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rFC Brings Good Luck for Horseshoe Crabs





Writer: Rohan Kumaran ‘25

Editor: Jasper Lincoln '25


Abstract

Horseshoe crabs (Order Xiphosura) are among the oldest animal species living on our planet today. They first evolved in the Paleozoic era 440 million years ago–– hundreds of millions of years before dinosaurs dwelled on Earth or the Pangea landmass broke apart–– but there has been little morphological evolution of the group since. While they belong to Phylum Arthropoda like true crabs, the horseshoe crab is more closely related to scorpions and spiders than crustaceans. There are four extant species of the horseshoe crab: Limulus polyphemus–– which is native to the Atlantic seashore off North America and the Gulf Coast–– and Carcinoscorpius rotundicauda, Tachypleus tridentatus, and Tachypleus gigas which are found in Asia and the Indo-Pacific region. Their survivability has been attributed to having ten eyes, a hard shell and exoskeleton, and evolutionarily miniaturizing and reducing basal metabolic needs for food and oxygen. The hemolymph in Horseshoe crabs has a unique composition of serine protease cascades which activate a clotting factor that precisely identifies bacteria and fungi. Since 1970, over a hundred thousand horseshoe crabs have been fished and ‘milked’ each year–– a euphemism for bleeding them of more than half of their hemolymph and leaving them for dead or as bait for whelk and eel fishing.


Introduction

Despite predating most species alive today, the horseshoe crab is not a rudimentary or antiquated organism. It is not under-evolved but rather evolved for longevity and survival. Xiphosura’s open circulatory system allows for blood to come in contact with many tissues, making the horseshoe crab vulnerable to bacterial infection and necessitating that their cerulean blood has antibacterial properties.

The two extant families of the horseshoe crab, Limulus and Tachypleus, are harvested for their production of Limulus Amebocyte Lysate and Tachypleus Amebocyte Lysate (LAL and TAL). The bacterial test in which horseshoe crabs are harvested, bled, and sometimes killed to develop can be produced from the blood of any one of the four extant species of Xiphosura in a laboratory (1).

The identification of a sophisticated coagulatory process in the horseshoe crab’s blood in 1964 quickly led to a double standard: the animals are recognized and harvested for the futuristic and surreal properties of their blood, but their rights and fair treatment are written off when the animal is diminished to an unfeeling poikilotherm. To understand what caused the overfishing and excessive bleeding of horseshoe crabs, it is important to understand the biochemistry of what makes them coveted bioprospects. Understanding the complexities of these biological mechanisms offers a framework for assessing the feasibility of synthetic alternatives, the economic value harvesting provides, and the ethicality of the harvesting and bleeding industry. There are clear economic incentives to harvesting horseshoe crab blood–– it can be sold for as much as $15,000 per quart, which is over $15 for just one single milliliter. The profitability, overfishing, and feasibility of harvesting Xiphosura have been a debate of cost-benefit analysis and moral obligation to animal rights. Is harvesting horseshoe crabs ethical, economically profitable, or even necessary with the development of synthetic alternatives?


Biochemicals of the Invertebrate Hemolymph

Though eye-catching, the cerulean color of the horseshoe crab’s hemolymph is not why the industry is out for their blood. The royal blue color is simply due to their possession of a different oxygen-carrying protein in the hemolymph. An iron cofactor in the heme prosthetic group of human hemoglobin gives it a rich, red color when oxidized. Conversely, the copper ion coordinated by histidine residues in hemocyanin–– the pigment and oxygen-transport protein in most mollusks and arthropods–– gives a rich sky-blue color when oxygen reversibly binds (2). This hue is unique to invertebrates. When mammalian veins appear bluish, it is neither due to hemocyanin nor as an indicator of low oxygen saturation of hemoglobin. It is simply because our skin tissues absorb the lower frequency red light and reflect the blue light back to our eyes (3,4).

