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Review Article Horseshoe Crabs

Review Article Horseshoe Crabs

Indian Journal of Geo-Marine Sciences Vol. 45(10), October 2016, pp. 1234-1244

Review Article

Horseshoe : biomedical importance and its potential use in developing health-care products

Vikash Kumar1*, Suvra Roy1, A.K. Sahoo1 & Vikas Kumar2 1Central Inland Fisheries Research Institute (CIFRI), Barrackpore, 700120, India 2Division of , Kentucky State University, Frankfort, KY, 40601, USA *[E-mail: [email protected]]

Received 10 February 2015; revised 23 March 2016

Horseshoe crabs have been a model for many biomedical science studies. Medicinal value of horseshoe crabs comes from its blue , eye and (chitin). Ability of blood to clot in the presence of bacteria, rendering the bacteria harmless has created its biomedical importance. Blood cells () carry Factor C, which binds (LPS), undergoes a structural reorganization, then auto-proteolytically activates itself to initiate the clotting pathway that eventually results in a proteolytic modification of the zymogen, coagulogen, which then self-polymerizes into the insoluble fibrils of the extracellular blood clot. Blood-clotting ability of the horseshoe makes it very valuable in testing for injectable medicines, vaccines and sterile medical equipment. Secondly, the pathways in the eyes of horseshoe crabs have led to many discoveries in human eye research. Furthermore, the outer shell of a is made primarily of chitin and being used as a coating for suture material and burn dressings, rapidly increases the wound healing, cutting the time by half.

[Keywords: horseshoe crab, amebocytes, Factor C, lipopolysaccharide, chitin]

Introduction Borneo, Malaysia1. They are a distant relative of polyphemus (Linnaeus, 1758), commonly external link, and are more closely related known as Atlantic horseshoe crabs along with to such as , and . Limulus albus (Bosc, 1802), Limulus americanus Although they look prehistoric, and ancient relatives (Leach, 1819), Limulus cyclops (Fabricius, 1793), of Limulus polyphemus were present 520 million Limulus occidentalis (Lamarck, 1801), Limulus years ago as evidenced by , this species has sowerbii (Leach, 1815), (Müller, only been around for about 20 million years, which is 1785), Limulus rotundicauda (Latreille, 1802), not enough time to consider this a "living (Leach, 1819), and Monoculus " as they are sometimes called. Despite polyphemus (Linnaeus, 1758), were originally inhabiting the planet for so long, horseshoe crab body classified as horseshoe crabs. They have wide forms have changed very little over all of those years. geographical distribution e.g horseshoe The strange anatomy of the horseshoe crab is one of crab (Carcinoscorpius rotundicauda) reported from this animal's most notable aspects. Unfortunately, the Bay of Bengal, Thailand, Malaysia, Philiphines, long, thin, spike-like tail of horseshoe crabs has given Borneo and Torres Straits; Atlantic horseshoe crab this species an unfavorable reputation. Many people (Limulus polyphemus) from Atlantic Coast of North view horseshoe crabs as dangerous because America from to Yucatan; Southeast Asian they have sharp tails but in reality, horseshoe crabs horseshoe crab (Tachypleus gigas) from Bay of are harmless. Their tails are used primarily to flip Bengal (North-East Coast), Thailand, Malaysia, themselves upright if they are accidentally Philiphines, Borneo & Torres Straits and Tri-spine overturned2. horseshoe crab (Tachypleus tridentatus) from Western The horseshoe crab plays a vital (little-known) role & Southern Japan, Taiwan, Philiphines & North in the human medication. An extract of the horseshoe KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE 1235

crab's blood is used by the pharmaceutical and production11, hemolymph coagulation12, melanin medical device industries to ensure that their formation13, and lectin-mediated complement products, e.g., intravenous drugs, vaccines, and activation14 involve in the process of immune defence medical devices, are free of bacterial contamination. in . In addition to these enzyme cascades, Horseshoe crabs have 10 eyes located all over their a variety of agglutinin-lectins and reactive bodies and most of the eyes are located on the back or producing and phagocytic systems cooperate with sides of the animal. Some eyes contain photoreceptors immune reactions to kill invading pathogens15. Figure such are located on their tails. The eyes found on the 1 shows the principal defense systems associated with back are having about 1,000 photoreceptor clusters or phagocytosis. Invaders detected by these systems are ommatidia, each with a lens, cornea and ultimately engulfed by phagocytes, such as photoreceptor cells. Horseshoe crabs have the largest macrophage-like, neutrophils like and dendritic cells, rods and cones of any known animal that are about and are then internalized as phagosomes and finally 100 times the size of humans and research work is killed10,16. going on to reveal the understanding of the horseshoe Antimicrobial peptides Phagocyte crab eyes working mechanism. Similarly, the outer Toll-like receptor system membranes know as chitin is mostly used for coating Protease cascade for suture material and burn dressings, rapidly Phagocyte Biosensor Lectin complement Invaders Biosensor Protease cascade increase the wound healing, cutting. This review network Biosensor highlights the fundamental mechanism, that involve Phenol oxidase system

