The Crab and the Hare: No Finish Line in Sight

Every time there are whispers of a new, dangerous virus that might become the next pandemic, scientists and researchers across government, the pharmaceutical industry, and universities rush to find the cure. While they are not always successful, sometimes preventative measures can be synthesized such as vaccines. Before these vaccines can be approved and released, they have to be tested. However, in an age where vaccine incredulity has gained momentum, the introduction of an inadequately tested vaccine not only risks public health, but can also amplify distrust, potentially undermining confidence in even the most rigorously vetted immunizations. One of these tests is for something called endotoxins, which are simply a type of poison that is inside bacterial cells. Endotoxins are responsible for toxic effects such as fever, multiorgan failure, septic shock, and sepsis. The modern testing for endotoxins involves using horseshoe crab blood and was actually found by accident. This blood testing replaced the original testing, named “rabbit pyrogen testing” (RPT), which used rabbits as subjects receiving clinical trials of medicine as human analogs because rabbits and humans have similar temperature responses when exposed to pyrogens. Endotoxins are a type of pyrogen; which is just a substance that can cause a fever in a living organism by stimulating the body’s immune system. Scientists moved away from RPT for a few reasons: the testing is scientifically limited, there are some non-human specificity elements, and superior alternatives were beginning to appear. But one reason catapulted all the others: public awareness. People were exceedingly upset about animal testing, from makeup to pharmaceuticals. This awareness and hostility that arose from it were not something organizations could afford to take lightly if they wanted to continue garnering respect and trust in their fields and among the public.

Modern testing involves using horseshoe crab blood as a ‘screener’, because the blood clots when exposed to endotoxins in order to protect the crab. This testing method is referenced as Limulus Amebocyte Lysate testing, or LAL. LAL testing has been irreplaceable for medical advances such as joint replacements, pacemakers, the flu shot, even testing the COVID-19 vaccine—that crab does a lot for us (Maloney et al.). But are we treating the horseshoe crabs humanely? Federal law does require some animals that are used for biomedical reasons to be treated humanely through the Animal Welfare Act, which mostly protects warm blooded animals like monkeys. This act doesn’t cover horseshoe crabs, leaving the Health Research Extension Act to cover other vertebrates that weren’t originally protected (Eisner).  In order to get all of this blood to test with, horseshoe crabs without visible injuries are strapped down to receptacles and hooked up to a line of other crabs, with a substantial amount of their blood harvested, drained right from them. Not all of their blood, but they can be drained of up to 30% of their blood at a time (Rozenbaum). The actual harvesting of the blood involves lab technicians piercing the crabs with a large gauge needle through the heart to drain them alive (Fultonk). This bleeding can sometimes last for as long as eight minutes, which may accidentally deplete the crab of more than half of their blood supply (Eisner). While many crabs do die in this process—which is an unfortunate expectation—around 15-30% of the approximately 700,000 crabs taken from beaches during spawning season die after being released back into the ocean, too. The bleeding process can injure the crabs or leave them disoriented and lethargic, which puts them at a higher risk of predation (Horseshoe Crab). 

When I was younger you could find me playing in the sand, chasing the birds, and flipping the upside-down horseshoe crabs over so they could return to their own home. My parents believe that a love of nature is crucial, to immerse oneself in it because you live in it. As I got older, I saw fewer and fewer crabs on the beach until one year when I went out on the sand; chillingly there were none. 

While medical testing is not going away anytime soon, some actions should still be taken to protect these indispensable animals. I am not saying we should abolish testing with crabs altogether; but am instead proposing that we turn to a synthetic method as the primary method because if we lose the crabs to extinction, our current precise and accurate way of testing for endotoxins will disappear forever, and that could lead to unnecessary challenges. Adopting alternative endotoxin testing methods is crucial for protecting horseshoe crab populations, ensuring ecological balance while still meeting biomedical needs.

