Horseshoe Crabs Are Bloody Amazing

horseshoe crab chasing endotoxin graphic ftloscience post

On the surface (of the beach), horseshoe crabs might not seem like interesting creatures. Armored crab-like stingrays that placidly slide across the sand, like forgotten relics of a lost age. But there is much more to this unassuming creature than meets the eye. For starters, if you’ve ever had a vaccine shot, then you already owe a debt of gratitude to these horseshoe crabs. For they have literally bled for you. The unique properties of their blue blood make them a truly remarkable asset to healthcare and medicine.

Inside the Horseshoe Crab

The horseshoe crab has a truly unique look: an outer carapace that covers the entirety of its body, five pairs of legs, a rigid tail, along with several groups of eyes thrown in. Despite being named a crab, they actually bear a closer relation to arachnids such as spiders and scorpions1. But we will put aside the fact that the horseshoe crab doesn’t look like a horseshoe, and isn’t really a crab, and focus on a single aspect of this organism: its blood.

Blue Blood

While horseshoe crabs look like extraterrestrial species on the outside, what’s contained within them is even stranger. Cut one open and a light-blue, almost alien-like liquid trickles out. The color of this ‘blood’ is due to the presence of a copper-containing protein–hemocyanin–that can bind to and transport oxygen. In humans, the equivalent is the iron-containing protein hemoglobin. It is the copper-oxygen bond in horseshoe crab blood that gives it a blue color, while our own iron-oxygen bond shows up as red.

Apart from being used for oxygen transport, the blue blood of the horseshoe crab also plays a role in their immune response. You see, they don’t possess an adaptive immune response like our own, that can ‘remember’ pathogens once we have been infected. They do, however, have an innate immunity that is maintained within their blue blood. And it has worked pretty well for them, seeing as they, as a species, have remained relatively unchanged for around 400 million years2!



Coagulogen: The Key Component

This innate immunity comes in the form of a type of cell found in its blood, known as an amebocyte. Amebocytes are able to detect endotoxins, which are chemicals found on the outer membrane of certain species of gram-negative bacteria.

When these amebocytes come into contact with endotoxins, they release the chemical coagulogen. Coagulogen then starts a cascade of defense systems in the horseshoe crab that can neutralize the pathogens. In addition, coagulogen coagulates (as its name suggests) forming a mass around pathogens. By doing so, it confines the bacteria to a local area, preventing its spread to other parts of the crab.


In addition to endotoxins, (1,3)-β-D-Glucan (a cellulose derivative found in most fungi species) is also a target for amebocytes.

Extracting and Using Horseshoe Crab Blood

Limulus Amebocyte Lysate (LAL)

It was only a matter of time before scientists realized that this innate immunity that served the horseshoe crab so well over its evolutionary history could be used to benefit humans. In the 1970s, horseshoe crab blood was put forward as a viable detector of endotoxins in contaminated samples3. Since the coagulation process was almost instantaneous, the reaction upon contact with endotoxins provided both quick and accurate results.

Soon after, a technique was developed to extract coagulogen, the ‘detection’ molecule itself, from amebocytes in the blood. Horseshoe crabs are now collected from beaches and ‘bled’, by puncturing their abdomen and collecting the blood that flows out. This blood is subject to a centrifuge process, concentrating the mass of heavier amebocytes at the bottom while causing the lighter mass of other blood cells to rise to the top. The amebocyte cells are collected and split open, causing coagulogen and other proteins to spill out.

This mixture of amebocyte components is known as Limulus amebocyte lysate (LAL), the commercially viable form that can be found in virtually all microbiology laboratories today. Limulus comes from the scientific name of the Atlantic horseshoe crab (Limulus polyphemus), although blood from any of the other 3 species (Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda) of crab is just as viable.

horseshoe crab blood circulation diagram blue blood heart
Diagram of the horseshoe crab circulatory system4.

Endotoxin Detection Applications

The main application of LAL is in endotoxin detection. Endotoxins are chemicals that are a by-product of a bacteria’s life cycle but can be extremely toxic to humans. Ingesting even small amounts of endotoxins can disrupt our immune system and lead to a range of diseases and complications, including death.

In a twist of scientific irony, it turns out that the best way to manufacture many life-saving drugs and vaccines is to enlist the help of bacterial cells. The same cells that are capable of producing said lethal endotoxins! When we make use of bacterial cell machinery to produce these biologic drugs, the final product must not contain endotoxins, which is where LAL comes in. LAL can quickly detect even minute amounts, making it ideal for ensuring the safety of therapeutics.

