Enter Limulus and an MBL scientist named Fred Bang.

This section explains how an MBL scientist discovered some amazing properties in horseshoe crab blood that enable has revolutionized ways to detect potentially lethal bacterial toxins and spawned a multi-million dollar industry.
Bang was studying the circulation of blood using horseshoe crabs when he found that one of his crabs died as a result of a Vibrio bacterial infection. The infection caused a strange disease in which almost the entire blood volume of the crab clotted into a semi-solid mass. Other bacteria had not produced this sort of reaction at all. Bang began to investigate further and found that only gram-negative bacteria produced this reaction. Furthermore, heat-treated bacteria (dead bacteria) continued to produce the reaction so it wasn't a pathological disease but something different. His original 1956 paper describing his first observations is available here.

Bang noted that the reaction he was observing was very similar to a well-known endotoxin reaction in mammals, the Schwartzman reaction. Back at Johns Hopkins University, he pushed to have this new phenomenon researched more intensively. Jack Levin, a hematologist, joined Dr. Bangs laboratory. What they eventually found was the "fire alarm" system that could be used to detect, with exquisite sensitivity, the fever-producing endotoxins that are so dangerous to people.

Limulus lives in an aquatic world; the sea. The sea is almost literally awash in gram-negative bacteria. Millions can be found in a single gram of sediment. Bacteria that are both harmless as well as pathogenic (disease-causing).

Why is horseshoe crab blood blue?

The oxygen-carrying pigment in horseshoe brab blood is a protein called hemocyanin. It is very similar to the hemoglobinmolecule we have in our blood. Hemoglobin gets it's red color (which makes our blood red) from the iron molecule in the center of the protein. Hemocyanin contains a copper molecule which results in a blue color.

Limulus is an arthropod, a close relative to spiders. In fact more closely related to spiders than to true crabs. Arthropods possess a semi-closed circulatory system. We mammals have literally thousands of miles of blood vessels that carry blood to our tissues through vast networks of capillaries. Bacteria entering our bodies through these capillaries are initially limited in the area they can infect, having to fight their way into the body through these narrow channels, all the while in contact with the white blood cells that are our first line of defense.

The circulatory system of Limulus is far more open. Large sinuses exist that allow blood direct contact with tissues. There are many wide open spaces and bacteria entering a crack in the shell of a horseshoe crab have easy access to large internal areas of the crab, a potentially deadly scenario. Over the course of it's hundreds of millions of years of interacting with the bacterial swarms it coexists with, Limulus, like us, has developed exquisitely sensitive means for detecting the presence of bacteria through the LPS they shed into their environment.

See the illustrations of Milne Edwards for a look at the eerily beautiful illustrations of the circulatory system of Limulus
Limulus is cold-blooded. It can't raise it's body temperature to kill off an infection. Nor does it have the vast confusing network of blood vessels to contain an infection. It needs to act quickly, and sometimes even rashly. The soldiers of the immune system in Limulus are it's single type of blood cell, the amoebocyte. As it's name implies it is an ameoboid cell (it has motility). The cell itself is often obloid in the blood stream and perform most of the normal functions associated with blood cells, engulfing foreign or dead cells, transport and storage of digested materials, repair of wound sites, etc. The cells appear oval when seen inside a living crab and they are packed with small granules. These granules contain clotting factors that are released outside the cell when it detects the bacterial endotoxin. When the hemocyte is in the presence of endotoxin it changes dramatically, so much so that it was originally believed Limulus had several types of blood cells. The compact shape changes to an irregular amoeboid shape with numerous cytoplasmic processes streaming in all directions. The cell discharges the granules of coagulogen which empty the cell.

It's a very sensible system. Imagine a horseshoe crab has sustained a small injury. Seawater comes into contact with the tissue and bacteria come into contact with the blood and begin to enter (ie infect) the body of the crab. small bits of the cell wall slough off as the bacteria propels itself through the blood. A Limulus blood cells detects this tiny fragment and responds by releasing the contents of the granules into the surround medium. These granules contain a clotting factor, called coagulogen. The thought is that by clotting the immediate surroundings very quickly, the invading bacteria can become enmeshed and therefore stopped! Larger clots may not only stop enmeshed bacteria but serve as a barrier to the outside environment in the case of a severed limb or large incision. Bang found these clots to be very stable and prevented even Brownian motion in trapped bacteria.

The bottom line to all this is that Limulus contains an exceeding sensitive means to detect the presence of bacterial endotoxins that can be detected by the formation of a gel-like clot. This may not strike one as significant until one understands the impact of endotoxins on our health and healthcare systems.

