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The Blue Blood of Horseshoe Crabs: A golden Standard for Endotoxin Detection in the Biomedical Industry

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Abstract

This essay will discuss the use of blood in industry. In specific, the use of horseshoe crab blood as an important resource in the biomedical community will be focused on. Limulus blood contains a certain type of cell called an amoebocyte that allows for blood to neutralize toxins through forming clots, making it especially useful during the creation of new medical devices and drugs. Although, the collection of horseshoe crab blood is dangerous to these animal populations and, if continued without stricter regulation, will be very unsustainable. Current steps in research are being taken to create artificial replacements for horseshoe crab blood. However, there may be some challenges and barriers that need to be overcome.

Introduction

Each year, hundreds of thousands of horseshoe crabs are harvested to be bled for the biotechnology sector as their blood contains important enzymes that are used to detect endotoxins. Many medical devices and drugs are required to be tested using derivatives from horseshoe crab blood before being approved for commercial use. There exist many factories where horseshoe crabs are lined up in rows, their blue blood draining into beakers, for our purposes.

The current bleeding practices are still quite novel and have yet to be fully replaced by a more sustainable, less harmful technique. The demand for endotoxin detection is very great as the medical industry continues to grow and so the horseshoe crab populations may not be able to keep up. Until a new technological advancement is made, horseshoe crabs will continue be sacrificed for science.

This essay will explore the history of horseshoe crabs in industry, how horseshoe crab blood may detect endotoxins and what this means for biomedical research. Moreover, the sustainability of horseshoe crab collection will be further examined with an emphasis on whether these animals may risk becoming extinct. This will lead into the final section focusing on the possibilities and challenges of artificial analogues replacing the harvesting of horseshoe crab blood for research.

History of Horseshoe Crabs in Industry

Before the start of the 1970’s, a very common practice was to use horseshoe crabs for farming fertilizer. The number of horseshoe crab deaths was dramatic, and the development of artificial fertilizers became more common than this traditional method.

Nowadays, horseshoe crabs are no longer used as fertilizers. However, they are now a form of very popular bait for fishing. Horseshoe crabs were once considered to not have much economic value, leading to their use in these fields. Nonetheless, recently, horseshoe crabs have become involved in a different industry altogether (Madrigal, 2014). 

Fig. 1 Image of a pre-historic looking horseshoe crab shell. [Adapted from Mehmood, 2019]

Discovery of Limulus Blood

Horseshoe crabs are marine animals that are a type of invertebrate called arthropods. In the 1970’s, the pharmaceutical industry that had previously been responsible for exploiting rabbits discovered the unique properties of horseshoe crab blood that allowed for the implementation of a new kind of innovation in the domain of drug production (Mehmood, 2019).

The sale of drugs is regulated based on vigorous tests that are taken on different organisms to detect whether the manufactured injection or device is an endotoxin carrier. Endotoxins, or lipopolysaccharides, are a polymer toxin generated by cell walls of bacteria (Munford and Hall, 1986). They are identified as gram-negative bacteria, recognizable by their red color during the technique scientifically used to differentiate bacteria: the gram staining procedure. These polymers are found in cell membranes and may be present in certain drugs. They are considered fatal in the case of an injection in an organism (Mehmood, 2019).

Fig. 2 Image of horseshoe crabs being bled for the use of their amebocyte lysate in Charles River Laboratory, South Carolina. [Adapted from Maloney et al., 2018]

Traditionally, rabbits were used as lipopolysaccharide detectors leading to the death of many of these animals during testing. However, recently, scientists discovered that horseshoe crabs possess an immune system that is particularly designed to react and fight against endotoxins (Mehmood, 2019). The specificity of horseshoe crab blood is defined by an open hemolymph circulatory system which allows for the blood to circulate freely without the necessity of blood vessels to carry the vital fluid to tissues (refer to Cox, 2017 in Appendix). This physiological aspect is interesting when considering the better immune response reactivity of this invertebrate in presence of external factors such as viruses or bacteria. When horseshoe crab blood is in contact with endotoxins, it generates a defensive mechanism induced by specific cells known under the name of amebocytes. When in contact with bacteria, amebocytes react by releasing a toxic substance, neutralizing the toxin. This induces “degranulation” and triggers the coagulation of the hemolymph to create a clot around the toxin (Armstrong, 1980; Maloney et al., 2018). Thus, industries have since collected the limulus amebocyte lysate from horseshoe crab hemolymph. Once collected, the cells are lysed to extract the specific enzymes responsible for this immune system reaction. A newly manufactured sample of medication is then tested with the extraction and if a clot forms, the sample is considered unsafe (Maloney et al., 2018).

