A collection of horseshoe crabs (Chelicerata: Xiphosura) in the National Museum, Prague (Czech Republic) and a review of their immunological importance

2015·7.8 MB·English

by  Petr Dolejš

5
Arachnologische Mitteilungen 49: 1-9 Karlsruhe,Juni 2015
A collection of horseshoe crabs (Chelicerata: Xiphosura) in the National Museum,
Prague (Czech Republic) and a review oftheir immunological importance
PetrDolejs&KaterinaVahousova
doi: 10.5431/aramit4901
Abstract.Thezoological collection ofthe National Museum, Prague (NMP) contains spirit (juvenile) aswell as dry
(mostlyadult) specimensofhorseshoecrabs(Xiphosura). Living horseshoecrabsareofimmunological importance
due to clotting agents present in their hemolymph. Here we summarize basic data about the mechanism ofthe
immunesystemofthesemarineanimalsand itsusein practice-theLimulusAmebocyteLysatetest-includingthe
laboratory assays and handling with the animals. In the NMP collection, 82 specimens (16 dryand 66 spirit) ofall
fourcurrently recognised living speciesofhorseshoecrabsare present.Theywerecollected in Indonesia, USAand
Vietnam in 1872-1998;Limuluspolyphemusfromthe USA isthe mostnumerousspecies inthe NMP.Thecollection
contains no type specimens but three historical mounted ontogenetic series are present.The largest part ofthe
horseshoecrabcollection is55 spiritspecimensfromthecollection ofVaclav Fric(1839-1916) whose preparations
wereintended mostlyforeducational purposes.
Keywords:identification key,LALtest,pharmacology,VaclavFric,Xiphosurida,zoologicalcollection
Horseshoe crabs are marine bottom-dwellers, gro- place in the food chain in coastal ecosystem - mol-
wingupto 85 cmlong.Theirprosomais coveredwith luscs,crustaceans,fish,leopard sharks,eels,migratory
a single unjointed carapace bearing two compound shorebirdsandseaturtlespreyonhorseshoecrabsdu-
eyes. Research on the compound eyes of horseshoe ring different stages oftheir life cycle (Shuster 1982,
crabs has led to a better understanding ofhuman vi- Keinath et ah 1987). Other features oftheir biology
sion and based on their results, Ragnar Granit, Hal- and ecologycanbe found in Sekiguchi (1988), Shus-
dan Keffer Hartline and GeorgeWaldwere awarded ter et al. (2003) andTanacredi et ah (2009).
the Nobel Prize in Physiology or Medicine in 1967 History ofthe taxonomy ofhorseshoe crabs was
(Nobel Media AB 2014). The opisthosoma bears summarized in Dunlop et al. (2012), their phyloge-
paired movable lateral spins and an unpaired telson. neticrelationshipswererevealedbyObstetal.(2012)
There are four Recent species described: Limuluspo- andtheircurrenttaxonomicalpositionwasrevisedby
lyphemus (Linnaeus, 1758) from the NorthAmerican Lamsdell (2013) as follows:
East coast, and Carcinoscorpius rotundicauda (Lat- Subphylum: Chelicerata Heymons, 1901
reille, 1802), Tachypleustridentatus (Leach, 1819) and Class: Xiphosura Latreille, 1802 (syn. Merostomata
Tachypleusgigas (O. F. Müller, 1785) all from South- Dana, 1852)
EastAsia.Allfourspecies are similarinterms ofeco- Order:Xiphosurida Latreille, 1802
logy, life history and serology. They burrow through Family: Limulidae Leach, 1819
the surfacelayersofmuddysubstrateandingestsmal- The diploid chromosome numbers (2n) of hor-
ler animals or scavenge.The so-called trilobite larvae seshoe crabs were published byIwasaki et al. (1988).