Coagulation cascade blood clotting and clotting factors, mechanism of eye clot system Melanin formation for biomedical research and potential use in health care products. Antimicrobial substances Phagocyte Fig. 1- The principal defense systems associated with Horseshoe crab model for innate immune system phagocytosis in invertebrates. The innate immune system is considered as first line of inducible host defense against bacterial, The major innate immune systems include; fungal, and viral pathogens3. This defense system is hemolymph , melanization mediated by essential for the survival and perpetuation of all phenoloxidase, the expression of antimicrobial multicellular organisms4,5. Invertebrates, which do not peptides mediated by Toll-like receptors and the possess immunoglobulins, have developed unique immuno deficiency (IMD) pathway, and the modalities to detect and respond to microbial surface lectin/complement pathway mediated by bacterial cell antigens like (LPS), lipoteichoic wall components. Invaders detected by these systems acids, lipoproteins, peptidoglycan (PGN) and (1 → 3) are ultimately engulfed by phagocytic cells, such as β-D-glucans6. Because both invertebrates and macrophage-like, neutrophil-like, or dendritic cells, respond to these substances, it is likely and then internalized, processed, and killed10. that a system recognizing these epitopes emerged at an early stage in the evolution of animals7,8. Hemolymph and circulating hemocytes Moreover, it is well known that various microbial cell The innate immune system of horseshoe crab is wall components elicit a variety of responses that mainly involved in defence response by employing depend on the species and cell type4,9. The major unique and highly efficient host defense systems17. biological host defense systems of invertebrates Hemolymph and hemocytes plays fundamental includes hemolymph coagulation system, Pro- defence mechanism during infection. Hemolymph phenoloxidase (pro-PO) activating system, lectin- plasma of this animal contains many soluble defense complement system, agglutinin-lectin system, molecules, such as , various lectins, and antibacterial, antifungal, and antiviral systems C-reactive , and thioester bond containing mediated by toll-like receptors and peptidoglycan proteins (α2-macroglobulins), in addition to a large binding (PGBP), reactive oxygen-producing numbers of granular hemocytes (amebocytes), which system and phagocytic system8,10. undergo a rapid degranulation on contact with 18 After antigen recognition, several mechanism i.e., . Hemocytes, which are composed of more toll-like receptor-mediated antimicrobial peptide than 99 % of circulating cells, contain a variety of 1236 INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016

