Immunological Survey of Planktonic Embryos and Larvae of the Starfish Asterina pectinifera, Obtained from the Sea, Using a Monoclonal Antibody Directed against Egg Polypeptides

9 Pages·1991·3.5 MB·English

by  S Ikegami

Reference: Biol. Bull 181: 95-103. (August, 1991)
Immunological Survey of Planktonic Embryos and
Obtained
Larvae of the Starfish Asterina pectinifera,
from the Sea, Using a Monoclonal Antibody
Directed against Egg Polypeptides
SUSUMU IKEGAMI, TOMOKO MITSUNO, MASANORI KATAOKA,
SATOSHI YAJ1MA, AND MIEKO KOMATSU1
Faculty ofApplied Biological Sciences. Hiroshima University, Kagamiyama 1-Chome.
Hiroshima 724, Japan; and 'Department ofBiology,
Higashihiroshima-shi,
Faculty ofScience. Toyama University. Toyama 930, Japan
Abstract. A monoclonal antibody, Kl, specifically rec- Introduction
ognizes polypeptides with apparent molecular masses of The development ofa number of starfish species has
56 and 58 kDa, in the egg ofthe starfish Asterinapectin- beendescribed (Oguroand Komatsu, 1988). Nevertheless,
ifera. The Kl antibody reacted with extracts prepared the embryos and larvae obtained from the sea have hith-
and
from ovaries, oocytes, morulae, blastulae, gastrulae, ertobeenunidentifiable, becausetheentireprocessoftheir
bipinnariae. Brachiolariae, testes, pyloricceca,bodywalls, development and metamorphosis is not sufficiently well
and tubefeet did not contain Kl-reactive antigenic mol- known. Furthermore,theidentification ofindividualspe-
ecules. Extracts ofeggs ofthe starfish Asterias amurensis cies by examination of external morphological features
and several sea urchin species did not react with the Kl alone tends to be equivocal. Therefore, specific methods
antibody. Amongthe members ofthegenusAsterina, ex- fordetectingchemical components presentexclusively in
tracts of brachiolariae of .1. batheri and A. minor were embryos and larvae ofone species are needed.
not reactive, whereas blastulae and brachiolariae of A. We report the discovery ofspecific antigenic polypep-
pseudoexigua padfica did contain an antigenic compo- tides that are present in eggs, embryos, and larvae of,4s-
nent. Unlike the antigenic peptides in A. pectinifera eggs, terina pectinifera. but not in those of other species be-
the apparent molecular mass ofthe antigen molecule in longingto the same genus. A single embryo orlarva in a
embryos and larvae ofA. pseudoexigua padfica was 41 complex plankton mixture can be detected by immuno-
tkiDoan,owfhiancthigreenpremsoelnetcsulaesr.emUasriknagbltehephKyllogaennteitbiocdyv,ariwae- alosgsiacyal(EaLnaIlSyAs)isorwiitmhmuannoebnlzoytmep-rloicnekdeurde,imumsuinngosaomrobneont-
havedeveloped anassaysystemthatdetectsembryosand clonal antibody that recognizes the unique antigen mol-
larvae ofA. pectinifera in complex mixtures ofbiological ecules present in embryos and larvae.
specimens obtained from the sea.
Materials and Methods
Received 10December 1990:accepted 1 April 1991. Animals
CaAFbSbWre.vicaatlicoinusm:-fArSeWe,seaartwiaftiecira;lsDe-awMaEteMr,,BDSuAl,bebcocvoi'nsesmoedriufmicaaltbiuomnino;f Adults ofthe following starfish and sea urchin species
Eaglemedium;EL1SA,enzyme-linkedimmunosorbentassay;PCS,fetal were collected, during their breeding seasons, along the
calfserum;FITC, fluorescein isothiocyanate; HAT.hypoxanthine-ami- coastoftheJapan Islands.Thestarfishwerecollectedfrom
nopterine-thymidine;IgG,immunoglobulinG;kDa,kilodaltons;NSW. the following areas: A. pectinifera off Asamushi in
normalseawater,PBS,phosphatebufferedsalinewithoutdivalentcations; Aomori Prefecture, off Otsuchi in Iwate Prefecture, off
TP-MSTFB.S,p0h.e5nyMlmNetahCyll,su1l0fomnAy/iTfnlsuo-rHidCel;(SpDHS,7.8s)odainudm0.d0o5d%ecTylweseunlfa2t0e.; Hashirimizu in Kanagawa Prefecture, off Sensui Island
95
96 S. IKEGAMI ET AL.
in Hiroshima Prefecture, and offGogo Island in Ehime blotted onto nitrocellulose sheets(Bio-Rad), asdescribed
Prefecture; Asterina pseudoexigua pacifica from the below.
