Once in A Blue… Blood?
Jeff Russell Ancheta
Old Dominion University
19 November 2018
Word Count: 2156
There were many theories of how the human visual system worked from Plato, Aristotle, Galen, etc. (Stanford, 1999). Scientists were only able to understand how human vision worked through studying the eye of the horseshoe crab which contained a neural network (Lie & Passaglia, 2009). They have found that horseshoe crabs are the perfect interactive model for understanding how the human visual system operates since horseshoe crabs have rods and cones that are 100 times bigger than the human eye. Hartline and Graham (1932) discovered that humans had the ability to “adapt visual sensitivity to ambient lighting conditions” due to visual adaptation and spectral sensitivity in the retina. The retina is the layer at the back of the eyes where the macular, which contains special light-sensitive cells, trigger nerve impulses that sends the image to the brain (Barlow, 2009).
There are four species of horseshoe crabs, the Limulus polyphemus which can be found in North America, and the other three which can all be found in Asia: the Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda (Walls, Berkson, & Smith, 2002). These arthropods have been around nearly 300 million years ago and survived the K-T (Cretaceous-Tertiary) event which paleontologists theorized for the mass extinction of dinosaurs (Sacred Heart University, 2018). Horseshoe crab has earned the title “living fossils” because fossil evidence shows that they have been inhabiting the planet for more than 350 million years (Kreamer & Michels, 2009). But how did such a small creature survive for millions of years? The answer is in their blue blood. The blue pigmentation of their blood is due to the copper that is present in hemocyanin (Wisniewski, 2018). The word hemocyanin is composed of two parts: the Latin word heme, which means “blood,” combined with the Latin element cyanin, which means “blue.” If the horseshoe crab ever becomes injured, the infected area becomes sealed off by the clotting agent called amebocyte lysate (Wisniewski, 2018).
The blood of the horseshoe crab has paved the way for endotoxin screening in the pharmaceutical and biomedical industries. Consequently, over the years the horseshoe crab population has begun to decrease, and the International Union for the Conservation of Nature listed the species as “vulnerable,” just a category short from being endangered (IUCN, 2016). Therefore, humans should not rely heavily on the use of horseshoe crab blood as it will have negative effects on the ecosystem, biomedical industry, and commercial fisheries. Instead, we should find other alternatives to horseshoe crab blood such as adopting the use of the recombinant Factor C (rFC).
Statistics show over 85% of people get vaccinated (National Center for Health Statistics, 2016). However, before a human gets vaccinated, every medical dose must be tested for contamination (Madrigal, 2014). Madrigal explains that in order to test for contamination in drugs, scientist use the unique blood of horseshoe crabs as it is “sensitive to toxins and bacteria.” “Of all marine species, horseshoe crabs have contributed the most to medical and physiological research” (US Fish & Wildlife Service, 2006). However, the necessity for the species’ blood in the biomedical industry has caused a decrease in their population (Frank ; Tang, 2018). In a world where infectious diseases are the leading cause of death and human’s dependent on horseshoe crab blood, it is imperative that we prevent the extinction of their species.
In order to draw blood from the horseshoe crab, the animal must go through an ordeal process that sometimes leave them traumatized (Frank & Tang, 2018). Armstrong & Conrad (2008) clarifies that horseshoe crabs receive a “direct cardiac puncture” and then hung upside down to let gravity help the blood ooze out of their heart and into a flask. Maloney, Phelan, and Simmons (2018) provide a photograph (figure 1) that illustrates how the bleeding process is conducted. This bleeding process usually kills about 150,000 horseshoe crabs (Kreamer & Michels, 2009) while the rest who make it out alive are sold to commercial fisheries (Cramer, 2018). Although there are few lucky ones that get returned to their natural habitat, studies have shown that “10% to 25% of the animals will die within the first couple days after bleeding” and the rest suffer from “behavioral defects” such as stunted growth and the inability to breed (Frank & Tang, 2018).