While the oxygen-transport proteins give Xiphosura a breathtaking color, the blood cells–– or amebocytes–– provide incredibly defensive antibacterial, antifungal, and clotting properties (5). The coagulatory serine protease cascade begins with exposure to endotoxin or fungus and ends in a gel clot formed by active clotting enzymes. Endotoxin, which is a lipopolysaccharide (LPS) within gram-negative bacteria’s outer membrane, can cause fever, septic shock, or death (5–7). Factor C is a biosensor in the blood of horseshoe crabs which can detect these microorganisms with a remarkable specificity of one part per trillion (8). This LAL clotting cascade found in the blood of Xiphosura also responds to (1→3)-β-glucans, which are found in the cell walls of fungi, using a Factor G (9). Despite different intermediate steps, Factor C and Factor G activate the same final clotting enzyme.

After Factor C has identified a bacterial invader, it will be cleaved either via autolysis or through chymotrypsin, the serine protease that begins the coagulation cascade. If Factor C is cleaved autolytically, then 𝛼-Factor C will be formed. If chymotrypsin cleaves the protein, 𝛽-Factor C is formed. The key difference between these two activated forms is that Factor B, which is the next molecule in signal amplification and the protein responsible for activating the pre-clotting enzyme to the clotting enzyme, preferentially acts as a substrate to 𝛼-Factor C; on the other hand, 𝛽-Factor C loses the ability to bind LPS and defend from bacteria. So, when gram-negative bacteria with LPS are detected by Factor C, and Factor C is cleaved via autolysis, Factor B can bind the 𝛼-Factor C and continue the serine protease cascade to activate coagulated into coagulin, a gel-forming protein that immobilizes microorganisms (10).

When a fungal attacker is recognized by Factor G, coagulogen is directly activated to coagulin. Factors C, B, and G are all zymogens in the trypsin family (11). The three serine protease zymogens, along with the pre-clotting enzyme coagulogen, form the cascade and gel clots that protect from bacterial and fungal invaders (12).


Synthetic Alternatives Mimic Cascades

Biochemists’ search for synthetic replicas of LAL stems from the search for economic and ecologically conservative alternatives to harvesting horseshoe crabs.  In the late 1990s, scientists from the National University of Singapore discovered that a synthetic recombinant Factor C (rFC) could be used as a biosensor for gram-negative bacteria and as an endotoxin test without harming or harvesting animals. Development through recombinant synthesis makes rFC an exact copy of Factor C found in lysate reagents, with the added benefit that its fluorogenic properties can better visualize a sample’s endotoxin concentration (13,14).

In 2010, Lonza Bioscience commercialized the first Recombinant Factor C assay, PyroGene™. It has been an uphill battle to get these assays cleared by the United States Food and Drug Administration, but in 2020, FUJIFILM Wako Chemicals was able to introduce a similar rFC assay, PYROSTAR™ into the U.S. market. As more assay products clear regulation and reach the market, greater specificity and precision are enabled. For example, FUJIFILM’s PYROSTAR focuses on eliminating false positives from (1→3)-β-glucans, which would incorrectly indicate traces of fungal microorganisms.

“It mimics traditional LAL reagents with three recombinant factors (Factor C, Factor B, and proclotting enzyme) and the pNA chromogenic group,” reads the page of PYROSTAR™ Neo, a newly marketed endotoxin detection reagent developed by FUJIFILM Wako Chemicals (15).

The synthetic assay’s mechanisms sufficiently replicate the serine protease cascade and activate clotting factors. Understanding the cellular biology mechanisms and efficacy of the synthetic alternative undermines the justification for violently harvesting the horseshoe crab. This nuance enables economic, ecological, and ethical comparison of LAL assay options.


Economic Incentives

The vampiric act of scraping horseshoe crabs off the seafloor, stacking them in fishing boats, and bleeding them to near death once had a rational explanation–– the profit margin. The practice began once it was deemed to be lucrative, but it continues despite the emergence of synthetic alternatives on the market. So, either harvesting crabs is cheaper than manufacturing synthetic LAL, the harvesting of natural LAL makes for higher quality and more profitable products, there are other roadblocks in ending the practice, or it is a senseless, violent act.

Unfortunately, it turns out that the bleeding of the horseshoe crab persists because of the latter two explanations. The synthetic LAL has actually been proven to be economical compared to the traditional LAL assay harvested from a bled horseshoe crab. Eli Lilly, a pharmaceutical company in the United States, produced its entire supply of COVID-19 antibody medications using synthetic rFC assays. According to an NPR exposé and testimony from Jay Bolden, the scientist and employee who urged Eli Lilly to adopt the synthetic LAL assay, the switch has actually been “cost-advantageous.” Bolden also mentioned that the synthetic alternative has been better from a quality standpoint. The only drawback has been obtaining the additional approval required by the U.S. Food and Drug Administration and the U.S. Pharmacopeia (16).