hatchfrom the eggs; the horseshoe crabs reaches ma- Recently, mitochondrial genome sizes (MGS) and
turityafter 9-12 years and the life span maybe up to a genome adenine-thymine base ratio (AT) ofhor-
19 years. Males differ from females in having modi- seshoe crabs have been studied (Lavrov et al. 2000,
fied the first two pairs ofwalking legs into claspers Baek et al. 2014) resulting in the following gene-
usedduringmating.Horseshoecrabs occupyacrucial tic characteristics - L. polyphemus'. 2n = 52, MGS
= 14985 bp, AT = 67.6%; C. rotundicauda'. 2n = 32,
MGS = 15033 bp, AT = 73.8%, T. tridentatus'. 2n =
tCPoezrterychMDOuRLseEepJuuSbm,l,iDcC;eipEra-kmruatsiomlv:eanpte1t7o4rf0_Z,dooColZleoj-gsy1@,9n3mN.a0tc0iz,onParlahMau9s-euHomrn-iNPaotcuerranlicHies,- M26G,MSGaSnd=A1T50ye0t6ubnpk,nAoTwn=.7I4n.a0d%d;itTi.ognigatso'.th2ins,=ho2r8-,
aKk.aas.tt,[email protected]Ä0,1,CConZt-ro5l61Ana0l2y,tCizcaelchLaRbeopruabtloircy;,EC-omnatili:prvoanPohuasromvaa. csleosthtoiengcraagbesntasreproefsegnrteaitnctlhieniircahleimmoplorytmapnhceasduseumt-o
submitted27.11.2014,accepted10.3.2015,online:29.5.201 marized below.
2 P.Dolejs&K. Vanousova
Currently, many museums are publishing ca- Endotoxin (LPS) is a part ofthe outer membra-
talogues of their collections (e.g. Jirouskova et al. ne ofGram-negative bacteria and is released during
2011, Chiarle et al. 2012, Dunlop et al. 2012, 2014, lysis of the cell or cell division. Most problems for
Kielhorn et al. 2012, Mlikovsky et al. 2013, Seiter the pharmaceutical industry are caused by non-pa-
&
Hörweg 2013). Thus, the aim ofthis paper is to thogenic Gram-negative bacteria amply represented
summarize the practical use ofhorseshoe crabs and in aquatic environments.The high occurrence ofen-
to provide further information about the zoological dotoxin cannot be excluded even in sterile products,
collections ofthe NationalMuseum in Prague. since endotoxinis abletowithstandsteam sterilizati-
on.MildGram-negativebacterialinfectionscantrig-
TheAmericanhorseshoecrah-aguardianof ger a pyrogenic response.The presence ofendotoxin
publichealth in the blood stream can cause fever, inflammation,
The study ofhaematology using the horseshoe crab and frequentlyirreversible shock(Joineret al.2002).
as a model is possible due to large blood cells (ame- Since humans are sensitiveto minute amounts ofen-
bocytes) andledto discoveryoftheLimulusAmebo- dotoxin, the importance oftesting for endotoxin is
&
cyte Lysate test (LAL test) byBang Forst (1953). apparent.
Theyobservedthatinfectionbygram negativebacte- In the early 1970s,a newerpyrogen testingtech-
ria caused intravascular coagulation in the horseshoe niqueusinghorseshoecrabs’bluecopper-basedblood
crab. Coagulation is activated by the cascade ofen- was developed. The LAL test would be 100 times
zymes located in the amebocytes and is triggered more sensitive than the rabbit testing methods used
by lipopolysaccharide (LPS, endotoxin) in the gram before (Novitsky 1984). Clot formation is initiated
&
negative bacterial cell walls (Levin Bang 1964a, by pico- to nanograms ofbacterial endotoxin (Mik-
1964b, 1968). The catalytic nature ofeach activated kelsen 1988). Because ofits superior reliability and
enzyme in the coagulation cascade serves in turn to simplicity (the test takes only one hour), the LAL
amplifythenextstep,resultinginahighsensitivityof testbecame aninvaluabletoolforthepharmaceutical
LAL to LPS (Iwanaga 2007). industry. Every drug and medical device certified by
Content ofmicroorganisms in the seawater can the Food and Drug Administration must be tested
reach up to 10^ bacteria/ml and 10^ virus/ml of bya LAL test (Walls &Berkson 2000).
