R&D 50 | Prospect Malaysia desiccation and submergence. wide rangesof salinity,temperature, coastal shallow habitat.Itcanalsotolerate of adversesituationsinitsestuarine and that thisanimalcanovercome allkinds survived forsuchalongperiod. Itseems how thehumblehorseshoecrab has become completelyextinct,it is awonder latecomers totheMesozoicera,have including dinosaursandmanyother 350 millionyears.Whilesomanyanimals, phenotypic change,evenafteraspanof archeal animalbodyhasnotshownany present shapeand,surprisingly,the so, thehorseshoecrabevolvedintoits years ago.After150millionor Precambrian seas,nearly600million named trilobites,whichlivedinthe from muddwellingprimitivearthropods, fossil'. and isconsideredtobetheoldest'living arthropod, isonesuchamazingcreature animals. Thehorseshoecrab,achelicerate to beextinct,stillflourishasliving number ofmarineorganisms,suspected all marineanimalspecies.Surprisingly,a 35,000 varieties,contributefour-fifthof animals moultastheygrow),withover limbs andanoutershell,whichthe the ocean,whereasarthropods(jointed phyla ofmarineorganismsarefoundin living andnonresources.About26 University MalaysiaTerengganu by AnilChatterjiandNoraznawatiIsmail A Valuable MarineCreature Horseshoe Crab – The horseshoecrabhasdescended The oceanisatreasuretroveofmany primitive spines.Inmodernhorseshoe whereas, thelastpairterminatedin started developingpinchingclaws, forms. Gradually,thefirstfourpairs abdomen, werepresentintheprimitive of walkinglegs,discontinuingatthe well definedappendages.Thefivepairs less abdominalsegmentsbutwithout crabs weresimilartotheaglaspids,with records showthattheoldesthorseshoe or freshwaterenvironments.Fossil are believedtohaveinhabitedbrackish The ancestorsofthehorseshoecrab possibly contribute totheevolutionary migration. Such characteristicscould the chanceofmortality duringtheir respective destinationsandreduce the animalstheirsafearrivalto their and dispersal.Thesemethods ensure their selectedhabitatforreproduction specialized methodsfordetecting distribution pattern.Theyhave highly conditions. and adapttodifferentenvironmental reflect theiramazingabilitytotolerate crab duringtheirdifferentstagesoflife habitats encounteredbythehorseshoe most partsofitslife.Thefluctuating inhabits thedeeperzoneofseafor it isgenerallybelievedthattheanimal cycle oftheanimalisnotyetknown, detailed knowledgeofthecompletelife salinity andtemperature.Although environmental conditionsincluding animal issubjectedtoawiderangeof During thisshorewardmigrationthe waters specificallyforbreedingpurposes. migrate totheshorefromdeeper bottoms fortheirbiogenicactivities.They seas orestuarieswithmuddysandy benthic community.Theyprefercalm developed fromtheseappendages. gills ofthehorseshoecrabwerepartly and overlappingplates.Theexternal specialized appendageswithbroad,flat crabs, thesefivepairsarehighly Horseshoe crabhabitat The horseshoecrabhasapatchy The horseshoecrabbelongstothe R&D Prospect Malaysia | 51 Modern researchers find the This principle has been useful in namely Carcinoscorpius rotundicauda, namely Carcinoscorpius rotundicauda, Tachypleus gigas and Tachypleus tridentatus along the coast of Malaysia. A record breaking large specimen of a T. tridentatus (7.5 kg) was collected for the first time along the coast of Paper Sabah. Although there are high degrees of similarity among all three Asian species of the horseshoe crab, T. gigas is morphologically more closely related to T. tridentatus than C. rotundicauda. Optic nerves horseshoe crabs’ unique physiology of the heart and the functioning of its optic nerves interesting. The crab has altogether ten eyes which are located on the upper surface of the body. Out of these, two pairs are known as true eyes though the rest of the organs are also sensitive to light. The compound eyes of the horseshoe crab can polarize light and the crystal lining cones can concentrate it tenfold. This is an adaptation for their continuous living in translucent and dark muddy waters. The vision is also sensitive to infrared and ultraviolet lights. These find to crabs horseshoe the help structures