The Wonder of Blood form the Genus Limulus

by Isaiah Robertson
BIOL/WATR 361, Spring 2015

Key taxa: Arthropoda, Merostomata, Xiphosura, Limulus

There comes a time when medicine, like most sciences, needs to look back to its roots of observation of the natural world to find new solutions to old problems. One such time, when in search of quality control procedures to insure the cleanliness of surgical equipment, the employment of a living fossil was necessary. The horseshoe crab, from the genus Limulus, can attribute its “living fossil” status to its amazing immune systems. This system is made up of their strong exoskeleton and their blood, which embodies some tools with potential in the medical field. Comparing Limulus blood to human blood is helpful to understanding how the bizarre blue blood of a Limulus functions. Similarly, comparing the blood clots helps better explain the way Limulus blood clots aid the crabs. The blood seems to exhibit potential antibacterial effects, however, and there is little to compare this to in humans. The blue blood of the Limulus has many unique properties that make it ideal for a number of quality control tests in the medical field.

Unlike humans, who have multiple kinds of blood cells, Limulus only has one cell type (Armstrong, 1979). These cells exhibit two forms. Like human leucocytes, white blood cells, Limulus blood cells are amoeboid that serve an important immunological role. However, when they are not actively serving an immunity purpose, they take a granular form and function like humans red blood cells by participating in a gas exchange role (Armstrong, 1979). Nevertheless, Limulus do not have hemoglobin like humans. Their blood contains a protein hemocyanin which serves the same function as hemoglobin but uses copper instead of iron providing the Limulus blood with its blue color (Sullivan, Bonaventura, and Bonaventura, 1974). Hemocyanins account for the blue blood, but they do not explain the unique chemical reactions that occur when Limulus blood comes in contact with a pathogen. Similarly to humans, Limulus blood cells are in an aqueous solution called plasma. Limulus blood cells secrete a compound called α2-Macroglobulin, which is responsible for clotting, into the plasma. This protein is common in most animals, but in the case of Limulus it binds with the surface proteins of most foreign cells (Armstrong et al., 1990). α2-Macroglobulin is present in high concentrations in the hemocoel of a Limulus and very reactive to foreign proteins. Therefore, instead of the amoeboid cells engulfing and breaking down pathogens, as seen in humans, they clot around any intrusion. Clots in Limulus blood were found to capture as little as one bacterium but usually two or more (Isakova & Armstrong 2003).

The movement of the Limulus blood cells is very different than human blood movement since all the blood cells serve the various purposes served by different human blood cells. Limulus blood cells do not move unless there is some sort of irritation that requires the cells to deal with an invasion. Armstrong (1979) compared movement of Limulus blood cells in vitro to movement of Limulus blood cells in vivo. When observing gill tissue, it was found that the cells in a Limulus are not typically motile. Instead they just float around, moving with the natural current of fluid within the hemocoel that carries them to exchange gases with the tissues of the crab. However, if a cell were going to be motile, it was found that Limulus blood cells need to degranulate, at least enough to form pseudopods for movement. Armstrong observed that this was mostly exhibited by cells on glassware where the cells were outside their typical environment. The Limulus blood cells quickly degranulated here and started moving around on their own. This degranulation is required for a clot to form as the globulin that forms the clot is also what granulates the Limulus blood cell (Armstrong & Armstrong, 2003). It is logical to assume that this evolved out of a necessity for the cells to be mobile when there are also clots forming. However, there is little other than theory to support this idea. In comparison to humans, the cells serve the same function as red blood cells until they are needed to combat a pathogen. Then they function as white blood cells.

One of the times these cells become motile is when there is an irritation in the Limulus. They need to degranulate when they find an invading cell as the globulin is needed to clot around the invader. Armstrong & Armstrong (2003) found that the blood clots of a Limulus, like in humans, clot at the area of a wound and aid in healing. They do this by forming a bridge between damaged tissues for new cells to grow along. In contrast to human clotting, Limulus blood also clots internally around pathogenic cells. Armstrong and Armstrong demonstrated that, unlike human blood clots which are made of platelets, Limulus blood cells contain globulin, clotting proteins in a crystalline form, which breaks down and leaves the cell, forming clots once outside of the cells. Then these polymers of protein either aid in healing or imprison pathogens keeping them from freely traveling about the animal. They further found that there are multiple compounds that bind to the protein fibers of the clot. These compounds serve many purposes, primarily aiding in clotting. Some seem to protect the clot from being destroyed by other compounds in the blood, while others seemed to increase rigidity. This has not been reported to be present in human cells. Clots are drastically different between humans and Limulus. It is particularly interesting that the Limulus clots internally as well as externally, when humans only clot at the site of a wound.

