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Stopping the sting

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Science  09 Nov 2018:
Vol. 362, Issue 6415, pp. 631-635
DOI: 10.1126/science.362.6415.631

Jellyfish almost killed Angel Yanagihara. Now, she is on a mission to save others from their fatal venom.

One of the deadliest box jellyfish, Chironex yamaguchii, collected by Angel Yanagihara off the Philippines.

PHOTO: ANGEL YANAGIHARA

On 17 June, several families were celebrating Father's Day here at Dalahican Beach, a popular bathing spot near Lucena, a city on Luzon island. A steady breeze blew across sand that looked like fine brown sugar. Children splashed in the dark green water. Suddenly, people started to scream as a toddler was lifted unconscious from the water, his lips pale. A witness recalled that dark lashes crawled across the toddler's thighs—the telltale marks of a jellyfish sting. The boy's family simply held him and cried. Shortly after, Prince Gabriel Mabborang, 18 months old, was dead—one of at least three children killed in the Philippines this summer by the stings of box jellyfish.

On a midmorning 3 weeks later, Angel Yanagihara, who studies jellyfish venom at the University of Hawaii (UH) in Honolulu, arrived at Dalahican Beach. After slipping into a full-body wetsuit, she slung a box over her shoulder, put on gloves, and walked into the sea. No reminders of the recent tragedy were present; children were playing in the shallows, clapping their hands to Filipino songs. “Hello! What's your name?” they giggled as Yanagihara, 58, walked by. Yanagihara spent almost 3 hours wading in waist-deep waters, hoping to catch box jellyfish for her studies of their venom. One of the nearly transparent animals swam to the surface, almost within reach, but then escaped as she approached. She emerged empty-handed, but villagers had brought her two specimens earlier that day.

Among the world's public health problems, jellyfish stings may seem trivial, affecting millions of people each year but known to kill only a few dozen. But many deaths may go unrecorded, and in some places, jellyfish stings take a real toll. Prince Gabriel was the second child killed on the same beach in the past year, and many people in the area bear the scars of nonfatal attacks. After news of the boy's death spread rapidly on social media, Lucena health officials invited Yanagihara to talk about jellyfish venom and how to save sting victims, a service she provided for free. She spoke at a basketball court by the beach, and as she flipped to her slide on first aid, cellphones rose in a wave, snapping photos.

Angel Yanagihara preparing for a dive off the shore of Honolulu. She decided to study jellyfish venom after being stung during a morning swim in 1997.

PHOTO: LAURA AGUON

Her message was clear—and controversial. Yanagihara has staked out one corner in a debate over how the venom of box jellyfish kills, stopping the heart in as little as 5 minutes. What she calls her unified field theory holds that the venom contains proteins that puncture red blood cells and release potassium, disrupting the electrical rhythms that keep the heart beating. Her conclusions, and the treatments she has based on them, emerged from 20 years of science that colleagues praise as thorough and imaginative. Yanagihara “has done a great favor to the field in doing systematic comparisons” of methods to collect and study the venom, says Kenneth Winkel, a former director of The University of Melbourne's Australian Venom Research Unit who is now at the university's Melbourne School of Population and Global Health.

But nobody has independently replicated Yanagihara's methods and findings or tested her treatments. Some jellyfish researchers say other compounds in the venom are the real killers and that different remedies—or none at all—are more likely to work. “Jellyfish venom is a graveyard for simplistic causation and therapy,” Winkel says.

Research that would resolve the debates is scarce. Worldwide, only about five research groups study jellyfish venom. Funders prefer to focus on bigger public health problems—although Yanagihara thinks the stings exact a much higher death toll than most people assume. So she and her few colleagues and competitors struggle on with small budgets to study the threat, develop remedies, and educate communities at risk.

