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Home , Crustacean, Horseshoe crab, Limulus amebocyte lysate, Mangrove horseshoe crab, Mesolimulus

Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 956-964

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 956-96 4 http://www.ijcmas.com

Review Article

Horseshoe and its medicinal values

Vikash Kumar*, Suvra Roy, A.K. Sahoo, B.K. Behera and A.P. Sharma

Central Inland Research Institute (CIFRI), Barrackpore- 700120 *Corresponding author

ABSTRACT

The medicinal value of comes from its ability to clot in the K eywo rd s presence of bacteria, rendering the bacteria harmless. Even though bacteria are usually destroyed by modern sterilization techniques, some bacteria, called endotoxins, may survive the sterilization process. This blood-clotting ability of the H orseshoe horseshoe crab makes it very valuable in testing for injectable medicines, vaccines crab, E ndotoxins, and sterile medical equipment. The horseshoe crab has also proved valuable to the B lood- medical field in another way. Studies involving the nerve pathways in the of clotting, horseshoe have led to many discoveries in human research. The outer

Chitin shell of a horseshoe crab is made primarily of . Scientists discovered that when used as a coating for suture material and burn dressings, chitin rapidly increased wound healing, cutting the time by half.

Introduction

Horseshoe crabs are marine of it is not really a crab. Horseshoe crabs the Limulidae and order or belong to the phylum, Arthropoda, along Xiphosurida, which live primarily in and with crabs, , and other around shallow waters on soft sandy with jointed legs, but their closest living or muddy bottoms. They occasionally come relatives are and . True onto shore to mate. They are commonly crabs have two pairs of antennae and a pair used as bait and fertilizer. In recent years, a of mandibles, or jaws; horseshoe crabs lack decline in the population has occurred as a these structures. Further, comparing the legs consequence of coastal destruction in of a true crab with the legs of a horseshoe Japan and overharvesting along the east crab reveals the other significant difference. coast of North America. may True crabs classified as decapod be present in the row of inhabiting , have five pairs of legs, which the waters of [1]. Because of their include a pair of claws. Horseshoe crabs origin 450 million years ago, horseshoe have seven pairs of legs under their helmet- crabs are considered living [2]. like shells; five of these seven pairs of legs are equipped with claws. In adult males, the Even though the horseshoe crab has a hard second pair of claws () has a shell and numerous with claws, “boxing-glove” appearance and is used to

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 956-964 grasp females during spawning. Horseshoe : Carcinoscorpius crabs also have four simple eyes on the top (O. F. Müller, 1785) of their instead of two as with the (extinct) true crab. Our North American species has been named Limulus – Limulus Species: Carcinoscorpius rotundicauda meaning “a little askew or odd” and Limulus polyphemus (Linnaeus, 1758) Polyphemus after the giant in Greek mythology. Mesolimulus walchi (extinct) Classification / history of the horseshoe crab Horseshoe crabs resemble crustaceans, but belong to a separate , Horseshoe crabs are among the world’s , and are closely related to oldest and most fascinating creatures. The . The earliest horseshoe crab earliest horseshoe crab species had already fossils are found in strata from the late inhabited Earth at least 200 million years period, roughly 450. The before the dinosaurs arrived or about 400 Limulidae are the only recent family of the million years ago. Horseshoe crabs are order Xiphosura, and contain all four living of the temperate . During the species of horseshoe crabs [3]: cold months, they lie half-buried in the ocean sediments. Horseshoe crabs have been  Carcinoscorpius rotundicauda, the observed mating from April through horseshoe crab, found in December, although the peak reproduction Southeast Asia period occurs during the highest in late May and early June, at the time of the full or

