Table Of ContentReference: Kiol Hull 183: 21 1-219. (October, 1992)
Cell Cooperation During Host Defense in the Solitary
Tunicate Ciona intestinalis (L)
VALERIE SMITH* AND CLARE M. PEDDIE
J.
School ofBiology andMedicalSciences, GattyMarineLaboratory.
L'niversitv ofSi Andrews, Fife. KYI6 SLB. Scotland
Abstract. Phagocytosis in the solitary ascidian Ciona ofthe host's ability to survive microbial infection orpar-
intestinalis was investigated using mixed and separated asitization. Invertebrates lack specific immunoglobulins
populations ofblood cells in vitro. Only the vacuolarand andspecialized Tlymphocytesin theirdefense repertoires,
granular amoebocytes were seen to ingest bacteria, and butareableto mount highlyefficientcellularand humoral
when the serine protease inhibitors, STI, orbenzamidine defense strategies against foreign agents (Ratcliffe el al.,
wereaddedtomonolayersofmixedcelltypes, uptakewas 1985;Smith, 1991). An understandingofthebiochemical
significantly reduced. Analyses carried out with isolated basis for recognition and blood cell activation in these
cells revealed that phagocytosis was enhanced by incu- animals is important in tracing the evolution ofimmune
bation of the bacteria in blood cell lysate supernatants capability and in the identification of suitable non-ver-
(CLS) that had been pre-treated with LPS. By contrast, tebrate models for experimental study. The deuteros-
pre-incubation ofthe bacteria in CLS preparations that tomes, and especially the ascidians, are a particularly sig-
were inhibited by benzamidine produced lower levels of nificant group because oftheir phylogenetically strategic
phagocytosis. Treatment ofthe bacteria with plasma also position close to the vertebrates (Bone. 1979).
failed to promote uptake, and there was no detectable Several workers have examined the cellular responses
agglutination ofthe bacteria by CLS. As lysate superna- ofascidians using both /// vivo (Smith. 1970; Anderson,
tants made from morula cells, but not other cell types, 1971; Parrinello et al.. 1977; Raftos and Cooper, 1991)
wereeffectivein promotingphagocytosis,weproposethat and in vitro methods (Rowley, 1981; 1982a) (see also re-
opsonins are derived from the morula cells, and that view by Pestarino, 1991). However, the responses ofthe
phagocytosis involves cooperation between different cell cells to foreign onslaught, particularly in vitro, have been
populations. Moreover, as the morula cells are the prin- studied only with mixed blood cell populations. There
cipal repositories ofprophenoloxidase and an associated have been no investigations ofseparated cellsorattempts
serine protease (factors which are activated by LPS but toassesstheextent ofinteraction betweenthevariouscell
blocked by STI orbenzamidine), prophenoloxidase orthe types.
protease may be involved in the opsonic phenomenon in Although immune reactivity in vertebrates entails co-
a similar way to that previously reported for arthropods. operation between thedifferent leucocytegroups, the im-
portance ofcell-cell cooperation in invertebrate host de-
Introduction fense has only been demonstrated for the arthropods
Tihnec rleeccouggnimtuiuoMn ouifnnounn--sseelnfiiss aa fluuiniduaaimnecniitiaali aassppteccit ouif (Soderhall et al.. 1986; Persson et al.. 1987; Anggraeni
an animal's immune network and a major determinant and Ratcliffe, 1991). In both crustaceans and insects, the
granular-type cells appear to mediate cell cooperation by
Recened 4 March 1992;accepted 7July 1992. providing the opsonins necessary for efficient uptake of
* Towhomrepnntrequestsandcorrespondenceshouldbeaddressed. bacteria by the phagocytes (Soderhall et al.. 1986; Ang-
Abbreviations: STI. Soyabean trypsm inhibitor. LPS. Lipopolysac- graeni and Ratcliffe. 1991 ). In crayfish, they also donate
chande;CLS.wholebloodcelllysatesupernatant;MAC.marineantico- factors which promote encapsulation (Persson et al.