defense molecules, which are located in two types of Table 1 Defense molecules found in hemocytes and hemolymph secretary granules viz large (L)-and small (S) (Fig. plasma of the horseshoe crab 2)19,20. L-granules selectively store more than 25 Proteins and Mass Function/ Localization defense components with molecular masses between peptides (kDa) specificity 8 and 120 kDa. These include clotting factors, a Coagulation clottable protein coagulogen, proteinase inhibitors, factors lectins, and antimicrobial proteins. In contrast, the S- Factor C 123 Serine protease L-granule Factor B 64 Serine protease L-granule granules contain at least six antimicrobial peptides Factor G 110 Serine protease L-granule and several proteins of molecular mass <30 kDa. Proclotting 54 Serine protease L-granule These peptides include large amounts of hairpin-like enzyme tachyplesin (17-18 amino acid residues, >10mg per Coagulogen 20 Gelation L-granule individual), tachystatins (41-44 amino acid residues), Protease inhibitors LICI-1 48 Serpin/factor C L-granule tachycitins (73 amino acid residues) and big defensins LICI-2 42 Serpin/clotting L-granule (79 amino acid residues), which are highly active enzyme against Gram-negative and -positive bacteria and LICI-3 53 Serpin/factor G L-granule fungi21,22. Trypsin inhibitor 6.8 Kunitz-type ND LTI 16 New type ND LEBP-PI 12 New type L-granule Limulus cystatin 12.6 Cystatin family L-granule 2 α2-Macroglobulin 180 Complement Plasma & L- granule Chymotrypsin 10 ND Plasma inhibitor Antimicrobial substances Anti-LPS factor 12 GNB L-granule Tachyplesins 2.3 GNB, GPB, FN S-granule Polyphemusins 2.3 GNB, GPB, FN S-granule Big defensin 8.6 GNB, GPB, FN L & S- granule Tachycitin 8.3 GNB, GPB, FN S-granule Tachystatins 6.5 GNB, GPB, FN S-granule Factor D 42 GNB L-granule Lectins Tachylectin-1 27 LPS (KDO), L-granule Fig. 2- Electron micrograph of horseshoe crab (T. tridentatus) LTA hemocytes, and major defense molecules that have been identified Tachylectin-2 27 GlcNAc, LTA L-granule in large and small cell granules (Source: Iwanaga and Lee 2005). Tachylectin-3 15 LPS (O- L-granule antigen) Various proteins and peptides that are identified in Tachylectin-4 470 LPS (O- ND T. tridentatus hemocytes and hemolymph plasma are antigen), LTA Tachylectin-5 380-440 N-acetyl group Plasma summarized in Table 1. Hemolymph plasma of Limunectin 54 PC L-granule Tachypleus tridentatus contains three predominant 18K-LAF 18 Hemocyte L-granule protein types, namely, (O2 transporter), aggregation 23 Limulin 300 HLA/PC, PE, Plasma C-reactive proteins (CRP) , and α2- macroglobulins24,25,26. Moreover, circulating SA, KDO LCRP 300 PC, PE Plasma hemocytes are extremely sensitive to bacterial LPS, TCRP-1 300 PE Plasma and respond by degranulating a number of granular TCRP-2 330 HLA/PE, SA Plasma components after LPS-mediated stimulation, which TCRP-3 340 HLA/SA, KDO Plasma results in the formation of hemolymph clot. This rapid Polyphemin ND LTA, GlcNAc Plasma TTA ND SA, GlcNAc, Plasma clotting response is believed to be important for the GalNAc animal’s host defense, which involves engulfing of Liphemin 400-500 SA Hemolymph invading microbes, and in addition prevents Carcinoscorpin 420 SA, KDO Hemolymph hemolymph leakage27. GBP 40 Gal Hemolymph KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE 1237