 Limulus polyphemus, the Atlantic new moon. Most spawning is at night horseshoe crab, found along the because of the protection afforded by American Atlantic coast and in the darkness. In spring, males arrive at the Gulf of Mexico shorelines first, followed by the females a  Tachypleus gigas, found in Southeast week or two later. Females average being and East Asia 30% larger than males and attract the males by releasing a pheromone, or natural  Tachypleus tridentatus, found in chemical stimulant, into the water. Southeast and East Asia Horseshoe crabs also use their relatively good vision to help locate potential mates. of the Horseshoe Crab The males patrol the near shore waters and use their pedipalps to attach to the Kingdom: Animalia of a female as she moves toward the beach. Phylum: Arthropoda They swim upside down, moving their legs Subphylum: Chelicerata and in a progressive wavelike Class: Merostomata (Dana, 1852) oscillation from front to back. Subclass: Xiphosura (Latreille, 1802) Order: Xiphosura (Latreille, 1802) Horseshoe crabs push their way along the Suborder: Limulina (Richter and Richter, bottom, digging small furrows in search of 1929) food. They use their first pair of legs as Superfamily: Limulacea (Zittle, 1885) feelers to determine the presence of prey. Family: Limulidae (Zittle, 1885) When the crab feels or smells a worm, clam, 957

Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 956-964 or dead , one of the claws picks it up and about 1000 ommatidia, plus a pair of median pushes it toward the heavy, spiny eyes that are able to detect both visible light projections that surround the mouth; the and light, a single endoparietal horseshoe crab has no nose; but the tiny eye, and a pair of rudimentary lateral eyes hairs on the spiny projections around its on the top. The latter becomes functional mouth are chemoreceptors, allowing the just before the embryo hatches. Also, a pair crab to smell prey. Since the horseshoe crab of ventral eyes is located near the mouth, as has no jaws to chew its food, it must bring well as a cluster of photoreceptors on the all of its legs together and use the spiny . Despite having relatively poor projections around its month than the first eyesight, the animals have the largest rods set of legs to crush the worm or clam. and cones of any known , about 100 Horseshoe crabs also have times the size of humans' [7, 8]. The mouth containing sand and small bits of gravel to is located in the center of the legs, where help grind their food. Horseshoe crabs their bases have the same function as jaws continue to grow for nine to ten years until and help grinding up food. It has five pairs they reach maturity. The young horseshoe of legs for , swimming, and moving crabs molt, or shed, their outer food into the mouth, each with a claw at the () often, until they reach sexual tip except the last pair. The long, straight, maturity, then molting slows, occurring only rigid tail can be used to flip it over if turned about once annually. The animals increase upside down, so a horseshoe crab with a in size from 25-30% with each molt by broken tail is more susceptible to desiccation pumping in water to expand their new shells, or . which will harden in approximately 24 hours. Males are sexually mature at their Behind their legs, they have book gills, sixteenth molt or ninth year. Females need at which exchange respiratory gases and are least 17 molts and mature in their tenth year. also occasionally used for swimming. As in Unlike the blue crab, this breaks out of its other arthropods, a true endoskeleton is old shell, the horseshoe crab crawls forward absent, but the body does have an out of its shell through a split that develops endoskeletal structure made up of along the junction of the dorsal (top) and cartilaginous plates that support the book ventral (bottom) surfaces. gills [9].

Horseshoe crabs face dangers from a variety Horseshoe crab normally swims upside of predators throughout their lifecycle. down, inclined at about 30° to the horizontal These include mollusks, crustaceans, fish, and moving at about 10–15 cm/s (0.22–0.34 leopard , , , turtles, and, mph) [10, 11, 12]. Despite this, they usually maybe most importantly, the man. A partial are found on the ocean floor searching for list of organisms known to prey on worms and molluscs, which are their main horseshoe crabs during various stages of food. They may also feed on crustaceans and their is given in Table 1 [4, 5, 6]. even small fish. Females are larger than males; C. rotundicauda is the size of a and behavior human hand, while L. polyphemus can be up to 60 cm (24 in) long (including tail). The The entire body of the horseshoe crab is juveniles grow about 33% larger with every protected by a hard carapace. It has two molt until reaching adult size [13]. compound lateral eyes, each composed of