seawater (Ammerman et al. 1984). Therefore, the The LAL test is employed to ensure that phar-
effective and robust innate immune system ofhor- maceutical products are endotoxin-free including
seshoe crabs is aprerequisite fortheirsurvivalin this bulk lot release testing, final product release testing
&
environment (Menzel et al. 2002, Tincu Taylor and raw materials testing (Rudloe 1983). Anyone
2004). It has been suggested that the clot formed who has ever encountered intravenous fluids, vacci-
through the activation ofthe cascades captures and nes or surgical implants has been protected against
immobilizes invading microorganisms; clot forma- contact with bacterial endotoxin by the blue blood
tion is triggered not only by an endotoxin released ofa horseshoe crab. The LAL test is used not only
by Gram negative bacteria but also by (l,3)-ß-D- to determinate harmful levels ofendotoxin in phar-
glucan (Morita et al. 1981) which is mainly present maceutical products, but is also the backbone of
in the cell walls ofyeast and other fungi (Iwanaga controllingendotoxininboth the process and equip-
1993, 2002, Muta et al. 1995). Moreover, the clot ment involved in producing pharmaceuticals and of
formed as a result ofactivation byendotoxin orglu- monitoringhigh puritywaterused as aprime source.
cans provides wound controlbypreventingbleeding Furthermore, the LAL test is the method ofchoice
and forming a physical barrier against additional forresearchers examiningthe clinicalorthe environ-
infection entry (John et al. 2010, Armstrong et al. mental effects ofendotoxin (Walls et al.2002).
2013).Thus, the Limulus clotting system is thought Currently, three principal LAL test methods
to be criticalforboth haemostasis andthe biological exist; the gel clot, turbidimetric and chromogenic
defence ofthis animal (Iwanaga2007).This gelation methods. The latter two are referred to as the pho-
reaction ofLimulusamebocytelysate hasbeenwide- tometric method for they require an optical reader.
lyemployed as arapid and simple method forendo- The gel clot assay is the simplest method ofdeter-
toxin testing. How then do horseshoe crabs protect mining the level ofbacterial endotoxin. In the assay,
public health? equal volumes ofLAL reagents are mixed with the
Horseshoecrabsincollectionsandmedicine 3
tested sample and clot formation is observed.At the
end ofthe incubation period the tube containingthe
mixture ofthe sample and LAL is inverted. Ifa gel
has formed and remains intact in the bottom ofthe
reaction tube after an inversion of180 degrees (Fig.
A
1), the test is positive. positive test indicates that
the concentration ofendotoxin in the tube is greater
thanorequaltothe sensitivityofLAL (Associatesof
Cape Cod Inc.2007a).
Both photometric methods require a standard
curve to determine the endotoxin level in the sam-
ple.The chromogenic assayis based on replacing the
natural substrate,coagulogen,byachromogenic sub-
strate. The chromogenic substrate is cleaved by the
serineproteasecoaguloseactivatedbyendotoxin,and Fig. 2: Bleeding of horseshoe crabs (photograph provided by
then the chromophore is released and is measured AssociatesofCapeCodInc.,reprintedwithpermission)
by spectrophotometry (Associates ofCape Cod Inc.
2011).The turbidimetric method is analogous to the
chromogenic method,but the turbidityis monitored
(Joiner et al.2002). CodInc.2007b).Furthermore,anendotoxin-neutra-
In the blood of the horseshoe crab, other com- lizing protein which has potential as an antibiotic as
pounds ofbiomedical interest have also been disco- well as an alternative endotoxin assay, and a number
vered. LAL is used for detecting l,3-ß,D-glucans ofotherproteinsthatshowanti-viralandanti-cancer
e.g.inpharmaceuticalproducts orin atestforfungal activityarebeingexplored (Valespietal.2000,Andrä
infection (Obadasi et al. 2004, Associates of Cape et al.2004,Tincu dcTaylor2004).
