horseshoe crab

Credit: Barry Falls

Blue bloods

For each COVID-19 vaccine shot in the arm, we have the horseshoe crab—and the collaboration between a Johns Hopkins immunologist and hematologist—to thank

Fear at first sight: That was Jack Levin's reaction upon meeting the creature that put him on the path to lasting scientific achievement. The year was 1963, two years before Levin would begin a 17-year stint on the faculty of the Johns Hopkins School of Medicine. He was a hematology fellow at this point, shipped up to Cape Cod for a summer research project at the Marine Biological Laboratory.

Levin met his new boss, Frederik Bang, and soon followed the then chair of Pathobiology at the Johns Hopkins School of Hygiene and Public Health into a primitive-looking laboratory containing a rudimentary "sea table" and a dingy aquarium tank. When the prehistoric-looking marine arthropods inside that tank moved, seawater splashed onto the floor.

"Pick one out," Bang said, pointing to the creatures. Levin knew nothing of Limulus polyphemus, or horseshoe crabs as they're commonly known. On first glance, the creature looks to be 10 claws set under a military helmet and backed by a long, malevolent-looking tail. They can grow to be 19 inches long.

"I was scared to death," he says.

Bang was having fun with the newbie. Despite appearances, horseshoe crabs are mostly harmless. Those 10 claws will grasp at you, but the result is more hug than bite.

Looks aside, the crab offered up another surprise. Bang put Levin to work in that lab without telling the young hematologist that because of a smidgeon of copper in Limulus' biological mix, its blood comes in a strange, almost alien, shade of milky blue.

That blood is central to the tale of scientific innovation that runs through that 1960s moment and right up into our present public health circumstances, with the world enduring a pandemic that has taken more than 3 million lives. The COVID-19 vaccines that are raising hopes for an end to that pandemic rank as 21st-century scientific miracles. The success of Operation Warp Speed was built on the latest advances in genomics and computing.

Because of a smidgeon of copper in Limulus' biological mix, its blood comes in a strange, almost alien, shade of milky blue.

Limulus polyphemus has a big role in the story, too, thanks to work that Bang and Levin began that day in Woods Hole, Massachusetts. The pair published a run of papers in the 1960s that revealed the bacteria-fighting secrets of blue horseshoe blood. Using these crab blood cells, Levin would then develop the Limulus amebocyte lysate, or LAL, test to screen for the presence of dangerous bacterial endotoxins in whatever sample you wanted to examine. The test has been a boon to public health since coming into wide use in the 1970s, saving millions of lives by ensuring the safety of countless drugs, intravenous fluids, vaccines, and medical devices. COVID-19 vaccines, too, need to pass horseshoe-crab muster before heading out to market.

And all this started in a lab with no hot water or working air conditioning.

"The conditions Bang and Levin worked in were so austere," marvels Jim Cooper, a Hopkins-trained nuclear pharmacist who played a key role in both developing the LAL test and turning it into a successful commercial product. "They had none of the lab equipment we take for granted now. They didn't have the computers we have. They didn't have software. To me, what they accomplished is all the more impressive because of that."

Serendipity is the word that comes up time and again in reflecting on these accomplishments. A couple of months before his death in 1982, Bang built his keynote address to a conference at the Woods Hole Oceanographic Institute around those five syllables. He argued that the LAL test came into being in ways that speak to the very nature of scientific innovation.

Bang—an infectious and parasitic diseases expert whose career was lined with notable discoveries about malaria, cystic fibrosis, and cancer—laid out in that talk "Three Rules of Serendipity." The first, "Take a fresh look at old phenomena," fits the speaker's own scientific modus operandi to a T. His insatiable curiosity about biological systems led him across countless disciplinary boundaries, and often into the workings of strange ancient creatures like the ones in his lab tank.

To say that Limulus polyphemus qualifies as "old phenomena" would be an understatement. Fossil experts guesstimate the age of the species at 450 million years, which means that horseshoe crabs were waddling around in dinosaur times. Despite the informal name, the species is more closely related to spiders than crabs. The Western Hemisphere's branch of the Limulus population is found today along the Atlantic Coast and in the Gulf of Mexico.

