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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
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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
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(“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).
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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)
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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
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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
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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
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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
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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).
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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.
Current and future experiments will offer us further in-depth views into the production and
release of the myotoxins, hemolytic toxins, and various other structures present in jellyfish
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neurotoxins. The denaturation effects of heat on proteins such as CfTX-A and CfTX-B will save
lives, and further research into the glycosylation effects on proteins may allow for development
of a compound that can prevent envenomation altogether.
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