believed that pouring vinegar onto a jellyfish sting would soothe the burn, but recent studies have suggested heating the area of envenomation instead. In one particularly promising set of studies, James Cook University in Australia had quite profound results from using heat to denature the proteins found in jellyfish venom, which has opened the door for further research into the base components of these neurotoxins and ways that they can be combatted before doing serious damage to a non-prey species (Carrette, Cullen, Peiera, Little, and Seymour, 2002). Discussion: Mechanism of Envenomation Jellyfish tentacles are incredibly thin – sometimes only a single cell thick – but can be up to three meters in length, and each jelly can have up to eighty free-floating tentacles (“Jellyfish by the Numbers”; Lewis and Bentlage, 2009). Studded along every square millimeter of each tentacle are a multitude cellular structures called nematocysts, microscopic coiled threads inside a protective cnidocyst shell made of collagen and keratin (Endean et al., 1993; Bloom, Burnett, and Alderslade, 1998). Each of these threads is coated with tiny hollow barbs that are filled with a noxious mixture of proteins designed to immobilize and kill prey before it has time to escape from the jelly’s tentacles (Albert, 2010). The threads can be retracted by the jelly back into the shell inside the tentacle, but the barbs will remain in the prey animal, allowing for maximum envenomation (Endean et al., 1993). Ossip 4 Ensuring that the maximum possible dosage of toxin is delivered is critical for the jellyfish, as such potent toxins are biologically expensive to produce – cnidarians can’t afford to waste their energy on unnecessary venom release. To prevent constant nematocyst firings, each cnidocyst shell has a hair-like projectile covering called a cnidocil. When a prey animal brushes against this cnidosil “trigger,” calcium ions inside the nematocyst’s capsule flood out, causing a shift in osmotic pressure, filling the thread rapidly with water and sending it shooting out of the cnidocyst shell into the prey animal. Jellyfish have evolved this mechanism so perfectly that the entire process can take place in less than one microsecond (See Figure 1; Bentlage et al., 2009; Brinkman, Aziz et al., 2012). Box jellyfish, along with other members of the cnidarian family, have a chemoreceptor for a specific protein that can effectively turn the nematocyst triggering mechanism off, so that the jelly won’t sting itself or other members of the same species when not hunting or in defense mode (Brinkman et al., 2014). Experiments are being conducted into the nature of this chemoreceptor, in the hopes of creating a preventative method to allow swimmers, surfers, and divers to share the waters with box jellies safely; these experiments will be explored at greater length later in the discussion. Figure 1: An illustration of nematocyst discharge (Pearson Education, 2014) Ossip 5 Chemical Analysis of Protein and Hormone Components of Cnidarian Toxin Because of the nature of box jellyfish nematocysts and their instabilities outside of a saltwater environment, collecting specimens for lab analysis has proven extremely difficult in the past. In order to collect intact, un-triggered nematocysts, tentacles are removed from the jelly, chilled in distilled water, stirred, filtered, and centrifuged. Dislodged, intact nematocysts are isolated and suspended in a buffer to be stored before laboratory tests (Chung, Ratnapala, Cooke, and Yanagihara, 2001). Because of this novel preservation technique of such delicate toxins, chemical analysis using fractionation and purification techniques, native polyacrylamide gel electrophoresis (NPAGE), nuclear magnetic resonance (NMR), and cation exchange chromatography were all combined to finally produce the first complete sequence of jellyfish toxin proteins at the Suntory Institute for Bioorganic Research in Japan in the early twentieth century (Nagai, Ito et al., 2000; Brinkman, Konstantokopoulous et al., 2014). This complete analysis of the chemical structure of these complex toxins revealed sixty one proteins and toxins composed of these proteins were identified – among them, two novel classes of bioactive proteins that had never before been seen – CfTX-A and CfTX-B (Brinkman, Aziz et al., 2012; Nagai, Sakamoto et al., 2000). These toxins carry with them a high concentration of porins, proteins that embed themselves into cells and create pores through which solutes and substrates can pass (Endean, 1987). Also injected into the prey species are a myriad of amines and hormones such as serotonin, caissarone, and histamine, which attack the nervous system and flood the body with an immediate anaphylactic overload (Chung et al., 2001; Endean, 1987). This causes calcium ions