agulent: MS. marine saline;CAC I, citrated cacodylate buffer. CAC II, 1987). Asyet, thecell signals involved have notbeen fully
cacodylate buffer; proPO. prophenoioxidase; L-dopa L-hydroxyophe-
nylanaline; MLS. morula cell lysate supernatant; CcLS. cell lysate su- characterized, but the prophenoloxidase activating
pernatantexcluding morulacells; pNA, p-nitroaniline. (proPO) system, contained within the granular and se-
21
I
212 V. J. SMITH AND C. M. PEDDIE
migranular cells or ti .alent, has been implicated cardium. The blood was drained from the perivisceral
in the phenom hall el a/., 1986; Anggraeni and pericardial cavities, care being taken not to contam-
and Ratcliffe. >roPO system in crustaceans inate the blood with pyrogens from the gut. The blood
and in Cascade ofserine proteases and (usually 1-2 ml per animal) was diMluted 1:1 with ice-cold
other specifically triggered by microbial marine anticoagulant (MAC) (0.1 glucose; 15 mA/tri-
mM mM
carbo viy lipopolysaccharides (LPS) orf3 1,3- sModium citrate; 13 citric acid; 10 EDTA; 0.45
.v.-ws by Soderhall. 1982; Soderhall and NaCl; pH 7.0) and processed immediately.
b; Smith and Soderhall, 1986; Johansson
andSoderhall, 1989;Smith, 1991). Becausephenoloxidase Cell separation
acthit\ occurs exclusively in the granular type cells, it The blood cell populations ofC. intestinalis were sep-
also representsa useful markerforthepurity ofseparated arated by a modification ofthe density gradient centrif-
cellsin functional evaluationsofimmune reactivity(Sod- ugation method described by Soderhall and Smith ( 1983)
erhall and Smith, 1983; Soderhall el al, 1986; Smith and and Smith and Soderhall ( 1991). Briefly, freshly collected
Soderhall, 1991; Chisholm and Smith. 1992). So far, few blood (approximately 2 ml), diluted 1:1 in MACwasspun
suitable markers have been identified for the cell popu- through preformed continuous gradients of60% Percoll
lations in other invertebrate phyla. (Pharmacia, Uppsala, Sweden) in 3.2% NaCl, at 1900 g
Recently, however, the separation technique devised for 10 min, at4C. Thecell typeswereidentifiedaccording
for crustacean hemocytes has been successfully applied to the criteria given by Rowley (1981). The phagocytes
to the solitary ascidian, dona intestinalis (Smith and and morula cells were then further purified by a second
Soderhall, 1991). Five cell bands were obtained, and the centrifugation on preformed gradients of 40% Percoll,
enzyme prophenoloxidase was found to reside primarily while the pigment cells and stem cells were enriched on
in the morula cells (Smith and Soderhall, 1991). Further secondary gradients of80% Percoll.
analyses haveestablished that phenoloxidase activityalso The separated cell populations were removed from the
occurs in the morula cells ofseveral ascidian species and gradients and washed once in 10 ml of marine saline
mM
in individuals ofC. intestinalis, as in arthropods, it exists (MS) (12 m.U CamClM2-6H2O; 11 KC1; M26 mA/
as a proenzyme which is activated by proteases or LPS MgCl:-6H:O; 45 tris; 38 mA/ HC1; 0.45 NaCl;
but notothercarbohydrates(Jackson etat., in press). Fur- pH 7.4)before resuspension in 800jil MS. Phenoloxidase
thermore, the enzyme is blocked by the serine protease and protease activities in the blood cell suspensions were
inhibitors, ST1 and benzamidine, (Jackson ctal., in press) determined for each cell band; L-dopa or S-2337 were
andseemstobeassociatedwithan LPS-sensitive protease used as described below.
