{"id":13706,"date":"2020-09-16T13:24:37","date_gmt":"2020-09-16T17:24:37","guid":{"rendered":"https:\/\/ncseagrant.ncsu.edu\/coastwatch\/?page_id=13706"},"modified":"2024-08-15T13:13:50","modified_gmt":"2024-08-15T17:13:50","slug":"blood-draw-at-the-horseshoe-corral","status":"publish","type":"post","link":"https:\/\/ncseagrant.ncsu.edu\/coastwatch\/blood-draw-at-the-horseshoe-corral\/","title":{"rendered":"Blood Draw at the Horseshoe Corral"},"content":{"rendered":"\n\n\n\n\n

Once researchers develop a COVID-19 vaccine that receives approval from the U.S. Food and Drug Administration, we undoubtedly will celebrate the news. Those scientists will deserve our gratitude. But we might also appreciate another important player: the American horseshoe crab (Limulus polyphemus<\/em>), a rugged coastal creature found along the eastern seaboard and portions of the Gulf of Mexico.<\/p>\n\n\n\n

Horseshoe crab blood is the basis for a gold-standard safety test used worldwide on vaccines and other injectable medicines, intravenous fluids, and implantable medical devices. Specifically, the test, or assay, identifies whether certain bacterial contaminants called endotoxins are present, and to what extent. Endotoxins can be detrimental to human health if they appear in high concentrations in the blood.<\/p>\n\n\n\n

In essence, the assay consists of a series of enzymes from the horseshoe crab\u2019s only blood cell, the amebocyte. Known as LAL, for Limulus amebocyte lysate, the extract reacts when it comes into contact with endotoxins, forming a telltale clot.<\/p>\n\n\n\n

\"\"
Jack Levin co-developed the Limulus amebocyte lysate test. Photo courtesy of Jack Levin<\/figcaption><\/figure>\n\n\n\n

In 2019, the researchers who developed the LAL assay \u2014 Jack Levin and Frederik Bang \u2014 won a Golden Goose Award<\/a>, which honors seemingly obscure research that ultimately has a significant impact on society.<\/p>\n\n\n\n

\u201cThe ability to measure in a precise way the amount of endotoxin in things we are exposed to every day has a societal benefit that I think few people recognize,\u201d said Brad Fenwick, a senior vice president at the science- focused publisher Elsevier, in a video<\/a> published by the Golden Goose Awards.<\/p>\n\n\n\n

Presently, there are four U.S.-based LAL manufacturers, three of which serve the U.S. market. Horseshoe crabs are gathered from Atlantic waters up and down the East Coast and transported to facilities for blood collection, then returned to the location they came from.<\/p>\n\n\n\n

Those companies have stated that they\u2019re committed to horseshoe crab welfare and conservation. \u201cOverall it\u2019s a sustainable industry if it\u2019s done properly,\u201d says Jennifer Mattei, a biologist who studies horseshoe crabs at Sacred Heart University in Connecticut.<\/p>\n\n\n\n

Still, the reliance on wild organisms \u2014 whose numbers have ranged from plentiful to precarious \u2014 has led researchers to pursue alternative strategies for endotoxin testing.<\/p>\n\n\n\n

One company in particular is taking a page out of the oyster grower\u2019s book: Greensboro-based Kepley Biosystems Inc., which received initial funding from North Carolina Sea Grant, aspires to build a network of horseshoe crab aquaculture facilities for the express purpose of blood collection.<\/p>\n\n\n\n

\u201cThere was an opportunity to benefit a species both critical to the ecosystem and to human health,\u201d says Kepley Biosystems Inc. founder and president Anthony Dellinger. \u201cThis aligns with our company philosophy to focus on projects that provide a net good to the public and the planet while addressing massive global markets.\u201d<\/p>\n\n\n\n

Ancient Mariners<\/h2>\n\n\n\n
\"Horseshoe
Horseshoe crabs come ashore on sandy beaches en masse to lay their eggs. Photo by Gregory Breese\/USFWS<\/figcaption><\/figure>\n\n\n\n