their way on cloudy days when the sun is not visible - a small patch of clear sky is enough for the animal to locate the sun. developing a compass for navigation in the Polar Regions where magnetic compasses are not reliable and celestial navigation is also difficult as the sun and stars are not visible in the long polar twilight. The design of eye structure of the horseshoe crab has also been successfully adopted in designing solar energy collectors. The optic nerve cells of the horseshoe crab are cross connected in such a way that when one nerve is stimulated, its partner nerve gets inhibited, resulting in an increase in contrast of the images. The diversified Paleozoic group is of the horseshoe crab, Limulus and Cancer mollucensis were reported from the same region. represented by only four species with confirmed discernible morphology and three genera of the horseshoe crab worldwide. It is interesting to note that among these four species, Limulus polyphemus and Tachypleus tridentatus occur in a north-south-north direction, whereas, Tachypleus gigas and Carcinoscorpius rotundicauda occur in an east-west-east direction (Plate 4). This includes Malaysia. It is an interesting phenomenon to have an abundance of three species of the horseshoe crab, In contrast, uniformly distributed rotundicauda in a mangrove area animals are subject to more competition animals are subject to more competition for their biological needs and have fewer opportunities to survive. The horseshoe crab has a difficult distribution pattern because the animal is totally dependent on two important physical stimuli – the tide and the lunar periodicities. There have been a number of attempts to study the distribution of the horseshoe crab in the recent past decades. For example, the occurrence of a species of the horseshoe crab, Tachypleus hoeveni, was reported for the first time off the coast of Moluccas. Later, the occurrence of two more species T. gigas migrating on sandy beaches for spawning advantage of the horseshoe crab for their very long survival. R&D 52 | Prospect Malaysia as speartips.Aftergrindingthebody, Indians usedthetailspinesofcrab humans. Itisbelievedthattheearly horseshoe crabhashadmanyusesfor radar systems. has alsohelpedindevelopingimproved presently incommonuse.Theprinciple provide sharpertelevisionimagesandis by GeneralElectricalCompanyUSAto based onthisprinciplewasdeveloped of thehorseshoecrab.Avideosystem on thefunctioningofopticnerves shared aNobelPrizeforhisnotablework Remedies andornaments For thepastseveraldecades, In 1967,ProfHKHartlineofUSA A studentofUMTworkingonahorseshoecrab a utensilforeatingfoodanddrinking Indians alsousedthehardcarapaceas for treatingrheumaticpaininIndia.The crab isboiledwithmustardoilandused Additionally, thedeadcarapaceof the appendagesofhorseshoecrab. Indians intheearlydaysusedtoeat in WestBengal,India.Itisbelievedthat pains andtheyaresoldbyfaithhealers are usedforhealingarthritisorotherjoint it. Ithasbeenreportedthatthetailtips their armsorprickforeheadswith different typesofpain.Theytiethetailon still usethetailpieceforrelieffrom inhabiting thenorth-eastcoastofOrissa and ponds.InIndia,someofthetribes it wasusedasfertilizerfortheirfields immunity fortheirfoetus. the eggmassofhorseshoecrabfor women inSingaporearesaidtoeat eat itaspartofeverydayfood.Pregnant meat oftheappendageswithsaucesand in thesouthernpartofChinablend are regardedadelicacywhilepeople and Borneo,theeggsofhorseshoecrab pieces totourists.InSingapore,Malaysia, dried horseshoecrabshellsasdecorative In Korea,fishermensellhundredsof horseshoe crabtomakesmallornaments. or ladle.Theyalsousethetailof of theT.tridentatusspeciesasahat water. the amoebocyteshapechangesand, In thepresenceofharmfulbacteria, of endotoxinsorharmfulbacteria. quickly whenexposedtominuteamounts blood clotting.Thecellsaggregate consisting ofelementsrequiredfor packed withlargerefractilegranules cytoplasm inthebloodcellsisdensely and withaprominentnucleus.The amoebocytes whichareellipticalinshape cells ofhorseshoecrabsareknownas system ofthecrab.Thewhiteblood that