Fig. 3 Scheme of limulus amebocyte lysate experiment to detect endotoxins. [Adapted from Tinker-Kullberg et al., 2020]

Evolutionary Adaptations of Horseshoe Crab Hemolymph

Horseshoe crab evolution has been discussed at length. However, there are many phylogenetic details that are yet to be discovered. Horseshoe crabs are part of the Limulidae arthropod family and make up four different species including Limulus polyphemus, Tachypleus gigas, Tachypleus tridentatus and Carcinoscorpius rotundicauda (Pati et al., 2017). Scientists theorize that these animals first appeared even before the dinosaur era, meaning that they have been in existence for over approximately 350 million years (Smith et al., 2002). This explains why, today, they are known as living fossils (Avise et al., 1994).

Fig. 4 Phylogenetic relationships emphasizing on the evolutionary aspect of the Arthropoda and Arachnida families. [Adapted from Bechsgaard et al., 2016] Horseshoe crabs are part of order Xiphosura.

The particularity of horseshoe crab hemolymph may result from an evolutionary adaptation that is still not very well-known. Nevertheless, the extraordinary immune capacity of horseshoe crab hemolymph proteins has been compared to arachnid’s hemolymph proteins (Smith et al., 2002). Results have shown that, despite the word “crab” in the name “horseshoe crab”, these animals are surprisingly related to some spiders and even scorpion species when considering the genetical scale. In fact, researchers have analysed the clotting process of arachnids and T.tridentatus and noticed similarities. The clotting pathway of T.tridentatus involves the recognition of lipopolysaccharide by factor C.

Factor C is responsible for activating Factor B which itself contributes to the activation of a pro-clotting enzyme. On the other hand, Factor G can detect a specific polymer known under the name of beta-1,3-glucan that is contained in lipopolysaccharide (Bechsgaard et al., 2016). This detection will also induce the pro-clotting enzyme function to catalyse the conversion of coagulogen to an insoluble component of the clot coagulin (Muta and Iwanaga, 1996). This clotting immune response prevents the spreading of bacteria by allowing a control of their proliferation. The comparison of the structure of the domains of Factor C (Fig. 5) with those of the T.tridentatus have shown similarities with the one of the scorpion Mesobuthus.

Fig. 5 Diagram of the coagulation pathway for T.tridentatus. [Adapted from Bechsgaard et al., 2016] In blue are represented the pathogen molecules, in brown the cascade molecules and in grey the end products. LPS is for lipopolysaccharide.

Indeed, researchers have noticed that the LCCL domain of factor sequence present at a specific position in Factor C of T.tridentatus is not observable in the scorpion Mesobuthus at that same position. Consequently, scientists concluded that the LCCL domain of factor C is specific to the T.tridentatus. As a result, arthropods and arachnids have shown similarities at a genomic scale of the Factor C involved in the launching of the clot cascade. Thus, based on that example, a hypothesis has been made anticipating that the clotting process of arthropods and arachnids are quite similar. This would imply a conservation of an innate immune response between these species (Bechsgaard et al., 2016).

Fig. 6 The factor C Domain structure of T.tridentatus. [Adapted from Bechsgaard et al., 2016] The presence of the LCCL domain that has been identified as the specific domain of T.tridentatus horseshoe crab is presented.

However, scientists discovered that arachnids lack the recognition protein of β-1,3-glucan. This particularity involves the fact that this classification of invertebrates should not be able to trigger the toll cascade process — unless they possess another specific molecule able to launch this process, but this is not yet known by scientists (Bechsgaard et al., 2016). The toll cascade is a signaling pathway in the immune system able to detect pathogens to induce their elimination. Knowing this specificity, we could say that horseshoe crabs did have a common ancestor with common immune system reactions as discussed previously. Nonetheless, the immune response has evolved and has been improved for some species such as the case of horseshoe crabs possessing “pathogen recognition molecules”. This particularity allows the Limudae to own a more sensitive and efficient immune system (Bechsgaard et al., 2016).  