Figure 1: Bleeding Process for Horseshoe Crabs (Maloney et al., 2018)
Though the U.S. Food and Drug Administration (FDA) has mandated a law that declares bled horseshoe crabs must be returned to their habitat within 72 hours after capture; the population is still dwindling (Botton & Ropes, 1987). Correspondingly, management plans on the conversation of horseshoe crab population have been made. However, most of the data on the horseshoe crab population gathered by the Atlantic States Marine Fisheries Commission (ASMFC), which are deemed to be “necessary information” for managing the species population, was either not gathered or was “not of sufficient quality and quantity” (Berkson & Shuster, 1999). Luckily, some fisheries have steered away from the use of horseshoe crabs in general and instead use the waste of other crustaceans as bait. The remaining fisheries who still use horseshoe crabs are those who have begun to synthesize artificial bait from the eggs of horseshoe crabs to mimic the attractant that allures eels and conches (Walls et al., 2002).
Scientists have also begun to synthesize similar chemicals in hope to eliminate the method. In order to synthetically attain these chemicals, scientists need to understand what biochemical components are found in LAL. Walls et al. (2002) have found that factor C, factor B, and proclotting enzyme Z are the major chemicals in the LAL test that allows the horseshoe crab to immobilize and engulf an endotoxin. It all begins with factor C which reacts with the endotoxin and activates factor B which is then responsible for recognizing the endotoxin. They further explain that if factor B recognizes the endotoxin to cause detrimental health problems, proclotting enzyme Z would be activated in order to seal off and kill the endotoxin. Analyzation of LAL biochemical components have led to genetically engineered recombinant cascade reagents (RCRs) as an alternative use of horseshoe crab blood (Mizumura et al., 2017). Mizumura et al. provide a flowchart (figure 2) that shows how the recombinant zymogens mimic the LAL test. From the horseshoe crab species Carcinoscorpius rotundicauda, a synthetic alternative has been made from their endotoxin-reactive component of C (Novitsky, 2009).
Figure 2: LAL Flow Chart Process (Mizumura et al., 2017)
Maloney et al. (2018) found that factor C reacts with the endotoxins and cause for coagulation in the horseshoe crab hemolymph, a fluid plasma in their blood. In 1997, Jeak and How were the first to manufacture a synthetic alternative to horseshoe crab blood called recombinant Factor C (rFC) (Maloney et al., 2018). Though rFC is the first laboratory-synthesized alternative, scientists from different institutions have reviewed and evaluated the rFC-based assay to be “equivalent to the LAL test both in its ability to quantifiably measure endotoxin and in its ability to detect endotoxins across a range of concentration” (Maloney et al., 2018) and is “more efficient and cost-effective” (Cramer, 2018).
As previously mentioned, horseshoe crab population has been continuingly dropping over the past years and are a category short from being listed as an endangered species (IUCN, 2016). Though synthetic alternative rFC has been in the market since the early 2000s, it has not been accepted by all pharmaceutical companies (Cramer, 2018). In order to preserve the remaining population, pharmaceutical and biomedical companies must all adopt the use of rFC. Not only will it save the horseshoe crab population but also save human lives as it is more effective and does not get false positive results until the LAL test (Maloney et al, 2018).
Ding and Navas (1995) studied the difference in RNA sequence of factor C between the Carcinoscorpius rotundicauda and Tachypleus tridentatus and have noted that factor C, a zymogen, is responsible for coagulation. The analyzation and cloning of the molecular sequence of factor C and synthetic substrates have opened the possibility for a LAL alternative (Ding and Navas, 1995). Cloning from the Tachypleus tridentatus zymogens has made a new possible method of measuring endotoxins called the Tachypleus Amoebocyte Lysate (TAL) test.
The TAL uses a portable kit called an endosensor which measures the endotoxin in liquid biosamples (Akbar, Kamaruzzaman, Jalal, & Zaleha, 2012). Akbar et al. (2012) explains that the method works by injecting the sample with TAL, and if there are any endotoxin present in the test sample, the solution will clot and turn into a gel-like substance. If not, the solution will stay in its liquid form. Scientists have expanded the list of species and organisms which they can extract biochemicals from; this is currently undergoing further research to trial if these biochemical components will lead to future alternatives (Walls et al., 2002).