MOST OF THE 4000 species of jellyfish cause only pain and discomfort when they sting humans. Only Cubozoans, or box jellyfish, of which some 50 species inhabit tropical and temperate seas around the globe, are fatal. They take their name from their cubic body, which has between one and 15 tentacles up to 3 meters long growing from each of the four corners. The tentacles are carpeted with hundreds of thousands of specialized cells, each harboring a capsule called a nematocyst that can fire a microscopic harpoon at speeds of more than 60 kilometers per hour. The harpoon carries a spiny hollow tube that injects venom after it strikes a victim (see graphic, p. 633).

Yanagihara, born in Alaska, hadn't planned to study jellyfish. But in 1997, the year she obtained her Ph.D. at UH for research on cellular ion channels, the jellyfish found her. One day that year, Yanagihara swam out to sea before dawn—“My father taught me to swim before I walked,” she says—when she encountered a swarm of box jellyfish some 500 meters offshore. She felt needles burning into her neck and arms and her lungs collapsing; her arms began to fail. She switched to a breathing technique she had learned for childbirth and clawed back to shore in agony, “like an automaton.” The pain kept her in bed for 3 days. After she recovered, she wanted to know what almost killed her.

In some cases, box jellyfish venom causes Irukandji syndrome, in which an overload of stress hormones and inflammation proteins produces pain and nausea for days, as well as high blood pressure that can lead to brain hemorrhage and death. Most sting casualties, however, die within minutes from cardiac arrest. The prevailing hypothesis 20 years ago was that the culprits are ion channel blockers, molecules that disrupt movement of ions in and out of cells. The blockage shuts down nerve and muscle cells, including those that keep the heart pumping.

To test the idea, Yanagihara followed a standard procedure for studying jellyfish venom: She dissolved the tentacles in water to release the nematocysts and broke them with a mortar and pestle or glass beads to release the venom. Then she exposed immature frog egg cells—a common model in cell physiology—to the venom and measured ion movement using electrophysiological techniques. But the experiments kept failing. After scrutinizing every part of her experimental setup, she began to wonder whether her venom preparation was too impure to reveal its secrets. She realized that crushing the nematocysts produced a crude mix of venom and cellular debris—akin to putting “a rattlesnake in a blender” to get its venom, she says.

GRAPHIC: V. ALTOUNIAN/SCIENCE

Taking a cue from a 1970s study, she developed a new method that uses citrate, an acidic compound, to dislodge the nematocysts without breaking them. She then puts them in a French press, in which a piston forcibly ruptures all the nematocysts at once. A minuscule harvest of venom squeezes out through a tiny outlet that filters larger cellular components.

Chironex fleckeri, one of the deadliest box jellyfish species, has left its mark on a patient's leg in North Queensland in Australia.

PHOTO: AUSCAPE/UIG/GETTY IMAGES

The yield is excruciatingly low: some 10 milliliters of venom from 1000 box jellyfish. (Yanagihara collects a species named Alatina alata, often called the sea wasp, en masse in Hawaii.) But the result, she says, is a much purer venom. In it she found not only ion channel blockers, but also many porins, proteins that puncture cells, allowing their contents to leak out. She suspected hemolysis—the destruction of red blood cells by porins—might be the fatal mechanism.

Studies supported that hunch. In a 2012 paper in PLOS ONE, Yanagihara and a colleague reported that venom of Chironex fleckeri, one of the deadliest jellyfish species, rapidly punctures red blood cells, causing them to leak a huge amount of potassium ions. A high level of potassium in the blood, or hyperkalemia, causes cardiac arrest, and when Yanagihara injected mice with high doses of venom, their hearts quickly stopped. The same happened when she injected only the porins from the venom.

In human jellyfish sting victims, however, autopsies show no signs of hemolysis, says Jamie Seymour, a prominent toxinologist at James Cook University in Cairns, Australia. He is skeptical that porins are the killers. In venom from C. fleckeri, his team instead found two distinct protein groups that specifically attack and kill human heart cells; those proteins are “the bit that will kill you,” he says.