And how is the blood ofthe horseshoe crab ob-
tained? Adult horseshoe crabs are collected by traw-
lers orbyhand-harvest and transported to the lab of
a biomedical company, where they are washed and
placedon arack.Horseshoe crabs arebledfromperi-
cardiumwith alargegauge needle-up to30%ofthe
animal’s blood is removed (Fig.2).Within 72 hours,
the bled horseshoe crabs are returned to the place of
capture and released alive (ASMFC 1998, Leschen
&
Correia 2010). Their blood volume restores in
about aweek.The amebocytes regenerate at a slower
rate, requiring up to four months before cell counts
equal to those obtained prior to bleeding (Novitsky
1984).
Mortality of horseshoe crabs after the bleeding
process was found to be 3-15 % (Walls 5c Berkson
2000).There are currentlyfive biomedicalcompanies
producing LAL in the United States. Each ofthem
has uniquebleedingmethods,methodofcapture,di-
stance and method oftravel to bleeding lab, holding
time andconditions,and methods ofreturn mostap-
propriatetotheirownsettingandsituation.Thus,the
impact ofthe blood extraction processes on survival
Fig. 1:The positive result ofa gel clot assay (photograph pro- of the horseshoe crabs varies between operations
videdbyBiogenix,s.r.o.,reprintedwithpermission) (Walls et al.2002).
4 P.Dolejs&K. Vanousova
The blood of horseshoe crabs can be extracted Materialandmethods
without killing the animals, but nowadays attention Methods for curating the horseshoe crab collection
is paid to long-term injury caused by the bleeding followed those of Dunlop et al. (2012). Specimens
process. Recent studies denote that the biomedical were determined by the key provided below that
bleeding process potentiallyled to several sub-lethal was composed based on characteristics published by
&
behavioural and physiological changes. The most Yamasaki (1988), Shuster Anderson (2003) and
obvious behavioural effects are immediate decreases Dunlop et al. (2012). Current nomenclature and the
in walking speed and latent reductions in both ove- Life Science Identifier numbers (Isid) were adopted
WoRMS
rall activity and the expression oftidal rhythms.The from (2014). The items belonging to one
greatest impact ofbleeding onLimuliisphysiologyis species are sorted chronologically from the oldest to
an immediate and sustained decline in hemocyanin the newest one. Data for each item are arranged as
NMP
concentrations (Anderson et al.2013). follows: inventorynumberunderthe acronym
Horseshoe crab blood is not only a backbone of (National Museum Prague), number of specimens
pharmaceutical industry, but it is also big business. and their sex (type ofpreparation),name ofthe coll-
On the world market, a quart of horseshoe crab ector, date ofcollection, locality - the current name
NGA
blood has aprice tagestimated at $15000,leadingto ofthe localitywas adopted from (2014); note
overallrevenues from the LAL industryestimated at ifany.Indrymaterial,totallength (TL) andcarapace
U.S. $50 million per year. According to the Atlantic width (CW) is provided. Ifthe telson is missing or
CW
StatesMarine Fisheries Commission,that $50 milli- broken,only is noted.