Native Americans used the meat from horseshoe crabs for food and used their shells as tools before European settlers started using them for other means (Kreamer & Michels, 2009). However, horseshoe crabs were considered a nuisance to commercial fisheries because of how often they get caught in fishing nets (Walls et al., 2002). The ease of harvest with minimal financial backing (usually by hand on the beaches) and a maturation rate of 10 years creates a “time lag for population recovery” (Berkson & Shuster, 1999). Those that were caught were used as fertilizer, food for livestock, or used as bait to catch eels and other crustaceans. Walls et al. (2002) have found that commercial fisheries prefer female horseshoe crabs because of the “certain chemical odors unique to the egg-laden females” and how it attracts eels and conches.
Not only do humans rely on horseshoe crabs but also shorebirds. Migrating shorebirds travel to breeding grounds located along the Atlantic Coast of North America. One of these locations happens to be Delaware Bay, where horseshoe crabs’ nest and lay their eggs (Berskon ; Shuster, 1999). However, the birds are not getting enough food as egg densities have thinned down by 98% (Crammer, 2018)! If horseshoe crabs become extinct, the population of shorebirds would correspondingly decrease. The eggs of horseshoe crabs are like cliff bars to shorebirds; it makes up the shorebird’s dietary supplements for their initial nesting period and helps sustain enough energy for their non-stop strenuous flight back home (Berskon & Shuster, 1999). Walls et al. provides a table (figure 3) that lists organisms known to prey on the horseshoe crab. It is apparent from the table that other marine life such as shrimp, conch, and eels rely on horseshoe crabs as a food source all of which are sought by commercial fisheries.
Figure 3: Organisms Known to Prey on the Horseshoe Crab (Walls et al., 2002)
Walls et al. (2018) have offered the idea of using invasive species such as rabbits. Invasive species can be plants, animals, or any other organisms that are non-native to the ecosystem and causes economic or environmental harm or poses a threat to human health. Rabbits are considered as an invasive species because of how fast they can reproduce and repopulate with four to twelve babies and gestation in only a month period (Soniak, 2015). Before the use of horseshoe crab blood, rabbits were the standard method for screening endotoxins via the Pyrogen Test (PT). The word pyrogen is composed of two parts: the Greek word pyro, which means “fire,” combined with the Greek element gen, which means “thing that produces or causes.” As such, the rabbit’s temperature was observed for signs of fever after injection of a certain chemical or drug (Cramer, 2018). The alternatives are not limited to just rabbits alone. Humans can extract the chemicals from other organisms such as sea cucumber, gorgonians (coral), and red algae in hopes that the biochemical that helps them regenerate aid humans into finding other synthetic alternatives (Walls et al., 2018).
The multi-use of horseshoe crabs has resulted in the decline of the species population. With this purpose in mind, scientists should not harvest horseshoe crab as “mortality rates from the bleeding process are reported to be as high as 20%” (Hurton, Berkson, ; Smith, 2009). To prevent the population from declining further, conservation plans must be placed. To start, according to Cramer (2018) and Berkson and Shuster (1999), monitoring and collecting basic population should be of high priority. Other scientists in Asia have set up protected breeding and nesting sites and believe that public education is the biggest factor in the management of horseshoe crab population (Berkson, Chen, Mishara, Shin, Spear, ; Zaldivar-Rae, 2009). If the general public realizes the consequences that follow the extinction of horseshoe crabs, it will expedite the process for a synthetic engineered alternative that is accepted by all pharmaceutical and biomedical industries. For now, we must turn to the adoption of the rFC as it is not only saving the lives of these animals but are also cost effective and more efficient that the LAL test.