to release from the cell through the cellular pores that the porin in the toxin has created, causing a condition called hyperkalemia. With a severe ionic shift into the bloodstream, electrical signals Ossip 6 between cells are overloaded and nerve cells begin firing randomly, causing widespread muscle spasms (Brinkman, Mulvenna et al., 2012). When the effects of muscle dysfunction in the heart cause cardiac arrest, the prey species dies – and all of this takes a matter of minutes (Chung et al., 2001; Jouiaei, 2015). In rare cases, individuals stung by certain species of box jellyfish, particularly in the waters southeast of the Philippines, experience a type of delayed response that can last for weeks, referred to as Irukandji Syndrome. Those effected will gradually develop a gamut of symptoms over the course of five minutes to two hours after envenomation, including lower back pain, muscle and limb cramping, headaches, nausea, vomiting, chest pain, and – oddly – a “sense of impending doom” (Bloom et al., 1998). The exact chemical cause of Irukandji Syndrome is unknown, but many believe that an excess of catecholamine may cause neurons to misfire. Catecholamine is a class of organic compound that includes hormones such as epinephrine, norepinephrine, and dopamine (Lewis and Bentlage, 2009). It is thought that following the initial anaphylactic response to envenomation, the body continues to produce an excess of these compounds that may somehow react with lingering toxins to cause Irukandji Syndrome (Bloom et al., 1998). Hymolytic Toxin and Myotoxin Chemical Analysis Two novel proteins were discovered in the analysis of box jellyfish toxin. The proteins – dubbed CfTX-A and CfTX-B – are hemolytic toxins, meaning they disrupt the ability of red blood cells to carry oxygen throughout the body (Nagai, Ito et al., 2000; Brinkman, Konstantokopoulous et al., 2014). These never-before seen toxins can be found in Chironex fleckeri, Carukia barnesi, Malo kingi, and another species of the box jellyfish cnidarian family, Carybdea rastoni. CfTX-A is found within the thread-like appendage of the nematocyst, but Ossip 7 CfTX-B is only found in the nematocyst chaperone cells, cnidoblasts (Nagai, Ito et al., 2000). Both toxins contain a high concentration of porins, causing cellular “leakage” throughout the prey species and allowing these complex venom proteins to enter cells directly (Nagai, Sakamoto et al., 2000). At the same time, complex myotoxins that disrupt the electrical signals that control muscle function and cause muscle spasms circulate through the body, neurotoxins bind themselves to chemoreceptors, blocking nerve functions, and a protein called CAH1 lyses red blood cells, depriving the body of oxygen, releasing calcium and magnesium ions into the blood stream, and preventing effective responses to these poisons (Jouiaei, 2001; Chung, 2001). CfTX-A and CfTX-B are novel proteins, never before characterized or synthesized in a lab setting, but they do share some similarities with other known protein toxins from the cnidarian family. CfTX-1 and CfTX-2 are another clade of neurotoxic, porin-containing proteins that cause cardiovascular shock, but are not identical to the novel proteins discovered in 2000 (Brinkman, Konstantokopoulous et al., 2014). The discovery of CfTX-A and -B indicate that there may be more diversity than previously thought in the composition of box jellyfish venoms, but all that are currently known are very similar in chemical structure (Jouiaei, 2001). Nematocyst Toxin Production and Chemical Activation The method of neurotoxin production in box jellyfish is currently unknown, but the protein-dependent process of glycosylation is being examined as one of the possible mechanisms. Glycosylation in jellyfish tentacles is a post-translational modification to selected proteins, which causes the addition of sugars to amino acids where they wouldn’t normally be found. Some snake species produce a nematocystic protein toxin called nematogalectin, which is stabilized in the collagen-keratin shell by glycosylation processes (Endean et al., 1993). A team of scientists led by Diane Brinkman at the Australian Institute of Marine Science in Queensland Ossip 8 found that some of the toxins produced by box jellyfish are chemically similar to nematogalectin, indicating that glycosylation may modify the proteins that make up these chemicals in such a way that explains some of their properties (Brinkman, Aziz et al., 2012). Another area of research is the triggering mechanism for the nematocyst and the cnidocil projectile that activates the cnidarian tube that injects venom into the prey. There is evidence that cnidoblasts, the partner cells to nematocysts, produce a chemical signal that activates or deactivates the nematocyst triggering mechanism in a jelly’s