undersurface ofstonesatthe intertidal zone ofKushimoto
inWakayama Prefecture;andAsterinaminorandAsterina
batheri along the coast ofthe Noto Peninsula in Ishi- Preparation ofantigens
kawa Prefecture. The sea urchins, Scaphechinus mirabilis, Mature eggs(3 ml packed volume) from A.pectinifera,
Hemicentrotus pulcherrimus, and Pseitdocentrotus de- shed from the isolated ovaries ofadults by application of
pressus, were collected off Sensui Island in Hiroshima 1-methyladenine at a final concentration of 150 ng/ml,
PSruegfaescthuirmea, oIfsflaKnidwiandoMiien PYraemfeacgtuucreh,irePsrpeefcetcitvuerley,.and off wstearretedc.olTlheectyedwer1ehwaasfhteerd,thbey sheottrlminogn,eint5revaotlmeonftAhSaWd
The plankton samples were collected by a diver and homogenized, in a Potter-Elvehjem glass homoge-
equipped with SCUBA, who towed plankton nets (100 nizer, at 0C in 12 ml 20 mA/Tris-HCl (pH 7.5) buffer
^m-mmesh; open mouth, 40 cm in diameter) horizontally with 0.1 mA/phenylmethylsulfonyl fluoride (PMSF). The
50 at discrete depths off Sensui Island in Hiroshima subsequent procedures were carried out at 4C, unless
Prefecture. otherwise stated. The homogenate was centrifuged at
18,000 X gfor 20 min. The pellet was resuspended in 15
Gametes, embryos, andlarvae ml 20 mA/Tris-HCl (pH 7.5)bufferwith 0.1 mAlPMSF,
Fertilizable eggs were induced to spawn from isolated and the suspension was centrifuged again. The superna-
ovaries offemale A. pectinifera and A. amurensis by ap- tants were combined, and solid ammonium sulfate was
plication of 1-methyladenine at a final concentration of added toa final ammonium sulfateconcentration of20%
150 ng/ml (Kanatani, 1973). SpawnedA. pectiniferaeggs (w/v). The mixture was stirred for 1 h, then centrifuged
werewashed several timeswith artificial seawater ASW), at 18,000 X gfor 20 min. The supernatant was removed,
Jamarin U (Jamarin Laboratory, Osaka),and insem(inated theammonium sulfateconcentration wasadjusted to 80%
at40-60 min afterthe startof 1-methyladenine treatment. (w/v) by adding solid ammonium sulfate, and 1 h later,
Formed embryos were cultured in ASW at 23 2C, the mixture was centrifuged as before. The precipitate
and theearlybrachiolariaewere reared in natural seawater wasremovedanddissolved in 21loadingbuffer,composed
(NSW), which waskept in thesun and changedeverytwo of50 mAl Tris-HCl (pH 7.4) with 0.1 mA/ PMSF. The
or three days. Embryos ofA. hathen were obtained sim- solution was then loaded onto a Whatman DE-52 ion-
ilarly. Embryos and larvae of hermaphroditic A. minor exchange resin column (2.5 X 3.3 cm), which had been
were obtained from acontainerofadult coloniescollected equilibrated with loading buffer. Step-wise elution was
duringthe breedingseason, duringJuneatthe Noto Pen- performed with solutions ofNaCl in loading buffer using
insula in Ishikawa Prefecture. Embryos and larvae ofA. 50 ml each of20, 150, 200, and 500 mAlconcentrations.
pseudoexiguapacificawerecollected from thebody cavity The fractions eluted with 150 and 200 mA/ NaCl were
of the adult during the breeding season, in July at Ku- pooled and dialyzed against phosphate-buffered saline
shimoto in Wakayama Prefecture. Eggs of sea urchins without divalent cations (PBS). The non-dialyzable frac-
were obtained by introducing 0.5 M KC1 into the body tion was frozen at -80C for future use.
cavity.
Eggs, embryos, and larvae were washed several times
ASW NSW ~80C Preparation oj nuclei
with or before being stored at until
required. Two thousand, five hundred (2500) A. pectinifera em-
bryos were washed twice with calcium-free seawater
Protein detenuinution (CaFSW, Jamarin Laboratory, Osaka; 4C)and oncewith
Protein wasdetermined by the protein-dye binding as- buffer A (4C), which consisted of 250 mA/ sucrose, 5
mA/ MgCl 40 mA/ NaCl, and 50 mA/ Tris-HCl (pH
say, with bovine serum albumin (BSA) as the standard :,
(Bradford, 1976). a7t.5)4.CTheinyw5emrlehboumffoegrenAizceodntwaiitnhinagDo1u%n(cwe/vh)omTorgietnonizXe-r
100. The homogenate was passed through a40-/um stain-
Sodium dodecyl sulfate-polyacrylamidegel less steel filter by gravity. The filtrate was centrifuged at
electrophoresis 1000 X gat 4C for 5 min. The nuclear pellet was resus-
Discontinuous sodium dodecyl sulfate (SDS)-poly- pended in 2.5 ml buffer A, followed by centrifugation at
acrylamide gel electrophoresis was performed with 5% 1000 X gfor 5 min. This processwas repeated once. The
stacking gel, as described by Laemmli (1970). The gels washed nuclear preparations were frozen at -80C for
were then stained with Coomassie brilliant blue R-250 or future use.
