Horseshoe Crabs Are Bloody Amazing

On the surface, horseshoe crabs are unexciting creatures. You might have come across one on the beach, a prehistoric scorpion/crab-like creature gliding slowly across the sand. But there is more to this unassuming creature than meets the eye. For starters, if you ever had a vaccine shot, then you owe a debt of gratitude to these horseshoe crabs; they have bled for you – literally. The unique properties of their blue blood make them truly invaluable to human health.

The horseshoe crab has a truly unique look: an outer carapace that covers the entirety of its body, five pairs of legs, a hard tail, and several groups of eyes thrown in. Despite its ‘crab’ nomenclature, they actually bear a closer relation to arachnids such as spiders and scorpions¹. But while the horseshoe crab doesn’t actually look like a horseshoe, and isn’t really a crab, they do possess an extremely special type of blood…

Unique Properties of Horseshoe Crab Blood

Cutting a horseshoe crab open reveals a light-blue, almost alien like liquid. 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. Copper-oxygen bond transitions causes the blue color, while iron-oxygen bond transitions show up as red.

Apart from being used to oxygen transport, the blue blood of the horseshoe crab also plays a role in their immune response. While they don’t possess an adaptive immune response, they do have an innate immunity that is maintained within their circulatory system. And it has worked pretty well for them, seeing as they have maintained their current biology for 400 million years²!

This innate immunity protects the horseshoe crab against pathogens, thanks to a type of cell found in its ‘blood’, known as an amebocyte. Amebocytes are able to detect endotoxins, chemicals found on the outer membrane of certain species of gram-negative bacteria.

When amebocytes come into contact with endotoxins, they release a clotting factor known as coagulogen. Coagulogen then starts a cascade of defense molecules that can neutralize the pathogens. In addition, it 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.

Limulus Amebocyte Lysate (LAL)

However, this innate immunity that served the horseshoe crab so well over its evolutionary history has now become the biggest factor in its decline in numbers. In the 1970s, horseshoe crab blood was put forward as a viable detector of endotoxins³. The instantaneous and visible reaction was of particular benefit, producing quick and accurate results.

Soon after, a technique was developed to extract coagulogen – the ‘detection’ molecule – from amebocytes in the blood. Horseshoe crabs are collected from beaches and ‘bled’, by puncturing their abdomen and collecting the blood that flows out. This blood is subject to a centrifuge, so that the heavier amebocytes form a concentrated mass at the bottom. These cells are split open or ‘lysed’, causing coagulogen and other proteins to spill out.

The result is a solution known as Limulus amebocyte lysate (LAL), the commercially viable form we know 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) is just as viable.

Applications of LAL

Its use is now widespread, especially in microbiology laboratories supporting the manufacture of biological, protein-based drugs. The bacteria that produce the drugs can also produce endotoxins, which can disrupt our immune systems even at small amounts, leading to a range of diseases and complications, some of which can be fatal.

Other applications of LAL include quality control for vaccines, intravenous drugs and implantable devices. Maintaining sterility in these applications is important, as endotoxins can produce more severe effects if they enter our bloodstream directly. Recent studies show that LAL can also be viable in environmental applications (air, water and soil quality) and even in space⁵!

What’s in Store for Horseshoe Crabs?

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 industry, that continues to depend on the chemical for its simplicity and effectiveness. Furthermore, the growth of biologics and vaccines mean that the demand for LAL will continue to rise.

These factors spell bad news for horseshoe crabs, which are 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 isn’t completely sustainable either – studies show that their mortality rate 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.

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 allows 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. This spells a new era of endotoxin detection, one that doesn’t depend on horseshoe crabs, while ensuring our drugs and vaccines are safer than ever.

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

Reference

1. Garwood, R. J., & Dunlop, J. (2014). Three-dimensional reconstruction and the phylogeny of extinct chelicerate orders. PeerJ, 2, e641.
2. Kin, A., & Błażejowski, B. (2014). The horseshoe crab of the genus Limulus: living fossil or stabilomorph?. PLoS One, 9(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 Thrombosis, 7(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 Science, 5, 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).