An alternative to horseshoe crab blood may save their species

By Heiloi Yip

Often in life, one must make difficult decisions involving sacrifices. In a hypothetical example, the blood of a species of marine arthropods contains medicinal properties, making it invaluable to the manufacture of medicines. However, extracting the blood will kill the arthropod in the process, and multiple members of the species need to be harvested to keep up with the high demand for their blood. Some may choose to throw the species ‘under the bus’ for the good of humanity, while others might immediately seek alternatives. Luckily, modern biotechnology may offer a compromise to this dilemma. 

Horseshoe crabs are marine arthropods that have existed for more than 200 million years as ‘living fossils’. They are found in warm shallow seas around North America and East Asia (Smith et al., 2016). Of all the unique features the horseshoe crab has, its blood is probably the most intriguing. For one, their blood is blue, which arises from hemocyanin containing copper ions. Secondly and more importantly, horseshoe crab blood contains Limulus amoebocyte lysate (LAL), which clots in response to a wide range of bacterial toxins (Maloney, Phelan & Simmons, 2018). 

The clotting response of LAL to toxins is so sensitive that it will be activated even by trace amounts, making it a very accurate tool for testing drugs for contamination. As a result, LAL is used extensively in pharmaceutical companies to screen vaccines and medications as part of quality control, including potential vaccines for COVID-19. The role of LAL in drug testing is so important that manufacturing of most pharmaceutical drugs must pass the LAL screening first. This has put high market value on LAL, being worth several thousand pounds per litre (Arnold, 2020; Fick, 2019).

With such high demand for LAL, strain has been put on wild populations of horseshoe crabs. Horseshoe crabs are taken from shallow coastal areas and transferred to a bloodletting facility. From there, a needle is inserted into the crab’s artery to extract a set amount of blood, after which the crab is released back to where it was retrieved (Fick, 2019). Unfortunately, the bleeding procedure has a moderate chance of killing the horseshoe crab in the process. With a significant proportion of blood being drained, combined with extensive amounts of time spent out of water, this puts the horseshoe crabs under stress and increases their chance of dying. Even after successful release, the crabs may still suffer from injuries and a shortened lifespan (Maloney, Phelan & Simmons, 2018). 

An assessment has been conducted on the population of an American species (Limulus polythemus) across multiple coastal areas of North America. Noticeable declines in crab population over time were observed in most of the areas being studied, a sign that overharvesting may be impacting the population of horseshoe crabs. Extinction of horseshoe crabs may prove devastating to the ecosystems, as their spawn is part of many organisms’ diets (Smith et al., 2016). Horseshoe crab populations all around the world are already facing pressures from overharvesting for bait and habitat destruction. Biomedical horseshoe crab harvesting only adds to this pressure. Consequently, one species of horseshoe crabs has been categorised as ‘vulnerable to extinction’ according to the IUCN, while another is assigned the ‘endangered’ category (IUCN, 2020). 

Perhaps the wild population of horseshoe crabs would not be put under as much risk if there was a more sustainable way of extracting LAL. One possible answer is to breed and raise horseshoe crabs in an aquaculture setting to keep up with high demands. Different designs of aquaculture systems have been developed, some of which produced healthy populations of horseshoe crabs, providing an alternative source of crabs for bloodletting (Waycott, 2020).

Another alternative could be achieved through biotechnology by directly synthesizing LAL, more specifically the agent in LAL responsible for the clotting behaviour. Recombinant factor C (rFC) is the specific protein in LAL that is responsible for toxin detection and clotting.  If the gene for rFC could be found and isolated, the gene can then be spliced into another eukaryote for mass production of rFC. One group at the National University of Singapore managed to extract the gene and spliced it into insect cells, where expression of the gene is then stimulated for mass production of the protein. This has the advantage that the end-product is much more concentrated in rFC than ‘natural’ LAL (Zhang, 2018; Maloney, Phelan & Simmons, 2018). 

Horseshoe crab bloodletting is still a common procedure in the production of LAL, and the demand will likely increase over the next few months as vaccines for COVID-19 start awaiting approval for mass production (Arnold, 2020). However, the advent of synthetic LAL has not swayed pharmaceutical companies towards switching. Even when presented the option to choose between ‘natural’ LAL and rFC, most companies stick to LAL simply due to a lack of trust in the newer alternative. However, there can be confidence that LAL will gradually be phased out by rFC over the next several years, just as bacteria had replaced pigs in insulin synthesis (Zhang, 2018). 

References:

Arnold, C. (2020). Horseshoe crab blood is key to making a COVID-19 vaccine—but the ecosystem may suffer. Available from: https://www.nationalgeographic.co.uk/science-and-technology/2020/07/horseshoe-crab-blood-is-key-to-making-a-covid-19-vaccine-but-the

Fick, L. (2019). Horseshoe Crabs Endangered by Biomedical Bloodletting. Available from: https://science.howstuffworks.com/life/biology-fields/horseshoe-crabs-endangered-biomedical-bloodletting.htm [Accessed 9 November 2020]

IUCN (2020). International Horseshoe Crab Day: a celebration of the flagship species for coastal habitat conservation. Available from: https://www.iucn.org/news/species-survival-commission/202006/international-horseshoe-crab-day-a-celebration-flagship-species-coastal-habitat-conservation

Maloney, T., Phelan, R., Simmons, N. (2018). Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLOS Biology, 16(10), p.e2006607. DOI: 10.1371/journal.pbio.2006607 

Smith, D., Brockmann, H., Beekey, M., King, T., Millard, M., Zaldívar-Rae, J. (2016). Conservation status of the American horseshoe crab, (Limulus polyphemus): a regional assessment. Reviews in Fish Biology and Fisheries, 27(1), pp.135-175. DOI: 10.1007/s11160-016-9461-y

Waycott, B. (2020). RAS key to horseshoe crab aquaculture for sustainably sourced medical resource. Available from: https://www.rastechmagazine.com/horseshoe-crab-aquaculture-key-to-sustainably-sourced-medical-resource/

Zhang, S. (2018). The Last Days of the Blue-Blood Harvest. Available from: https://www.theatlantic.com/science/archive/2018/05/blood-in-the-water/559229/

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