IMMUNODETECTION OF STARFISH LARVAE 97
Monoclonal antibodyproduction Rad) were incubated with undiluted culture supernatant
A 7-week-old BALB/c female mouse was injected in- awtit3h7diCstiflolred2wahtewri,ththgeeyntwleersetitrhrienng.waAsftheerd,afborrie1f0wmaisnh
traperitoneally with 100 jxgprotein in complete Freund's each, in two changesofexcess 10 mA/Tris-HCl (pH 7.4),
taidojnusvaonft.50FMogurpraontedinthweenrefiavdemwineiesktserleadte.r,Abnodosttherreeindjaeycs- a0t.537A/CNawiCtlhahnodrs0er.a0d5i%shTpweereoxnid2a0,sea-ncodnjiungcautbeadtegdoaftoran2tih-
after the second booster, the mouse was sacrificed, its mouse IgG (Bio-Rad) diluted 1:3000 in the same buffer
mo(sXupiDltn1-e,0Mei7nnEwsiMwp1t)lahe,smelmnrwyiecDetmeulholloovlbuemsetd/ca,mcsyoceaee'rlnlsluodsmmm,(otaShdbPceieu-flt2clie)sclc)al.o(stanTittahoalaneilnrofoaiwuftnesigdiEooa5tnogo0flwe%fa1ussmpXeoec,lady1rfie0rotiru8h/em1-8d twushstaaiostnphgpiwenaHdgs2baOygua2srieinadn,nsdisttnorg4i-pwbcsilhtowlhceokrrwoea-ttahlees-rsnaaanpyinhetdtrdohscfoteolrrli.lppusTelwrohesoeerxierdsedtaarrsicieptesid.aocnitAnifvwatiiaetrsry.
ylene glycol-6,000. To stop the fusion, 20 ml D-MEM obtParionteedinbbyloetlsecotfrooopchyotreest,icegtgrsa,nsefmebrryforso,maSnDSd-lpaorlvyaaecwreyrle-
wceanstraidfduegdatdiroonpa-twi2s5e0, aXngdfcoelrl5pemlilnet.sTwhereeceplrlecpieplilettastewderbey amide gel to nitrocellulose sheets and then treated as
resuspended in 5 ml D-MEM containing 5% fetal calf specified above, except that the IgG solutions, diluted 1:
serum (FCS, Microbiological Associates), incubated for 100to 1:1000 in PBS, wereused forantibodyincubations
instead ofhybridoma culture supernatant.
12-18 h. then plated in 98-well microtiter dishes at 200
l/l/well in HAT medium (complete D-MEM containing
1 X 10~4A/hypoxanthine, 4 X 10~7A/aminopterin, 1.6 Immunofluorescence microscopy
X 10~5A/thymidine, and 10% FCS). Halfofthe medium EmbryoswerewashedtwicewithASW, fixedwith 100%
wasremoved from eachwell and replaced with fresh HAT methanol for 10 min, immersed in 100% ethanol for 10
medium after2,4, 7, and 10days. The supernatantswere min andthenembedded in polyesterwax(BDH). Sections
harvested when theclonesbegan to exhaust their medium 7 jim thick were placed onto 0.1% (w/v) amylopectin-
and were screened byprotein blotting, asdescribed below. coatedcoverslipsand incubatedwith IgGsolutiondiluted
Hybridomas that showed reactivity were expanded and 1:100 in PBS for 1 h, at room temperature, after which
subcloned by limited dilution. they were washed in PBS for 30 min. Fluorescein iso-
Hybridomas (1 X 107 cells) were injected intraperito- thiocyanate (FITC)-conjugated goat anti-mouse IgG
neally intoa BALB/c mousewhich, 1-2 weeks previously, (Tago),diluted 1:200inPBScontaining2% BSAwasthen
at 8 weeks old, had received an injection of 0.5 ml added and the sections were incubated, in the dark, at
2,6,10,14-tetramethylpentadecane. Onetotwoweeksafter room temperature, for 1 h, afterwhich they were washed
the injection of hybridomas, the ascites were collected with PBS for 30 min. Coverslips were mounted in a so-
and centrifuged at 1500 X gfor 30 min. The supernatant lution of20% glycerol in PBS, sealed, and viewed under
( 1 ml)wasdialyzed, at 4C, against 50 mA/Tris-HCl (pH a Nikon TMD-EP2 photomicroscope. Photographs were
8.6) with 0.15 A/ NaCl. The non-dialyzable fraction was then taken using Kodak Tri-X pan ASA 400 film.