Fig. 7 The Toll intracellular cascade process. [Adapted from Bechsgaard et al., 2016] The dark blue represents pathogen molecules, the green is for recognition molecules, light blue is for signaling molecules (signals carriers towards target cells) and the red box is for the transcriptional factor (transcription of antimicrobials peptides).

These two comparative examples between arthropods and arachnids’ immune system response are not the only and studies are still led to discover more about hemolymph evolution of these living fossils.

Mechanism of Limulus Amebocyte Lysate

The Primitive Immune System of Horseshoe Crabs

Horseshoe crabs are such successful living organisms that they have been able to survive billions of years, mainly thanks to their immune system. In human blood circulation, the blood travels through a closed circulatory system from the heart to the arteries and eventually into the smallest capillaries, where oxygen and carbon dioxide diffuse from the vessels and tissue. However, the system of horseshoe crabs is rather different. Horseshoe crabs have hemolymph flowing through their bodies in the place of blood and, once it leaves the heart and arteries, it enters the lacunar system (Göpel and Wirkner, 2015). In other words, the hemolymph in horseshoe crabs travels through a brief vascular system where compounds such as oxygen will diffuse directly into the tissues and organs. This is also called an open circulatory system as the blood vessels do not form a closed circuit. This method of hemolymph transportation is rather dangerous as any bacterial infection would likely spread very rapidly throughout the entire organism. Therefore, a strong immune system is essential for the survival of this species.

The immune system of horseshoe crabs is highly sensitive to bacterial invasion, especially to gram-negative bacteria which have lipopolysaccharide (LPS-endotoxin), a type of pyrogen that is responsible for fever, present on their cell walls (Armstrong and Rickles, 1982). To combat the presence of these bacteria, horseshoe crab hemolymph may form gel clots that traps the bacteria. The mechanism of this immune activity is an interesting field of study and has led to this special property of horseshoe crab blood being applied to the medical industry.

Unlike hemoglobin in the red blood cells of vertebrates, the oxygen carrier in horseshoe crabs is hemocyanin, which is suspended directly in the hemolymph. Based on cell morphology, there is only one single type of cell present in the hemolymph: amebocytes (John et al., 2010). This cell has an ovular, plate-shaped structure and possesses granules, a type of vesicle that contains release factors, in its cytoplasm (Iwanaga et al., 1992) (Fig. 8).

Fig. 8 A scanning electron micrograph of amebocytes. [Adapted from Iwanaga et al., 1992]

The granules within the cell can be classified in two types: large (L) and small (S) granules (Fig. 9). The L-granules contain at least 20 protein components including four clotting factors and one antimicrobial factor (anti-LPS factor) whereas the S-granules exclusively contain the other antimicrobial substance that accomplish this activity. During immune response reactions, these granules will become disrupted and excrete antimicrobial substances. This process is called degranulation. Generally, L-granules will be the first to be released and as they degranulate more rapidly compared to the S-granules. However, nearly all granules will undergo degranulation in the final stages of immune activity (John et al., 2010).

Fig. 9 A cross section showing the organelles in amebocyte. D. small granules. L. large granules. [Adapted from Iwanaga et al., 1992]

Coagulation Cascade in Horseshoe Crabs

Initially, when an amebocyte comes into direct contact with bacteria, the exocytosis of both L-and S-granules will rapidly trigger degranulation. This response signifies the start of the coagulation cascade (Armstrong and Rickles, 1982). The coagulation process mainly involves three serine protease zymogens — a type of enzyme that can cleave peptide bonds — that include Factor C, Factor B and a pre-clotting factor (Muta and Iwanaga, 1996).

Firstly, Factor C and Factor G, which are considered biosensors, will be released from granules and will then be automatically activated by the presence of endotoxins (LPS) and (1, 3) β-D-glucans respectively. These activator compounds are common cell wall components found in gram-negative bacteria.