ondollarindustryrequiresthebloodofapproximate-
ly500,000 horseshoe crabs (ASMFC 2013). Identificationkeyforlivingspecies
Fortunately, companies producing LAL realize 1) A pair of finger-like projections on the genital
that a stable population ofhorseshoe crab is essential operculum (Fig. 3); the first entapophyseal pit on
not onlyfor the pharmaceutical industrybut also for opisthosoma triangulate (left p, right q ); telson
survival ofother marine animals that have a symbi- shorter than the body; distributed in the western
otic relationship with the horseshoe crab. The LAL Atlantic along the eastern seaboard of North-
industry has taken steps to make the LAL test syn- CentralAmerica Limuluspolyphemus
thetically or to find methods to improve the sensiti- — No finger-like projections on the genital oper-
vity ofLAL, which would eliminate the use oflive culum (Fig. 4); the first entapophyseal pit on
horseshoe crabs for the LAL reagent (Thorne et al. opisthosomaelongate (l); telson atleastaslongas
2010).Withgrowingconcernoverdecliningpopula- the body; distributed in the Indo-Pacific waters
tions,itis obvious thatitwillbe achallengetoensure ofSouth-EastAsia Tachypleinae (2)
thathorseshoe crabs manage to fulfilallthese diverse 2) Telson longer than the body, smooth, rounded in
needs and at the same time to ensure their sustaina- profile (withoutanyventralgrooveordorsalkeel);
ble population forthe future. all opisthosomal marginal spines short (Fig. 12);
The situation is bit different in Asian horseshoe total length up to 35 cm in males and 40 cm in
crabs. The Tachypleus Amebocyte Lysate (TAL) is females Carcinoscorpiusrotundicauda
derived from the two Tachypleus species - T. triden- - Telson as long as the body, triangular in profile
tatus and T.gigas. Nevertheless, only two species, L. (usuallywithaventralgroove anddorsalkeel);the
polyphemus and T. tridentatus are mentioned in the firstthree (infemales) orall (in males) opisthoso-
EuropeanPharmacopeia,theUnited States Pharma- mal marginal spines long; usuallylarger animals
copeia and in the Japanese Pharmacopeia. In Asia, Tachypleus (3)
TAL is manufactured in China andJapan (Wang et 3) Total length 45-70 cm in males and 55-85 cm
al. 2007). However, most ofthe animals used come in females; mid-dorsal part ofopisthosomal pos-
from areas in SoutheastAsiawhere harvestingregu- terior margin with three short immovable spines
lationshave notbeenestablishedorenforcedandless pointing posteriorly (Fig. 5); anal angles (from
is known regarding their horseshoe crab collection ventral view) with thorn-like spines on both
and handling practices. It is believed that most hor- outer lateral margins (Fig. 6); males with a pair
seshoe crabs die post-bleeding, some as bait, some ofstrong indentations to the anterior rim ofthe
as food,and some due to the bleedingprocess itself. prosoma (Fig. 13) T.tridentatus
Horseshoecrabsincollectionsandmedicine 5
- Total length 25-40 cm in males and 25-50 cm
in females; mid-dorsal part ofopisthosomal pos-
terior margin with one short immovable spine
pointed posteriorly and with no spines on either
side (Fig. 7); anal angles (fromventralview) usu-
allywith smooth outer lateral margins (although
theyrarelyhave small spines on the margin) (Fig.
8);maleswithsmoothmarginsoftheanteriorrim
ofthe prosoma (Fig. 14) T.gigas
Systematiclist
Subfamily: Limulinae Leach, 1819
Genus: Limulus O. F.Müller, 1785
Figs 3-8: Diagnostic characters of horseshoe crabs. 3: Limu-
luspolyphemus, a pairoffinger-like projections on the genital
Limuluspolyphemus (Linnaeus, 1758)
operculum;4:Asian horseshoecrabs,nofinger-likeprojections
NurMn:PlsiPd6:Em-ar2i4n6e0s,pe1ci$es(.dorrygm:attaexrniaamle,:T1L50=51348 cm,CW opanrtthoefgoepniisttahloospoemracluplousmt;er5i:orTamcahrygplienuwsittrhidtehnrteateuss,homritdi-dmomrosva-l
= 20 cm), collected by Dr. Palacky in 1872 in North able spines pointed posteriorly; 6: T. tridentatus, anal angles
(fromventral view)withthorn-likespineson both outerlateral
America.