Despite what their name suggests, horseshoe crabs aren\u2019t actually crabs. More closely related to spiders and scorpions, they hail from an ancient lineage predating the oldest dinosaurs by a couple hundred millennia. Based on fossil evidence, horseshoe crab physique has remained relatively unchanged for nearly 450 million years.<\/p>\n\n\n\n

\u201cThey\u2019re amazing to look at, because they have a very unique body plan that we don\u2019t see in other organisms,\u201d says Mattei, who runs a horseshoe crab tracking program called Project Limulus<\/em><\/a>.<\/p>\n\n\n\n

\"\"<\/a><\/figure>\n\n\n\n

The creature\u2019s tank-like structure consists of a front shell hinged to a back shell, where a pointy tail called a telson pokes out. Despite its menacing appearance, \u201cit\u2019s not a weapon,\u201d says North Carolina Sea Grant marine education specialist Terri Kirby Hathaway. \u201cIf the animal gets turned upside down, it uses that tail to push into the sand and turn itself back over.\u201d The telson also functions as a rudder in the water and has patches of specialized cells that detect light.<\/p>\n\n\n\n

A glance under the carapace reveals six pairs of appendages for locomotion and foraging and so-called book gills \u2014 thin sheets of tissue with a leathery cover \u2014 used for breathing and propulsion.<\/p>\n\n\n\n

American horseshoe crabs are coastal species from Maine to northeast Gulf of Mexico states, as well as along Mexico\u2019s Yucat\u00e1n Peninsula. While the animals have different habitat requirements at different stages of their life cycle, spawning adults depend on sandy beaches where they come ashore annually, typically during spring high tides. Females dig pits in the sand where they deposit tens of thousands of eggs, which males then fertilize.<\/p>\n\n\n\n

\"The
The underside of a horseshoe crab. Photo courtesy of University of Georgia Marine Extension and Georgia Sea Grant<\/figcaption><\/figure>\n\n\n\n

It takes 10 years for a horseshoe crab to mature to an adult, which can reach at least 20 years of age, according to Mattei. These long-lived arthropods are integral to their ecosystems. For starters, loggerhead turtles eat them, and a host of organisms such as barnacles, marine snails called slipper shells, and anemones reside on horseshoe crab shells.<\/p>\n\n\n\n

Their eggs, meanwhile, nourish various invertebrates, finfish, and migrating shorebirds. For example, during a 9,000-mile migration from the tip of South America to the Canadian Arctic, Atlantic Coast red knots touch down on eastern beaches to refuel. It\u2019s no coincidence that Delaware Bay, the second-largest staging area for migrating shorebirds in North America, is also a horseshoe crab spawning hotspot.<\/p>\n\n\n\n

\u201cThere\u2019re some organisms that are really important when they\u2019re abundant, and the horseshoe crab is one of them,\u201d Mattei says.<\/p>\n\n\n\n

Luck of the Draw<\/h2>\n\n\n\n
\"Spawning
Spawning female horseshoe crabs usually come ashore with a male attached. Photo by Chris Engel<\/figcaption><\/figure>\n\n\n\n

Never in his life had Jack Levin seen a horseshoe crab when he arrived at the Marine Biological Laboratory in Woods Hole, Massachusetts, during the summer of 1963 to study the organism\u2019s blood.<\/p>\n\n\n\n

At the time, Levin was a research fellow in hematology at the Johns Hopkins University (JHU) School of Medicine and Hospital, where he was studying blood clotting in rabbits. Levin\u2019s advisor had suggested he collaborate with a JHU colleague named Frederik Bang, who spent summers at Woods Hole. Bang was \u201ca pioneer in applying marine biology to medical research,\u201d the New York Time<\/em>s noted<\/a> after his death in 1981.<\/p>\n\n\n\n