With the presence of the now active Factor C, written Factor , another enzyme that was released during granulation, i.e. Factor B, will also become active (Nakamura et al., “Lipopolysaccharide-sensitive serine-protease zymogen (factor C) found in Limulus hemocytes”).

The newly activated enzyme, Factor , will then trigger the transition of the pre-clotting enzyme to the clotting enzyme. The active clotting enzyme is then able to activate coagulogen, a protein in L-granules that forms clots. This protein will develop a specific type of insoluble gel to trap the toxins (Muta and Iwanaga, 1996). The LPS-mediated coagulation cascade process is represented in Fig. 10.  

Fig. 10 Defense systems in horseshoe crab hemocytes. [Adapted from Muta and Iwanaga, 1996] 

The Role of Factor C as a Biosensor

The serine protease zymogen factor C is intensely involved in the initial stages of the coagulation cascade. It is a two-chain glycoprotein, weighing in total 123 kDa, which is composed of a heavy chain (80 kDa) and a light chain (43 kDa). Upon activation when interacting with endotoxins present on the bacterial cell membrane, the light chain will cleave into an A-chain (7.9 kDa) and a B- chain (34 kDa), which are linked via disulfide bonds (Tokunaga et al., 1987). This activation is a result of interaction of the NH2 terminal of factor C on the B chain and the essential component of LPS-lipid A (Ariki et al., 2008).

As a result, the activated Factor C has the new form of Factor , which is a three-chained serine protease. In an experiment, Triton X-100 was used to destroy LPS micelles, and the response of factor C was analyzed. The observations indicated that this process strongly inhibited Factor C. However, it did not influence the function of the activated form, Factor . This suggests that there is a high correlation between the activation of factor C and the presence of LPS (Nakamura et al., “Interaction between Lipopolysaccharide and Intracellular Serine Protease Zymogen”). The activation of biosensor Factor C indicates the official start of the coagulation cascade, and in the later stages, this enzyme also serves as an indicator and activator protein for Factor B.

The Role of Coagulogen in the Coagulation Process

During coagulation, the most important enzyme to consider is definitely coagulogen, which is the main component of clotting proteins in the amebocyte (Shigenaga et al., 1993).

Studies have shown that coagulogen plays a similar role as fibrinogen in mammalian blood, where blood coagulation is dependent on the induced polymerization of fibrinogens. Just like human fibrinogen which forms clots at the surface of phospholipid at the site of injury, coagulogen only forms gel on the surface of the pathogen — gram-negative bacteria (Armstrong and Rickles, 1982). At the final stage of coagulation, coagulogen is converted to coagulin through the activation of the pre-clotting factor. The activated clotting factor may cleave the coagulogen protein into a two chain-form of coagulin, while excising a fragment called peptide C (Armstrong and Rickles, 1982). The two chain-form of coagulin formed by the monomers is called the AB polymer. This polymer consists of an NH2 terminal — N terminal — on the A-chain and a COOH terminal — C terminal — on the B-chain.

The AB-chain is linked by two covalent disulfide bonds (Armstrong and Rickles, 1982). Interestingly, the N-terminal sequence found in the coagulin chain are Ala- Asx- Thr, which is homologous when comparing to the N-terminal residues of non-human fibrinogen, which could demonstrate a potential evolutionary link. On the other hand, the C-terminal residue of the coagulin chain has been discovered to be phenylalanine, which is the amino terminal of a peptide chain. Therefore, it is highly possible for coagulogen to consist of a single polypeptide chain (Nakamura et al., “A clottable protein (coagulogen)”).

After peptide C has been released by the cleavage, the chain form of coagulin is expressed by having a hydrophobic cove on the chain terminal. This hydrophobic head will interact with the hydrophobic tail of the other molecule (Osaki and Kawabata, 2004). This polymerization will result in a coagulin homopolymer (Fig. 11) and will eventually become the coagulin gel formed at the surface of the bacteria.