Akbar, J., Kamaruzzaman, B., Jalal, K., ; Zaleha, K. (2012). TAL – a source of bacterial endotoxin detector in liquid biological samples. International Food Research Journal, 19(2), 423-425. Retrieved from: http://www.ifrj.upm.edu.my/19%20(02)%202012/(6)IFRJ-2012%20Akbar.pdf
Armstrong, P., ; Conrad, M. (2008). Blood collection from the American horseshoe crab, limulus Polyphemus. Journal of Visualized Experiments, 20. doi: 10.3791/958
Berkson, J., Chen, C., Mishra, J., Shin, P., Spear, B., ; Zaldivar, J. (2009). A discussion of the horseshoe crab management in five countries: Taiwan, India, China, United States, and Mexico. Biology and Conservation of Horseshoe Crabs, 465-475. doi: 10.1007/978-0-387-89959-6_29
Berkson, J. ; Shuster, C. N. (1999). The horseshoe crab: The battle for a true multiple-use resource. Fisheries, 24(11), 6-10. doi: 10.1577/1548-8446(1999)024;0006:THCTBF;2.0.CO;2
Cramer, D. (2018). Inside the biomedical revolution to save horseshoe crabs and shorebirds that need them. The National Audubon Society. Retrieved from: https://www.audubon.org/magazine/summer-2018/inside-biomedical-revolution-save-horseshoe-crabs
Ding, J. L. ; Navas, M. A. (1995). Molecular cloning and sequence analysis of factor c cdna from the Singapore horseshoe crab, carcinoscorpius rotundicauda. Molecular Marine Biology and Biotechnology, 4(1), 90-103. Retrieved from: http://www.horseshoecrab.org/research/sites/default/files/rFC%20%28Mol%20Marine%20Biol%20%26%20Biotechnol%29.pdf
Frank, J., ; Tang, A. (2018). Why horseshoe crab blood is so expensive. Business Insider. Retrieved from: https://www.businessinsider.com/why-horseshoe-crab-blood-expensive-2018-8
Hurton, L., Berkson, J., ; Smith, S. (2009). The effects of hemolymph extraction volume and handling stress on horseshoe crab mortality. Biology and Conservation of Horseshoe Crabs, 331-346. doi: 10.1007/978-0-387-89959-6_21
Kremer., G. ; Michels, S. (2009). History of horseshoe crab harvest on Delaware Bay. Biology and Conservation of Horseshoe Crabs, 299-313. doi: 10.1007/978-0-387-89959-6_19
Liu, J. S., Passaglia, C. L. (2009). Using the horseshoe crab, limulus Polyphemus, in vision research. Journal of Visualized Experiments, 29. doi: 10.3791/1384
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), 1-10. doi: 10.1371/journal.pbio.2006607
Maloney, T., Phelan, R., ; Simmons, N. (2018). Horseshoe crab bleeding process. Photograph. doi: 10.1371/journal.pbio.2006607
Mizumura, H., Ogura, N., Aketagawa, J., Aizawa, M., Kobayashi, Y., Kawabata, S., ; Oda, T. (2017). Genetic engineering approach to develop next-generation reagents for endotoxin quantification. Innate Immunity, 23(2), 136-146. doi: 10.1177/1753425916681074
Mizumura, H., Ogura, N., Aketagawa, J., Aizawa, M., Kobayashi, Y., Kawabata, S., ; Oda, T. (2017). LAL process. Flow chart. doi: 10.1177/1753425916681074
Novitsky, T.J. (2009). Biomedical applications of limulus amebocyte lysate. Biology and Conservation of Horseshoe Crabs, 315-329. doi: 10.1007/978-0-387-899959-6_20
Soniak, M. (2015). Are rabbits as prolific as everybody says? Mental Floss. Retrieved from: http://mentalfloss.com/article/29870/are-rabbits-prolific-everybody-says
Walls, E. A., Berkson, J., ; Smith, S. A. (2002). The horseshoe crab, limulus polyphemus: 200 million years of existence, 100 years of study. Reviews in Fisheries Science, 10(1), 39-73. doi: 10.1093/oxfordjournals.jbchem.a123337
Walls, E. A., Berkson, J., ; Smith, S. A. (2002). Organism known to prey on the horseshoe crab. Table. doi: 10.1093/oxfordjournals.jbchem.a123337