It’s a June summer evening in Delaware, and as Slaughter Beach slowly empties of beachgoers, a different crowd takes their place. It’s horseshoe crab mating season, and thousands of these prehistoric creatures are emerging onto beaches all along the eastern US coastline.

This emergence also signals open season for horseshoe crab harvesting. Each year, an estimated 1 million of these crabs are collected for their blood, which has unique properties that have probably saved your life at some point. But what is it that makes these creatures’ blood so special?

Are they actually crabs?

Despite their name, horseshoe crabs are not crabs at all. They belong to a separate group of arthropods, called chelicerates, which are more closely related to spiders. They are one of the oldest existing species and have lived on Earth for approximately 450 million years, surviving five mass extinctions, all while remaining largely unchanged. In fact, they are so old their blood isn’t based on iron like ours, but rather copper, making it bright blue.

It’s this blood that spurred the creation of the horseshoe crab fishery industry. Since the 1970s, the pharmaceutical industry has relied on horseshoe crab blood to ensure vaccines and other injections are pathogen-free, thanks to their unique immune response.

Why is horseshoe crab blood so special?

While we have a neat and tidy closed-circulatory system, consisting of blood vessels, horseshoe crabs have an open-circulatory system; their blood sloshes freely around inside their body cavity. While this allows these bottom dwellers to withstand the high pressures of the ocean floor, it leaves them vulnerable to infections that can rapidly spread.

To combat this, the horseshoe crab has amoebocytes (their version of our white blood cells) packed with granules of coagulogen. When an amoebocyte encounters endotoxins found in the cell membrane of a bacterium, it releases these granules, triggering an enzymatic cascade that results in the formation of a gel around the invading endotoxin. This clot immobilises the offender, rendering the pathogen harmless.

This clotting mechanism is used by scientists as a convenient visual confirmation of endotoxins in a sample. Thus, the Limulus Amebocyte Lysate (LAL) extract was born (or the Tachypleus Amebocyte Lysate (TAL) test when harvested from the Asian, tri-spine species). Prior to this test, scientists would inject large numbers of rabbits with the drug to be tested and wait to see if they developed an infection. The LAL test was believed to be a less wasteful and cruelty-free process. This, however, is not the whole story. 

How is horseshoe crab blood harvested?

Horseshoe crabs are caught, bled in a lab and then released back into the wild. But while we walk away from donating blood happy and healthy, around 15–30% of these horseshoe crabs die. This is because about 30% of their blood volume is harvested (the average human blood donation is about 10%). Studies show that this bleeding can reduce mating activity, lower immune function and increase vulnerability to predation.

Additionally, harvesting practices are poorly regulated in many areas. An investigation by NPR found that, in the US, fishermen hired by bleeding companies were handling horseshoe crabs badly and, in some cases, even violating harvesting laws. Internationally, horseshoe crabs are also popular targets for smugglers due to the high price fetched by their blood at around $16,000 (£11,770) per litre.

These effects point to an industry contributing to an ongoing decline in global horseshoe crab stocks due to a combination of overfishing, climate change and habitat loss. In their former stronghold in the Delaware Bay, numbers have dropped by two-thirds and in China the region’s own species of horseshoe crab (the tri-spine horseshoe crab) has declined by 90% in some areas, a trend reflected throughout Asia.

These trends were thought to have been made worse with the rapid surge in demand for LAL testing for Covid-19 vaccines. To date, the tri-spine horseshoe crab is classified as endangered, while the American species is listed as vulnerable.

What are the alternatives to wild harvesting?

The Covid-19 surge spurred some groups to consider aquaculture. Anthony Dellinger’s group at Kepley BioSystems found that aquacultured horseshoe crabs could be bled at 10% of their volume up to 24 times a year, with 100% survival. According to their 2020 study, around 60,000 crabs could exceed current biomedical demand.

So why haven’t more companies adopted this approach? Horseshoe crabs are expensive to farm and take 10 years to mature. Furthermore, captive nutritional demands are difficult to meet, accounting for mortality within 3–4 months of captivity. Dellinger attributes his group’s success, in part, to their focus on proper nutrition.

Are there alternatives to horseshoe crab blood?

The other option is to synthetically recreate the assay ourselves. And that’s precisely what happened in the late 1990s when biologists at the University of Singapore developed recombinant factor C (rFC), modelled off the starting point in LAL’s clotting cascade, clotting factor C.

Studies have found rFC assays to be comparable to, and in some cases even more effective than, LAL for bacterial endotoxin testing.

Proponents of the synthetic alternative have also highlighted that not only would rFC remove the pharmaceutical industry’s reliance on a dwindling resource, but the production process means rFC can be produced consistently around the world with less variation between batches than wild-harvested LAL. rFC testing also costs less to run, takes half the time and produces less waste.

Another, less common, alternative is recombinant cascade reagents (rCR), the difference here being that the reagent contains not just factor C, but also factor B and the pro-clotting enzymes derived from horseshoe crab blood.

ToxiSense is another alternative in development. Recent University of Pennsylvania graduate Aaravind Krishnan has spun out a company to develop a plant-based alternative for endotoxin testing. He started his work at the age of 14 when he learned about horseshoe crab harvesting and is working with the plant Arabidopsis thaliana, which can be genetically engineered to respond to endotoxins by emitting light. The new alternative is set to be a bargain too, at just $1 per test.

Where have these alternatives been adopted?

While Europe, China and Japan have all adopted the recombinant reagents, the US has been dragging its feet, stating there is not enough evidence to prove that they’re a viable alternative to the traditional LAL assay. Despite this, Eli Lilly has been using rFC since 2016 after enduring lengthy additional procedures to achieve FDA approval. It currently conducts 80% of its testing with rFC, and most notably produced all its Covid-19 vaccinations using the synthetic alternative.

Fortunately, in November 2024, the US Pharmacopeia finally issued guidelines on the use of recombinant reagents for endotoxin testing . It’s a move that will hopefully spur other pharmaceutical companies to follow Eli Lilly. However, given the cost and retraining involved, it may only be feasible for large pharmaceutical companies at the moment. The more recently developed rCR assay holds promise too, but with a shorter history and less published research supporting its use, it is still gaining momentum within the field.