carriesoxygenviathecirculatory to haemocyanin,acopperbasedmolecule The bluecolourofthebloodisattributed are alsoknownasbluebloodedanimals. exposed toair.Hence,horseshoecrabs is whiteincolourbutitturnsbluewhen generally knownashaemolymphwhich Diagnosing endotoxins In China,peopleusethebigcarapace The bloodofthehorseshoecrabis horseshoe crabever reported The largestspecimen ofa R&D Prospect Malaysia | 53 In recent times, the biomedical With the help of lysate, the pyrogen The amoebocytes of the horseshoe of endotoxins. This process is popularly of endotoxins. This process is popularly known as Limulus Amoebocyte Lysate or LAL test. Dr R Nandan, an Indian scientist based in USA, was the first to patent the LAL test in 1956. properties of the horseshoe crab have assumed greater importance due to the unanticipated marketing of Limulus Amoebocyte Lysate as an endotoxin tester in food, drug and pharmaceutical industries. The lysate test has been proved to be a better substitute for the rabbit vaccine test because of its efficiency in terms of quick results and costs. test is simple, sensitive, accurate and easy to perform. The test does not require any special type of equipment. The test sample (0.1 ml) is mixed with lysate (0.1 ml) in a sterile test tube and incubated in a water bath at a constant temperature of 370 C for a period of two hours. If the solution is clear, liquid, and runs down the wall of the test tube, the sample is considered to be free from gram-negative bacteria. But, if the solution turns into a firm opaque gel and sticks to the bottom of the test tube when inverted, the sample is considered to be contaminated with gram-negative bacteria. crab have a clotting enzyme - lysate which clots and forms a firm opaque gel in no time when it is exposed to minute amounts of endotoxin or bacterial pyrogens. The rate of gelation of lysate is directly related to the concentration of endotoxins, which in turn, is dependent on the protein content of lysate. In lysate, the enzymatic activity basically controls the rate of gelation at a particular LIMULUS POLYPHEMUS POLYPHEMUS LIMULUS (MULLER) Coast of North Atlantic to America from Maine Yucatan CARCINOSCORPIUS ROTUNDICAUDA (LATREILLE) Bay of Bengal, Thailand, Malaysia, Philiphines, Borneo & Torres Straits. However, the pioneer research on In human beings, the presence of The LAL test these endotoxins is generally assessed with the help of a rabbit vaccine test. This method is not very sensitive and does not produce accurate results and it results in the death a large number of rabbits. In India, approximately 50,000 rabbits are sacrificed every day for the purposes of the detection of endotoxins. In addition, maintaining a rabbit colony requires large amounts of money. The other limitation in the rabbit vaccine test is that it takes about 8-10 days to get a clinical report and this delay to confirm results may result in the death of the patient. endotoxin diagnosis with the help of a reagent prepared from the blood of the horseshoe crab has opened a new chapter in the history of the diagnosis Bay of Bengal (North - East Coast), Thailand, Malaysia, Philiphines, Borneo & Torres Straits TACHYPLEUS GIGAS TACHYPLEUS (MULLER) Western & Southern Japan, Western & Southern & Taiwan, Philiphines Malaysia North Borneo, TACHYPLEUS TACHYPLEUS TRIDENTATUS (POCOCK) This process is followed by the Blue blood of horseshoe crab degranulation of the amoebocyte and release of all the clottable proteins or coagulogen that immobilizes and engulfs the endotoxins to form a firm gel or clot. Endotoxin is present in the triple layered cell wall of gram-negative bacteria which is composed of polysaccharide lipids and smaller peptides (lipopolysaccharide or LPS). The amount of endotoxins in a cell is reported to be 10-4 g. The release of endotoxins in the surrounding medium is due to the rupture or lysing of the bacterial cell wall. The toxicity of endotoxin is basically due to the presence of lipid-A fraction with LPS. The endotoxin has an extraordinary resistance to a wide range of temperature and to all types of