NMP margins; 7: Tachypleusgigas, mid-dorsal part ofopisthosomal
19/1960/2564,6juveniles (spiritmaterial), posterior margin with one short immovable spine pointed
collectedbyan unknown collectorin 1886 inWoods posteriorly and with no spines on either side; 8: T. gigas, anal
Hole (4r3F25”N 70°40’20”W), USA; ex. coll. V. angles (from ventral view) with smooth outer lateral margins;
te=telson
Fric.
NMP
19/1960/2745, 30 juveniles (spirit mate-
rial), collected by an unknown collector in 1886 in
Woods Hole (4r31’25”N 70°40’20”W), USA; ex.
NMP
coll.V.Fric. 19/1960/3100, mounted ontogenetic se-
NMP
P6E-3903, mounted ontogenetic series ries containing 4 eggs and 9juveniles (spirit materi-
containing 18 eggs and 4juveniles (spirit material), al),unknown origin; ex.coll.V. Fric; Fig. 11.
NMP
donated byA. S. Packard toNJ.MBaPrrande from whose P6d-8/2003C,W1 malformed 9 with broken
inheritance it arrived in the in 1894; Fig. 9. telson (drymaterial, = 29 cm) and 1 6(dryma-
NMP P6E-2462, mounted ontogenetic series terial,TL = 39 cm,CW = 19 cm),unknown origin.
NMP
containing 4 eggs and 6 juveniles (spirit material), P6d-254/2003, 1 female carapace (dry
collected by an unknown collector on 24 Septem- material, TL = 61 cm, CW = 29 cm), collected by
ber 1896 in New York, Long Island (40°37’00”N Mr. Morawitz in 1957 in New York, Long Island
73°50’20”W),USA; Fig. 10. (40°37’00”N 73°50’20”W),USA.
NMP NMP
19/1960/2580,7juveniles (spiritmaterial), P6E-2794, 1 9 and 2 juveniles (dry ma-
collected by Brimley Bros Raleigh in February 1904 terial,TL = 37, 4 and 3 cm, CW = 18, 2 and 2 cm),
inUSA; ex. coll.V.Fric. collected by D. Collins in 1968 in Naples, Florida
NMP
19/1960/2905, 1 juvenile (spirit materi- (26°08’30”N 81°48’30”W),USA.
al), collected by an unknown collector on 25 Janu- Other material: 1 c5in poor condition (drymate-
ary 1913 inWoods Hole (4r31’25”N 70°40’20”W), rial,TL = 38 cm,CW= 21 cm) and2 damaged (pro-
USA; ex. coll.V.Fric. bablyfemale and male) carapaces (drymaterial,TL =
NMP P6E-2461, 1 9 (drymaterial,TL = 56 cm, ? and 39 cm,CW = 25 and 19 cm),unknown origin;
CW
= 27 cm), collected by an unknown collector in packed in a newspaper “Narodni politika” [National
1913 in NorthAmerica; ex.coll.V. Fric. politics] from 22 March 1929.
NMP
19/1960/2280, 1 juvenile (spirit material),
unknown origin; ex. coll.V.Fric.
NMP
19/1960/2281, 1 trilobite larva (spirit ma-
terial),unknown origin; ex. coll.V.Fric.
6 P.Dolejs&K. Vahousova
Fig.9:MountedontogeneticseriesofLimuluspolyphemus(NMPP6E-3903),inheritanceofJ.Barrande
Fig.10:MountedontogeneticseriesofLimuluspolyphemus(NMPP6E-2462),fromtheformerzoologicalexhibition
Subfamily:Tachypleinae Pocock, 1902 NMP P6E-3086, 1 young 9 (dry material, TL
CW
Genus: Carcinoscorpius Pocock, 1902 = 61 cm, = 32 cm), collected by K. Vopafil in
1998 in VIETNAM; originallylabelled as ''Limulus
Carcinoscorpiusrotundicauda (Latreille, 1802) grandis\
urn:lsid:marinespecies.org:taxname:238267
NMPP6E-3085,1 c3(drymaterial,TL= 33 cm,CW Tachypleusgigas (O. F.Müller, 1785)
= 15cm),collectedby WünschinMay1973inTuan urn:lsid:marinespecies.org:taxname:238271
J.
