The Biology of the Venom of Hapalochlaena Maculosa
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Hapalochlaena maculosa, commonly known as the blue-ringed octopus, is a golf ball-sized cephalopod inhabiting the waters around Tasmania and southeastern Australia with a highly potent neurotoxin that it uses as a predatory and defensive mechanism. H. maculosa does not actually synthesize its venom, but rather, the neurotoxin (known as maculotoxin) is produced by a bacterial symbiont of the octopus that lives in its salivary glands. While not overly aggressive, H. maculosa has been known to bite humans when they disturb the usually reclusive octopus. Tetrodotoxin (TTX), the principle component of maculotoxin, inhibits the nervous system by binding to sodium channels on nerve cells to prevent the flow of sodium and release of neurotransmitters. Recent findings have shown that there are sodium channel variants that are either immune or resistant to tetrodotoxin. In most humans, however, victims of the blue-ringed octopus’s neurotoxin will enter into increasingly dangerous stages of paralysis, which will often end in death of the victim without medical assistance. Sustained medical care for the duration of the toxin’s effects will improve the likelihood of a victim’s survival, but mortality rates are still staggeringly high despite current medical efforts and attempts to find an antidote.
Australia is home to many of the most venomous animals in the world. Many people know that the bites from many of Australia’s snakes and spiders or the stings from its jellyfish and scorpions can be lethal, and oftentimes images of a person writhing in agony as a toxin courses through his or her body come to mind. However, one of Australia’s deadliest creatures has a toxin that works in a very different way. The venom of Hapalochlaena maculosa, or the blue-ringed octopus, causes relatively little pain, but its effects are much more terrifying for the victim. This venom, known as maculotoxin, is an unusual venom both in how it has come to be the weapon of the blue-ringed octopus and in how it works.
H. maculosa is a member of Class Cephalopoda and Family Octopodidae. The octopus is made up of a main body, or head, in which there is a well-developed brain, a mouth region that houses the beak, and a pair of eyes. Surrounding the mouth are eight or ten tentacles which are lined with suckers. Within the mantle cavity are the visceral organs, including the venom apparatus (Halstead et al.
, 1990). The blue-ringed octopus is not very large, with an average body size of about the size of a golf ball. The octopus seldom exceeds 20 centimeters in length when fully extended, and the average mass of a mature octopus is only about 38 grams (Sutherland, 1994). The octopus is generally a light tan color with dark brown bands all over its body and dark blue rings overlaying the bands. When it becomes threatened, the blue rings become a dazzling peacock blue and the rest of its body darkens significantly.
Distribution and Human Encounter
H. maculosa can be found in the ocean waters along Australia’s southeastern shore and around Tasmania. The octopus generally inhabits shallow tidal pools, and it generally lives a solitary lifestyle. However, it has been found that these octopuses will sometimes be seen in large groups as they leave their tidal pools after a region has experienced a heavy rain (Sutherland and Tibballs, 2001). The blue-ringed octopus tends to be found at depths no greater than 20 meters, which is likely explanation for why they are so commonly found in tidal pools. H. maculosa seems to prefer to hide itself within small containers or shells that it finds. This can make the octopus a very dangerous threat to unsuspecting children who might pick up a pretty shell or an old soda can and find one of these creatures inside.
For Dr. Roy Caldwell of the University of California at Berkeley, this tragic scenario very nearly became a reality. While on a rubble collecting dive to study mantis shrimp behaviors, Dr. Caldwell’s daughter sat in a boat, sorting through the rubble her father would bring up to her in a bag. At the end of the day, the daughter told her father that one of the oysters had had “something soft and squishy inside.” Upon cracking open this oyster, Dr. Caldwell was shocked to discover an agitated female blue-ringed octopus that was brooding its eggs. As Dr. Caldwell then noted, “This was a potentially lethal blue-ringed octopus that [his] unsuspecting daughter had handled just minutes earlier” (Caldwell, 2000).