detergents. simultaneously, long processes extend from one amoebocyte to the other and appear in a pseudopod-like fashion. R&D 54 | Prospect Malaysia exhaustion, gastro-enteric symptoms, this medicinefor thetreatmentofmental There areanumber ofapplications haemolymph ofthehorseshoe crab. therapies, ispreparedfromthe by DrCHerringinhomoeopathic practices. Limulusmedicine, introduced crab isalsoimportantinhomoeopathic crab. part ofthehaemolymphhorseshoe has alsobeendiscoveredinthelymph vitamin B12deficiencyinhumanbeings, an importantprotein,whichcandetect pharmaceutical products.Veryrecently, viral vaccines,andvarious tests fortestingradiopharmaceuticals, Administration ofUSApermittedlysate (Plate 8). which isextremelyreactivewithlysate bacterium, possessinganendotoxin (Neiserria gonorthea)isagram-negative the bacteriumresponsibleforgonorrhea recently, ithasalsobeendiscoveredthat efficiently withthehelpoflysate.Very bacterial meningitiscanalsobedetected urinary infection,infectionoftheeyeand and drugproducts.Endotoxaemia, endotoxin inhumans,animalinjectables as anend-producttestingprocedurefor most countriesandiscommonlyused regulatory authoritiesandindustriesin use. Thelysatetestisrecognizedbydrug drugs whichareintendedforhuman with parenteralpharmaceuticalsand in vitrotestforendotoxinsassociated pg/ml. Ithasbecomeabasisforthe (gram-negative bacteria),evenupto100 method forthedetectionofendotoxins currently apromisingandsensitive less thananhour. of abillionthgramendotoxininjust sensitive thatitcandetectonemillionth concentration ofendotoxin.Lysateisso Homeopathic healing The haemolymphofthehorseshoe In 1970,theFederalFoodandDrug The endotoxintestingbylysateis tract tothebloodcirculatorysystem endotoxins fromthegastro-intestinal radiation. the vascularandhaemodynamiceffectsof helpful inprovidingprotectionagainst regression ofmalignanttumoursand also beenreportedtoinducenecrosisand to differentpathogens.Endotoxinshave increases non-specificbodyresistance in ourimmunologicalsurveillanceand is beneficialinproducingstimulation A littleLPSofgram-negativebacteria gram-negative bacteriaandhumanbeing. important symbiosisexistsbetweenthe and Neisseriaceae.Formutualbenefit,an (mainly Eschrichiacoli),Bacteriodaceae Psudomonadaceae, Entererobacteriaceae negative bacteriabelongtothefamiliesof bacteria. Themostcommongram- human beingisahouseforgram-negative cholera, anddrowsinessafterseabathing. The preventionofabsorbing The gastro-intestinaltractofthe necessary precautions. reported, inspiteoftakingallthe where a30%mortalityratehasbeen gram-negative bacteriaissepticaemia most commonexampleoftheeffect circulatory collapseorevendeath.The coagulation, haemorrhage,hypertension, the disseminationofintravascular surveillance system. metabolic systemandtheimmunological the bloodcells,hormonalsystem, especially thehypothalamusofbrain, extremely toxicandaffectsthebody, enters intothebloodstream,itbecomes endotoxin ofthesegram-negativebacteria by anychanceevenasmallfractionof the detoxificationofendotoxins.But,if regulated mechanismswhichinvolve is mainlycontrolledbytheliver- plastic cartridgehasfourchannels a pressofbutton.Everythumb-sized tests onathumb-sizedcartridgewith crew toperformcomplexlaboratory PTS isahandhelddevicethathelpsthe will deliverresultsquickly.TheLOCAD- although maynotidentifythebacteria, Test System-theLOCAD-PTS–which, Application DevelopmentPortable process takesdays. microorganisms foundonboard.This used Petridishestocultureandidentify are sick.Previously,thestationastronauts know quicklywhatiswrongwhenthey laboratories inspaceandtheyneedto rapid deviceastherearenoclinical The astronautsrequireasensitiveand held diagnosticdevicesforastronauts. cells andhaveincludedtheminhand- enzymes fromhorseshoecrab'sblood USA hasrealizedtheimportanceof and SpaceAdministration(NASA)of Diagnostic device These endotoxinscanresultin A newdevice,calledLab-on-a-Chip Recently, theNationalAeronautics R&D Prospect Malaysia | 55 It has also been observed that been observed that It has also cell replacement Organogenesis and The same is true for successful The eggs of the