Chau, Vjnh Ha Long (20°55’40”N 106°59’40”E), NMP P6E-3904, 1 juvenile (spirit material), coll-
VIETNAM; Fig. 12. ected by an unknown collector in 1898 in Maluku
INDONESIA;
(3°50’S 129°50’E), originally la-
Genus: Tachypleus Leach, 1819 belled as Limulus moluccanus.
NMP P6E-3120, 1 9 (dry material,TL = 40 cm,
CW
Tachypleustridentatus (Leach, 1819) =19 cm), collected by Dr. Jerman on 17 De-
urn:lsid:marinespecies.org:taxname:238270 cember 1933 in Gunung Pantaicarmin, Sumatera
NMP P6E-2838, 1 6 (dry material, TL = 60 cm, Barat (L22’60”S 100°34’30”E),INDONESIA.
CW = 28 cm), collected by Dr. Hlozanekin 1988 in NMP P6E-3121, 1 (5(dry material,TL = 31 cm,
VIETNAM; Fig. 13. CW = 15 cm), collected by Dr. Jerman on 11 No-
Horseshoecrabsincollectionsandmedicine 1
Fig.11:MountedontogeneticseriesofLimuluspolyphemus(NMP 19/1960/3100),ex.coll.V.Fric
Figs 12-14: Males of horseshoe crabs in relation to one another. 12: Corcinoscorpius rotundicauda (NMP P6E-3085), note short
opisthosomalmovablemarginalspines;13:Tochypleustridentatus(NMPP6E-2838),noteapairofstrongindentationstotheanterior
rim ofthe prosoma (arrowheads); 14: Tachypleusgigas (NMP P6E-3121), notesmooth marginsoftheanteriorrim ofthe prosoma;
scalebar= 10cm
vember 1934 in Gunung Pantaicarmin, Sumate- cal bleeding process on the American horseshoe crab,
ra Barat (r22’60”S 100°34’30”E), INDONESIA; Limuluspolyphe7nus.-BiologicalBulletin225: 137-151
Fig. 14. AndvriätsJk,yGTa,riKdoeclhP,MMHajJer&leBAr,aJnedrealnabRu,rgRiKdg2e00R4,PBainosphEy,siNcoa-l
characterizationoftheinteractionofLimuluspolyphemus
Acknowledgements
We would like to thankJason A. Dunlop (Museum für eEnudrootpoexainnJnoeuurtnraalliozfiBngiopcrhoteemiinstwrityh27l1i:po2p0ol3y7s-a2c0ch4a6ri-dedo.i-:
Naturkunde, Berlin) for supplying us with important li-
10.1111/j.l432-1033.2004.04134.x
tmearnautsucrre,ipgtr.aWmemaarlsocotrhraenctkiMoinschaanedlausZeefumlänckoomvmäen(tBsiogteontihxe, ArmstrongMT,RidesFR&.ArmstrongPB2013Capture
oflipopolysaccharide (endotoxin) by the blood clot: a
s.r.o.,Prague)andVeronikaWills(AssociatesofCapeCod, comparative study.-PLoS ONE 8(e80192): 1-9-doi:
Inc.,EastFalmouth) forpermissiontousethefigures.This
10.1371/journal.pone.0080192
wthoerCkzweacshfRienpauncbilailcly(sDuKppRoVrtOed20b1y4/M1i4n,iNsattriyoonfalCMulutsueruemo,f ASMFC 1998 Interstate Fishery Management Plan for
Horseshoe Crab.Unpublished report. Horseshoe Crab
00023272).
PlanDevelopmentTeam,Atlantic StatesMarine Fish-
eriesCommission,Richmond.57pp.-Internet:http://
www.asmfc.org/uploads/fde/hscFMP.pdf (March 6,
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