contained within the blood cells (Jackson and Smith, un-
publ. obs.). Preparation oj hloodcell mono/avers
The present investigation was conducted to examine Blood cell monolayers from individuals ofC. intestin-
the cellular defenses ofurochordates and, by using sepa- aliswereprepared bya modification ofthemethodsgiven
rated cell populations, to assess the extent ofany cell co- in Smith and Ratcliffe (1978) and Soderhall ct al. (1986).
operation involved. We chose to examine phagocytosis Forthe mixedcell monolayers, 1.2 ml offreshlycollected
because it is a universal process which serves in surveil- blood, diluted 1:1 with MAC. waswashed oncewith 5 ml
lance against invading micro-organisms in nearly all an- ofMS, and resuspended in 1.2 ml MS. Two hundred mi-
imals. We show that uptake ofbacteria is achieved only crolitres ofthis mixed cell suspension was laid onto each
bythe phagocyticamoebocytesand isstrongly influenced clean, pyrogen-free. glass coverslip in tissue culture grade
by factors (opsonins) derived from the morula cells. In sterile six-well trays (Sterilin. Feltham, England), and the
addition, by treating the cells or opsonizing preparations coverslipswere incubatedat 20C for60 min toallowthe
with LPS or serine protease inhibitors, we explored the cellstosettle, attach and spread on theglasssurface. Each
possibility of a link between the opsonic effect and the coverslip was then washed twice with 1 ml of MS and
phenoloxidase and protease activities contained within moistened with a fresh 0.5 ml volume ofMS.
the morula cells. For separated blood cell monolayers, the cells were re-
moved from the gradients with a pasteur pipette, washed
Materials and Methods once in MS. and resuspended in fresh MS at a concen-
Animal.', tration ofca 1 X 108 ml"'. To each coverslip was added
200 ^1 ofthe cell suspension and the cellswere incubated
Specimens ;i C'. intestinalis were collected from the and washed as above.
west coast ofScotland and maintained in aerated circu-
lating seawater (32 2%; 10 2C) until use. The ani- Preparation ofbloodcelllysatesupernatants
mals were blotted dry to remove any excess seawaterand The diluted blood (about 5 ml) from 4-5 large (7.5-14
bled by removal of the tunic and puncture of the peri- cm long) animals was pooled, and the cells were pelleted
TUNICATE IMMUNITY 213
by gentle centrifugation (800 g, 10 min, 4C). The su- reaction was terminated by the addition of 100 ^1 of50%
pernatant was discarded, and thecells were washed twice acetic acid, the samples spun at 2500 g(10 min), and the
in ice-cold citrated cacodylate butter (CAC I) (10 m.U absorbances read against a blank, consisting of buffer,
sodium cacodylate. 100 m.U trisodium citrate. 0.45 .U substrate and elicitor. at 405 nm. Enzyme activity was
Nad: pH 7.0) before homogenization with a glass piston calculated as nmol p-nitroaniline (pNA) released per
tissue grinder in citrate-free cacodylate buffer (CAC II) minute per mg protein.
m(10Mm.U sodium cacodylMate; 50 m.U MgCl: -6H:O: 50
CaCl: 6H2O; 0.45 NaCl; pH 7.0). The homoge- Protein
nate was then spun (40,000 g, 20 min. 4C) and the re-
sulting supernatant, designated CLS. used as enzyme Protein in the various cell lysates and plasma was de-
termined bythe method ofBradford (1976); bovineserum
source.
For some phagocytosis experiments (see below), cell albumin was used as standard.
lysate supernatants were prepared from separated cells.