Fig. 11 A molecular model of the polymerization of coagulogen. [Adapted from Armstrong and Rickles, 1982]

The coagulation cascade is a significant inspiration to modern pharmaceutical industries for its great ability to detect and react in the presence of endotoxins. The modern application of horseshoe crab blood’s innate immune system response is called the Limulus Amebocyte Lysate test (LAL). This endotoxin test is widely used in medical and pharmaceutical industries as mentioned in the previous sections (John et al., 2010).

Sustainability of Limulus Blood Harvesting

Although horseshoe crab blood has allowed for many developments in biotechnology and healthcare, the bleeding of these animals may pose long term challenges that must be considered. Currently, hundreds of thousands of horseshoe crabs are bled each year for the amebocyte lysate in their blood. In the United States, roughly 8 – 15 % of harvested animals, equating around 50 000 crabs per year, die from the procedure, with the remaining being released back into their habitats (Zhang, 2018) (Table 1).

Table 1 Number of horseshoe crabs harvested for LAL production and assuming 15% mortality, the estimated number lost due to the bleeding process each year. [Data was taken from ASMFC. Table was adapted from Gauvry, 2015]

In Southeast Asia, it is rare if any of the crabs harvested for TAL survive as, after bleeding, they are sold into other markets (e.g., food market) (Gauvry, 2015). It can easily be seen that, over time, if humans continue to interfere with horseshoe crab populations, these prehistoric creatures may become extinct and take their miraculous blood along with them.

To protect the incredible discovery of limulus blood and what it means for science, it is important to evaluate how the health of these animal populations and the ecosystems they thrive in will be impacted in the years to come.

Human Population Growth and what it means for Horseshoe Crabs

As humans continue to further develop medical technology through the creation of new drugs and innovative devices, expected longevity of our species is increasing. However, prolonging human lives come with a cost to horseshoe crabs. With the creation of these new devices and drugs, more and more limulus blood will be needed to test for endotoxins (Gauvry, 2015).

Without considering human influence, horseshoe crabs already have many hurdles to overcome in nature to ensure their populations remain stable. From birth, approximately only 3 out of 100 000 horseshoe crabs will survive their first year alive as juveniles are food for numerous predators (Tanacredi et al., 2009; Gauvry, 2015).  After 10 to 12 more years, a horseshoe crab finally reaches maturity, where it can live without too much worry of predation for up to another 10 years (Gauvry, 2015). The largest threat to adult horseshoe crabs are humans (Tanacredi et al., 2009).

However, it is important to remember that horseshoe crabs do not only provide resources to humans. Limulus crab eggs and juvenile horseshoe crabs are essential in maintaining ecosystem stability by acting as prey for other species. One of these such relationships is with shorebirds who rely heavily on the nutrients provided by the millions of horseshoe crab eggs laid each year as they migrate through Limulus crab habitats (Tanacredi et al., 2009) (refer to Fig. 12). In addition, the shells of horseshoe crabs provide protection to numerous different species of mussels and barnacles, creating a small ecosystem in itself (Krisfalusi-Gannon et al., 2018) (refer to Fig. 13).

Fig. 12 Red Knot Shorebirds feeding next to a horseshoe crab. [Adapted from “Modeling a Future for Horseshoe Crabs and Red Knots” | U.S. Fish & Wildlife Service, 2016]
Fig. 13 A horseshoe crab’s shell provides shelter to many animals such as muscles and barnacles. [Adapted from “Horseshoe Crab” | Barrier Island Ecology UNCW, 2020]  

If horseshoe crabs are harvested irresponsibly, considering the notable amount of time it takes for a horseshoe crab to reach maturity, there will not be enough adult crabs to reproduce eventually. This will cause the relationships between Limulus crabs and shorebirds, among others, to vanish and natural order would be placed in a position of disequilibrium.

The Possible Extinction of Horseshoe Crabs

The IUCN explains that the main threats to horseshoe crabs are overharvesting for food, bait and biomedical testing along with habitat loss (Botton and Shin, 2020). All these factors, as well as many others, will influence the likelihood of species extinction.