PAP 40 Protein A Hemolymph clotting is unclear35. (1→ 3) β-D-glucan 168 Pachyman, Hemocyte In crustaceans the clotting reaction are binding protein cardlan characterized and observed that TGase-mediated Others Transglutaminase 86 Cross-linking Cytosol cross-linking of a specific plasma clotting protein 36,37 (TGase) (CP) . The crayfish CP, has been biochemically and 8.6 kDa protein 8.6 TGase substrate L-granule functionally characterized36,38. It is a very high density Pro-rich proteins 80 TGase substrate L-granule lipoprotein (VHDL)38 consisting of two identical 210- (Proxins) 36 Limulus kexin 70 Precursor ND kDa subunits held together by disulfide bonds . Each processing one of the 210-kDa subunits has both lysine and Hemocyanin 3600 O2 transporter Plasma glutamine side chains, which are recognized and (PO activity) become covalently linked to each other by TGases36. Toll-like receptor 110 ND Hemocyte Clotting is induced when a TGase is released from (tToll) 2+ L1 11 Unknown L-granule hemocytes or tissue becomes activated by the Ca - L4 11 Unknown L-granule content in plasma, and starts cross-linking the plasma CP molecules into large aggregates. The hemocytes Mechanism of clot formation also contain components of the so-called and animals have evolved prophenoloxidase activating system (proPO system), efficient molecular mechanisms to form clots through that constitutes an important part of the immediate a sequential process using blood components. This is immune response in crustaceans39,40. Components of vital in preventing loss of blood in case of injury and the proPO system cause degranulation and lysis of as defence mechanism against certain microbes. All hemocytes, and as a result more proPO components vertebrates have similar coagulation systems based on and TGase are released39,40. In this way, the proPO the proteolytically induced aggregation of fibrinogen system could affect the clotting reaction by causing into insoluble fibrin28,29. The fibrin aggregates, which the release of TGase activity. However, the proPO initially are noncovalently associated, are further system and the clotting reaction do not appear to share stabilized by intermolecular covalent crosslinks a common activation pathway, as the proPO system is formed by a proteolytically activated transglutaminase activated by a proteolytic cascade [triggered by (TGase), factor XIIIa. TGases (EC 2.3.2.13) are Ca2+- microbial polysaccharides], and the initiation of the dependent enzymes capable of forming covalent clotting reaction requires no proteolytic processing bonds between the side chains of specific lysine and (only Ca2+, which activates the TGase)36. glutamine residues on certain proteins30,31. Among the In lobster the N terminus of the fibrinogen (the CP invertebrates, which is a more diverse group, different homologue in lobster) was reported to have sequence coagulation mechanisms seem to have evolved, and similarity to vitellogenins (VTGs)41, which are detailed information on the coagulatory mechanisms proteins expressed only in females of egg-laying at the molecular level is lacking in most groups. One animals (vertebrates as well as invertebrates)42,43. exception is the hemocyte (blood cell)-derived Besides having similar functions, the CP does not clotting cascade in horseshoe crabs, which has been appear to share any characteristics with fibrinogen or characterized in detail32. The clotting system in coagulogen, the proteins forming clots in vertebrate horseshoe crab is activated by microbial animals and horseshoe crabs, respectively. This lipopolysaccharides or β-1,3-glucans, and it has some indicates that the crayfish CP36,38 and its homologues resemblance to the vertebrate coagulation system, as it in other crustaceans37,41 constitute a separate group of is based on a proteolytic cascade leading to the blood CPs35. Recently Sahoo et al.44 reported conversion of a soluble protein (coagulogen) into an hemolymph clot in shimp, p.monodon against the insoluble aggregate (coagulin). However, the proteins white spot syndrome virus infection. Further, author participating in the Limulus clotting system are all hypothesized that hemolymph clot may progress to from the hemocytes and are not homologous to the melanin formation which is having antimicrobial vertebrate plasma coagulation proteins. A TGase has activity. been characterized and cloned from Limulus hemocytes, but it does not appear to recognize the Hemolymph clotting system in horseshoe crab coagulogen as a substrate33,34, and its role during The hemolymph-clotting phenomenon was first 1238 INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016