Bang had done earlier research on horseshoe crab blood clotting. Levin was to continue in that vein, conducting basic research into similarities between the horseshoe crab\u2019s blood cell \u2014 the amebocyte \u2014 and human platelets, which help blood clot.<\/p>\n\n\n\n

Bang acquainted Levin with his study subject by pointing to a tank containing horseshoe crabs and instructing him to, \u201c\u2018Pick one out,\u2019\u201d recalls Levin, now on the faculty at the University of California School of Medicine, San Francisco. \u201cOf course, I was absolutely thunderstruck, sure that my fingers would either be crushed by one of their claws or stuck by their tail.\u201d (Neither happened.)<\/p>\n\n\n\n

\"One
One female horseshoe crab may lay a few egg clusters each evening over the course of several nights. Photo by Gregory Breese\/USFWS<\/figcaption><\/figure>\n\n\n\n

As Levin pursued his research, he noticed that samples of liquid horseshoe crab blood left in the lab overnight had coagulated by the next morning, despite the addition of standard anticoagulants. He wondered: Could his glassware be contaminated? From his studies at JHU, he knew that bacterial endotoxins caused rabbit blood to clot. Maybe endotoxins were responsible for horseshoe crab blood clotting as well.<\/p>\n\n\n\n

Further experiments supported Levin\u2019s hunch \u2014 endotoxins prompted blood coagulation. He also learned that clotting was the result of an enzymatic reaction. Once a clot forms, the amebocyte unleashes other factors that work to destroy the source of infection, Levin explains.<\/p>\n\n\n\n

The adaptation is phenomenal if you consider the horseshoe crab\u2019s habitat, he says. \u201cAn animal like Limulus<\/em> is crawling around the bottom, getting bumped, damaged, and they have to have a mechanism that immediately controls bacterial infection,\u201d Levin says. \u201cThe amebocyte is an all-purpose cell. It aggregates, traps the bacteria, produces clots around them, and then is capable of releasing substances that will kill or immobilize the bacteria. It\u2019s a much more effective all-purpose cell than anything we humans have.\u201d<\/p>\n\n\n\n

Levin went on to develop a way to extract the clotting factors from the amebocyte. In 1987, the LAL assay became the standard method of screening for bacterial endotoxins. Until that point, a test that required the use of live rabbits had been used.<\/p>\n\n\n\n

\u201cNot only was the Limulus test more sensitive, but it was relatively easy to do,\u201d as well as practical, Levin says. Overall, \u201cit was clearly enormously better.\u201d<\/p>\n\n\n\n

Out of the Wild<\/h2>\n\n\n\n
\"Charles
Charles River Laboratories in Charleston, South Carolina, is one facility that collects horseshoe crab blood, which appears blue upon air exposure. Photo by Timothy Fadek<\/figcaption><\/figure>\n\n\n\n

The collection of horseshoe crabs for the biomedical industry ranks second to their commercial use as bait in the eel and whelk fisheries, according to the Atlantic States Marine Fisheries Commission, or ASMFC, which oversees the management of horseshoe crabs from Maine south through Florida\u2019s East Coast.<\/p>\n\n\n\n

The ASMFC assumed that role in 1998, following several years of dramatic growth in horseshoe crab bait landings. Since then, landings have declined and become fairly stable. In 2018, close to 660,000 horseshoe crabs were harvested for use as bait. Whereas 100% of bait crabs die, the ASMFC estimates a 15% mortality rate from bleeding the animals for LAL production.<\/p>\n\n\n\n

Although the collection of horseshoe crabs for biomedical purposes has increased since reporting began in 2004, the number has remained relatively consistent over the past several years, hovering around 500,000.<\/p>\n\n\n\n

Yet, these pandemic times beg the question: Will COVID-19 vaccine production have a measurable impact on the demand for horseshoe crab blood?<\/p>\n\n\n\n