The large number of variables that affect horseshoe crab population make it very difficult to estimate if or when these animals will go extinct. Considering the current situation of horseshoe crab harvesting in the United States, it is estimated that, within the next two decades, the demand for the LAL test will reach unsustainable levels (Krisfalusi-Gannon et al., 2018). The American Horseshoe crab is listed as a species vulnerable to extinction and the tri-spine horseshoe crab, found in Asia, is already considered endangered by the IUCN (Botton and Shin, 2020). The threat of extinction of these animals is evident and humans need to deeply consider how our practices affect the safety of these animals.

It has been found that after bleeding, horseshoe crabs exhibit changed behavior, such as lethargy and unresponsiveness which can lead to reduced spawning, further affecting population health (Krisfalusi-Gannon et al., 2018). These post bleeding effects are not taken into consideration with the approximate 15 % mortality rate of the collection process. The stress undergone by these animals is highly conducive of long-term effects.

Since the beginning of the twenty-first century, horseshoe crab harvesting has been increasing substantially each year and is expected to continue increase. It is therefore essential that alternative methods for endotoxin detection are considered as well as possible ways to better protect horseshoe crab populations and their breeding grounds. Hence, there may still be hope for these amazing creatures as researchers continue to strive for more sustainable techniques such as artificial blood substitutes to replace the need for limulus blood.

Artificial Analogues of Limulus Blood

As previously stated, the current situation regarding Limulus Blood and the biopharmaceutical industry is an important challenge that places horseshoe crabs and their ecosystems in great danger. The populations of all four species of horseshoe crab are in decline because of human activities and the biopharmaceutical industry is estimated to cause at least 130 000 deaths of horseshoe crab every year overall due to the bleeding process, the release of the arthropods and their potential sale as bait (Maloney et al., 2018). 

Endotoxin Detection with Recombinant Factor C (rFC)

Fortunately, there exists an artificial analogue called recombinant Factor C (rFC) which is currently improving the situation and could potentially replace limulus amebocyte lysate (LAL) for the detection of endotoxins. In fact, the recognition of endotoxins with LAL is a cascade system initiated by the enzyme named Factor C. In 1997, two researchers — Ling Ding Jeak and Bo How — from the National University of Singapore cloned, for the first time, the DNA of a Factor C molecule to synthesize recombinant Factor C (Maloney et al., 2018). It was the first laboratory-synthesized endotoxin detection technology that did not exploit animals. Since then, there have been many other groups studying the different species to find the best rFC test for the detection of endotoxins.  

The recombinant Factor C test uses a single protein synthesized from the cloned DNA as its active ingredient.

Fig. 14 Comparison between LAL cascade and Recombinant Factor C technologies for endotoxin detection. [Adapted from Cardonnel, 2019]

The test begins by the binding of the endotoxin — with zymogen protein — which activates the rFC molecule. Then, it cleaves a synthetic fluorogenic substrate — small fluorophore peptide — resulting in the generation of a fluorogenic compound. The fluorescence of the product is then compared with the fluorescence of the initial sample to determine the concentration of endotoxins (Williams, 2019).

Another possible alternative to detect endotoxins exist. Although it is not literally an analogue of limulus amebocyte lysate, the monocyte activation test (MAT) can be performed to detect endotoxins and many other pyrogens. In fact, monocytes release cytokines in presence of pyrogens, it is basically the process causing fever in humans. Then, the concentration of cytokines is measured to determine the presence of endotoxins (Schindler et al., 2009). It is similar with the pyrogen test on rabbits that was performed before the development of LAL technology (their fever was measured).