Seymour says he has unpublished evidence that Yanagihara's technique for collecting venom deactivates the heart toxins along with other components. Winkel, too, is skeptical. He doesn't contest that porins puncture red blood cells, but agrees with Seymour that hemolysis is not usually seen in sting victims. Porins should be tested on heart cells and tissues, he says, to find out whether they directly affect the heart.

Yanagihara acknowledges that jellyfish venom contains other toxins, including molecules that break down lipids and proteins, but her studies convinced her that porins are the main and fastest killer. Recently, she and U.S. military researchers began to study how the venom affects piglets, which are physiologically much closer to humans than mice are. At a 2017 meeting in Florida, the group presented results showing they could reproduce both rapid death and Irukandji syndrome, depending on the dose of venom injected; the as-yet-unpublished findings also supported Yanagihara's porin hypothesis.

That hypothesis pointed to a remedy. In the PLOS ONE paper, Yanagihara showed that zinc gluconate inhibits porins and prolongs survival when injected into mice that had received a lethal dose of porins. Later, she found that copper gluconate works even better.

On the basis of those findings—and heeding instructions from the U.S. Department of Defense, which had funded her work—Yanagihara developed two patented products under the brand name Sting No More to counter jellyfish envenoming. A spray helps remove tentacles clinging to the skin; it contains urea, which is thought to make tentacles less sticky, and vinegar, which older studies and Yanagihara's own work had shown can deactivate unfired nematocysts. A cream containing copper gluconate is then applied to inhibit the injected venom. The products are used by U.S. military divers and sold on her website; dive shops in Hawaii carry them as well. She says she has yet to recoup her startup costs, in part because she gives the products away in developing countries.

Yanagihara has also developed simpler ways to test how well her products and other interventions inhibit porins, including a bioassay consisting of human blood suspended in agar (a gelatin derived from seaweed) overlaid with a membrane from pig intestine. A live tentacle placed on the membrane immediately pierces it and injects venom into the agar; blood cells destroyed by porins show up as white patches against the vibrant red. Winkel calls the test “the closest we have to human skin and blood, short of getting an experiment on human volunteers,” and Yanagihara says it confirms her treatment's effectiveness.

“I was really impressed by the scientific rigor” in Yanagihara's methods, says jellyfish ecologist Thomas Doyle at University College Cork in Ireland. In 2016, he worked with Yanagihara to test treatments for several species in Irish waters, including the lion's mane (Cyanea capillata) and the Portuguese man-of-war (Physalia physalis), which resembles a jellyfish but belongs to a different class. Doyle and Yanagihara showed that treating stings with seawater and ice, as recommended in Irish guidelines that Doyle helped draft in 2008, actually worsens sting injury. He is now pushing to revise those guidelines.

HERE IN TALAO-TALAO, the day before her talk, Yanagihara's hotel room smelled of vinegar. Neat rows of empty spray bottles stood beside a big plastic box on the floor. Her Filipino collaborator poured 23 liters of vinegar into the box, followed by a base solution—made separately by mixing water with a blue powder—and voilà, the Sting No More spray was ready. They pumped the solution into the bottles with a long siphon, ready to be handed out.

Her talk offered an unexpected chance for a real-world test. As she started to speak, a young man who had heard about her quest for box jellyfish walked in with a live one the size of a baseball cap. Wearing only boxers—he had just come from the beach—he held the relatively harmless cubic top in his hand, at arm's length, the tentacles dangling to his knees. The audience froze in tension, while Yanagihara grabbed her spray. The man thrust the jellyfish into a bag and then jumped back when a tentacle grazed his hand. It hurt so badly that he wanted to scratch his hand off, he said. Yanagihara quickly applied her spray and cream. Three minutes later, the man said the pain had eased. He sat through the 90-minute talk.

So far, Yanagihara has only such anecdotal evidence—along with hundreds of testimonials, she says—that her products work. Together with a clinician and two nurses in Hawaii, she has started a clinical trial in which 48 volunteers will be stung on both arms with centimeter-long pieces of tentacle from A. alata—small enough to cause only minor damage at the sting site. One arm will then be treated with vinegar and a hot pack, the other with either Yanagihara's products or a combination of vinegar and a cold pack. (Yanagihara says she will take no part in the data collection and analysis.)

