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Límulus

What beats inside blue blood

Text by Agustín B. Ávila-Casanueva

Drawings by Mateo Pizarro

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Limulus is a living fossil. The latter is a way of saying that limulus is very resistant, that it has endured geologic changes, predators, preys and parasites without having to adapt to them. When we analyze this arthropod – its closest relatives are spiders, not crabs – we see that what has made it so successful is a strong defense. Limulus polyphemus is quite a strange being; its common names, “sauce-pan,” “horse foot crab,” “sea tank” and “marine cockroach” give us an idea of its physique and characteristics. Virtually all its body is covered with a hard shell, but the strongest point of its defense is in its blood.

The limulus has blue blood, which is probably why it is known in the U.S. as “king crab.” But it didn’t get this attribute due to royal decent, it is purely a physiological issue… chemical, actually. In our blood, the cells in charge of transporting the oxygen to the rest of the body – the red blood cells or erythrocytes – use a protein called hemoglobin that acts as a small box, storing oxygen and linking it to an iron atom. This oxidized iron, as we know it, is red, oxide color, which is why our blood looks like it doews. The limulus however, does not use iron, it prefers copper to link oxygen to the blood cells, giving it the blue color of oxidized copper.

Its blood is more than oxidized copper, the only cells it contains are called amebocytes because they have their own motility, like amoebas do; the cells not only transport oxygen to the entire body, they are reactionary xenophobes and due to their high sensitivity, they’re an even better defense than the flashy shell that dresses the limulus.

In the 1950s, an American scientist by the name of Fred Bang 1 studied blood circulation using the limulus as a model. During one of his experiments, he noticed that one of the horseshoe crabs that he was working with died in a very strange manner: virtually all the volume of its blood had coagulated into a single gelatinous mass. This death, which seemed to be taken out of a science fiction film, intrigued Bang so that he changed his research and found out what had been the cause of such a reaction in the blood of a deceased limulus.

Fred Bang found the answer. The death was caused by an infection of the bacteria Vibrio cholera, the cause of cholera in humans. But limulus did not die of cholera, the infection never really occurred. Bang found out about this when he exposed the blood of the limulus to the dead cells of the Vibrio and they kept causing the same coagulating effect, hence it wasn’t something the Vibrio did, but something it contained.

Vibrio has a large molecule called lipopolysaccharide or endotoxin in its exterior membrane as one of the many blocs that conform it. As its name indicates, this molecule is a toxin to many animals, and in humans it unleashes heavy fevers that function as a preventive alarm; fever is a way the body has of saying “I found a stranger that wants to harm us, I will try to kill it before something happens to us.” Fever, white blood cells – that eat the toxic intruders -, and an intricate network of minute blood vessels and capillary that maintain an infection isolated from the rest of our body, are a few of the immune defenses we have. On the other hand, limulus only has one.

The limulus lives in the sea, a soup of microorganisms that are willing to explore every new environment they come across. Thus, a small crack in the limulus’ shell terribly exposes it to all these microorganisms; it is true that not many might harm him, but it must be careful with those that do. Limulus has cold blood, which means that its temperature cannot increase to cause a fever and its circulatory system is classified as open, which means there is no small web of capillary to segregate an infection. Thus, the horseshoe crab coagulates.

This is when the amebocytes come into play, the blue cells found in the blood of the limulus. Besides transporting oxygen, they’re in charge of detecting intruders that might harm them, like the ones that contain toxins in their exterior membrane. When the amebocytes find this toxin, they secrete a coagulation factor – coagulogens – that causes the amebocyte that detected it as well as all that surrounds it to coagulate, and they go from being a motile cell to forming a dense and insoluble gel. This is how the infection is controlled.

In the lab, Fred Bang exposed the limulus’ blood to other species and genders of bacteria and found that all those that shared this type of toxin in their exterior membrane, known as Gram-negative bacteria, caused the same immediate and coagulating reaction. These bacteria may also cause febrile reactions in humans and provoke an even worse infection. Bang had found a practical way of knowing if that type of bacteria was present in certain environments.