The epicenter of that population is in the Delaware Bay, where several small towns hold annual festivals honoring their prehistoric neighbors as natural wonders of the first order. Each spring, a mating ritual occurs in which horseshoes emerge from the ocean in astounding numbers, clambering up onto the sand under a romantic full or new moon. The fecundity of female horseshoes is astonishing. Over the course of a few sex-filled days, each one can lay between 80,000 and 100,000 eggs. Those eggs are packed with nutrition, which is why so many hungry migrating shorebirds join the party.

A .gif depicting horseshoe crabs washing up on a shore

Image credit: Getty

Spectacular as this scene is, however, that milky blue blood gives Limulus an even stronger claim to natural-wonder status. Bang found his way to that blood because he was a big believer in the medical value of marine invertebrates, arguing that "transparent" sea creatures held great potential for generating insights into the workings of human biology.

During the summers of 1953 and 1954, Bang conducted a series of what he dubbed "naive experiments" with Limulus polyphemus. The coastal waters where horseshoe crabs live are chock-full of bacteria, and Bang was curious about which of the many pathogens in that water posed a threat to horseshoes and what sort of defenses the creature had developed against them.

The work involved injecting crabs with various types of bacteria to identify which substances were toxic and at what levels. In writing up preliminary thoughts in a 1956 issue of the Johns Hopkins Bulletin, Bang was struck by the way Limulus blood clotted amid some bacterial invasions. He speculated that the clotting happened so as to trap pathogens inside biological bubbles that served like detention centers, keeping infectious substances from spreading.

Bang stored bacterial samples from those tests in a freezer at Woods Hole. Then he did … nothing. Nearly a decade passed, until one day he called on Lockard Conley, the chief of Hematology at Hopkins, and asked to borrow one of his young researchers.

No one seems to remember what sparked Bang's sudden renewed interest in Limulus. Did he stumble across those old samples in a freezer? Did he see something in the literature or on a sea table that brought those gloppy horseshoe-blood bubbles to mind? In any case, Conley soon sent Jack Levin up to Cape Cod to help Bang solve the hematological mysteries of this ancient sea creature.

"Bang quickly became a hero of mine," Levin said. "He had an incredible mix of curiosity, passion, and scientific instinct. How many researchers out there who are working at his level are going to drop everything to think the way he did about these horseshoe crabs? The answer is either 'Next to nobody' or 'Nobody.'"

The COVID-19 pandemic is the latest chapter in a story that runs through all of human history. Viruses and bacteria are the deadliest enemies of our species. Think smallpox, which killed some 300 million people in the 1900s alone. Traces of that virus have been found in 3,000-year-old Egyptian mummies. The infamous Black Death was a bacterial plague that wiped out more than half of Europe's population in the 1300s.

Only in recent centuries have humans found effective ways to fight these enemies. The first vaccine appeared in the late 1700s when Edward Jenner inoculated a 13-year-old boy against smallpox by exposing him to a less dangerous vaccinia virus, or cowpox. Modern syringes that made vaccine administration safer and faster showed up half a century later. But these tools opened the way for a bacterial counterattack in the form of a mysterious and sometimes fatal "injection fever" that arose after patients received shots.

Frederik Bang was struck by the way Limulus blood clotted amid some bacterial invasions.

In the 1920s, biochemist Florence Seibert identified the culprit as endotoxins. These molecules line the cell walls in a class of disease-inducing bacteria known as "gram negative." Her work on what would become intravenous therapy led her to develop a test for the presence of endotoxins that relied on the common European rabbit. Put simply, the rabbits were inoculated with a substance that could contain a pyrogenic (fever-producing) contaminant. If one was present, the rabbit's temperature went up.

In its time, the "rabbit test" was widely used by pharmaceutical companies and medical device makers. But it was also costly and cumbersome. Levin administered a few rabbit tests early in his career and hasn't forgotten about the damage the animal's claws can do to researchers trying to measure their temperature by way of the rectum.