horseshoe crab are In most Asian countries, the local heart. This clearly indicates that peri- indicates This clearly heart. that molecules contain fluids vitelline growth and the differentiation stimulate organs. of specific fluid of the developing peri-vitelline horseshoe crab showed embryo of pro- of both anti- and the presence useful in cardiac angiogenic activities therapies. This is an disorders and cancer since inhibition important observation, way to ”suffocate” of angiogenesis is a by depriving them developing tumours and oxygen supply. of vascular irrigation and economic This is of biomedical research. cancer for especially importance, a popular method therapy will soon be medical to handle several dreaded diabetes and complications including degenerative diseases related to the heart. In vitro organ development and stem cell biology are two of the approaches that will underline the success of such a strategy. However, in order to achieve ideal organ growth, differentiation and/ or regeneration, it would be essential to identify, purify and employ specific molecules, either individually or in various combinations. applications of stem cell biology wherein one needs to provide a defined milieu to naive stem cells so as to coax them to take one or the other developmental pathway. The possible source for such novel molecules is, again, the amazing horseshoe crab. The PVF of the Indian horseshoe crab contains peptide(s) capable of positively influencing differentiations of specific organs. Such peptides are likely to be present in minute quantities and may be as proteins. Threat of extinction reported to be eaten by striped bass, striped killi fish, silver perch, northern king fish, Atlantic silverside and flounders. The appendages and gill hooks of the young limuli are sought after by the puffer and cat fish. Plovers, sand pipers and gulls are also considered to be regular predators of the horseshoe crab eggs and larvae in the beach nest. The other predators causing considerable damage to the horseshoe crab population are tiger sharks, leopard sharks, loggerhead turtles, rays and sword fish. The horseshoe crab also has a great fear of mosquitoes and will die if bitten by a mosquito. fishermen are not fully aware of the economic importance of the horseshoe crab and discard them. This is because the horseshoe crabs damage their fishing nets when they are trapped. Fishermen usually catch these crabs and throw them on the shore, leading to a high mortality rate of the animals. In addition, It has been observed that peri-vitelline The peri-vitelline fluid contains fluid contains The peri-vitelline proteins such as hemagglutinins and as hemagglutinins proteins such may have an hemocyanin, which embryogenesis. important role during embryo Studies on chick developing fluid showed that the peri-vitelline biological active contained important early compounds that influenced development. vertebrate embryonic cultured in vitro Chick embryo explants system as several are used as a model development features of chick embryo to mammals, are remarkably similar and these including human development embryos are more amenable to in vitro culture and experimentation. fluid tends to stimulate several aspects of chick embryonic development. For example, peri-vitelline fluid stimulates the development and differentiation of specific organs such as the brain and uses of peri-vitelline fluid. It has been It has fluid. of peri-vitelline uses helps fluid peri-vitelline that the observed may of β-cells that in the proliferation in human insulin production be used in may help in developing beings. This drugs. effective anti-diabetic The LOCAD-PTS is meant to provide The LOCAD-PTS is The egg of the horseshoe crab is The egg of the horseshoe An embryo of a horseshoe crab with fluid and the effect of fluid on chick developing embryo An embryo of a horseshoe crab with fluid and the effect of fluid on chick developing Peri-vitelline fluid quicker warnings about any potential quicker warnings about only 15 minutes health threats. It takes from station to identify