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, hundreds of thousands of horseshoe crabs are rounded up and
brought to the lab - not to be killed, but for their blood to be carefully extracted.
These animals, often called living fossils, are one of the ‘oldest’ creatures on the planet.
They have remained nearly unchanged since they first appeared on earth over 450 million years
ago. This is due to some exceptionally effective adaptations, and genes that code for remarkable
molecules that have allowed the horseshoe crab to survive, just as it is, for so long.
One of these ancient compounds is the reason that hordes of these animals are dredged up
from the ocean, jabbed with a hypodermic needle, and their blue blood drained,
processed, and sold. Their blood, made blue from a copper-based oxygen-carrying molecule, is so
valuable that it is the basis for a multi-million dollar pharmaceutical industry. So valuable,
that a single liter of it goes for around $16,000 - one of the most valuable liquids on earth.
Our reliance on these animals puts immense pressure on a fragile ecosystem,
and 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, and why can we only seem to get it from this one creature?
The American horseshoe crab (on screen: Limulus polyphemus) is an ancient, aquatic arthropod.
They belong to their own class of animals, called Merostomata, and are not actually 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.
The modern horseshoe crab as we know it has technically only been around for 20 million years,
but some of its early relatives, like Limulus darwini, existed around 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.
Here’s some perspective on just how long ago horseshoe crabs came into existence. Pangea,
the supercontinent of the past 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 into, and emerged from, 2 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 reason natural selection has preserved them,
pretty much as they are, is their hardy body plan.
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 9 eyes - 2 compound eyes which
act much like our own eyes, 5 secondary simple eyes on top of their shell which can detect UV
light, and 2 ventral eyes located on their underside, perhaps to help with orientation.
Along with their complex circulatory system, 5 pairs of gills, and 12 bristled legs, evolution
created them to be creatures extremely well adapted to their particular environmental niche.
But beyond the physical traits that we can observe, much of their survival is due to
something we can’t see - their incredible, but simple immune system. It’s protected them as a
species from bacterial infection for eons. It works in an entirely different way from ours,
and in the late 1960s, we began to harness its power for ourselves.
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 revolutionise drug safety testing forever.
Pretty much every creature in the world is vulnerable to bacterial
infection - and the horseshoe crab is no exception. Once an infection begins,
bacteria can reproduce quickly, and many give off toxins which 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 your nerves, paralyze you, and even kill you.
Toxins like this are called exotoxins. They are released from live bacteria into the
surrounding environment during an infection. But, bacteria don’t have to release these
exotoxins in order to be dangerous. In fact, they don’t even have to be alive.
Once a bacteria is killed within the body, they sometimes release endotoxins.
Endotoxins are the lipid portions of lipopolysaccharides (LPSs) that are
part of the outer membrane of the cell wall of some bacteria. 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 can be deadly.
But, fighting off these types of infections is what immune systems are for.
Immune systems have developed to protect all different kinds
of organisms from foreign pathogens. And during the course of evolution,
two different kinds of general immune system 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, and
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 the identification of general threat.
The basis for a horseshoe crab’s immune response are cells called granular amoebocytes.
When bacteria come into contact with a horseshoe crab’s blood,
they trigger an enzyme cascade, mediated by these amoebocytes,
which 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 very effective way for the horseshoe crab to defend itself from infection.
And, as researchers began to realize in the 1960s, it’s a very effective way for
us to detect the presence of toxins in places where we really, really don’t want them to be.
When creating injectable healthcare products like vaccines, medical implants, and IVs, it is
imperative that they are free of any invading microbes. It’s easy enough 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 was present
before sterilization, the endotoxin will remain, and can lead to severe consequences if injected.
Historically, pharmaceutical companies got around this problem with huge colonies
of rabbits - needed for what’s called the rabbit pyrogen test.
To see if a product or drug is contaminated with endotoxin, three (unlucky) rabbits would
be injected with a small amount of the drug or product in question and monitored
for four hours. Rabbits have a similar pyrogen tolerance to humans, so if any develop a fever,
the batch would be considered to be contaminated with bacterial endotoxin. This is an effective
way of preventing endotoxins from accidentally being injected into the public, but because it
is an in-vivo test - meaning done inside a living organism- it’s very time-consuming and expensive.
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 way of spotting contamination - a much cheaper, easier, and faster test.
This in vitro test is called the LAL test - limulus amoebocyte lysate. Limulus being
limulus polyphemus, the american horseshoe crab. This test has become the worldwide standard for
screening for bacterial contamination. It is capable of detecting 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 that for the LAL test.
Upon exposure to endotoxins, the amebocytes undergo a rapid enzyme cascade
that causes the cells to stick together and form a thick clot.
This clot can form in around 90 seconds, giving a nearly instant result.
We’ve never found anything that is as sensitive in detecting endotoxin than
the horseshoe crab’s amoebocytes. If there are dangerous bacterial endotoxins—even
at a concentration of one part per trillion - a clot will form and can be detected.
This is great news for us. Pretty much every single person who has ever had an injection
of any sort has been protected because of this compound from this strange, ancient creature. The
only problem is that for this test to be readily available, pharmaceutical companies need a large
supply of the blood of live crabs - which, as you’d guess, is not such great news for the crabs.
In theory, the process of extracting blood from the horseshoe crabs does not kill them.
It’s sort of like blood donation, albeit a nonconsensual one. And once their blood is taken,
the crabs are released in 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 - and 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.
Between 10 and 30 percent of the bled animals, according to varying estimates, actually die.
And 30% of the animals per year dying equates to losses in the hundreds of thousands.
And this isn’t just bad for the horseshoe crab, but for the entire ecosystem in which they live.
Many other species of 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 certainly been trying - and luckily for the
crabs they have started to have some success.
In 1995, scientists from the National University of Singapore were finally able to
identify and isolate the gene responsible for the endotoxin-sensitive protein called Factor C – the
most important component in the LAL test – and produce it in yeast. Several years after that,
they were able to create 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 the synthetic protein as an alternate endotoxin detection until 2015,
and the FDA in the US did not approve the first drug that used an endotoxin test based on the
synthetic protein until 2018. And 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 that its safety is still unproven.
For now, we still need the horseshoe crab and their baby blue blood.
But as more and more studies come out that demonstrate the safety of the synthetic
version of the endotoxin test, the horseshoe crabs can breathe a bit of a sigh of relief.
While they still face threats from overfishing for bait and habitat destruction,
the adoption of this technology will relieve at least one major pressure.
Our medical need for horseshoe crabs is what 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
first days of the horseshoe crab’s ancestor, they have faced - and survived - all FIVE
mass extinctions. These extinction events are defined as the loss of least 75 percent
of species, happening in the geological blink of an eye. Volcanoes erupting, oceans warming,
ice sheets forming, or oceans acidifying - the great die-offs result from a perfect
storm of multiple calamities. The horseshoe crab and its ancestors were one of the few
creatures to survive - but if so many things die, how does life rebound to flourish again?
This is the question that researchers at the University of Oslo are trying to understand,
and is the focus of the documentary “Breakthrough: Recovering From Extinction” on CuriosityStream.
They are pioneering an investigation about what survived, and what emerged
after the largest mass extinction on our planet, 252 million years ago..
This is one of many paleontology documentaries on CuriosityStream, which are all really good.
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