\u201cIt was estimated that the production of 5 billion doses of vaccine will require less than a day\u2019s production from three U.S.-based LAL manufacturers,\u201d according to Allen Burgenson, who chairs the ASFMC\u2019s horseshoe crab advisory panel and works for Lonza Walkersville Inc., which produces LAL in Maryland.<\/p>\n\n\n\n

\"Red
Red knots are migrating shorebirds that feast on horseshoe crab eggs. Photo by Gregory Breese\/USFWS<\/figcaption><\/figure>\n\n\n\n

Even so, the global bacterial endotoxin testing market is expected to double by 2024 based on growth in the pharmaceutical, medical device, and healthcare facility industries, says Glenn Gauvry, founder and president of the Ecological Research & Development Group Inc., a nonprofit horseshoe crab conservation organization.<\/p>\n\n\n\n

The American horseshoe crab population as a whole appears to be generally stable, according to the ASMFC, aside from the New York region, where it\u2019s declining. In North Carolina, the population also looks healthy. \u201cIt does seem like, over time, it has been increasing\u201d in the state based on the available data, says Kristen Anstead, a stock assessment scientist with the ASMFC.<\/p>\n\n\n\n

In Asia, however, the future is bleaker. There, a version of the assay is made with blood from the two horseshoe crab species of the genus Tachypleus<\/em>. One of those species has been listed as endangered<\/a> by the International Union for Conservation of Nature, or IUCN. There is insufficient data<\/a> on the other to determine its conservation status.<\/p>\n\n\n\n

If it turns out that Gauvry is right and demand for bacterial endotoxin testing spikes in coming years, the industry might have to lean more heavily on American horseshoe crabs. \u201cWhether Limulus<\/em> can handle that is in question,\u201d Gauvry says. \u201cRight now, it seems that it can, but it\u2019s something that we\u2019re going to have to keep an eye on.\u201d<\/p>\n\n\n\n

Coralling Crabs<\/h2>\n\n\n\n
\"A
A host of organisms, such as barnacles, marine snails, and anemones, reside on horseshoe crab shells. Photo by Chris Engel<\/figcaption><\/figure>\n\n\n\n

Like Jack Levin, Anthony Dellinger of Kepley Biosystems Inc. had never seen a horseshoe crab until 2018, when he began a Limulus<\/em> research project, supported by a North Carolina Sea Grant minigrant.<\/p>\n\n\n\n

He had worked with LAL as a student in biology labs and in other research posts, but he was unaware of its origin in horseshoe crabs. \u201cI didn\u2019t even know that the animal existed,\u201d Dellinger says.<\/p>\n\n\n\n

Eventually he learned about the organism \u2014 and saw an opportunity. He and his team could raise horseshoe crabs through aquaculture and bleed them in-house for LAL. That way, they could help curb wild collection. If they bled crabs at regular intervals, they could potentially increase exponentially the amount of LAL that can be produced per crab each year.<\/p>\n\n\n\n

\"Anthony
Anthony Dellinger, founder of Kepley Biosystems Inc., envisions creating a network of horseshoe crab aquaculture facilities for blood collection. Courtesy of Kepley Biosystems Inc.<\/figcaption><\/figure>\n\n\n\n

The N.C. Sea Grant minigrant enabled the Kepley team to acquire several horseshoe crabs, construct an aquaculture apparatus, and generate preliminary data.<\/p>\n\n\n\n

\u201cOne role of our minigrant program is to stimulate innovation, and Kepley\u2019s horseshoe crab aquaculture idea struck us as a unique concept worth supporting,\u201d says John Fear, North Carolina Sea Grant\u2019s deputy director.<\/p>\n\n\n\n

Dellinger adds that the minigrant \u201callowed us to get to a serious stage.\u201d Indeed, after their initial project, his team received funding from the National Science Foundation to continue their husbandry research.<\/p>\n\n\n\n

Working with University of Georgia Marine Extension and Georgia Sea Grant, Dellinger and his collaborators constructed four outdoor pens from PVC pipe and crab trap wire in a pond located at the 4-H Tidelands Nature Center on Jekyll Island.<\/p>\n\n\n\n