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Breeding the nerve cord and extends almost the entire length of its body. On average, the rate During the breeding season, horseshoe crabs of the horseshoe crab is 32 beats per minute. migrate to shallow coastal waters. A male It has eight pairs of slit-like openings, or selects a female and clings to her back. The ostia, each opening having two valves female digs a hole in the sand and lays her through which the blood enters the heart while the male fertilizes them. The from the pericardial chamber. The blood is female can lay between 60,000 and 120,000 pumped forward and escapes through three eggs in batches of a few thousand at a time. pairs of aortae, one pair of cerebral arteries, Shore birds eat many of the eggs before they and a frontal artery. Two types of hemocytes hatch. The eggs take about two weeks to exist in the blood of the horseshoe: an hatch. The larvae molt six times during the abundant number of granulocytes and a few first year. Raising horseshoe crabs in cyanocytes. The granulocytes are captivity have proven to be difficult. Some responsible for the rapid clotting of blood, evidence indicates mating only takes place especially in the presence of bacterial in the presence of the sand or mud in which endotoxins. the horseshoe crab's eggs were hatched. Neither what is in the sand that the crab can Horseshoe crab importance in human sense, nor how they sense it is known with medicine certainty [14].

The The Horseshoe crab is really important to the medical community. The horseshoe crab In the horseshoe crab, there are several large has a simple, but the amazing immune nerves that supply the crab with information. system. When a foreign object (bacteria) From the anterior portion of the , a pair enters through a wound in their body, it of optic nerves runs directly to the two almost immediately clots into a clear, gel lateral eyes. There are also 8 pairs of haemal like material, thus effectively trapping the nerves that extend into the body of the crab. bacteria. If the bacterium is harmful, the These contain motor and sensory fibers and blood will form a clot. Horseshoe crabs are are distributed mainly to the membrane and proving to be very helpful in finding other tissues. The sixth pair sends branches remedies for diseases that have built to the heart and intestine. All of the haemal immunities against penicillin and other nerves are essentially the same, except the drugs. first one, the lateral line nerve. It runs close to the surface, just outside the bases of the The horseshoe crab has the best- appendages, begins to branch at the base of characterized immune system of any long- the 6th leg. It extends the whole length of lived . The study of immunity in the branchial chamber, sending one small horseshoe crabs has been facilitated by the branch to the base of each of the five gills. It ease of collecting large volumes of blood is a purely sensory nerve and supplies the and from the simplicity of the blood. skin lining the channel along which water is Horseshoe crabs show only a single cell type carried to the gills. in the general circulation, the granular amebocyte. The plasma has the salt content The of sea water and only three abundant proteins, , the respiratory The horseshoe crab's heart is a long tube that protein, the C-reactive proteins, which lies along the opposite side of the body of function in the cytolytic destruction of 959

Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 956-964 foreign cells, including bacterial cells, and commercialization, and use of LAL have α2-macroglobulin, which inhibits the been an important improvement in the proteases of invading pathogens. Blood is pharmaceutical industry. Prior to the use of collected by direct cardiac puncture under LAL, compounds were tested for the conditions that minimize contamination by presence of endotoxins in a variety of ways (endotoxin, LPS), a that involved living animals or living parts product of the Gram-negative bacteria. A of animals [19]. Thus, LAL provides a large animal can yield 200 - 400 ml of means to detect endotoxins without having blood. Unlike , horseshoe crabs to kill or disable animals [19]. do not have in their blood, but instead use hemocyanin to carry oxygen. Limulus Amebocyte Lysate is extremely Because of the present in useful in detecting those toxins that cause hemocyanin, their blood is blue. Their blood – the bacterial “pyrogens” or contains amebocytes, which play a role endotoxins. Endotoxins occur as a structural similar to white blood cells of vertebrates in component of the cell wall of a large group defending the organism against pathogens. of bacteria known as Gram negative [20]. Amebocytes from the blood of L. Most aquatic bacteria are of the Gram- polyphemus is used to make Limulus negative variety, as studies at the Woods amebocyte lysate (LAL), which is used for Hole Oceanographic Institution have shown the detection of bacterial endotoxins in that contains over 1 million Gram- medical applications. The blood of negative bacteria per milliliter and that horseshoe crabs is harvested for this purpose almost 1 billion bacteria can be found per [15]. Harvesting horseshoe crab blood gram of sand near the shore [21]. Thus, the involves collecting and bleeding the horseshoe crab habitat contains vast animals, and then releasing them back into amounts of endotoxin, making it no the sea. Most of the animals survive the coincidence that the horseshoe crab evolved process; mortality is correlated with both the a vital system to protect itself against amount of blood extracted from an endotoxins. The horseshoe crab blood individual animal, and the stress experienced includes amebocytes that contain the during handling and transportation [16]. clotting enzymes and other factors with the Estimates of mortality rates following blood ability to immobilize and engulf an harvesting vary from 3-15% [17] to 10-30% endotoxin [19]. When exposed to endotoxin, [18]. the amebocytes change shape, adhere to the sides of the vascular channels, and from the The LAL test represents one of a number of resultant gel clot [4]. This phenomenon is at pharmacological significant, chemical the heart of the LAL assay, as the formation constituents found in marine flora and fauna of a clot shows the presence of endotoxin. [19]. A wealth of significant compounds has been isolated from marine animals. These The major use of LAL today is in the include compounds derived from the sea detection of endotoxins in pharmaceutical cucumber used in anti- chemotherapy, products [20]. Since its original description, from gorgonians used for birth however, it has also been used in the control, against peptic ulcers and asthma and diagnosis of endotoxemia in conjunction lowering blood pressure, as well as with cirrhosis, cancer, , eye compounds derived from red algae that can disease, dental problems, gonorrhea, prevent atherosclerosis [19]. The discovery, boutonneuse fever, and water-quality

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 956-964 analysis [21], as well as urinary tract mates during the spawning season. Each infections [20]. In addition, new applications has about 1,000 receptors or for LAL continue to be found, including the ommatidia [24]. The cones and rods of the detection of bacterially contaminated , lateral eyes have a similar structure to those fish, and dairy products, including frozen found in human eyes, but are around 100 items [20, 22]. times larger in size. The ommatidia are adapted to change the way they function by Study of blood chemistry day or night. At night, the lateral eyes are chemically stimulated to greatly increase the For a study of the plasma, blood cells are sensitivity of each receptor to light. This immediately removed from the plasma by allows the horseshoe crab to identify other centrifugation and the plasma can then be horseshoe crabs in the darkness. The fractionated into its constituent proteins. The horseshoe crab has an additional five eyes blood cells are conveniently studied on the top side of its prosoma. Directly microscopically by collecting small volumes behind each lateral eye is a rudimentary of blood into LPS-free isotonic saline (0.5 M lateral eye. Towards the front of the NaCl) under conditions that permit direct prosoma is a small ridge with three dark microscopic examination by placing one of spots. Two are the median eyes and there is more LPS-free cover glasses on the culture one endoparietal eye. dish surface, then mounting those cover glasses in simple observation chambers Each of these eyes detects ultraviolet (UV) following cell attachment. A second light from the sun and reflected light from preparation for direct observation is to the moon. They help the crab follow the collect 3 -5 ml of blood in an LPS-free lunar cycle. This is important to their embryo dish and then explanting fragments spawning period that peaks on the new and of aggregated amebocytes to a chamber that full moon. Two ventral eyes are located near sandwiches the tissue between a slide and a the mouth but their function is unknown. cover glass. In this preparation, the motile Multiple photoreceptors located on the amebocytes migrate onto the cover glass telson constitute the last eye. These are surface, where they can readily be observed. believed to help the brain synchronize to the The blood clotting system involves cycle of light and darkness. The research aggregation of amebocytes and the into their eyes has helped the study and formation of an extracellular clot of a understanding of how the human eye works. protein, , which is released from the secretory granules of the blood cells. The horseshoe crab (also known as the king Biochemical analysis of washed blood cells crab), is a hard-shelled invertebrate found on requires that aggregation and degranulation the sea floor in warmer climates. Despite its does not occur, which can be accomplished name, the horseshoe crab is not actually a by collecting blood into 0.1 volumes of 2% crab (or ) and is in fact more Tween-20, 0.5 M LPS-free NaCl, followed closely related to arachnids such as spiders by centrifugation of the cells and washing and scorpions. The horseshoe crab is a with 0.5 M NaCl [23]. primitive-looking with a hard, brownish-green exoskeleton and a spike-like Horseshoe crabs have a total of 10 eyes (Fig tail. Horseshoe crabs have bright blue blood 1) used for finding mates and sensing light. and it gets clotted when it comes into The most obvious eyes are the 2 lateral contact with foreign bacteria. compound eyes. These are used for finding 961