By the 1960s, medical advances were also pushing at the limits of what the rabbit test could handle. In nuclear medicine, for instance, newly invented concoctions used to perform imaging tests had a shelf life measured in hours. It took at least a full day for the results from rabbit tests to come in.

In handing the horseshoe baton off to Levin, Bang put his second rule of serendipity into play: "Remain naive, but carry knowledge of past experiences."

Unlike Bang, Levin came to the project with no special interest or background in marine biology. Levin's mission was to study the blood-­coagulation mechanisms at work in Limulus polyphemus. His work immediately hit a roadblock. The blood samples Levin prepared began clotting in a matter of hours. "They'd look fine when I left the lab, but the next morning was another story," he recalls. "You can't study blood cells in that condition—the blood needs to stay fluid."

He tried every anticoagulant trick in the book. Nothing worked.

Limulus polyphemus has been through tough times over its 450-million-year existence. The species has survived as many as five different mass-extinction events.

Levin had done some research on blood coagulation in rabbits. He knew that bacterial endotoxins could kick that process into gear. What if things worked the same way in this marine arthropod as they did in that mammal? The next time he drew horseshoe crab blood, he made sure that his glass tubes and other tools were free of bacterial contamination. The blood didn't clot this time. Now he was off and running, with a new focus on the horseshoe crab's defenses against endotoxins.

The discoveries came fast and furious in the years that followed. The key to the coagulation system lies with mobile blood cells called amebocytes that release proteins in response to the appearance of gram-negative endotoxins. He soon realized that the system is phenomenally sensitive, picking up the tiniest traces of contamination.

Levin then set out to transform these revelations into something useful. Soon, he was washing horseshoe amebocytes with an organic compound to produce liquid lysate from the amebocytes that contained all the secrets of the horseshoe's defense mechanism against endotoxins. If you dropped a bit of medicine into that lysate, you could examine it a few hours later for the telltale gel that signaled coagulation and provided proof positive that a sample was contaminated.

Levin has received numerous honors over the years for the run of discovery and innovation that led to the invention of the Limulus amebocyte lysate test. In 2019, the American Association for the Advancement of Science gave him its Golden Goose Award. Fittingly, that prize celebrates the way basic research into natural phenomena can sometimes yield great dividends for human health and wellness.

Limulus polyphemus has been through tough times over its 450-million-year existence. The species has survived as many as five different mass- extinction events. More recently challenges arose by way of humankind. In the late 1800s, horseshoe crabs became a popular agricultural fertilizer and livestock feed. Historical photos show farm fields near the Delaware Bay blanketed with thousands of crab carcasses.

That threat eased as better, cheaper fertilizers came on the scene. Another threat arose in the 1980s with the increasing popularity of conch and eel in regional cuisines in various parts of the world. Horseshoe crabs are commonly used as bait in both of those fisheries. Another challenge lies in the way residential and/or commercial development can encroach on or disrupt horseshoe crab habitats.

Is modern medical science and its LAL test a threat as well, especially given the sudden need to test billions of doses of new vaccines? The answer to that question is counterintuitive. Jim Cooper has been involved with the LAL testing industry since the 1970s, when he played a key role in shepherding the test out of the laboratory and into the marketplace.

"The COVID vaccines are really just a drop in the bucket," he says. "That can be hard for people to get their heads around if they don't know how this industry works, but it's true."

The process of creating LAL tests begins with specially trained and equipped fishing crews that catch horseshoes at nighttime in the warmer months. The work has been done in catch-and-release fashion by rules Cooper helped to establish in the 1970s with approval from the Food and Drug Administration. Once on shore, the crabs are trucked to nearby facilities where blood is drawn for five to eight minutes. The crabs are then returned to the water, by regulation within 36 hours—usually, it happens in less than a day.

About 450,000 horseshoes are harvested this way yearly, and some crabs die as a result of this ordeal. Mortality rates from bleeding are difficult to measure precisely after crabs return to the wild, but the number most widely accepted by researchers is 15%. That's roughly 2% of the estimated 20 million horseshoe crab population in the Delaware Bay, Levin says, a mortality number much smaller than the annual death toll inflicted by the bait industry.