loaded onto a column (0.6 X 10 cm) ofProtein-A cellu-
lofine (Seikagaku Kogyo, Tokyo), which had been equil- ELISA assay
0Ai.fbr1ta5etreAwd/awNsiahtCihln,5g0wtmihetAh/iTM3mr0miumsnl-oHg5Cl0lomb(Aup/lHTirn8.iG6s)-w(HiICtglhG)0(.pf1rH5ac8At./i6o)NnawwCialt.sh b6o2i.Fl5ievdmeAf/tohTror2uissma-inHndCli(n5(01p00Hm)l6o.8oS)cD,ySt2e%ss,aSmeDpgglSse,abnourdffe5emr%bcr2oy-nomtseariwcneairpne-g
eluted out by 0.05 sodium acetate (pH 4.0) and 0.15 toethanol (Laemmli. 1970). One hundred microliters of
A/NaCl. The pH ofthe IgG solution was adjusted to 7.2 each ofthe extracts, at a dilution of 1:2000 in PBS, were
by adding 1 A/ Tris-HCl (pH 9.0). The protein concen- addedtoawellofa microtiterplateforELISAassay(Pro-
tration was adjusted to 500 Mg/ml, and then the IgG so-
lution was frozen at -80C for future use. bind, Falcon), the content of the lysate in a well being
equivalentto0.25 oocytes,eggs, orembryos, andabsorbed
for2 h at 37C. The solution wasremoved, and the wells
Immunoblotting were treated at room temperature with 200 n\ PBS with
For screening hybridomas, nitrocellulose blots were 1% BSA for30 min. The IgG solution wasdiluted 1:1000
prepared by electrophoretic transfer from preparative in PBS, a 100-^1 aliquot was added to each well and in-
SDS-polyacrylamide gels ofthe 150 and 200 mA/ NaCl cubated for 2 h at 37C. The wells were washed twice
fractions obtained by Whatman DE-52 ion-exchange with T-TBS buffer, composed of0.5 A/ NaCl, 10 mA/
chromatography, performedasdescribed above, followed Tris-HCl (pH 7.4), and 0.05% (v/v) Tween 20, and then
by blocking in a solution of 10 mA/Tris-HCl (pH 7.4), three times with distilled water. One hundred microliters
0.5 A/ NaCl and 3% gelatin. Nitrocellulose strips (Bio- ofa solution ofhorseradish peroxidase linkedtogoat anti-
98 S. IKEGAMI ET AL
against A. pectinifera egg components with molecular
abcdef abcdef
masses of 188 or 133 kDa. All ofthese antibodies were
(kDa)
found to bind to some components ofA. amurensiseggs.
One clone produced an antibody that recognized com-
ponentsof.-1. pectiniferaeggs,butnotthoseofA. anniren-
sis eggs, in an ELISA assay. This specific antibody, Kl,
bound to A. pectinifera egg components with molecular
masses of58 and 56 kDa in an immunoblot assay (Fig.
1 ). The immunoreactive components were also present
in extracts ofA. pectinifera oocytes and embryos at the
32-cell morula, the 256-cell early blastula, mid-blastula
30- (10 h after fertilization), and early gastrula (24 h after
A
fertilization) stages. Protein binding experiments in-
dicated that the Kl antibody was ofthe IgG class.
The minimum number of eggs detectable by immu-
noblot analysis was then determined as follows. Graded
amounts ofegg lysate were loaded into slots ofan SDS-
polyacrylamide gel. After electrophoresis, the gel was
(5FMiggpurroete1i.neIamcmh)unwoebrleoetlsecotfr.olphMocrnemsiedpeocntinSiDfSe-ra12ex.t5r%actpso.lTyahceryexltarmaicdtes wbliotthtetdheonKtloaantniibtordoyc.elTluhleosleysfailtteerobtthaatinweadsftrhoemnopnreobsiexd-
gels.GelswereeitherstainedwithCoomassiebrilliantblue(A),ortrans- teenth ofan egg and loaded into a slot gave two bands at
blottedontoanitrocellulosefilter,whichwasprobedwithculturemedium molecular masses of58 and 56 kDa. The intensity ofthe
conditionedbyaKl antibody-producinghybndomacellline(B). Lanes bandswasclosetothelimitofdetection (data notshown).
a,b,c.d,e.andfcorrespondtotheextractofoocyles,eggs, 32-cell-stage To determine whether the antigenic molecules were
morulae, 256-cell-stageblastulae. mid-blastulae(10hafterfertilization)
andearlygastrulae(24 h after fertilization), respectively. polypeptides, we incubated 5 ^g egg extract protein with
0.001 to 0.01 units of papain at 37C for 30 min and
subjected the reaction mixture to SDS-polyacrylamide gel
mouse IgG, diluted 1:3000 in PBS with 0.05% Tween 20 electrophoresis. The gel was blotted onto a nitrocellulose
and 1%gelatin, wasaddedthen incubated for2 hat37C. filter, which was probed with the Kl antibody. The 58-
The wells then were washed twice with T TBS followed
by three times with distilled water.
Two hundred microliters of substrate solutions con- abode abcde
taining H:O: and o-phenylenediamine were added, left (kDa)
for 10-30 min, and then the reaction was stopped by the
addition of 100 ^1 4 N H:SO4. The absorbance at 492
nm was measured within 30 min ofthe sulfuric acid ad-
dition.
When the primary antibody was replaced with preim-
mune serum, orwasomitted, the result wasa completely
negative one.