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Table.1 Organisms Known to Prey on the Horseshoe Crab, Limulus polyphemus

Group Species Group Species Semipalmated plover (Charadrius Fish Silver perch (Bairdiella semipalmatus) (eggs) chrysura) (eggs and larvae) Black bellied plover (Pluvialis Weak fish (Cynoscion regalis) squatarola) (eggs) (eggs and larvae) Pectoral sandpiper (Calidris Northern kingfish melanotos) (eggs) () (eggs and larvae) (Calidris minutilla) (Menidia (eggs) menidia) (eggs and larvae) Semipalmated sandpiper (Calidris Summer flounder pusilla) (eggs) (Paralichthys dentatus) (eggs and larvae) (Calidris alba) (eggs) Winter flounder (Pseudopleuronectes americanus) (eggs and larvae) Laughing gull (Larus articilla) (eggs) Leopard (Triakis semifasciatus) (adults) Boat tailed grackle (Cassidix major) (eggs) (eggs) Red knots (Calidris canutus) (eggs) Puffers () (juveniles) Ruddy turnstones (Arenaria Devil ray (Mobula interpres) (eggs) hypostoma) gull (Larus argentatus) Sword fish (Xiphiidae) (adults) Greater black backed gull (Larus (Mugilidae) (eggs and marinus) (adults) larvae) Arthropods Sand ( Striped bass (Morone septemspinosa) (eggs) Saxatilis) (eggs) (Uca pugnax) (first and White perch (Morone second tailed stages) americana) (eggs) Blue crab ( sapidus) American (Anguilla (juveniles) rostrata) (eggs and larvae) Green crab (Carcinides maenus) Mollusks (Melongena spp.) (adults) (juveniles) crab ( spp.) (juveniles) Reptiles Loggerhead sea turtle (Caretta caretta) (adults) Amphipods (larvae) [4, 5, 6]

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Figure.1 Horseshoe crab eyes location

In other words, this stuff’s ideal for Acknowledgements detecting impurities and in human medicine, it is used to develop products like Authors are thankful to all the Central pharmaceutical drugs. Horseshoe crabs have Inland Fisheries research Institute (CIFRI) 2 large compound eyes located on the top of for ample help and support. We are also the shell. These eyes are made up of a thankful to central library of CIFRI for thousand light sensors that see in shades of same. gray. The crab combines all these separate sensors together as an image that they see. References These eyes are probably used for finding a mate. The compound eyes are larger and 1. Kōichi Sekiguchi (1988). Biology of have an optical nerve that is easy to identify Horseshoe Crabs. Science House. ISBN making the crabs ideal for studying how an 978-4-915572-25-8. eye works. The eyes have the ability to 2. Attaya Kungsuwan, Yuji Nagashima & detect UV light and are sensitive enough Tamao Noguchi et al. (1987). that the horseshoe crab sees as well at night "Tetrodotoxin in the Horseshoe Crab as it does during the day. Scientist and Carcinoscorpius rotundicauda Inhabiting researchers are putting effort in horseshoe Thailand" (pdf). Nippon Suisan Gakkaishi crab eyes to know the mechanism how their 53 (2): 261–266. eyes work so that it can give some light in doi:10.2331/suisan.53.261. human eyes working mechanism. 3. Sadava D., Heller H.C., Hillis D.M. and