tentacles. It is believed that jellyfish may use a primitive form of somatic sensory feedback to determine whether or not their nematocysts are ready to strike – this may be the reason that jellyfish cannot sting themselves, but little is known about the chemical structure of these chemical signals, and how they may work in a feedback loop with the nervous system of a box jelly (Albert, 2010). Despite all of the laboratory developments that have allowed for complete analysis of the cnidarian toxins carried by box jellyfish, little is known about the exact formation, replication, and function of these complex compounds (Currie, 1994; Brinkman, Aziz et al., 2012). The last two decades have seen an exponential rate of advancement in chemical identification technology, and it is likely that within the next two decades, we will understand the mechanisms of toxin production and have synthesized effective antivenins. Effects of Temperature on Compound Stability With 600,000 box jellyfish stings reported worldwide in 2015, experiments are underway to determine the best methods to counteract stings and prevent injuries (“Jellyfish by the Numbers”). One of the more promising experiments was conducted at James Cook University in Queensland, Australia in 2001. A team of scientists isolated the protein structures of toxins Ossip 9 collected from intact nematocysts and subjected them to various temperatures, ranging from four degrees Celsius to fifty eight degrees Celsius for various lengths of time. They found that raising the temperature of the toxin to fifty degrees Celsius, even for two minutes, resulted in nearly double the length of time from exposure to death in the prey species – in this experiment, freshwater crayfish. It was also discovered that prolonged exposure to less extreme temperatures decreased the potency of box jellyfish toxin – a twenty-minute exposure to just forty Figure 2: Effect of heat and time of exposure on lethality of box jellyfish venom (Carrette et al., 2002) degrees Celsius significantly increased the length of time from exposure to death. At just under fifty degrees Celsius, a twenty-minute exposure will prevent death altogether in crayfish. (See Figure 2). This prolonging effect is the result of protein heat denaturation – the delicate structures of proteins lose their shape and function when exposed to heat. Box jellyfish live in temperate waters, so even forty degrees Celsius can be enough to significantly reduce the number of effective protein toxins injected into the site of envenomation, and the longer the exposure, the more denaturation can occur. This explains the exponential decrease in lethality of cnidarian toxin at high temperatures, as illustrated in Figure 2 (Carrette et al., 2002). While increasing the time from toxin exposure to death of the prey species may seem like just a way of drawing out a painful demise, in reality, when the animal unfortunate enough to wander into a box jellyfishes’ tentacles is a human, applying heat to the wound may buy the victim precious minutes to receive antivenins and stop the effects of the toxins (Carrette et al., 2002). Ossip 10 Future Experiments The work done by Teresa Carrette and her team at James Cook University was successful in proving that protein denaturation by heat is be an effective way to treat box jellyfish stings in freshwater crayfish, but it’s far from being a perfect technique to prevent severe venom effects in human victims. New research from the Roscoe Bay Marine Biology Research Lab in Vancouver, Canada has promising methods of discovering the chemical structure of a cnidosil trigger activating signal, and observing its effects on chemoreceptors in the tentacles of box jellyfish. Ultimately, the goal of this project is to develop a synthetic chemical or treatment that could deactivate or neutralize the toxins produced (Albert, 2010; Currie, 1994). Although there is a lot of work left to do and plenty of mysteries to unravel before we fully understand the mechanisms and chemical processes of box jellyfish venom production, biochemical research experiments such as these show great potential and will continue to increase our understanding of these creatures. Conclusion: Box jellyfish are some of the most venomous animals on the planet – and yet, they’re hardly animals. Cnidarians such as jellyfish have no brains and only a primitive nervous system, but have somehow developed an elegant and complex mechanism for defense and predation. Although our technologies have made incredible strides in the last several decades towards safely identifying and understanding the components of cnidarian toxins, we are just beginning to scratch the surface of composition, replication, and function of these biochemical assays. 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