Results
Mice were immunized with the pooled 150 and 200
mAl NaCl eluates obtained by Whatman DE-52 ion-ex-
changeresinchromatography toproduce monoclonalan-
tibodies that would react with components present in A.
pectinifera eggs, but not with any components of A.
amitrensis eggs. The spleen was dissociated from the im-
munized mice. The cells were fused to myeloma cells Figure 2. ImmunoblotsoftissuesofadultAslerinapectinijera. The
platedataclonaldilution. From thisfusion, 30hybndoma extracts(5^gproteinseach)wereelectrophoresedonSDS-10%acrylamide
clonesgrew. Hybndoma cloneswere selected byscreening ogenlts.oGneiltsrowceerlelueliotsheeransdtaipnreodbweidthwiCtohomthaessKilebarnitlliibaondtybl(uBe),(Aa)sodresbclroitbteedd
antibodies secreted in the media with the ELISA and im- in Materialsand Methods. Lanesa,b,c,d,andecorrespondtoextracts
munoblot assays. Thirteen clones produced antibodies ofovary, pyloriccecum, testis, body-wall and tube-foot, respectively.
IMMUNODETECTION OF STARFISH LARVAE 99
Aabcde Babcde analysisofthesefractionsshowedthatp58/56werepresent
in the cytoplasmic, but not in the nuclear fraction, as
(kDa)
shown in Figure 4.
Embryoswerefixedwith methanol, embedded inpoly-
ester wax, sectioned, and the sections subjected to im-
munofluorescence staining. The Kl antibody stained the
94-
cytoplasm, but not the nucleus ofan 8-cell-stage embryo
67- (Fig. 5a). Figure 5bshowsasection ofabipinnaria(4days
after fertilization) that was stained with the Kl antibody.
Almostall oftheembryoniccellscontainedsomeantigen
43-
molecules.
Occurrence ofantigen molecules in other
30- Asterina species
We attempted to survey the distribution ofantigens in
the eggs and embryos of some sea urchin and starfish
Figure 3. ImmunoblotsofextractsofAsterinapectiniferaeggsand species. Lysates were prepared from eggs of5". mirabilis,
embryos-Theextracts(5jigproteineach)wereelectrophoresedonSDS- P. depressus. H. pulcherrimus, andA. amurensisandwere
b1l0u%ep(oAl)yoarcrbylloatmteiddeongetlos.nGietlrsocweelrlueleoistehearnsdtapirnoebdewditwhitChootmheasKslieanbrtiilblioadnyt subjected to immunoblot analysis; 10 ng of lysate were
(B)asdescribed in Materialsand Methods. Lanesa. b, c. d. and ecor- loaded into each slot. The analysis showed that no mol-
respondtotheextractofeggs, 1-day-oldearlygastrulae, 2-day-oId mid- eculesreactivewiththe K1 antibodywerepresentinthese
gastrulae. 3-day-old late gastrulae and 4-day-old early bipinnariae, re- sea urchin and starfish eggs (Fig. 6).
spectively. Next,weexaminedembryosofspeciesbelongingtothe
genus Asterina: A. minor, A. pseudoexigua pacifica, and
and 56-kDa forms were no longer detectable on the im- A. batheri. Development ofthese species is direct, with
munoblot, and prominent degradation products with
molecularmassesof49, 46, 43,and40 kDawereobserved
(data not shown). Treatment ofan identical amount of Whole embryo Nucleus
extractwithahigheramount (0.1 unit)ofpapain resulted abcdef bcde
in total loss ofimmunoreactivity. These results demon-
(kDa)
strate that the components recognized by the antibody
were proteinaceous. The immunoreactive 58- and 56-kDa
proteins were designated p58/56.
Distribution ofthe antigens in tissues, oocytes and 58-L
embryos ofA. pectinifera
56-T
Several tissues were excised from adult A. pectinifera,
and their extracts were examined, by immunoblot anal-
ysis, to determine whether Kl-reactive antigens were
present. Antigenic p58/56 were found to be present in
ovarian extracts ofovaries, whereas testes, pyloric ceca,
and tube-feet contained no antigenic components (Fig.
2). Next, we surveyed extracts of hatched embryos and
larvae by immunoblot analysis and found that p58/56
were present in all the samples examined; the amount of
p58/56, on a protein weight basis, was almost constant
throughout early embryonicdevelopment upto theearly Figure 4. Immunoblots ofwhole and nuclear extracts ofAsterina
bipinnaria stage (Fig. 3). pectiniferaembryos. Theextracts(5 ngproteinseach)wereelectropho-
resed on an SDS-12.5% polyacrylamide gel. The gel was blotted onto
Subcellular localization oftheantigens in A. pectinifera nitrocelluloseandprobedwiththeK.1 antibody,asdescribedinMaterials
eggs and embryos and Methods. Lanesa, b,c,d,e. andfcorrespondtotheextractsatthe
32-cell morulae, 256-cell earlyblastulae, 512-cell early blastulae, 1024-
Embryonic extracts were separated, by centrifugation, cellearlyblastulae, mid-blastulae(10hafterfertilization)andearlygas-
into nuclear and cytoplasmic fractions. Immunoblot trulae(24 hafterfertilization), respectively.
