Amoebocytes: An Indicator of Bacterial Contamination

Diya Adhikary, Amity University Kolkata

Can you name an animal that is even older than the Dinosaur? Well, one such animal is the Horse Shoe Crab! ‘Horseshoe crab’ (Limulus polyphemus), as the name suggests, is a marine and brackish water arthropod with a rounded head that resembles the shoe of a horse. It is a prehistoric animal that has been found to exist for more than 400 million years. The blood of this arthropod is bright blue and comprises immune cells that are remarkably sensitive to toxic bacteria. The special cells in horseshoe crab blood known as amoebocytes were discovered by Frederick Bang (1950’s). The cells attach to bacteria resulting in the formation of a viscous gel that prevents the bacteria from invading the crab’s body. Bang realized that these cells could be used in pharmaceutical drugs for the detection of the presence of bacteria. Blood from horseshoe crabs is presently being harvested by biomedical companies for the production of Limulus Amoebocyte Lysate (LAL). Surprisingly, NASA is trying to apply the use of LAL in space to assist the diagnosis of astronauts.

Horseshoe crabs are more closely related to trilobites (existing 544 million years ago). Trilobites disappeared at the end of the Paleozoic era but horseshoe crabs survived several mass extinctions and have kept the same body plan for millions of years. Horseshoe crabs referred to as living fossils look like prehistoric crabs but are more related to spiders, ticks, and mites than they are to crabs. Limulus polyphemus inhabits the eastern coastline of North and Central America. The other three species inhabit the coastlines of Japan, India, and Indonesia. 

Anatomical Features

Horseshoe crabs belong to the phylum Arthropods (animals having an articulated body and limbs), class Merostomata (legs attached to the mouth). They have a tank-like structure that consists of a front shell (prosoma), a backshell (opisthosoma), and a spike-like tail (telson). Its body is protected by a smooth, hard carapace. Despite having sharp tails they are harmless. The function of their tail is to flip them back over in case they get overturned by a wave. The head holds most of the organs (heart, brain, and mouth). Their legs help in locomotion and feeding. There are only four living species of horseshoe crabs: —Limulus polyphemus (Eastern coast of North and Central America), Tachypleus tridentatus (Indo-Pacific species), Tachypleus gigas (Indo-Pacific species), and Carcinoscorpius rotundica (Indo-Pacific species). In terms of overall dimensions, females are about 20-30% larger than males of this species.

The blue coloration of their blood

Their blood contains copper-based hemocyanin to carry oxygen. Hemocyanin (copper is the central metal atom) is a protein that similarly carries oxygen as hemoglobin. This gives the blood a blue shade of color. Hemocyanin transports oxygen throughout the body of the invertebrates (arthropods and mollusks). This respiratory pigment has a high molecular weight, pH, temperature, and ionic concentration that modulate the oxygen affinity. The sub-unit of hemocyanin tends to form aggregates. The copper [Cu(I)] is bound directly to the amino acid side chain, which is opposite to the metal being bound to the prosthetic group in the case of the hemoglobin.

Blue blood cells function

The blue blood cells (amoebocytes) aid in defending the crab against pathogens like bacteria that are omnipresent in seawater. When an amoebocyte comes into contact with a pathogen, it releases a chemical. This causes the local blood to clot. Researchers believe this to be a mechanism for isolating dangerous pathogens. The amoebocytes in horseshoe crab blood solidify when they come in close contact with endotoxins. Endotoxin is a pervasive and sometimes deadly product of bacteria that kicks immune systems into gear, which at times results in fever, organ failure, or even septic shock. The substance derived from horseshoe blood is called Limulus Amoebocyte Lysate (LAL) which quickly became nearly as valuable as gold. The components of LAL are part of the primitive immune system. They not only bind and inactivate bacterial endotoxin, but also help in forming the clot as a result of activation by endotoxin (enzymatic gelation reaction). It provides wound control by preventing bleeding and by forming a physical barrier against additional bacterial entry and infection. 

Coagulation cascade in horseshoe crabs

• The amebocyte lysate consists of a mixture of naturally occurring proteins that are involved in the endotoxin detection process. 
• Factor C acts as the principal biosensor by binding with bacterial endotoxins. It activates another protein called Factor B.
• Factor B then converts a pro-clotting enzyme to a clotting enzyme.
• The clotting enzyme further catalyzes a reaction that causes a change in viscosity, turbidity, or color that is detected to ascertain the concentration of endotoxins in the sample.

An alternative endotoxin test

The presence of endotoxin in drugs, needles, or anything that comes into contact with human blood often brings about serious problems. Researchers used rabbit models to test the presence of endotoxins. The material of interest was injected into the rabbits and based on the observations (how their immune system reacted), they concluded. The amoebocytes in horseshoe blood proved to be a game-changer. Instead of conducting time-consuming tests on rabbits, horseshoe crab amoebocytes could be added to a sample of a substance and if the sample started to clot, then the presence of endotoxins would stand to confirm.

Importance of horseshoe crabs

• They are an important part of the ecology of coastal communities.
• For small birds like Red knot, the eggs of horseshoe crabs serve as a vital food source during their long migration.
• Many fish species also feed on horseshoe crab eggs.
• For humans, horseshoe blood provides an important chemical that is needed for testing the sterility of the surgical equipment.
• Studies involving the nerve pathways in the eyes of horseshoe crabs have led to many discoveries in humans.
• Research on the compound eyes of horseshoe crabs has led to a better understanding of human vision.
• They are used in several fisheries.

Prospect

They are extremely important to the biomedical industry due to their unique, copper-based blood containing the substance “Limulus Amebocyte Lysate” (LAL). It is one of the wonders of evolution that the horseshoe crab utilizes endotoxin as a signal for wound occurrence and as an effective defense against infection. Every year the Atlantic horseshoe crab (Limulus polyphemus) arrives on the shore of Delaware Bay to spawn. In addition to the gradual climate changes impacting all ecosystems, commercial demand of Atlantic horseshoe crab blood in industrial endotoxin testing and its steady use as eel and whelk bait has put the future of this long-lasting species into question. In response, regulations have been adopted to enhance the traceability and record-keeping of horseshoe crab harvest. However, these regulations do not put a restriction on LAL harvest. Still, sometimes, lethal biomedical bleeding process and associated behavioral changes pose a risk to horseshoe crab viability after bleeding and being returned to the waters. Regulators and environmentalists are apprehensive about the current trends and overfishing of this marine arthropod that will significantly impact the surrounding ecosystem.

Also read:Prader-Willi Syndrome Awareness month, 2021

REFERENCES:-

1. Horseshoe Crab History | Sacred Heart University
2. https://myfwc.com/research/saltwater/crustaceans/horseshoe-crabs/facts/
3. Mechanism in the Clot Formation of Horseshoe Crab Blood during Bacterial Endotoxin Invasion (scialert.net)
4. BACTERIAL ENDOTOXIN TESTING: 10 reasons to choose recombinant factor C | bioMérieux industrial microbiology (biomerieux-industry.com)
5. Krisfalusi-Gannon J, Ali W, Dellinger K, Robertson L, Brady TE, Goddard MKM, Tinker-Kulberg R, Kepley CL and Dellinger AL (2018) The Role of Horseshoe Crabs in the Biomedical Industry and Recent Trends Impacting Species Sustainability. Front. Mar. Sci. 5:185. Doi:  https://doi.org/10.3389/fmars.2018.00185
6. https://biosyncorp.com/klh/introduction_to_hemocyanins/

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