Venom Apparatus and Use
H. maculosa tends not to be overly aggressive, but it will not hesitate to bite if its escape route is impeded (Sutherland, 1994). This bite contains a potent neurotoxin commonly known as maculotoxin. This toxin is produced by the two large salivary glands within the octopus’s mantle cavity. They are connected by a salivary duct that passes through the brain to the buccal mass where the saliva can be injected into predators, prey, or the surrounding water. H. maculosa generally feeds on small crabs, which are immobilized using the octopus’s venom. In an experiment conducted by Sutherland and Lane in 1969, it was shown that the octopus would attack crabs in two different ways. If the octopus had not eaten in several days, it would latch onto the crab and inject its venom while struggling to hold the crab. On the other hand, if the octopus was relatively well-fed, it would merely salivate into the water near the crab, then sit at a distance and wait while the crab became immobilized before it would approach its meal (Sutherland and Tibballs, 2001).
Venom of the Blue-ringed Octopus
While the official venom of H. maculosa is maculotoxin, this name is just a common name for the substance obtained directly from the octopus’s salivary glands. Many experiments have been conducted to determine the exact chemical composition of the toxin, but currently no one has been successful at collecting a sample of pure saliva of sufficient quantity for significant testing. All recent studies conducted on maculotoxin have used a solution made with the octopus’s entire salivary gland as a substitute for the pure saliva (Sutherland and Tibballs, 2001).
Researchers have known for over thirty years that maculotoxin is very similar to tetrodotoxin (TTX), but it was not until 1978 that tetrodotoxin crystals were isolated from H. maculosa venom (Fuhrman, 1986). Using these crystals, Sheumack, Howden, Spence, and Quinn were able to determine that tetrodotoxin and maculotoxin are the same toxin through nuclear magnetic resonance studies (Sutherland and Tibballs, 2001). While other chemicals such as hyaluronidase – a chemical that may help the spread of the toxin in tissues – found by Sutherland and Lane in 1969 may add to the total composition of maculotoxin, tetrodotoxin is the primary toxic component of H. maculosa’s venom.
Evolution of Tetrodotoxin
The origin of tetrodotoxin in H. maculosa is an area of some debate. At one time, it was thought that tetrodotoxin was restricted to sea creatures. Today, it is known that tetrodotoxin is found in a wide variety of animals both on the land and in the sea. The first known sources of tetrodotoxin were the puffer fishes of the genus Tetraodon (Johnson, 2002). Other tetrodotoxin-using animals include the Atelopus frogs, the Gobius newts, and various other animals (Fuhrman, 1986). All of these creatures have the common trait of tetrodotoxin-resistant sodium channels. This characteristic is essential to the leading hypotheses behind the evolution of tetrodotoxin as a venom or poison in each of these animals.
The current research on the evolution of tetrodotoxin tends to support the idea that there is a symbiosis between bacteria and tetrodotoxin-resistant animals. According to William H. Light, such a relationship would provide food and shelter for the bacteria, while the animal would receive the benefit of a powerful neurotoxin contained within its tissues which could be used for defense or hunting (Johnson, 2002). In the case of H. maculosa, tetrodotoxin-producing bacteria would live in the posterior salivary glands. Strains of the family Vibrionaceae, Pseudomonas, and Photobacterium phosphoreum produce tetrodotoxin or anhydrotetrodotoxin, making them likely candidates for this symbiotic relationship (Johnson, 2002).
Sodium Channel Interactions with Tetrodotoxin
Tetrodotoxin is classified as a neurotoxin because it is a toxin of the nervous system. Specifically, tetrodotoxin binds to the sodium channels and physically blocks movement of ions through these channels. Without being able to move sodium ions through the sodium channels, the nervous system is effectively shut down. Exactly how tetrodotoxin binds to the sodium channel is not known for certain, but several hypotheses have been suggested for probable attachment and blocking.