One, designated MLS, wasprepared from the morulacell Preparation of bacteria
band, whilea second, thecontrol (CcLS), was made from The bacterium, Psyelirobacter immobilis, formerly
all the cell bands excluding the morula cell band. In both Moraxella sp. (NCMB 308), was cultured and prepared
cases, the cell bands were removed from the gradients, as described in Smith and Ratcliffe (1978). Washed bac-
pooled where necessary, washed in 10 ml of MAC, and teriawere suspended in MS at a concentration of5 X 108
resuspended in 1 mlaliquotsoffreshcitratedCACIbuffer. mT1 and either used immediately or pretreated with var-
T4hCe,trweosussupsepnednesdioinns60we0r^e1 sofpuCnAaCt I8I,00angdfhoorm1o0gemniinzeadt pioruessecnetlaltilyosnatteo stuhpeecrenlalst.ants, as described below, before
with a glass piston tissuegrinder(8 min, 4C) beforecen-
trifugation at 14,500 g for 20 min (4C). Protein in the Phagocytosis assays in vitro
CcLS was adjusted to match that ofthe MLS, and phen-
oloxidase and protease activities in each ofthe lysate su- The optimal incubation time for bacterial uptake by
pernatants were determined as below. blood cells ofC. intestinn/iswas investigated by challeng-
ing monolayers prepared from mixed cells with 100 n\ of
Preparation o/plasma washed bacteriain MS(i.e., 5 X 107permonolayer). Con-
cloolnUlgne)dctaielndudtfeprdloambcleiodnodidinvt(ioadupiapclreo-mxceoilmddaitpueomllyysci2azermdbloanpnaeirtmeaalntsiubm(ea4sl-.)7Twchames tmorniolnastrroeocc3ekiihvnegadtp12l0a00tC^f1.oroOmfnMfoSrt,hteainmbdaesaiilsnltcoeufrlvttahulirssesreawxnepgreiernigimnefcnrutobma(ts3ee0de
Resultsbelow), thecell:bacteriaculturesin allsubsequent
samples were immediately centrifuged (800 g. 10 min). experiments were incubated for 2 h.
the supernatants decanted and used as plasma. To examine the effect ofserine protease inhibitors on
the phagocytic response, monolayers ofmixed cells were
Measurement ofenzyme activities incubated with either 100 n\ ofSTI in MS (togive a final
Phenoloxidase activity was determined spectrophoto- concentration of0.143 mgmr')or 100/nl ofbenzamidine
metrically at 490 nm after treatment with trypsin or, for (final concentration 14.3 m.A/ in MS) before addition of
controls, CAC II buffer; L-dopa was used as substrate as thebacteria. Controlsreceived 100M' MS, andallcultures,
in Smith and Soderhall (1983a, 1991). Enzyme activity except one ofthe controls which wasgiven 100 ^1 ofMS.
wasexpressed as the change in absorbance at 490 nm per were overlaid with 100 ^1 of bacteria and incubated as
min per mg protein. above.
Protease activity was assayed according to the method The uptake of bacteria by individual cell types was
of Soderhall (1983). Briefly, 100 n\ of blood cell lysate studied with monolayers prepared from separated cellsas
supernatants of C. intestinalis were pre-incubated with described above. To determine whether the uptake of
an equal volume of lipopolysaccharide (LPS) (from Es- bacteria by isolated phagocytes is influenced by factors
chericia colt 01 1 1:B4. phenolic extractionM), at a concen- present in the cells and entails cell cooperation, mono-
tration of 1 mg ml ' in tris buffer (0.1 tris; 50 m.U layers of separated phagocytic amoebocytes were chal-
HC1; 10 m.U CaCl:-6H:O; pH 8.0). For controls. CLS lenged with bacteria pre-incubated in different blood cell
was mixed with 100 n\ of tris buffer. After 10 min at lysate supernatants.
20C. 200 n\ of tris buffer, and 100 n\ of the chromo- Firstly, the bacteria were pre-treated in CLS by incu-
genic substrate, Bz-Ile-Glu (-O-piperidyl) Gly-Arg- bating 100 ^1 ofwashed bacteria (5 X 108 ml'1 in CAC
pNA-HCl (S-2337) (Kabi-Vitrum. Stockholm. Sweden) II)in4mlofCLS(protein concentration ofapproximately
(1 mg ml 'in tris buffer) were added to each tube, and 0.01 mgml '). Previously, theCLS hadbeen reactedwith
the samples were incubated for a further 60 min. The 100 n\ of LPS (1 mg m'r1 in CAC II) (10 min. 20C).