germs swabbed other surfaces. The instrument panels and so well that LOCAD tests have worked leave the NASA plans to permanently station. device onboard the and each channel contains a dried contains channel and each blood cells crab of horseshoe extract dye. In the presence and a colourless extract and fungi, the dried of bacteria to a green to turn the dye reacts strongly the few minutes, confirming colour in a more toxic materials - the presence of of dye, the more the presence the green samples. microorganisms in the also surprising and valuable. It is larger also surprising and valuable. It is larger than the sperm, greenish in colour, and surrounded by an extra cellular egg. The fluid between the outer envelops and embryo is known as peri-vitelline fluid. The peri-vittelline fluid contains important primitive types of proteins. An attempt was made to investigate the R&D 56 | Prospect Malaysia of removalblood withoutsacrificing of mangroves, and ensurethetechnique breeding beaches, banthedeforestation for constructionpurposesfrom the beaches, bantheremovalofsand gravel protecting sanddunevegetation onthe the coaststocontrolbeacherosion, ensure regularplantingoftrees along degradation. Environmentalists should from destructionandenvironmental to protecttheirbreedingbeaches purposes, effortsshouldbemade migrates towardstheshoreforbreeding Malaysia Terengganu. of MarineBiotechnologyUniversity techniques standardizedattheInstitute crab isalsooneoftheimportant blood withoutsacrificingthehorseshoe The non-destructiveextractionofthe crab withthehelpofelectricalstimuli. for artificialbreedingofthehorseshoe Terengganu hasdevelopedthetechnique Aquaculture, UniversityMalaysia conditions. TheInstituteofTropical salinity andotherenvironmental can toleratedifferencesintemperature, environments astheanimalishardyand breed thehorseshoecrabunderconfined from extinction.Itispossibletorearand essential toprotectthisvaluablecreature significant inbiomedicalresearch,itis crabs Conservation ofhorseshoe found alongthecoastsofMalaysia. on thethreespeciesofhorseshoecrabs comprehensive biotechnologicalstudies There isanurgentneedtocarryout developmental stagesoftheembryo. were limitedtoitsmorphologyandthe mass production.StudiesonT.gigas blood, itsreproductivebiologyand studied thecharacterizationofitsblue from UniversityPutraMalaysiahave researchers inMalaysia,particularly horseshoe crabshasbeenminimal.Some biotechnological potentialofthe ground,. of theirhabitat,especiallybreeding result ofthedegradationanddestruction horseshoe crab in several places is also the The decreaseinthepopulationof national monumentforhorseshoecrabs. measures bydedicatingacoastas subsequently implementedconservative the horseshoecrab(T.tridentatus)and to realizethedecliningpopulationof of theUSA.Japanwasfirstcountry horseshoe crabpopulationalongthecoast in theconsiderabledepletionof Amoebocyte. Thisactivityhasresulted year forthepreparationofLimulus brooder crabsaresacrificedevery extinction allovertheworld. horseshoe crabforfoodalsothreatensits the indiscriminateexploitationof As thehorseshoecrabregularly As thehorseshoecrabhasbecome In Malaysia,researchonthe In theUSAitself,about50,000 pharmaceutical productsfromhorseshoe that areactively involvedinproducing or semifinalproducts tocompanies is financialgaininexporting the final for researchandotherpurposes, there the localdemandofhorseshoe crab people ofMalaysia.Besidesmeeting role inimprovingtheeconomy ofthe becoming extinct. will protectthisvaluablecreaturefrom of thehorseshoecrab.Thesemeasures and disturbingthenormallifecycle This marinecreaturecanplay avital Collection ofblueblood horseshoe crab insemination ofa Artificial society. important programme forthebenefitof play avitalrole inimplementingsuchan support fromthelocalauthorities will administrators. Theactiveand sincere medical specialists,aswell research architects, marketingsurveyspecialists, comprise economists,urbanspecialists, implementation. Theteamshould well asthepolicmakerstoensure dedicated andcompetentteam,as crabs onacommercialscale. Such aprojectwouldrequire