100 S. IKEGAMI ET AL
Detection ofA. pectinifera embryos in the ocean
As A. pseudoexigna pacifica never swims or drifts at
any stage ofits life cycle (Komatsu el ai, 1990), its em-
bryos and larvae should not be present in specimens col-
lected from the sea with a plankton net. A. pectinifera,
however, startsitsplanktonic life atthe late blastula stage
and then sinkstothebottomatthelatebrachiolariastage.
Therefore, we would expect that an ELISA system, with
the Kl monoclonal antibody as the detecting antibody,
could be used to quantify embryos and larvae ofA. pec-
tinifera in a complex planktonic sample.
We assessed the sensitivity ofthe assay system forem-
bryosofA. pectiniferaby testinggraded quantities ofem-
bryos. Five thousand (5000) embryos, at each develop-
mental stage, were boiled in 1 ml SDS sample buffer, the
lysateswerediluted 1:5000to 1:40,000in PBS,anda 100-
jul aliquot was added to a well of a microtiter plate for
ELISA. The absorbance decreased as development pro-
gressed, although on a per protein weight basis, the ab-
sorbancewasnearlythesameforall stagesoftheembryos
examined. The protein content ofa single oocyte, egg, or
embryo at the mid-blastula stage (9 h after fertilization)
was 0.30 /*g; that ofa single embryo at the mid-gastrula
stage (24 h after fertilization) was0.21 ^g; and that ofan
earlybipinnaria(72 hafterfertilization) or mid-bipinnaria
(96 h after fertilization) was0.10^g- The absorbance val-
ueschanged linearlywithconcentration overthe following
Figure 5. Immunotluorescence microscopy ofsections ofAstcrina
pectiniferaembryosusingtheK1 antibody. AandBcorrespondtoa32-
cell morulaandanearly bipmnana. respectively. Barindicates 100/jm. A bed B abed
ThemagnificationofB isthesameasthatofA.
(kDa) a
nobipinnariastage, unlikethatofA. pectinifera. A. minor
is hermaphroditic (Komatsu, 1976), and when spawning
occurs, theanimalsclingtooneanotheralongthemargins 94-
oftheir bodies, or they are imbricated with others (Ko-
67-
matsu elai, 1979). Eggsthatwerespawnedspontaneously
andfertilizedwerecultured in the laboratoryand allowed
to develop into brachiolariae, which were then used for 43-
immunoblot analysis. A. pseudoexignapacifica is ovovi-
viparous(Komatsu el al.. 1990), and we were ableto col-
lect early blastulae, wrinkled blastulae, and brachiolariae .
from the adult ovary. Brachiolariae ofA. batheri (Kano 30-
and Komatsu, 1978) were obtained by rearingartificially
inseminated, spontaneously spawnedeggs. Theseembryos
were boiled in SDS sample buffer and loaded onto a gel,
which was electrophoresed and immunoblotted as de- Figure6. ImmunoblotsofeggsofScaphechinusmirahilix(a), Pseu-
scribedabove. Noneofthepeptidesfrom thebrachiolariae (liiccnlroti/s ilcprcxsus (b), Hemicentrotus pulclicrimits (c), Asterias
ofA. minor or A. batheri reacted with the Kl antibody amurensis(d),andAstcrinapectinifera(e).EggsweresolubilizedinSDS
t(Fuilga.e,7)o.rBubrtawchhieonlalryisaaeteosfoAf.eaprsleyubdlaosetxuilgaen,awpraicnikfliceadwblearse- /esulagemcpptrlroeotpebhiuonfrfewesrae,sd,alnoadanddaendthoaelnnitqoeuioettahceorhfsltthaaenienseoadfmpwSliDteSh-sCo1lo0uo%tmiaopsnosleiyqeaucibrvryailllaleimnaitndtetobgleu1le0,
separated on a gel, a band at 41 kDa was detected on the (A)orblotted ontonitrocelluloseand probedwiththe K.1 antibody(B).
blot (Fig. 8). asdescribed in Materialsand Methods.
IMMUNODETECTION OF STARFISH LARVAE 101
A B embryos or larvae present in a sample. We collected
planktonic samples from 6 tons ofwater from the sublit-
(kDa) a b c a b c toral zone ofHikonoura (1 or 3.5 m above thebottom of
m
thesea, ca. 100 offSensui Island, ca. 3423TV, 133 24T)
at high tide on November 22, 1987. The samples were
spun down at 1000 X g for 10 min, and the precipitate
was lysed in SDS sample buffer. Aliquots ofthe lysates
werediluted 1:1000 in PBS, addedtowellsofamicrotiter
94- plate, and assayed by the ELISA system. The observed
valueswerecorrectedwith referencetoapreparationcon-
taininga known numberofA. pectinifera embryosat the
67- mid-gastrula stage mixed with a standard amount of
plankton lysate. The estimated numbers ofA. pectinifera
embryosorlarvaeinthethreeplankton samplescollected
separately from 6 tons each ofwater were 30, 130, and
43-
130, assumingthat the planktonic embryoswereall mid-
gastrulae. As described above, these figures are the min-
imum numbers of embryos and larvae present in the
specimens, since the response oflysed larvae at later de-
velopmental stages was low compared with that of the
mid-gastrulae, as shown in Figure 9.