214 V. J. SMITH AND C. M. PEDDIE
Further 100 n\ sanv1 i^iia were incubated in CLSdilution showingunequivocal agglutination. Theliter
CLSthat hadb<: with 100jtlCACII instead was assessed visually and also at 570 nm using an agglu-
of LPS. Secor. > bacteria were incubated in tination program on a microplate reader (Dynatech).
CLS that h: d with benzamidine. Forthis,
mM
4mlofCI vith 1 mlof100 benzamidine Statisticalanalyses
inCAC I .ial concentration of20mAl)before
add -ind incubation with bacteria as above. Differences in levels of uptake of the bacleria by the
Tubes AC II bufferwassubstituted fortheben- cells between treatmenls, and differences in enzyme ac-
zamidine ^-re run in parallel as positive controls. Other tivities in the cell lysate supernalanls belween conlrols
bacterialsamples(100n\)wereincubated in4mloffreshly and experimenlals. were analysed by Ihe Sludenls /-lesl.
prepared plasma forcomparison with theCLStreatments. Differences were considered significanl when P < 0.05.
Finally, bacteria were pre-incubated either in morula cell
lysatesupernatants(MLS)orin lysatesupernatants made Results
from all cell types excluding the morula cells (CcLS). In
Cellseparation
each case, 500 ^1 ofthe respective cell lysate supernatant,
pre-treated with 50 ^1 ofLPS, were incubated with 50 M' The blood cells of C intestinalis were separaled by
ofbacteria. densily gradient centrifugation, yielding six dislincl bands
All bacteria:lysate, bacteria:buffer, or bacteriaiplasma ofcells (Table 1). Scrutiny ofIhese cells under phase con-
mixtures were incubated for 1 h at 20C. The bacteria Irast optics revealed eighl. morphologically differenl cell
were then washed three times in MS, resuspended in 0.5 lypes. They were identified, accordingloIhelerminology
ml of fresh MS, and incubated with the phagocytes as given by Rowley (1981, 1982a), as signel ringcells (band
above. In every case, controls received bacteria pre-in- 1), non-vacuolar hyaline amoebocyles (band 2), vacuolar
cubated in CAC II buffer only. Protease activities in the amoebocytes (band 3). granular amoebocyles (band 3),
lysate supernatants were determined as above. Protease. morula cells (band 4), compartment cells (band 4), pig-
rather than phenoloxidase, activity was used as a marker ment cells(band 5). and small, undifferentiated cells(band
for morula cell products because the activity ofthis en- 6)lhal resemble vertebrate lymphocytes, and lhalwedes-
zymegenerally showslessvariation between animals(data ignate stem cells because of their similarity to Ihe pro-
not shown). Cell viability was checked by trypan blue genitor cells of other invertebrate species (Table I). We
exclusion in all experiments, and bacterial uptake was were unable to distinguish a band ofcells corresponding
determined as described below. lo the retractile amoebocytes described by Rowley (1981,
1982a, b), although the reasons for this are unknown.
Evaluation ojbacterial uptake Only Ihe vacuolar and Ihe granular amoebocyles oc-
After incubation, the monolayers were gently washed cupying band 3 were seen lo ingesl bacteria in vitro, so
all the cells collected from this band are classified as
with three 1 ml changes of MS, fixed in 2 ml of 10%
seawater formalin, and observed under phase contrast phagocytic amoebocytes (see below). Likewise, as the
morula and compartment cells differed from each olher
opticsofa LeitzDiaplan microscope. Intracellularbacteria
only in lerms ofIheir degree ofvacuolation, they are re-
were distinguished from extracellular forms according to
tpheerccernitteargieaogfibvelnooidncSelmlistchonatnadinRiantgcloinfeTeor( 1m9o78r)e.ianngdesttehde pfeorrrteidontoofceoallcehctcielvleltyypaesimnowrhuollaebcellolso.dTishsehorewlnatiinveTapbrloe-
bacteria wasdetermined by countingat least 200cellsper I. Theseare unlikelytorepresentabsolutevalues, however,
cmoivneersdliopn. dFuoprliecaactehmeoxnpoelraiymeenrts,foprhaegaocchyatnoismiasl,waasnddewtietrh- aasllaognenuemibcesrtiomfulfaactlioorns,. iarneclukdnionwgnbotdoyasfifzeec,tsdeiafsfoenr,entainadl
the opsonization assays, uptake was assessed for 12 ani- Sbwlionoedhacreltl.c1o9u7n9t;sRianftaossciadnidanCsoo(pSmeirt.h,199119)7.0; Biggs and
mals using six different lysate supernatants.