The team monitored 40 crabs over the course of several months. \u201cThis was something totally different than I\u2019ve ever worked with,\u201d says marine resource specialist Lisa Gentit, who assisted with the fieldwork.<\/p>\n\n\n\n

For comparison, Dellinger\u2019s team also investigated crabs raised indoors in a recirculating aquaculture system.<\/p>\n\n\n\n

Their results indicated that the indoor system was superior to the outdoor pens. For one, 100% of the 24 indoor crabs survived. Meanwhile, seven of the crabs raised outside died, likely because water temperatures got too warm and dissolved oxygen got too low, according to Gentit.<\/p>\n\n\n\n

Outdoor aquaculture also posed logistical challenges. \u201cIt was really difficult to find [the crabs] in the pens, because they will bury up into the mud and sand,\u201d Gentit explains. \u201cSo, you had to basically dive under and feel around in the dirt, in the sediment, to find them.\u201d<\/p>\n\n\n\n

\"Working
Working with the University of Georgia Marine Extension and Georgia Sea Grant, the Kepley Biosystems Inc. team built outdoor aquaculture pens for horseshoe crabs. Photo courtesy of University of Georgia Marine Extension and Georgia Sea Grant<\/figcaption><\/figure>\n\n\n\n

Dellinger maintains that outdoor aquaculture has advantages, but it \u201cintroduces uncontrollable variables, like weather and temp,\u201d he says. In indoor systems, crabs can be closely monitored and their diets controlled.<\/p>\n\n\n\n

As Gauvry of ERDG sees it, raising crabs and bleeding them all in the same facility could help preserve their well-being.<\/p>\n\n\n\n

\u201cAnytime that you take a wild animal out of its natural habitat, you\u2019ve interjected stress,\u201d says Gauvry, who serves with Dellinger on an IUCN work group focused on the horseshoe crab trade. \u201cAnthony\u2019s approach eliminates that. These animals become acclimated to the environment that they\u2019re in,\u201d at least theoretically, he says.<\/p>\n\n\n\n

Dellinger\u2019s team has also been developing a nutritional regimen that has so far proven to ensure horseshoe crab vitality and LAL quality. At this article\u2019s writing, the journal Frontiers in Marine Science<\/em> was reviewing a paper on that work.<\/p>\n\n\n\n

Dellinger envisions ultimately building multiple horseshoe crab aquaculture facilities for LAL production. Growers could be trained to raise the animals, while traveling phleboto- mists could circulate around the facilities to bleed them on a rotating basis, he explains.<\/p>\n\n\n\n

Achieving that goal would require substantial investment, Dellinger acknowledges. And it\u2019s possible that, in the meantime, an alternative assay that already exists \u2014 one that negates the need for horseshoe crabs altogether \u2014 will become more widely adopted. That assay uses material called recombinant Factor C, or rFC, but its use in endotoxin testing is not standard in the U.S.<\/p>\n\n\n\n

For his part, Dellinger says that demand for LAL \u201cis likely going to increase\u201d and thinks Kepley could satisfy that market. \u201cWe\u2019ve always been of the opinion that if you could do it very thoughtfully and consciously and optimize the aquaculture, then there\u2019s not necessarily a need to transition away from LAL, especially if you can use a fraction of the crabs,\u201d he says. \u201cYou would still have this perfect end product, just in a more sustainable form.\u201d<\/p>\n\n\n\n

Access the PDF version of this article<\/strong><\/a>.<\/strong><\/p>\n\n\n\n

Supplemental content for grade 6-12 educators accompanies this article<\/a>.<\/strong><\/p>\n\n\n

Related<\/h2>
\n Fall 2020 Issue<\/span>\n\t\t\t\n\t\t\t\n\t\t<\/svg><\/span>\n <\/a>\n <\/div><\/div>\n\n","protected":false},"excerpt":{"rendered":"

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