Despite offering lower costs and equal or greater performance for established pharmaceutical and biotechnology companies, the regulatory approval process proves to be a large hurdle for promoting the synthetic alternative. For many, piercing the soft tissue around the horseshoe crab’s heart and drawing blood at $15,000 per quart is more profitable than spending time and effort lobbying for rFC and synthetic LAL approval in the U.S. FDA or Pharmacopeia (1,17).


Regulation and Ethics

While some sources say that horseshoe crabs are milked for 30% of their blood, others claim it can be for 50% or more (18). Overfishing of the horseshoe crab in its indigenous regions is a problem for the ecosystem and food network that they are part of.

Regulation surrounding bleeding is not tightly controlled–– but the distribution of the synthetic alternative is–– resulting in yet another inconsistency. There is a lack of FDA approval of the synthetic rFC alternative because of concerns that it originates from a single-source supplier, lacks inclusion in worldwide pharmacopeias, and is hard to license because while LAL is a blood product, rFC technically is not–– it’s synthetic (14)

While there is tight regulation making the synthetic alternative inaccessible, there is a lack of control to prevent harm to horseshoe crabs and their populations. No regulatory bodies take initiative in protecting these ‘living fossils.’ Fishermen claim that the bleeding industry is disparate as the horseshoe crabs are not eaten, and science and research entities claim that the harvesting is not in the research stage and that the animals are not warm-blooded, hoping to absolve themselves from responsibility (16).


Source: NPR

Aside from the regulations that prevent the overfishing of the horseshoe crab from an institutional level, records demonstrate that fisherman handling causes harm and violates harvest laws. For example, holding a horseshoe crab by the tail damages the structure, making it harder for them to upright themselves if flipped by a wave. The effects of overfishing have reduced the populations at spawn–– especially for female horseshoe crabs–– suggesting that populations across the globe are stressed by the burden that fishing imposes on Xiphosura (16,17). Many harvesting companies admit to this behavior and see no shame in it.

The red knot bird usually has a diet of mussels, clams, and snails, but in certain regions of the coastal United States like Delaware and Maryland, its diet can switch to be composed almost exclusively of the eggs of horseshoe crabs. The red knot population becomes threatened when the harvesting of horseshoe crabs is overindulgent. When horseshoe crab populations were depleted by 88%, red knot populations decreased by 70% (18).

In addition to the detrimental effects bleeding has on the horseshoe crab population or the ecosystems they support, bioethicists are also concerned with the animal’s rights. Common concerns include whether the animals are harmed or their lives are substantially placed at risk. It seems 10-30% of horseshoe crabs die as a result of the bleeding process, but these reports might misrepresent how many are displaced or threatened as a result of the harvesting (19).


“There is little evidence of pain in millipedes, centipedes, scorpions, and horseshoe crabs but there have been few investigations in these groups…much relates to the level of mortality following bleeding and release and hence, the sustainability of the populations (20).”


Oftentimes, if horseshoe crabs are not killed by or after the bleeding process, they are sold to be killed and used as bait in the whelk and eel fishing industries (18). While studies are incomplete and data is inconclusive as to whether or not the arthropods are harmed or their lives are put at risk, one thing is for certain–– that they are returned to their ecosystems as the shell of a creature that once thrived.


Conclusion

The horseshoe crab, an ancient marvel predating most species on the face of the earth, embodies resilience through its defensive antibacterial hemolymph and clotting factors used for testing antibiotics and vaccines. While the exploitation of the species for its Limulus Amebocyte Lysate disrupts ecosystems and is an ethically ambiguous practice, the advent of synthetic alternatives offers a promising solution. Recombinant Factor C assays mimic the crab's clotting cascade with greater antibacterial properties and lower production costs. As regulatory bodies approve the assays, the violent harvesting will hopefully go obsolete. Striking a balance between economic interests and ethical responsibilities remains imperative in determining the fate of these remarkable creatures. The campaign to protect these living fossils while using their biochemistry to advance human health requires not just scientific advancements but a collective commitment to ethical liability and conservation efforts.






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