Every year hundreds of thousands of horseshoe crabs arrive on the beaches of the Atlantic coast of America to lay their eggs. And every year, thousands of horseshoe crabs are rounded up and brought to the lab. It is because of their blood. These animals, often called living fossils, are among the ‘oldest’ creatures on the planet.
They have remained nearly unchanged since they first appeared on earth over 450 million years ago. Due to some exceptionally effective adaptations and genes that code for remarkable molecules. That has allowed the horseshoe crab to survive, as it is, for so long. One of these ancient compounds is why hordes of these animals are dredged up from the ocean, jabbed with a hypodermic needle, their blue blood drained, processed, and sold.
Why is horseshoe crab blood so expensive?
Horseshoe crab blood is made from a copper-based oxygen-carrying molecule that is so valuable. It is the basis for a multi-million dollar pharmaceutical industry. So valuable that a single liter of it goes for around $16,000. It is one of the most valuable liquids on earth.
Our reliance on these animals puts immense pressure on a fragile ecosystem. So far, scientists have struggled to recreate this compound and its effects in the lab. What is it about this primitive compound that we need so badly?
The American horseshoe crab Limulus polyphemus is an ancient aquatic arthropod. They belong to their class of animals, called Merostomata, and are not crabs. They are more closely related to scorpions, with their predecessors diverging from their arachnid cousins around 480 million years ago. Some recent studies even suggest they are arachnids.
Their incredible immune system protects them as a species from bacterial infection for eons. In 1968, two researchers at the Marine Biological Laboratory in Massachusetts observed that blood cells from horseshoe crabs vigorously clot in the presence of bacterial endotoxin. When they published their paper, they had no idea that what they found would revolutionize drug safety testing forever.
Every creature in the world is vulnerable to bacterial infection – and the horseshoe crab is no exception. Once a disease begins, bacteria can reproduce quickly, and many give off toxins that damage specific tissues in the body.
Botulism, for example, is an illness caused by a neurotoxic protein produced by a bacteria called Clostridium botulinum. The toxin can affect nerves, paralyze and even kill you. Toxins like this are called exotoxins. They are released from live bacteria into the surrounding environment during infection. But, bacteria don’t have to release these exotoxins to be dangerous. They don’t even have to be alive.
Once bacteria are killed within the body, they sometimes release endotoxins. Endotoxins are lipopolysaccharides (LPSs) lipid portions that are part of some bacteria’s outer cell wall membrane. The endotoxins are released when the bacteria die, and the cell wall breaks apart.
This toxin is a pyrogen – a fever-causing agent. If it gets into the bloodstream, it can lead to septic shock and be deadly. But, fighting off these infections is what immune systems are for. Immune systems have been developed to protect all different organisms from foreign pathogens. During evolution, two different general immune systems emerged within multicellular organisms.
Humans and many other vertebrates have adaptive immune systems that protect us by strategically mounting a defense against invading bacteria. It is activated by exposure to pathogens. It uses an immune memory to learn about the threat and enhance the immune response accordingly. But many invertebrates, including horseshoe crabs, don’t have this adaptive immunity. Instead, they have an innate immune system, which attacks based on identifying the general threat.
The basis for a horseshoe crab’s immune response are cells called granular amoebocytes. When bacteria contact a horseshoe crab’s blood, they trigger an enzyme cascade mediated by these amoebocytes. It causes the blood in the immediate area of the infection to clot into a gel. The gel surrounds and isolates the infection from the rest of the crab, and the pathogens are neutralized. The clotting from granular amoebocytes is a simple but effective way for the horseshoe crab to defend itself from infection.
Creating injectable healthcare products like vaccines, medical implants, and IVs must be free of invading microbes. It’s easy to sterilize the solutions or devices by blasting them with heat, radiation, or gas that is deadly to bacteria. But killing bacteria isn’t enough to make these products safe. If certain bacteria were present before sterilization, the endotoxin would remain. And it can lead to severe consequences if injected.
- Historically, pharmaceutical companies got around this problem with huge colonies of rabbits – needed for the rabbit pyrogen test.
Three rabbits would be injected with a small amount of the drug or product in question and monitored for four hours to see if a product or drug is contaminated with endotoxin. Rabbits have a similar pyrogen tolerance to humans. So if any develop a fever, the batch would be considered contaminated with bacterial endotoxin. It is an effective way of preventing endotoxins from accidentally being injected into the public.
So when researchers noticed the clotting effect of the horseshoe crab’s amoebocytes in the presence of endotoxin. They realized it could be an in vitro spotting contamination, a much cheaper, easier, and faster test. This in vitro test is called the LAL test, Limulus amebocyte lysate. It is Limulus polyphemus, the American horseshoe crab. This test has become the worldwide standard for screening for bacterial contamination. It can detect endotoxin at significantly lower levels than the rabbit pyrogen test.
Today, every drug certified by the FDA must be tested using LAL, as do surgical implants such as pacemakers and prosthetic devices. After the horseshoe crabs are brought to the lab, the tissue around their heart is pierced with a needle. And up to 30 percent of their blood is drained.