Two alternative methods of medical testing that show similarly great promise to LAL (horseshoe crab) testing are Recombinant Factor C (rFC) and Recombinant Cascade methods. Despite arguments against them, these current alternatives are available for use, and they work just as well as LAL, if not better. Recombinant Factor C (rFC) and Recombinant Cascade reagents (rCR) are two alternative methods that do not use horseshoe crab blood (Endotoxin Testing Alternatives).  LAL testing is undoubtedly the “gold standard” in pharmacology, but how do the alternative testing methods compare? In a six-year study conducted between 2014 and 2019, researchers from Microcoat Biotechnologie GmbH evaluated the performance of Limulus Amebocyte Lysate (LAL) and recombinant Factor C (rFC) assays in detecting bacterial endotoxins. Findings revealed that rFC-based assays were not only comparable to LAL in meeting the acceptance criteria outlined by compendial bacterial endotoxin testing, but in many cases, they demonstrated superior endotoxin recovery rates. Unlike LAL, which can be affected by sample impurities and environmental conditions such as pH and temperature—factors that disrupt its complex reaction cascade—rFC assays offer a more targeted and reliable approach. By eliminating all non-essential components of Horseshoe Crab blood, rFC methods achieve high specificity for endotoxins, making them a finer alternative to traditional LAL testing (Piehler, Maike, et al.).

Alongside rFC, recombinant cascade reagents (rCR) offer a second strong alternative to traditional LAL testing for bacterial endotoxin detection. Like LAL, rCR uses the same key clotting factors—Factor C, Factor B, and the proclotting enzyme—but produces them through a recombinant process, making the method completely animal-free. Unlike single-protein recombinant methods, rCR replicates the full enzymatic cascade of the natural LAL reaction, allowing it to achieve broad sensitivity across diverse sample types and greater assay robustness. It uses a traditional kinetic assay format on an absorbance reader, making it compatible with existing lab workflows. Importantly, companies adopting rCR must undergo the same rigorous validation and equivalency testing required for rFC, ensuring regulatory confidence. By combining ethical advantages with strong technical performance, rCR also stands out as a reliable and innovative option for endotoxin testing. Together, rFC and rCR demonstrate that recombinant technologies not only uphold the standards of traditional LAL testing—they often surpass them in specificity, consistency, and ethical responsibility (Endotoxin Testing Alternatives).

Given the concerns surrounding rFC and rCR, you might be wondering whether there’s another species suitable for medical testing that wouldn’t face the same risk of extinction. In fact, this debate has already partially unfolded in discussions about the use of rabbits. 
Way back when before LAL testing, we used rabbits, in what we called “rabbit pyrogen testing” (RPT). From the 1940s to late ‘70s, rabbits were used to test for endotoxins (Biomedical Use of…). The Rabbit Pyrogen Test (RPT) is a biological assay that detects the presence of pyrogens by measuring temperature changes in rabbits after intravenous injection of a test sample. The test produces qualitative results and has relatively low sensitivity. Its reliability can be affected by factors such as reduced responsiveness in rabbits due to repeated testing or stress during the procedure (Pyrogen Testing). Despite its long-standing use, RPT poses ethical concerns and practical challenges, leading to the exploration of alternative methods through other animals. The main reason they were used is that rabbits have easy to identify symptoms of endotoxin poisoning; they might develop a fever, go into respiratory stress or shock. After a certain “usage period”, the rabbits are killed (Preller). This specific type of testing started to slow in the 1970s because of the horseshoe crab, but not for one reason alone. 