Comparison between LAL and rFC Technologies

Recombinant Factor C tests are currently estimated to be comparable or better than limulus amebocyte lysate detection of endotoxins. This conclusion is supported by many scientific studies comparing the two technologies in terms of efficacy, sensitivity, applicability and reliability (Maloney et al., 2018). Indeed, rFC trials can detect gram-negative bacteria endotoxins that laboratories are tracking with LAL. Like LAL assays, they can detect endotoxins from different ranges of concentration and quantify their measure. Furthermore, in contrast with LAL, rFC are specific to endotoxins while LAL detect many pyrogens and substances from gram-positive bacteria such as peptyglodican, exotoxins, simple polysaccharides or dithiols (Maloney et al., 2018). In fact, rFC do not contain glucan sensitive Factor G and thus eliminate many false positive results found with LAL. Also, rFC technology is considered to have a high rate and reliable sensitivity in picogram quantities for the detection of various endotoxin structures (Abate et al., 2017). Another important aspect to consider is the range of applicability offered by those tests. And again, rFC detection shows great results in many settings. Three currently commercialized synthetic rFC correlate from 94.4 percent in water testing from different sources (lakes, springs, mineral and deionized water). LAL and rFC are also similar in detecting airborne endotoxins and at various stages of manufacturing therapeutic solutions (Reich et al., 2014). Nonetheless, endotoxins detection is not small business, biomedical industry must ensure safety of substances and equipment and ensure people’s safety. The reliability of rFC tests is another point to consider. rFC technology demonstrates a high rate of specificity for endotoxins and not any false positive has been observed yet, while LAL is reactive to proteases and phospholipids for instance. Furthermore, it could even be better than LAL in many aspects. LAL can lead to potential false negatives since its sensitivity can be inhibited by several buffers and solvents (Maloney et al., 2018). Actually, LAL is more subjected to the interferences present in complex samples or to lot-to-lot variability than rFC since it uses a unique protein (Hassan et al., 2017).

However, the most important and reliable study ever made for the general adoption of rFC was led by a consortium of 14 biopharmaceutical manufacturers — including Bayer, Merck and Co., Lonza, Biogen — known as the BioPhorum Operations Group (BPOG). They used similar protocols in 21 laboratories and compared 37 different reagent combinations with rFC and LAL (Maloney et al., 2018). Fortunately, the study delivered a hopeful conclusion for horseshoe crabs. Globally, rFC technology allows a high detection of endotoxins and is estimated to be comparable or better than LAL under various buffer conditions (Bolden et al., 2017). This study proves the ability of commercially available rFC technologies to replace LAL tests.  

 Conclusion

This essay established the uses of horseshoe crab hemolymph in industry. This animal of prehistoric age has revolutionized the biomedical industry. The discovery of its blood properties has shown to be beneficial in pharmaceutical production by providing a new and efficient method to test for the presence of endotoxins in medications without having to test directly on living animals. The sampling of horseshoe crab hemolymph allows for the extracting of an enzyme capable of inducing a clotting process if endotoxins are present.

The living fossil’s blood most likely results from an evolution progression in arthropod abilities in terms of immunity. The open circulatory system observed in horseshoe crabs actually increases the risk of a bacterium spreading throughout the organism. Thus, it is imperative that the immune system be sufficiently reactive. The particular abilities of horseshoe crab hemolymph are provided by unique cells called amebocytes, which contain immune system response actors, such as clotting factors and antimicrobial substances. These factors are essentials when it comes to the detection of endotoxins as they are responsible for launching the coagulation cascade and thus the neutralization of these toxins. This principle is notably used in the biomedical industry under the form of the LAL and TAL tests.

Currently, human activity is threatening horseshoe crab populations. The expansion of the biomedical industry poses the risk of a potential extinction of these prehistoric animals. If horseshoe crabs were to disappear, it would not only be terrible for the medical industry, but it would also be detrimental from an ecological perspective as nature’s balance would be thrown out of equilibrium. However, there exist modern efforts to limit the exploitation of horseshoe crabs and to create artificial analogues for Limulus blood.

The principal alternative method is based on recombinant Factor C technology which relies on cloned DNA of the Factor C molecule in limulus species. It allows to detect specifically endotoxins and shows great results comparable or even better than LAL under many conditions. It could effectively replace the need for bleeding horseshoe crabs but progresses slowly into the biopharmaceutical industry.

Until these artificial analogues become more widespread, it can be expected that many new medical discoveries will still rely on LAL testing before being released. Throughout history, there have been times when horseshoe crabs were considered of little value. It is important, now more than ever, that we recognize how these animals occupy a position of great ecological, adaptational and medical importance and how they have undoubtedly saved the lives of millions of people. Perhaps it is now our turn to ensure that their populations also strive.

Appendix

Cox, Elizabeth. “Why do we harvest horseshoe crab blood?.”, 21 Sept. 2017, TED-Ed, YouTube, https://www.youtube.com/watch?v=VgEbcQxFUu8&t=178s.

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