It is not a stretch to then question how simply clotting around an invasion is effective. Clotting would block blood flow. Killing pathogens often takes much more than starving, and the animal would never be able to survive with blood clotted around every foreign cell to ever enter a crab. However, blocking blood flow is not as much of a problem in a Limulus as it is in humans since horseshoe crabs have open circulatory systems. Having open cavities to flow in allows for clots that are otherwise too large to fit through vessels and would block flow. Unfortunately, an open circulatory system eventually would have clot problems. Clots would get stuck in gills for example. This, also, does not solve the problem of too many clots. Isakova & Armstrong (2003) explained why this is not a problem. In this study, Limulus clots were placed in several treatments: an agar based growing medium, typical for bacterial growth; simulated seawater, which is isotonic to Limulus blood; sterilely extracted Limulus blood; and Limulus plasma, isolated by centrifuging so as not to have any hemocyanins. In the growing medium the bacterial cells reproduced rapidly as is expected for bacteria in a growing medium — the only exception being that the bacteria was trapped within the Limulus blood clot. When incubated in simulated seawater the bacteria grew within the clot as well; however, they grew at a typical pace for seawater. In Limulus blood, however, the bacterial cells showed signs of death in 1-2.5 hours and were all completely dead within 8 hours. This also happened in the isolated Limulus plasma. This fact implies that there is a compound present that is killing bacteria in Limulus blood. Isakova & Armstrong (2003) were not able to identify the compound responsible for the bacterial death in this study, and no study since then has seemed to identify it either. Even though it is not known what the compound is, it is believed that this compound binds to the fiber of the clot (Armstrong & Armstrong, 2003). Consequently, blood clots only need to exist for about 8 hours and then can breakdown. This is helpful in the fact that it allows a Limulus to exist without a lethal number of clots.

This aggressive clotting is what makes Limulus blood so helpful in the medical industry. The blood can be used to identify the presence of pathogens. This is very helpful as quality control, where medical equipment is bathed in Limulus blood. If it clots it is not clean enough and needs to be re-cleaned. If the blood does not clot the equipment can be rinsed clean and be ready to use for surgery. Other times it is used to look for pathogens in spinal fluid. Sullivan & Watson (1974) examined what could affect the sensitivity of Limulus lysate. They did an assay to find out at what concentrations Limulus lysate could detect exotoxins, by adding varying amounts of Limulus blood to a small amount of endotoxin. Sullivan & Watson (1974) also tested multiple treatments to see what other factors affect the sensitivity of Limulus lysate. They found that Limulus blood collected from crabs in warm weather provided more sensitive blood and that extracting lysate with chloroform increases sensitivity by removing several inhibitors. This process made the use of Limulus blood more feasible for use in the medical field and proves its effectiveness. It is the shear strength of the immune system of Limulus that is harnessed when applying these methods to medicine.

The unique blood of a Limulus provides the horseshoe crab with an impressive immune system, which is a fantastic addition to the tools at the hands of today’s medical men and women. Its unique makeup of blood, with a single cell type and entire network of compounds contribute strongly both to the strength of its clots and the death of invading cells, makes this system a formidable one. This essay observed what chemically makes this blood blue, as well as, its makeup of cells. It also discussed the movement of these cells and the clotting they facilitate. These were all compared to human blood to make things a little easier to understand. Similarly, the clots themselves were also compared to human clots which proved to be a large part of what makes Limulus blood so effective at preventing infection. This also provides it with some uses to the medical field briefly discussed. Not only is this blood a uniquely potent clotting agent it also seems to be antibacterial. Even though, this is not yet well understood, it proves very helpful to the crab and potentially humans in the future of medicine. The immune system of a horseshoe crab is truly a force to be reckoned with and one that humans, despite their efforts do not fully understand and have not fully utilized.

References Cited

• Armstrong, P.B., J.P. Quigley & F.R. Rickles. 1990. The Limulus blood cell secretes α2-macroglobulin when activated. Biological Bulletin 178: 137-143.
• Armstrong P.B. 1979. Motility of the Limulus blood cell. Journal of Cell Science 37: 169-180.
• Armstrong P.B. & M.T. Armstrong. 2003. The decorated clot: binding of agents of the innate immune system to the fibrils of the Limulus blood clot. Biological Bulletin 205: 201-203.
• Isakova V. & P.B. Armstrong. 2003. Imprisonment in a death-row cell: the fates of microbes entrapped in the Limulus blood clot. Biological Bulletin 205: 203-204.
• Sullivan, B., J. Bonaventura & C. Bonaventura. 1974. Functional differences in the multiple hemocyanins of the horseshoe crab, Limulus polyphemus L. Proceedings of the National Academy of Sciences 71: 2558-2562.
• Sullivan, J.D. & S.W. Watson. 1974. Factors affecting the sensitivity of Limulus lysate. Applied Microbiology 28: 1023-1026.