Anything that goes into your body during surgery, by injection, or for therapy, has to be free of bacteria. If not, the recipient will get an infection. Not only must this material be sterile (meaning no living bacteria are present) but it must be pyrogen-free.! As was demonstrated long ago, our bodies, like the bodies of horseshoe crabs , respond to the presence or endotoxin, not just the bacteria. The industry of ensuring that injectable drugs, irrigation fluids, surgical tubing, etc are free of bacterial endotoxins is a big business. In the past, companies maintained large rabbit colonies. Rabbits, like use, are sensitive to endotoxin and if a suspect sample of saline injected into a rabbit caused a fever then it was contaminated. No fever, no contamination. This method was not only expensive (it isn't cheap to keep thousands of rabbits) it is also slow. A rabbit test might require 48 hours to obtain a result. A Limulus amoebocyte lysate (LAL) assay can take as little as 45 minutes. A suspect sample is mixed with reconstituted LAL and allowed to sit in a small tube. After 45 minutes the tube is inverted and if a clot has formed it will stick to the top of the inverted tube.

LAL is a multi-million dollar business. It received FDA approval in the 1970's for use in the testing of drugs, blood products, intravenous fluids, and disposable pharmaceutical devices and in 1983 was registered in the U.S. Pharmacopeia. The lysate is produced by extracting blood from the crab. This is done using a non-lethal method where blood is taken from a large dorsal blood sinus, the pericardium. The crabs are returned to the water within 24 hours and completely recover.

Jack Levin demonstrates the removal of blood from a Limulus. This procedure does not permanently harm the animal.

The blood is a milky-blue color due to the copper-pigmented hemocyanin molecule which turns blue upon contact with oxygen the same way our blood turns red. The blood is a mixture of liquid serum and suspended amoebocytes. Of course, the conditions for extracting blood must be sterile and pyrogen-free else the amoebocytes would immediately do their job and form a clot. If these conditions are met, the blood can be centrifuged and the result is a separation of blood cells from the serum. A small whitish pellet forms at the bottom of the tube. Technicians pour off the serum annd rinse the pellet with saline. It is then resuspended and added to the collection of amoebocytes. Eventually pyrogen-free, distilled water is mixed with the suspesion of blood cells. This causes the cells to absorb fresh water and balloon until they eventually burst (or "lyse" - hence "lysate"). This releases the coagulogen into solution.

The resultant solution is filtered to remove cellular debris and then freeze-dried to form a white powder of the lysate. This lysate is then packaged and sold to be reconstituted as the assay described above.

Stanley Watson was a microbiologist at the Woods Hole Oceanographic Institution next door to the MBL who used this lysate originally as an assay in his research with marine bacteria. He realized the potential value of LAL and his company, Associates of Cape Cod, is now doing a multi-million dollar business in horseshoe crabs.

A few decades ago, there was a bounty on Limulus as it was perceived to be a threat to the shellfish industry. The work of Bang and the resultant market developed by Watson and others has turned this animal into a valued resource. Not only is this commodity renewable and sustainable but the methods are non-lethal to the animal as well. This is a good example or basic research providing additional leverage in the conservation of Limulus and the aquatic environments it inhabits. Who knows where the next discoveries may lead. The line between basic research and applied science is indistinct and quite often it is the unexpected discoveries that are the most rewarding.

[NEXT: Peter Armstrong and new discoveries]

References used in this section

1. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of Horseshoe Crabs, p.334

2. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of Horseshoe Crabs , p.334

3. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of Horseshoe Crabs , p.338

4. Mürer, E.H., Levin. J. and Holm, R., 1975. Isolation and studies of the granules of the ameobocytes of Limulus polyphemus, the horseshoe crab. J. Cell Physiol., 86: 533-542

5. Armstrong, P.B. 1979, Motility of the Limulus Amebocyte, Biomedical Applications of the Horseshoe Cran (Limulidae), 73-92.

Quigley, J.P., Corcoran, G., Armstrong, P.B., A Hemolytic Activity Secreted by the Endotoxin-Challenged Horseshoe Crab: A Novel Immune System Operating at the Surface of the Carapace. , Biological Bulletin, 193: 273 (October 1997)

6. Milne, Edwards, H., Historie naturelle des Crustacea., Paris, 1834-40

7. Milne, Edwards, H., L'Anatomie des Limules, 1873

8. Sargent, William., The Year of the Crab., W.W. Norton & Company 1987