This discovery, found during a basic research project, became of great medical importance. During different medical procedures, serum, vaccines, anti-inflammatory, antibiotics and various other liquids enter our body, these must – as doctors should – first and foremost, follow the Hippocratic Oath, “never do harm,” this includes not only being free of bacteria, but also toxins that unleash unwanted reactions in the body, like fever.

The limulus’ blood, or more like the limulus amebocyte lysate (LAL) – a solution that contains only amebocytes without the blood of the horseshoe crab – becomes a useful tool to detect this type of unwanted intruders. Since the 1970’s, LAL has been used in the pharmaceutical industry to prove that each batch of products destined to enter the human organism is free of pathogens and toxins. A small sample of the testing product is poured into a test tube along with the LAL; after 45 minutes, the tube is turned; if there is a clot at the bottom, it must be discarded, if not, it may be distributed in hospitals. The astronauts of the International Space Station use the coagulogen to better detect if there are any bacteria or fungi present in their own space lab before getting to work.

Now, how does the pharmaceutical industry obtain enough blue blood to make these tests? Limulus tends to live in the Gulf of Mexico and along the north Atlantic coastlines. Many pharmaceuticals collect these arthropods in the coasts of Delaware and New England to take them to labs close to the coast, extract a liter of blood a puncture in an artery, they allow them to recuperate for a period of two to three days, and then place them back in the sea. This procedure must take place in sterile conditions to prevent bacteria contamination that would kill the limulus.

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This procedure has been done for several decades and it does not cause grave discomfort to the limulus, although reports show that a little less than 10% of the limulus that are collected do not survive the procedure.

Until researcher Christopher Chabot 2 and his working group in New Hampshire (U.S.) further analyzed what happened to the limulus after they were bleed. A liter of blood represents almost a third of the total blood of the horse foot crab; this, added to the capture, transportation and drainage caused by being out of the water, creates stress in the animal.

Chabot and his collaborators captured fifty-six limuli in the coasts of New Hampshire, transported them to his lab near the coast, bleed them and put them back in the ocean; however they were in enclosures to be monitored or their movement was measured with cameras 24 hours a day.

These researchers found what one would suspect happens to someone, arthropod or not, that gets a third of blood extracted from their natural environment and is unsettled: the sauce-pan seem stressed, they reduce their walking speed to almost half, they’re disoriented, and they even change to a preferably nocturnal or diurnal lifestyle, when in normal conditions, they’re active throughout the day. But the changes don’t stop there: even though the blood volume is recuperated almost three days after the extraction, the level of amebocytes is not until three months later. The main defense, the one that has helped maintain the limulus as a living fossil for millions of years, does not go back to its normal levels after three months.

Although the fishing industry has been banned from catching limulus – they are used as bate to catch sea snails, which are used in various stews -, the capture to obtain LAL has increased 76% since 2007 in the New England area, and the total population of these arthropods has systematically decreased throughout the years.

The limulus is a wonder of evolution and its blood has proved to be almost as useful to us as it is to it; we have become the vampire of that species and it does not seem to adapt to that. The work of Chabot and his collaborators seeks to alert us about the stress that is being inflicted on this species; to control the amount of animals collected and completely stop the activity during mating season.

Let us hope that the limulus has enough defenses in its blood to allow it to resist the time it needs to readapt to our environment and that we may not exterminate it.

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1. Article on Fred Bang and the history of blue blood: http://hermes.mbl.edu/marine_org/images/animals/Limulus/blood/bang.html 2. Rebecca L. Anderson, Winsor H. Watson, and Christopher C. Chabot. Sublethal Behavioral and Physiological Effects of the Biomedical Bleeding Process on the American Horseshoe Crab, Limulus polyphemus. The Biological Bulletin, December 2013

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