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. And now, CuriosityStream has partnered
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The first thing I thought of upon seeing the video title was what I learned in my module lol. (the thing about the rabbit test and the LAL test)
but yay for Singaporean research