Primarily, the horseshoe crab blood is more sensitive to the presence of endotoxins. It’s also just more convenient than administering a bunch of shots to rabbits and writing down how they behave. The blood is less expensive to obtain, allowing LAL to be deemed an overall better test. Governing bodies such as the FDA wanted to replace the rabbit pyrogen testing with the LAL testing more rapidly, but there were arguments over different aspects of whether it actually worked, so serious validation testing had to occur over a span of 20-30 years before it was implemented as valid test (Liebsch). Let us not forget another reason why this testing may have switched: the fuzz factor. Most people would agree that bunnies are cute, right? And seeing an animal—a hopping, floppy-eared puffball at that—would make many individuals feel adversely about what is being done to these seemingly cute animals, leaving them with feeling they would rather act in accordance with looking after or protecting them (Knight). With crabs and other animals that have an obscene number of legs, many are pushed away by the “creepy crawly” aspect of them. Considering that horseshoe crabs are more closely related to arachnids than actual crabs, the fear that many hold towards them shouldn’t be brushed off as baseless. Just for the record—horseshoe crabs are fairly defenseless, too. They can’t hop away quickly like a rabbit, in fact they can’t move very fast at all. The one quick way they can move is forcing water through their gills to propel themselves, and if stuck out of the water they have to crawl everywhere, dragging that big shell around with them. Most of the time, animals that people don’t necessarily interact with regularly or are afraid of are not at the forefront of their minds as notable animals to save, if they cross someone's mind in the first place.

With the switch from rabbit pyrogen testing to horseshoe crab blood, there is still unrest between animal loving communities and those who want the crabs for the profit. Synthesized alternatives to animal testing have been in the works for some time now, but aside from the fact that corporations are slow to trust, removing animal testing entirely is proving to be a challenge. One challenge is simply the fact that animal testing is present. With the presence of a practice that is considered taboo despite its commonplace in society today, the public opinion of animal testing is closely divided, with some 47% favoring the practice, while a nearly equal share (50%) oppose it (Funk). The doubt stirred by this practice hurts corporations' abilities to gain trust from people. Even if everyone had a decided opinion on the subject, the murky opinions make movement in any direction, whether it be to abolish the practice or upscale it, difficult.

One of the reasons companies have been loath to cease horseshoe crab use is the profit from doing so compared to the profit from using synthetics.  A quart of horseshoe crab blood has an estimated price tag of $15,000 although some researchers say it is impossible to put a rigid figure on it (Fultonk). To utilize the crabs, researchers need to obtain permits and adhere to regulations, and not necessarily pay obscene amounts of money for developing something that may or may not work the way they want it to. But, why would they need to pay when some alternatives–like rFC–have been around for nearly 20 years? Profit is unfortunately still the answer here. rFC was patented and licensed to only one company, which may be one reason for the reluctance of drug companies to adopt its use (Schweitzer). Competitive industries don’t compete if they’re “relying on one source for an essential testing reagent with no competition to temper the cost” (Schweitzer). Pharmacopeia requires case-by-case validation for usage of the alternatives, which is an immensely expensive and time consuming process—not necessarily the kind of cause you see people lining up to donate for. Many people hesitate due to the extensive validation burden imposed by pharmacopeial standards. Any alternative must demonstrate fitness for use along with comparability between their new testing and LAL testing. USP emphasizes that this is normal testing; validation must be tailored to the specific product and testing context (Chapter 86 Frequently). Alongside these standards, supplemental data may need to be provided for regulatory authorities which is lengthy and expensive in and of itself. As outlined in industry guidance, demonstrating equivalence to compendial methods like LAL requires but is not limited to identification of a potentially suitable alternative methodology, demonstration that the method is equivalent and applicable as a replacement for a standard compendial method, development of user specifications for equipment selection, and qualification of the method in the laboratory. Along with these broader progress markers, there are subsections to each area of testing that make the data collection process alone tedious (USP Chapter 1223 Validation).

Today, a portion of companies say that today it’s no longer necessary to use horseshoe crabs, and that "[it is] beyond time to transition to a sustainable alternative for this critical step in drug- safety testing” (Michigan). Some companies have even found synthetic alternatives to be more cost-efficient and effective, although others still say that LAL producers are purposefully stalling the approval process of synthetic alternatives to protect their market, which with the medical advances of the past few decades, has morphed into a growing monetary enterprise. 