Berenbaum M. (2009). Life: the Science of

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Biology (9th ed.). W. H. Freeman. p. 683. Limulus polyphemus". Ohio Journal of ISBN 978-1-4292-1962-4. Science70 (5): 276–283. 4. Shuster, C. N., Jr. 1982. A pictorial review 14. Lesley Cartwright-Taylor, Julian Lee & of the natural history and ecology of the Chia Chi Hsu (2009). "Population structure horseshoe crab, Limulus polyphemus, with and breeding pattern of the mangrove reference to other limulidae. pp. 1–52. In: horseshoe crab Carcinoscorpius Physiology and Biology of Horseshoe rotundicauda in ". Aquatic Crabs: Studies on Normal and Biology 8 (1): 61–69. doi:10.3354/ab00206. Environmentally Stressed Animals. 15. David Funkhouser (April 15, 2011). "Crab (Bonaventura, J., C. Bonaventura, and S. love nest". Scientific American 304 (4): Tesh, Eds.). New York: Alan R. Liss, Inc. 29.doi:10.1038/ scientificamerican0411-29. 5. Keinath, J. A., J. A. Musick, and R. A. 16. http://www.horseshoecrab.org/med/med.ht Byles. 1987. Aspects of the biology of ml Virginia’s sea turtles: 1979–1986. Virginia 17. Lenka Hurton (2003). Reducing post- Journal of Science, 38:.329–336. bleeding mortality of horseshoe crabs 6. deSylva, D. P., F. A. Kalber, Jr., and C. N. (Limulus polyphemus) used in the Shuster, Jr. 1962. and ecological biomedical industry (M.Sc. thesis). Virginia conditions in the shore zone of the Polytechnic Institute and State University. River estuary, with notes on other 18. Crash: A Tale of Two Species – The species collected in deeper water. Benefits of Blue Blood", PBS. University of Delaware Marine Labs. 19. Mikkelsen, T. 1988. The Secret in the Blue Report 5. Lewes, Delaware. Blood, Science Press. 7. Horseshoe crabs UK Natural History 20. Novitsky, T. J. 1984. Discovery to Museum website 2012 commercialization: the blood of the 8. Anatomy: Vision - The Horseshoe Crab horseshoe crab. Oceanus, 27: 13–18. 9. Horseshoe Crab, Limulus polyphemus ~ 21. Rudloe, A. 1983. The effect of heavy MarineBio.org bleeding on mortality of the horseshoe crab, 10. The biology of cartilage. I. Invertebrate Limulus polyphemus, in the natural cartilages: Limulus cartilage environment. Journal of Invertebrate 11. S. M. Manton (1977). The Arthropoda: Pathology, 42: 167–176. Habits, Functional Morphology, and 22. Walls, Elizabeth A. Berkson, Jim and Smith Evolution. Clarendon Press. p. 57. ISBN Stephen A. 2002. The Horseshoe Crab, 019857391X. LCCN 77005466. Limulus polyphemus: 200 Million Years of 12. Carl N. Shuster, Jr. & Lyall I. Anderson Existence, 100 Years of Study. Reviews in (2003). "A history of skeletal structure: Fisheries Science, 10(1): 39–73. clues to relationships among species". In 23. Armstrong P., Conrad M. (2008). Blood Carl N. Shuster, Robert B. Barlow & H. Collection from the American Horseshoe Jane Brockmann. The American Horseshoe Crab, Limulus Polyphemus. JoVE. 20. Crab. Harvard University Press. pp. 154– doi:10.3791/958. 188. ISBN 978-0-674-01159-5. LCCN 24. Anatomy of the Horseshoe Crab, Maryland 2003056640. Department of Natural Resources. 13. E. D. Vosatka (1970). "Observations on the Retrieved 3 December 2014. swimming, righting, and burrowing movements of young horseshoe crab,

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