30-
Discussion
Figure7. ImmunoblotsofAstcriiitibatherihrachiolariae(a),Asterina Mochizuki and Hori (1980) examined the phylogenetic
minorbrachiolariae (b), and Asterinapectinifera oocytes (c). Embryos relationship between fiveAsterina species by the enzyme
andoocytesweresolubilizedinSDSsamplebufferandanaliquotofthe
samplesolution, equivalent to 5 ng protein, wasloaded ontoeach lane
ofSDS-10% polyacrylamidegel,electrophoresed,andthenstainedwith A B
Coomassie brilliant blue (A) or blotted onto a nitrocellulose sheet (B).
The sheet was probed with the K.1 antibody, asdescribed in Materials (kDa) a
and Methods.
ranges: 0.0125 to 0.05 individuals per well for oocytes,
eggs, and mid-gastrulae(24 hafterfertilization);and0.025 94-
to0.1 individualsperwell forearlybipinnariae(72 hafter
fertilization) and mid-bipinnariae (92 h afterfertilization). 67-
Itisnot known atwhich stage ofdevelopmenttheamount
ofthe antigenic p58/56 in an embryo or larva becomes 43-
lower than the detection limit ofthe ELISA system with
the Kl antibody. However, we succeeded in rearing five
early brachiolaria ofA. pectinifera (12 daysafterfertiliza-
tion) by feeding bipinnariae in the laboratory. They were 30-
all loaded intoaslotofan SDS-polyacrylamidegel, which
wassubjected, afterelectrophoresis, toimmunoblot anal-
ysis. The sensitivity of the immunoblot analysis is ap-
proximately five times lower than that ofthe ELISA sys-
tem used in this study. There were no immunoreactive
abamnodusntonoftph5e8/sl5o6t p(rdeasteantnoitn ashloawrvna),atstuhgegemsitdi-nbgiptihnantartihae terFiingauprsee8u.doeIxmimguuanopbalcioftiscaofeaArsltyebrliansatupleaceti(nbi)f,erwarionokclyetdesbl(aa)s,tualnadeA(cs)-.
stage decreased to less than 10% as development pro- and brachiolariae (d). Embryos and oocytes were solubilized in SDS
gressed to the early brachiolaria stage. sample buffer,andan aliquotofthesamplesolution, equivalent tofive
Although neitherembryos nor larvae at various devel- oocytesorembryos, wasloaded ontoeach laneofSDS-10% polyacryl-
opmental stagescould bequantified bythe ELISA system, ablmuiede(A)gelo,reblleocttterdopohnotreosaedn,itarnocdeltlhuelnosestsahieneetd(wB)i.thThCeoosmheaestsiweasbrpirlolbiaendt
it could be used to estimate the minimum number of with the Kl antibody, asdescribed in Materialsand Methods.
102 S. IKEGAMI ET AL.
25 time as members of the plankton community before
moving into the benthic environment and undergoing
20 metamorphosis. Therefore, the Kl antibody-reactive
componentsdetected bythe ELISAassay in theplankton
specimen were very unlikely to have been derived from
embryos or larvae ofA. pseudoexiguapacifica. Although
we did not analyze the plankton specimens collected in
c
nnj 10 the field by immunoblot assay, thisassay can discriminate
between embryos ofA. pseudoexigua pacifica and those
05 of A. pectinifera. according to the differences between
molecular masses of the immunoreactive components.
This enables A. pectinifera embryos and larvae in the
mixed planktonic specimens to be identified unambigu-
0013 0025 0050 0100
ously.
Number of individuals per well A. pectinifera differs from the otherAsterina species in
that the size and yolk content ofits eggs are small, and
Figure 9. Measurement ofantigen content in extracts ofAsterma that larval development, at both bipinnaria and brachio-
pleatcelignaisfterrualoaoec(yAt)e,sa(nO)d,4e-gdgasy(-o)l,d1e-adralyy-ohlidpienanralryigaaest(rDu)lauesi(An)g,a3n-dEaLy-IoSlAd laria stages, is indirect (Oguro and Komatsu, 1988).
system with the Kl antibody. The method ofmeasurement ofantigen Therefore, this speciescan usually be identified accurately
contentisdescribed in Materialsand Methods. by microscopic examination ofa larval specimen. How-
ever, there are huge numbers of diatoms in planktonic
samplescollected from wateralongtheJapan Islands, and
inhibition test, usingrabbitanti-.-l.pectinij'erahexokinase these makethedetection ofsmall numbersofstarfishem-
antisera. The antisera inhibited the activity from hexo- bryos and larvae almost impossible. Therefore, an im-
kinase ofA. bat/ieri, Asterina coronatajaponica, and A. munological search for embryos and larvae in a complex
pseudoexigua pacifica by 50-54% and that ofA. minor plankton sample is particularly useful when their popu-
by 30%. In light of these results, Mochizuki and Hori lation is thin.