There is an undeniable need for a safety test that is at least as effective as LAL testing, if not more so, to ensure that medical treatments remain safe and effective without relying on the declining population of horseshoe crabs. Some regulatory bodies and individuals argue that the synthesized alternative does not help, stating that the man-made alternative doesn’t cut it (Delbert). While the alternatives have been found to be helpful in testing for endotoxins, the testing with actual blood is broader, helping to detect more potential issues in the medicine. To even think about synthesizing something like blood, despite the medical marvels of the modern world, is still too far a reach. In today's world, blood cannot be copied. Circling back to the opinion that synthetics shouldn’t be encouraged—at least as strongly—is this lack of replicability of the blood. This factor alone is partly why so many feel that the crabs are the only option to test these drugs. Even the USP believes this to some degree, when it “decided to relegate synthetic crab blood to a separate chapter from the rest of the endotoxin testing standards” (Delbert). This means that the synthetic blood got uncategorized as an acceptable endotoxin test, because the USP doesn’t believe that the synthetic fits the standards that other tests have fit.
 

While the testing with real blood is broader, it may be too broad. Research by Jeak Ling Ding of the National University of Singapore shows that rFC is more efficacious than LAL in many respects (Schweitzer). Because the LAL assay is derived from a living organism, the makeup of its blood-based components shifts with the seasons and must be accounted for. The test’s mechanism relies on a chain of reactions involving several molecules that are sensitive to changes in temperature, pH, and the presence of proteins, which can lead to false positives (Schweitzer). Synthesized alternatives without animal parts have been in production for a long time, but corporations are slow to trust them, and they might lead the way for companies to be seen as more principled. Even with the low number of current synthetic alternatives in testing, some are doing well, and are closer to being ready for use than others. Some researchers have found suitable alternatives to replace LAL testing, and one currently being pushed for use is Factor C. Factor C, or rFC, is “an FDA-approved alternative to crab blood, and it does what crab blood does. It offers the same safety testing and can protect species, slows environmental damage and brings other benefits such as supply reliability” (Michigan). This alternative is actively being pushed by some researchers such as Timothy Cernak, a medicinal chemist at the University of Michigan’s College of Pharmacy. Cernak and a growing number of wildlife conservationists, scientists, environmentalists, investors and others are calling for change, for the pharmaceutical industry to turn to the alternative—which we know would “not only would spare the crab population and protect the ecosystem, but create a more reliable, predictable supply chain for the substance needed to test so many medical products for safety” (Michigan).

As I have argued in this paper, what’s being done to protect these crabs now isn’t enough; and steps need to be taken not only to protect the crabs but to make progress towards perfecting testing using synthetic materials. Advancing synthetic testing methods not only relieves harvesting pressure but also safeguards the species’ long-term viability. Preserving healthy horseshoe crab populations enable scientists to deepen their understanding of immune responses—insights that translate towards human medicine. These intertwined strategies of conservation and synthetic innovation create a positive feedback loop, reinforcing both ecological balance and biomedical progress.

As long as modern practices continue, horseshoe crabs will continue to be threatened, putting pressure on their entire species as well as the ecosystem—horseshoe crabs have been dated at least 445 million years back (Evolution of…), making them older than some dinosaurs. These living fossils are essential to the balance of the ecosystem, and without them it could crumble, possibly leading to an untimely and ultimate collapse. In 100 years, maybe even 50, expressing regrets over what could have been done will not be enough; action needs to be taken to safeguard these crabs, securing their future as well as our own. Without change, crabs will continue to be hauled out of the oceans by the thousands, with an estimated minimum of “130,000 [crabs dying] each year in the process” (Cronin). Until we see decisive action or a change of heart from people in power, I fear this topic will remain on the hotplate, with horseshoe crabs’ hearts perpetually under the needle and their species at rising risk of extinction.

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Reflection

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