The Atlantic horseshoe crab population has been listed as "vulnerable" by the International Union for Conservation of Nature across a broad range stretching from the Gulf of Mexico to the northeastern United States. The Delaware Bay population seems to have stabilized after a sharp decline in the early 1990s. Numbers of spawning females counted in annual surveys have been on the rise since the early 2000s.

The fact that Limulus blood ranks as an indispensable tool in protecting human health has spurred efforts to study and protect the species. The surveys that are now conducted annually didn't happen in pre-LAL times. The bait fishery has been outlawed in South Carolina and operates under new and stricter limits in New Jersey, Delaware, Maryland, and other states. Bird lovers concerned about the health of those migrating shorebirds also contribute to the push for best practices that ensure the species' future viability.

A little Limulus blood also goes a long way. The blood collected from those harvested crabs is transformed into 70 million units of a 21st- century version of the lysate invented by Levin. It goes even further because of the way LAL tests are conducted—not on individual vials, or even vats of medicine but rather in spot-test fashion at manufacturing facilities. That way, small numbers of tests clear vast quantities of product.

"Think about walking down a hospital corridor and seeing all the IV bags, all the medicines being injected," Cooper says. "Then think about all the hospitals, pharmacies, and clinics across the country—and around much of the world." Those 70 million LAL test units were enough to ensure the safety of that global supply chain before the COVID-19 pandemic.

A wild card looking forward is the recent development of LAL tests that use recombinantly derived reagents in place of real horseshoe blood. If widely adopted, those tests might significantly reduce the need for biomedical horseshoe crab harvests. So far, the tests are making more inroads in Europe than in the United States, where just a handful of firms are using or experimenting with them. Cooper says researchers and regulators still have concerns about whether this recombinant-factor approach has progressed to the point where its sensitivity levels equal those of traditional LAL testing. He predicts the traditional tests will remain a dominant product for "the foreseeable future."

Frederik Bang's third rule of serendipity—"Ask new questions and seek new answers"—brings the story of LAL back to the 1960s. Cooper was in the midst of his Hopkins training to become a pharmacist then, working under the mentorship of Henry Wagner, a pioneering nuclear medicine specialist.

Nuclear medicine was still in its infancy, but it was already clear that newly developed radio­active drugs had great potential as imaging and diagnostic tools. When Wagner heard that Jack Levin was giving a talk at the School of Medicine about his work on horseshoe crabs and LAL testing, he sent Cooper to listen in.

The fact that Limulus blood ranks as an indispensable tool in protecting human health has spurred efforts to study and protect the species.

"Jack's talk was so compelling, just riveting," Cooper recalls. "When I came back to the lab, I was almost jumping up and down. That day, I started writing up a protocol to study the possibilities for developing a new test" that would work not just in nuclear medicine but in the broader drug industry. There were no grant applications involved in that process, he adds—a reflection of the way things worked in different times.

"The study we did with Jack was published less than a year later." That work drew quite a bit of attention in what was then called the radiopharmaceutical industry. One firm hired Cooper to consult on the creation in 1971 of the first-ever commercial LAL facility, housed at first in an old oyster-processing plant in Chincoteague, Virginia.

By that time, Cooper was working at the U.S. Public Health Service, where he was soon assigned to collaborate with the FDA on its approach to testing and regulating these radiological medicines. The LAL test still had a long way to go before it would dislodge the rabbit test. Convincing regulators that it's time to do away with a proven tool they'd been using for four decades is never an easy job, Cooper notes.

LAL passed each new hurdle, finally emerging as the gold standard in endotoxin testing in the 1970s and then becoming standard operating procedure in the 1980s—some three decades after Frederik Bang decided to spend a part of his summer taking a naive look at the blood of a prehistoric sea creature.

Near the end of his 1982 lecture on serendipity, Bang said, "In this day of cost benefits, perhaps the administrator may recognize that although the cost of serendipity is moderately high and failure is not infrequent, the benefits" to human health can turn out to be "many times greater."

Jim Duffy is a writer living on Maryland's Eastern Shore.