suggested that A. pectinij'era and A. minor, but not the Thisstudywasaimedatdeterminingthespawningsea-
otherAstrinaspecies, belongtoconvergent lineages. Mat- son ofA. pectinifera at specified regions along the coast
suoka(1981)arrived at a similarconclusion asa result of ofthe Japan Islands. Ten percent ofthe total number of
electrophoretic analysis of hexokinase and six other en- adult A. pectinifera caught on 29 October 1987, at Hi-
zymes from five Asterina species. In our study, a peptide konoura off Sensui Island, contained ripe ovaries with
capable of recognizing the Kl monoclonal antibody, full-grown oocytes. This fell to 3% on 15 November and
which isreactivetoA. pectiniferap58/56, wasfound only to 1.7% on 19 December. These observations suggest that,
inembryosand larvaeofA. pseudoexiguapacificaamong at this sampling site, many adult A. pectinifera spawned
theAsterina species examined. Since little information is at the beginning ofNovember. Forthree years, spawning
available regardingthe function and structure ofthe pep- at this location has been observed to occurin November.
tides containing the K1 epitope, the reason for the phy- Our results, obtained by immunological detection using
logenetic variation of the peptides in these two species an ELISA system, forA. pectinifera embryos or larvae in
remains unclear. plankton samplescollectedatthislocationon22 Novem-
So far, A. pseudoexigua pacifica has been found only ber 1987, agree well with these observations. However,
in Sabiura, ofFKushimoto in Wakayama Prefecture (Ko- inspection of the ovaries ofadults collected from other
mm
matsu el a!., 1990). It istiny (its body is only 5 long) areas along the coast of the Japan Islands revealed that
and ovoviviparous. Development of this species, from the breeding season of this species is quite diverse, oc-
fertilization to the completion ofmetamorphosis, occurs curring in May in Tokyo Bay in Kanagawa Prefecture,
withinthegonad. Thejuveniles, measuringapproximately June in Ago Bay in Mie Prefecture, July and August in
0.5 mm in total length, crawl out ofthe gonad and pass thesouth-western partoftheInlandSeaofJapan in Ehime
through the gonoduct. According to the observation of Prefecture. September in Mutsu Bay in Aomori Prefec-
Komatsu and Kano, made in July and August of 1972, ture, and November in the Sea ofYatsushiro in Kuma-
the maximum numberofjuvenilesreleased from asingle moto Prefecture. Thespawningseason in most otherparts
adult was 1288 (Komatsu elal, 1990). On the otherhand, ofJapan is, as yet, unknown. The spawning season in a
A. pectinifera is extremely abundant along the Japan Is- specified region oftheseacould bepredicted byanalyzing
lands; an individual spawns more than 100,000 eggs, and plankton specimens, sampled periodically, with the im-
it has embryonic and larval forms that live for a short munological assay method as described in this paper.
IMMUNODETECTION OF STARFISH LARVAE 103
In summary, we have developed an assay system that Kanatani, H. 1973. Maturation-inducing substance in starfishes. Int.
detects embryos and larvae ofA. pectinifera in complex Rev Cytol. 35: 253-298.
biological specimens. The resultsofthe this investigation Kanok,rnYi.aTh.,alahnedriMG.olkoo.maDtt-sv,u.Gr1o97w8t.hDiDffeevre.l2o0p:me1n07t-o1f1t4.hesea-star,As-
suggest that immunological techniques are useful tools komatsu, M. 1976. Wrinkled blastulaofthesea-star.Asterinaminor
for taxonomic identification ofeggs, embryos, or larvae Hayashi. Dev. Growth Differ. 18:435-438.
in plankton populations. komatsu, M.,Y.T.kano,andC.Oguro. 1990. Developmentofatrue
ovoviviparousseastar,Asterinapseudoexiguapacifica Hayashi. Biol.
Acknowledgments Bull 179: 254-263.
komatsu. M., Y'. T. kano, H. Yoshizawa, S. Akabane, and C. Oguro.
Thiswork was supported in part by aGrant-in-Aid for 1979. Reproduction and development ofthe hermaphroditic sea-
Cancer Research from the Japanese Ministry of Educa- star,AsterinaminorHayashi. Biol Bull. 157: 258-274.
tion, Science and Culture, and by grants from the Nissan Laemmli, II. k. 1970. Cleavage ofstructural proteins during the as-
Science Foundation and from the Japanese Fisheries sembly ofthe headofbacteriophageT4. Nature221: 680-685.
Agency to S.I. We thank Professor C. Oguro, Toyama Matsuoka, N. 1981. Phylogenetic relationshipsamong five speciesof
University, for his valuable suggestions and encourage- sBtiaorcfihsehm.ofPhtyhseio/g.en7u0s.B:A7s3te9r-i7n4a3:.an electrophoretic study. Comp.
ment.
Mochizuki, Y., and S. Hori. 1980. Immunological relationships of
starfish hexokinases: phylogenetic implication. Comp. Biochem.
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