One hypothesis that was accepted for a long time before being disproved was proposed by Kao in 1986. Kao suggested that the guanidium group of tetrodotoxin binds to a receptor within the sodium channel and the rest of the tetrodotoxin molecule serves as a plug for the opening (Auyoung, 1999). While this idea was proven to be incorrect by later experimentation, it is quite possible that the guanidium group is involved in the binding of TTX to the sodium channel. In various experiments conducted by Mosher, alterations to the guanidium end of tetrodotoxin resulted in inactive or greatly weakened toxins while alterations to the C-6 and C-11 ends had little effect on the toxicity (1986).
The tetrodotoxin molecule is composed of a guanidium group, a pyrimidine ring, and five other ring systems (Johnson, 2002). Figure 1 shows a molecular model for tetrodotoxin. The guanidium group has a positive charge which is stabilized through resonance with the negatively charged Oxygen atom. This negatively charged oxygen atom is also one of six hydroxyl groups present in the molecule. The pyrimidine ring is composed of part of the guanidium group, the two Carbon atoms drawn in the background of Figure 1, and the Carbon atom that joins the guanidium group and these two Carbon atoms.
With new knowledge of the structure of the sodium channel, current hypotheses are leaning toward something similar to a scenario proposed by Choudhary et al. (2003). This group proposed that the guanidium group binds to the selectivity filter of a voltage-gated sodium channel while the six hydroxyl groups form hydrogen bonds with various amino acids in the sodium channel. The selectivity filter is a structure at the base of the P-loops which are peptide chains that connect the fifth and sixth segments of the four domains of the á-subunit. Figure 2 shows the á-subunit as it would appear if it could be taken out of its customary circular conformation. Choudhary et al. used a series of experiments involving single amino acid mutations in the voltage-gated sodium channels to determine their hypothetical tetrodotoxin binding arrangement. In addition, they found that the C-11 hydroxyl group of TTX is important to tetrodotoxin binding in contrast to the studies done by Mosher eight years earlier.
Tetrodotoxin Resistance and Immunity
H. maculosa and other animals are only able to use tetrodotoxin as a weapon or defense mechanism because they have evolved to be immune to the toxin. Tetrodotoxin is a very powerful neurotoxin, but it is also highly specific. Even a single mutation in the amino acid sequence of the á-subunit of a sodium channel can cause immunity or resistance to tetrodotoxin. William H. Light noted that a single point mutation in Fugu rubripes, a type of pufferfish, has rendered the species immune to tetrodotoxin (Johnson, 2002).
While little research has been done involving H. maculosa’s immunity to tetrodotoxin, there have been several recent studies involving a newly evolving resistance to tetrodotoxin in the garter snake Thamnophis sirtalis. These snakes readily eat newts that contain tetrodotoxin in their skin, but unlike other animals that would die from such a meal, these snakes merely become less active. T. sirtalis has evolved sodium channels that are resistant to tetrodotoxin. These tetrodotoxin-resistant sodium channels come as a genetic tradeoff for the snake, however, as these channels also seem to conduct action potentials at a slower rate than tetrodotoxin-susceptible channels (Huey and Moody, 2002). It is also interesting to note that the total resistance of these snakes is geographically varied due to individual genetic variation (Geffeney et al., 2002).
These observations could explain why tetrodotoxin resistance is not more prevalent in other organisms. Unless the benefits of having the tetrodotoxin-resistant sodium channels outweigh the cost of having slower channels, it seems more likely that most animals would have the faster, tetrodotoxin-susceptible sodium channels. However, for defensive and predatory tetrodotoxin-using organisms and their predators, these adaptations would prove to be essential.
Symptoms of Tetrodotoxin Intoxication
When a person is bitten by H. maculosa they might not feel anything, and at worst, they might feel a pinprick from the penetration of the octopus’s beak through the skin. The first sign of tetrodotoxin envenomation is a slight tingling around the lips and tongue starting fifteen minutes to several hours after the bite. Continually increasing paresthesia (a sensation of the skin such as burning, tingling, or tickling without apparent cause) of the lips, tongue, face, and extremities will also develop (Johnson, 2002). Other early symptoms include salivation, nausea, vomiting, and diarrhea with abdominal pain (Benzer, 2001).