identified as a prominent defense system in the in the mammalian blood clotting system47. horseshoe crab (Limulus polyphemus) by Bang45. Interestingly, the NH2-terminal portions of zymogen When Gram-negative bacteria invade the hemolymph, factor B and of proclotting enzyme contain a small hemocytes detect LPS molecules on their surfaces46, compact domain containing three disulfide bonds, and then release, via rapid exocytosis, the contents of called the clip domain10,19. A similar clip domain has L- and S-granules47. These released granular also been reported in the NH2-terminal proenzyme components include two biosensors, named factors C regions of Drosophila-derived serine proteases. and G. These two factors are serine protease Moreover, the folding pattern of the three disulfide zymogens and are autocatalytically activated by LPS bridges located in the clip domain is identical to that or (1→ 3)-β-D-glucan, which are major components of big defensin, which was recently identified as an of the cell walls of Gram-negative bacteria and fungi, antimicrobial peptide in T. tridentatus hemocytes. As respectively. In 1996, Tamura et al.48 reported that the COOH-terminal end of the clip domain in hemocytes contain a (1→ 3)-β-D-Glucan binding proclotting enzyme constitutes a hinge region protein, which differs from factor G as it does not susceptible to proteolysis, the clip domain, in the same participate in the hemolymph clotting cascade. One of manner as defensin, might be released during the the authors of this review has previously described in activations of serine protease zymogens, in order to detail LPS and (1→ 3)-β-D-glucan-mediated clotting act as an antimicrobial substance. In fact, the clip cascades and their molecular structures, and the domain derived from the prophenoloxidase activated functions of the five clotting factors, factor C, factor serine protease of freshwater crayfish has an G, factor B, proclotting enzyme, and clottable antimicrobial activity similar to that of human β- coagulogen, which all participate in clotting defensin60. Thus, the clotting cascade could also cascades17,19,27,47. Figure 3 illustrates the LPS and (1→ produce antimicrobial agents, and thus provide a dual 3)-β-D-glucan-mediated clotting cascades of the action clotting and killing system against invaders61,62. hemolymph of T. tridentatus21, and includes limulus intracellular coagulation inhibitors (LICI), which act 49 Lipopolysaccharide β-1,3-Glucan as regulators of the cascade reaction . These clotting (LPS) cascades both involve four serine protease zymogens, factors C (123 kDa), B (64 kDa), G (110 kDa), proclotting enzyme (54 kDa), and coagulogen (20 Factor C Factor C Factor G Factor G 50,51 kDa) . In the presence of LPS or synthetic lipid A LICI 1 analogs, factor C is autocatalytically activated to an LICI 3 active form, factor C17,52. Factor B zymogen is then Factor B Factor B activated by factor C to its active form (factor B), which activates proclotting enzyme to clotting Proclotting Clotting enzyme enzyme53. Clotting enzyme then converts coagulogen enzyme to an insoluble coagulin gel, which is composed of LICI 2 non-covalent homopolymers, through head to tail Gelation 54 Cell agglutination Coagulin Coagulogen interaction . On the other hand, factor G zymogen etc. consisting of two heterosubunits and is autocatalytically activated in the presence of (1→ 3)- Fig. 3- LPS- and (1→ 3)-β-D-glucan mediated clotting cascades β-Dglucan, in the absence of any other protein55. The found in horseshoe crab (T. tridentatus) hemocytes. LICI, resulting active factor G activates proclotting enzyme (Limulus intracellular coagulation inhibitor). directly, resulting in coagulin gel formation56. Recently, Osaki et al.57 found that non-covalent The biochemical principle of the so called limulus coagulin homopolymers are cross-linked by bridging test, which is used for detecting bacterial endotoxins are shown in figure 3. The method was developed by hemocyte cell surface proteins, named proxins, in the 63 presence of hemocyte-derived transglutaminase58,59. Levin and Bang based on a finding that a trace This indicates that cross-linking is important at the amount of endotoxin coagulates the hemocyte final stage of hemolymph clotting to facilitate () lysate of the American horseshoe crab, hemostasis and wound healing, as has been reported Limulus polyphemus. This gelation reaction has been widely employed as a simple and highly sensitive KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE 1239

assay for endotoxins (LPS). The limulus test is 3-15% to 10-30 %67. The LAL test represents one of a dependent on the protease cascade reaction shown in number of pharmacological significant, chemical the figure, and is being used extensively in constituents found in marine flora and fauna68. A combination with new technology10,12,64,65. wealth of significant compounds has been isolated from marine animals. These include compounds Potential application of horseshoe crab in human derived from the sea cucumber used in anti-cancer medicine chemotherapy, hormones from gorgonians used for The horseshoe crab has the best-characterized birth control, against peptic ulcers and asthma and immune system of any long-lived invertebrates. When lowering blood pressure, as well as compounds a foreign object (bacteria) enters through a wound in derived from red that can prevent their body, it almost immediately clots into a clear, gel atherosclerosis68. The discovery, commercialization, like material, thus effectively trapping the bacteria. If and use of LAL have been an important improvement the bacterium is harmful, the blood will form a clot. to the pharmaceutical industry. Prior to the use of Horseshoe crabs are proving to be very helpful in LAL, compounds were tested for the presence of finding remedies for diseases that have built endotoxins in a variety of ways that involved living immunities against penicillin and other drugs64. animals or living parts of animals68. Thus, LAL The study of immunity in horseshoe crabs has provides a means to detect endotoxins without having been facilitated by the ease in collecting large to kill or disable animals64. volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and α2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 ml of blood (Fig. 4). Unlike vertebrates, horseshoe crabs do not have in their blood, but instead use hemocyanin to carry oxygen. Because of the present in hemocyanin, their blood is blue. Their blood contains amebocytes, which play a role similar to white blood cells of vertebrates in defending the organism against pathogens. Amebocytes from the blood of L. polyphemus are used to make (LAL), which is used for the detection of bacterial endotoxins in medical applications. The blood of horseshoe crabs is harvested for this purpose66. Harvesting horseshoe crab blood involves collecting and bleeding the animals, and then releasing them back into the sea. http://www.wired.com Most of the animals survive the process; mortality is Fig. 4- Extraction of blood from horseshoe crabs correlated with both the amount of blood extracted from an individual animal, and the stress experienced Limulus Amebocyte Lysate is extremely useful in during handling and transportation. Estimates of detecting those toxins that cause fever – the bacterial mortality rates following blood harvesting vary from pyrogens or endotoxins. Endotoxins occur as a 1240 INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016