Seymour questions whether Yanagihara's antiporin cream can save lives, and he argues that her vinegar-based spray may even harm sting victims. In a 2014 paper in the journal Diving and Hyperbaric Medicine, he and his colleagues reported that vinegar causes nematocysts that have already fired to release more venom. He now recommends no treatment at all for sting victims suffering cardiac arrest, except cardiopulmonary resuscitation, which can help keep blood pumping to the brain until the heart starts to beat again. “I don't care if they are screaming in pain 20 minutes later,” Seymour says, “as long as they are alive.”

In a letter in the same journal, Yanagihara, along with a statistician, criticized Seymour's vinegar study for flaws in the design and statistics; a group of Australian physicians published a critical letter as well. Yanagihara has also blasted “wildly extrapolative” reports of the study by Australian media, which claimed vinegar might kill.

SETTLING THE DEBATE will require more research on venom pathology and treatment—plus funding, which might be easier to win if researchers could point to hard numbers on the toll of stings. Studies and media reports often cite an estimate of 150 million stings each year worldwide and 20 to 40 deaths in the Philippines annually. Those figures surfaced in a 2008 report from the U.S. National Science Foundation, but what they are based on is unclear. In a 1998 review, clinicians estimated that jellyfish kill up to 50 people in the Philippines every year, “based on personal experience,” without further explanation. More recent studies tallied at least two dozen fatal and severe jellyfish stings in Malaysia and Thailand combined since 2000, almost all in tourists from abroad.

Most researchers believe the real number is much higher. The Philippines has a long, populated coastline dotted with estuaries where box jellyfish like to breed. In almost every coastal community Yanagihara has visited, locals lifted their shirts, sleeves, or pants to show scars from stings and recalled the deaths of friends and family from jellyfish. Many such cases don't make it into official statistics. Seymour says he had the same experience in the Southeast Asian nation of Timor-Leste 20 years ago: Villagers “said they get stung all the time but didn't bring the victims to the hospital,” he recalls. “They pointed to a tree and said they just buried them there.”

Angel Yanagihara gives a presentation about box jellyfish biology and stings at a rural health unit in Tagalag, a town on Samar island in the Philippines. Education and first aid can help reduce injuries and deaths, she says.

PHOTO: NOEL SAGUIL

Yanagihara and her collaborators are examining health surveillance records and surveying villagers and health workers in the Philippines. “We can triangulate these results to get a better idea of the burden,” says Catherine Pirkle, a UH public health epidemiologist on the project. Getting the study underway wasn't easy. The National Institutes of Health twice rejected a grant application, Yanagihara says, and local institutes and health units initially were lukewarm as well. Part of the problem may be that many communities accept the danger as part of life. “Although our fishermen and children are often stung by box jellyfish, we don't think it's a serious problem,” says Reil Briones, Talaotalao's village chief, who was stung by a jellyfish at age 11 and carries a scar on his arm.

Yanagihara says the sentiment is now changing. On her latest trip, she spoke to full rooms of policymakers, health workers, and researchers, and many asked to collaborate with her team. Photos of Prince Gabriel circulating on social media may have played a role. “It's a big issue if people are dying from jellyfish,” says Janet Gendrano, who leads the Disaster Risk Reduction and Management Office in Lucena. She says the tragedy was a wake-up call and wants to join the survey project; once the data are in, her office might propose an ordinance requiring beach resort operators to take first-aid training for stings and to put up warning signs.

Yanagihara hopes the study will get jellyfish the attention they deserve. “If you are a pony on this racetrack of human suffering,” as many believe jellyfish stings to be, “you want to stand down,” she says. “But I have nothing but evidence to the contrary.”

  • * Yao-Hua Law is a science journalist in Kuala Lumpur.

Correction (14 November 2018): This story has been updated to correct number of tentacles box jellyfish can have.

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