The amoebocytes in the blood are then extracted from the LAL test. Upon exposure to endotoxins, the amebocytes undergo a rapid enzyme cascade. It causes the cells to stick together and form a thick clot. This clot can form in around 90 seconds, giving a nearly instant result.
Scientists have never found anything as sensitive in detecting endotoxin as the horseshoe crab’s amoebocytes. If there are dangerous bacterial endotoxins, a clot will form and be detected even at a concentration of one part per trillion. It is excellent news for us.
Every single person who has ever had an injection of any sort has been protected. It is because of this compound from this strange, ancient creature. The only problem is that pharmaceutical companies need a large supply of the blood of live crabs for this test to be readily available.
In theory, extracting blood from the horseshoe crabs does not kill them. It’s like blood donation, albeit a non-consensual one. And once their blood is taken, the crabs are released to a new location. So they do not accidentally get caught a second time, ensuring they have a chance to recover. Their blood volume rebounds in about a week.
The LAL industry states that there are no long-term ill effects for the crabs. They measured mortality rates of less than 3%. But conservationists tell a different story. According to varying estimates, 10 and 30 percent of the bled animals die. And 30% of the animals per year dying equates to losses in the hundreds of thousands. This isn’t bad for the horseshoe crab but for the entire ecosystem they live.
Why do we harvest horseshoe crab blood?
Many other animals rely on the horseshoe crabs’ eggs for food, like shorebirds and turtles. So the obvious question is – why haven’t scientists made a synthetic alternative to LAL? Since the 1970s, they have been trying – and luckily for the crabs, they have started to have some success.
In 1995, National University of Singapore scientists finally identified and isolated the gene responsible for the endotoxin-sensitive protein called Factor C. The essential component in the LAL test and produced in yeast. Several years later, they created a rapid endotoxin test based on this recombinant protein.
But despite these advances, these synthetic tests are still not widely available. They have been adopted extremely slowly due to regulatory and safety concerns. Europe did not recognize synthetic protein as an alternate endotoxin detection until 2015. The FDA in the US did not approve the first drug that used an endotoxin test based on synthetic protein until 2018.
Earlier this year, the American Pharmacopeia, which sets the scientific standards for drugs and other products in the U.S., declined to place the synthetic protein on equal footing with crab lysate, claiming its safety is still unproven. For now, we still need the horseshoe crab and their baby blue blood. But as more and more studies demonstrate the safety of the synthetic version of the endotoxin test. The horseshoe crabs can breathe a bit of a sigh of relief.
How did horseshoe crab’s blood evolve?
The modern horseshoe crab has technically only been around for 20 million years. But some of its early relatives, like Limulus darwini, existed about 150 million years ago and look nearly indistinguishable from today’s horseshoe crabs. The iconic body plan has been around even longer, emerging around 450 million years ago. The changes within the horseshoe crab group have been shockingly minor in the big picture of evolution.
Pangea, the last supercontinent, formed 335 million years ago and began to break apart about 175 million years ago. The non-avian dinosaurs emerged 245 million years ago and were wiped out 66 million years ago. The earth descended and emerged from two completely different ice ages since these crabs came about. The world has changed so much since then. And all the while, horseshoe crabs have been here, crawling along the seafloor, standing the test of time. Some of the reasons natural selection has preserved them.
Their hard shell, called a carapace, is an exoskeleton so strong that only sharks or turtles can penetrate it. And guiding them through the ocean depths are nine – 2 compound eyes that act much like human eyes.
- 5 simple secondary eyes on top of their shell can detect UV light.
- 2 ventral eyes are located on their underside, perhaps to help with orientation.
Along with their complex circulatory system, five pairs of gills, and 12 bristled legs, evolution created them to be creatures exceptionally well adapted to their particular environmental niche.
Will horseshoe crabs go extinct?
Horseshoe crab still faces threats from overfishing for bait and habitat destruction; adopting this technology will relieve at least one significant pressure. Our medical need for horseshoe crabs has started to push these animals towards extinction in recent decades. But this is not the first time they have faced such a profound threat.
Since the horseshoe crab’s ancestors’ first days, they have faced and survived – all FIVE mass extinctions. These extinction events are defined as losing at least 75 percent of species in the geological blink of an eye. Volcanoes are erupting, oceans are warming, ice sheets are forming, or oceans are acidifying. The great die-offs result from a perfect storm of multiple calamities.
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Kōichi Sekiguchi, Biology of Horseshoe Crabs. Science House. ISBN 978-4-915572-25-8.
Stine Vestbo; Matthias Obst; “Present and Potential Future Distributions of Asian Horseshoe Crabs Determine Areas for Conservation.”
Smith, D.R.; Beekey, M.A.; “Limulus polyphemus.” IUCN Red List of Threatened Species. IUCN. 2016: e.T11987A80159830.
Attaya Kungsuwan; Yuji Nagashima; Tamao Noguchi; “Tetrodotoxin in the Horseshoe Crab Carcinoscorpius rotundicauda Inhabiting Thailand.”