structural component of the cell wall of a large group of bacteria known as gram negative69. Most aquatic bacteria are of the gram-negative variety, as studies at the Woods Hole Oceanographic Institution have shown that contains over 1 million Gram- negative bacteria per milliliter and that almost 1 billion bacteria can be found per gram of sand near the Separation of amoebocytes shore70. Thus, the horseshoe crab habitat contains vast Horseshoe crab Extraction of blood amounts of endotoxin, making it no coincidence that the horseshoe crab evolved a vital system to protect Pharmaceuticals products Food products Diagnosis/ treatment itself against endotoxins. The horseshoe crab blood includes amebocytes that contain the clotting enzymes Clinical diagnosis and other factors with the ability to immobilize and .Body and mental exhaustion 68 engulf an endotoxin . When exposed to endotoxin, Eye disease .Drowsiness after sea bathing .Gastroentric symptoms the amebocytes change shape, adhere to the sides of Spinal meningitis the vascular channels, and form the resultant gel .Pain in the body clot71. This phenomenon is at the of the LAL Urinary infection assay, as the formation of a clot shows presence of endotoxin. The major use of LAL today is in the Rheum atism 69 detection of endotoxins in pharmaceutical products . Since its original description, however, it has also Fig. 5- Use of Limulus amebocyte lysate (LAL) been used in the diagnosis of endotoxemia in conjunction with cirrhosis, cancer, meningitis, eye The blood clotting system involves aggregation of disease, dental problems, gonorrhea, and water-quality amebocytes and the formation of an extracellular clot analysis70, as well as urinary tract infections69. In of a protein, coagulin, which is released from the addition, new applications for LAL continue to be secretory granules of the blood cells. Biochemical found, including the detection of bacterially analysis of washed blood cells requires that contaminated meat, fish, and dairy products, including aggregation and degranulation does not occur, which frozen items72. can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, Blood chemistry followed by centrifugation of the cells and washing For study of the plasma, blood cells are with 0.5 M NaCl73. immediately removed from the plasma by centrifugation and the plasma can then be fractionated Principle of Limulus test into its constituent proteins. The blood cells are Limulus test, a test for detecting nano gram of conveniently studied microscopically by collecting bacterial endotoxins, was invented by Levin and Bang small volumes of blood into LPS-free isotonic saline based on their finding that a trace amount of endotoxin (0.5 M NaCl) under conditions that permit direct coagulates hemocyte lysate of the horseshoe crab, microscopic examination by placing one of more Limulus polyphemus63. This gelation reaction has been LPS-free cover glasses on the culture dish surface, widely employed as a simple and very sensitive assay then mounting those cover glasses in simple method for bacterial endotoxins. The original method is observation chambers following cell attachment. A qualitative or semi-quantitative; the presence of second preparation for direct observation is to collect endotoxin is determined by reading the formation of gel 3 -5 ml of blood in a LPS-free embryo dish and then clot after incubation of a sample with the hemocyte explanting fragments of aggregated amebocytes to a lysate at 37 °C for 1 hour (Limulus gelation test). chamber that sandwiches the tissue between a slide During the past decade, the studies on molecular and a cover glass (Fig. 5). In this preparation, the mechanism of hemolymph coagulation in horseshoe motile amebocytes migrate onto the cover glass crab, established a protease cascade described above. surface, where they can readily be observed. Because the Limulus lysate contains all the enzymes described above, the Limulus test reacts with (1,3)-β-D- KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE 1241