As the toxin spreads, paralysis becomes increasingly prevalent as tetrodotoxin shuts down the nervous system’s sodium channels. Paralysis begins between four and twenty-four hours after the initial venom injection (Benzer, 2001). Extremities become paralyzed first, followed by loss of brain stem reflexes. The loss of respiratory muscle action is the most serious threat. Respiratory muscle paralysis can cause death of an untreated victim between four and six hours after being bitten (Benzer, 2001). If the victim survives respiratory muscle paralysis, cardiac dysfunction occurs, the victim may go into a coma due to dysfunction of the central nervous system, and seizures may develop (Benzer, 2001). Until the victim enters this last stage, he or she may be fully conscious despite being completely paralyzed and unable to communicate (Johnson, 2002).
Treatment for Tetrodotoxin Victims
Treatment for victims of H. maculosa is still entirely symptomatic. No anti-venom has been developed because unlike many toxins, tetrodotoxin is not a protein. If this were the case, antibodies could easily be developed to create an antidote to reverse the toxin’s effects. While no successful antidote studies have been conducted using maculotoxin, the actual venom of the blue-ringed octopus, monoclonal antibodies have been isolated which have been successful at treating lethal doses of tetrodotoxin in mice (Benzer, 2001). Anticholinesterase drugs have also been proposed as a possible treatment for H. maculosa envenomation. Torda, Sinclair, and Ulyatt found that muscle power restoration was accelerated using an anticholinesterase drug (Sutherland and Tibballs, 2001). Benzer suggests Neostigmine (Prostigmin) as a possible anticholinesterase for extreme tetrodotoxin intoxication (2001). Anticholinesterase drugs prevent the destruction of acetylcholine, which can assist neuromuscular junction transmission.
At present time, emergency response for tetrodotoxin victims focuses on the ABC’s, or airway, breathing, and circulation care. If possible, the airway should be secured through intubation to provide oxygen, and an IV should be established to provide fluids and heart regulatory treatments (Benzer, 2001). The victim’s cardiovascular system should be supported until the toxin has been removed from the body. Most victims of tetrodotoxin envenomation recover completely if they survive the first twenty-four hours of intoxication, though effects may last for several days. Despite good medical assistance, however, mortality rates may still be as high as fifty to sixty percent (Benzer, 2001).
Tetrodotoxin as an Anesthetic
Due to tetrodotoxin’s ability to inhibit a neuron’s sodium channel, its use as an anesthetic has become a possibility. Tetrodotoxin would be useful as a long-term anesthetic because it takes effect relatively quickly, and its effects last for around twenty-four hours. In an experiment done by Schwartz et al., tetrodotoxin was found to be a good long-term anesthetic in the rabbit cornea without any noticeable side effects (1998). Anesthetic effects on the rabbit cornea lasted varying amounts of time depending on the concentration of the toxin. At a concentration of 10 mM, tetrodotoxin was useful as a topical anesthetic for up to eight hours after injection (Schwartz et al., 1998).
H. maculosa may be a small and beautiful member of Australia’s faunal population, but it is also one of the deadliest. Maculotoxin, the venom of the blue-ringed octopus, is a potent neurotoxin made up primarily of tetrodotoxin. The tetrodotoxin is produced by a bacterial symbiont that co-evolved with H. maculosa due to its tetrodotoxin-resistant sodium channels, which tetrodotoxin usually inhibits. Victims of tetrodotoxin intoxication may not suffer a horribly painful death, but their death will also be a very slow and terrifying one unless medical assistance is quickly provided. While no antidote to tetrodotoxin is known at this time, attempts are still being made to find a treatment. With proper knowledge of the dangers of H. maculosa and its powerful venom, this small octopus can be a spectacular addition to Australia’s amazing collection of wildlife.
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