Its use is now widespread, especially in quality control laboratories supporting the manufacture of biological, protein-based drugs, vaccines and even medical devices. Recent studies show that LAL can also be viable in environmental applications (monitoring air, water and soil quality) and even in space5!



What’s Next for Horseshoe Crabs

Sustainable Harvesting of Blood?

At the time of writing, a measly 2 mL of LAL will set you back a few hundred dollars. This is by no means a deterrent for the biotech industry, which depends on the chemical for its simplicity and effectiveness. Furthermore, the growth of biologic drugs and vaccine development means that the demand for LAL will continue to rise.

These factors spell lousy news for horseshoe crabs, which are typically captured, harvested for their blood, and then thrown back into the ocean. While this sounds like a painful process, the crabs usually survive the ordeal and can slowly regenerate their blood. But this process might not be completely sustainable; studies show that the mortality rate of harvested crabs increases by 8% within two weeks of bleeding6.

For the first 20 years after the commercialization of LAL, there were no regulations controlling the harvest of horseshoe crabs. Due to worries about unsustainable fishing permanently damaging the ecosystem, however, strict limits are now in place.

horseshoe crab beach sea
Goodbye, horseshoe crabs: It is clear that the future calls for alternative methods of endotoxin detection.

Future Direction and LAL Alternatives

While the Food and Drug Administration and other regulatory agencies continue to adopt LAL as the standard of endotoxin detection, other promising alternatives are becoming available. Since 2016, a new endotoxin test involving a synthetic version of LAL has been in use. The new test is based on a genetically engineered Limulus clotting factor C, the enzyme that starts the coagulation process in LAL and is responsible for endotoxin sensitivity.

Recombinant forms of LAL allow its production in the laboratory, without the need to harvest horseshoe crabs. While the shift has been slow, the acceptance of alternatives spells good news for horseshoe crabs, who have seen their populations decline in recent years. We need to enter a new era of endotoxin detection, one that doesn’t depend on horseshoe crabs, while ensuring our drugs and vaccines are as safe as ever.

Cover graphic: artwork of the amoebocytes cells from horseshoe crabs detecting bacteria by Melanie (@nanoclustering)

Reference

  1. Garwood, R. J., & Dunlop, J. (2014). Three-dimensional reconstruction and the phylogeny of extinct chelicerate orders. PeerJ2, e641.
  2. Kin, A., & Błażejowski, B. (2014). The horseshoe crab of the genus Limulus: living fossil or stabilomorph?. PLoS One9(10), e108036.
  3. Iwanaga, S., Morita, T., Harada, T., Nakamura, S., Niwa, M., Takada, K., … & Sakakibara, S. (1978). Chromogenic substrates for horseshoe crab clotting enzyme. Pathophysiology of Haemostasis and Thrombosis7(2-3), 183-188.
  4. Krisfalusi-Gannon, J., Ali, W., Dellinger, K., Robertson, L., Brady, T. E., Goddard, M. K., … & Dellinger, A. L. (2018). The Role of Horseshoe Crabs in the Biomedical Industry and Recent Trends Impacting Species Sustainability. Frontiers in Marine Science5, 185.
  5. Novitsky, T. J. (2009). Biomedical applications of Limulus amebocyte lysate. In Biology and conservation of horseshoe crabs (pp. 315-329). Springer, Boston, MA.
  6. Walls, E. A., & Berkson, J. (2003). Effects of blood extraction on horseshoe crabs (Limulus polyphemus).

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2 Responses

  1. Sean says:

    Thank you! I’m delighted to hear you enjoyed the read. As of right now, draining of the crabs blood is still the dominant approach but who knows? If the population dwindles and/or recombinant techniques improve there is a good chance the focus will shift toward more sustainable means. That’s the story of the century isn’t it!?

  2. scott L says:

    Great read, Sean! I remember reading abit on this and you really expanded my understanding. YAY! I’m glad that a new approach of creating recombinant forms of LAL have been discoevered and hopefully this puts alot less pressure on the draining of blood from these cute crabs hehe. Do you happen to know if the synthetic process of creating LAL based on Limulus clotting factor C more difficult or expensive than extractions from horseshoe crabs? XD.

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