glucan as well as endotoxin. The latter activates factor new and full moon. Two ventral eyes are located near C, whereas the former activates factor G; both pathways the mouth but their function is unknown. Multiple converge on proclotting enzyme, ensuing its activation photoreceptors located on the constitute the last and hydrolysis of a chromogenic peptide substrate12. eye. These are believed to help the synchronize The chromogenic substrate used for specific assay of to the cycle of light and darkness. The research into bacterial endotoxins is Boc-Leu-Gly-Arg-p-nitroanilide their eyes has helped the study and understanding of (pNA). The sequence of this substrate originates from how the human eye works. the sequences located close to the site cleaved during the gelation of coagulogen by Limulus clotting enzyme. Chitin The chromogenic substrate is hydrolyzed by clotting Chitin, a cellulose-like component from the shell enzyme to release pNA. By measuring the absorbance of the horseshoe crab, is non-toxic, biodegradable and of released pNA at 405 nm, endotoxin concentration in used in contact lenses, skin creams and hair sprays. It the samples can be determined. Endotoxin is also used to make chitin-coated sutures and wound concentration can also be determined by measuring the dressings for burn victims. The chitin-coated sutures absorbance at 545 nm after the diazo coupling of pNA, reduce healing time by 35% to 50%. When chitin is when a yellowish color in samples interferes with the processed, another substance, called chitosan, is measurement at 405 nm64. The methods described produced and can be used as a raw material to above are a 100 times more sensitive than the limulus manufacture a variety of important products. gelation test and are very reproducible. If this technique is to be applied to blood samples, however, the Conclusion activities of limulus test-interfering factors in the Horseshoe crabs are chelicerates, distant relatives samples, such as thrombin, blood coagulation factor Xa, of spiders. They are often referred to as living fossils, and α1-antitrypsin, need to be abolished. To remove as they have changed little morphologically in the last such interferences, various methods have been studied 445 million years. There are four extant species of and applied to blood samples, such as pretreatment with horseshoe crabs. The species Limulus polyphemus chloroform, ether, acid, or alkali and heating65. occurs only along the eastern coast of the USA. The other three species, Tachypleus tridentatus, Horseshoe crabs eye anatomy and importance Tachypleus gigas and Carcinoscorpius rotundicauda Horseshoe crabs have a total of 10 eyes used for live along the coast of the Indo-West Pacific. In Asian finding mates and sensing light. The most obvious waters, habitat degradation especially the loss of eyes are the 2 lateral compound eyes. These are used spawning and nursery grounds, marine pollution and for finding mates during the spawning season. Each human exploitation have resulted in a decline in has about 1,000 receptors or horseshoe crab populations. ommatidia74. The cones and rods of the lateral eyes Horseshoe crab having bright blue blood contains have a similar structure to those found in human eyes, blood cells amebocytes carry protein called coagulogen but are around 100 times larger in size. The which plays an important role in blood clotting and ommatidia are adapted to change the way they trapping of bacteria when it comes into contact with function by day or night. At night, the lateral eyes are foreign bacteria. The mechanism of blood clotting and chemically stimulated to greatly increase the entrapment of foreign bacteria drawing attention to sensitivity of each receptor to light. This allows the many researchers and many healthcare products are horseshoe crab to identify other horseshoe crabs in the been getting developed like pharmaceutical drugs etc. in darkness. The horseshoe crab has an additional five human medicine. Horseshoe crabs have 2 large eyes on the top side of its prosoma. Directly behind compound eyes located on the top of the shell. These each lateral eye is a rudimentary lateral eye. Towards eyes are made up of a thousand light sensors that see in the front of the prosoma is a small ridge with three shades of gray. The crab combines all these separate dark spots. Two are the median eyes and there is one sensors together as an image that they see. These eyes endoparietal eye. Each of these eyes detects ultraviolet are probably used for finding a mate. The compound (UV) light from the sun and reflected light from the eyes are larger and have an optical nerve that is easy to moon. They help the crab follow the lunar cycle. This identify making the crabs ideal for studying how an eye is important to their spawning period that peaks on the works. The eyes have the ability to detect UV light and 1242 INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016

are sensitive enough that the horseshoe crab sees as 16 Greenberg, S. and Grinstein, S., Phagocytosis and innate well at night as it does during the day. Scientist and immunity, Curr. Opin. Immunol., 14(2002) 136-145. 17 Iwanaga, S., Miyata, T., Tokunaga, F. and Muta, T., researchers are putting effort in horseshoe crab eyes to Molecular mechanism of hemolymph clotting system in know the mechanism how their eyes work so that it can Limulus, Thrombosis Res., 68(1992) 1-32. give some light in human eyes working mechanism. 18 Iwanaga, S., Kawabata, S. and Muta, T., New types of clotting factors and defense molecules found in horseshoe crab Acknowledgements hemolymph: their structures and functions, J. Biochem., 123(1998) 1-15. Authors are thankful to all the Central Inland Fisheries 19 Muta, T. and Iwanaga, S., The role of hemolymph coagulation research Institute (CIFRI) for ample help and support. in innate immunity, Curr. Opin. 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