Phenoloxidase and prolease activities significantly
above control values were found only in the morula cell
band (P = 0.016 and 0.045, respectively). Slightly en-
The p -e ofbacterial agglutinins in blood cells of hanced protease activities were detectable in Ihe phago-
C. intestiiii. is investigated as follows. Serial two fold cyliccell band although thesewere not significantly higher
dilutions . ' nf CLS in CAC II were made in U- lhan Ihe conlrols (P = 0.461, Table I). Whelher ihis was
bottomed mic "c irays(Sterilin). Control wellsreceived a resull of contamination by products released by the
50 M! ofCAC II instead ofthe CLS samples, and 50 n\ of morula cells during bleeding orseparation is not clear. In
P. immobilix (ca 5 < 10s ml ' in CAC II) was added to general, enzyme activities in the morula cell band were
every well. The trays were incubated at 20C for 24 h, approximately three limes higher lhan in Ihe olher cell
and the tilers were recorded as the reciprocal ofthe last bands or Ihe controls (Table I). Measurement of phe-
TUNICATE IMMUNITY 215
noloxidase and protease activities in the signet ring cell 60 a 100
band was prevented by low protein yields. Spot assayson 50-
thisband, performedasdescribedbySoderhalland Smith -95 <
(1983), indicated that enzyme activity was not detectable 1
(data not shown). -90 .3
2
Phagocytosis by mixedcell monolayers ofC. intestinalis 20-
-85
in vitro 10-
Cell viability remained at about 96% for 2 h and at ~"~*
80 Phagocytosis
over 90% for 3 h under all the experimental conditions 0.0 1.0 2.0 3.0 --o-- Viability
(Fig. 1). Uptake ofthe bacterium P. immobilis on mixed Time(h)
cmeillnmtoonoalamyaexrismiuncmreoafse3d6f%roatm 2abhou(tFig1.%1i).n Tthheerefiarfstte3r0, culFtiugruersef1r.omVCiaiboinlaityinatnesdtipnhalaigso.cyTthoesisexipnemriixmeendtbslowoedrecelrlepmeoantoeldayfievre
uptake declined to about 23% at 3 h (Fig. 1). This per- times.ThevaluesrepresentmeanwithbarsrepresentingSE. Phagocytosis
ceived drop in phagocytic activity may represent the de- ofthe bacteria Psychrobacter immobilis was determined under phase
tachment of phagocytes from the monolayers over pro- contrast optics as described in Materials and Methods. Viability was
determined by trypan blueexclusion.
longed incubation. Accordingly, 2 h was taken to be the
optimal incubation time in terms of both cell viability
and uptake in all subsequent experiments.
immobilis was achieved only by the phagocytic
Treatment ofthe mixed cell monolayers with the serine
amoebocytes collected from band 3 of the Percoll gra-
porfoutpetaaskeeionfhibbaicttoerr,iaSfTr1,oms3ig9nitfoic1a6n.t6l%y odveeprre2shse(dPt=he0.l0e2v9e)l dients. Under these culture conditions, about 5% ofthe
(Table II). A similareffectwasobservedwithbenzamidine. c3e9ll%swoferteheseceelnlstoweirnegessteebnacttoecrioant(aTianblientIrIaI)c,elwlhuelraerabsacatbeoruita
where the level ofphagocytosiswas reduced to 13.4% over
tvihaebislaitmyerteimmaeinpeedriaotda(bPou=t09.60%09)or(bTeatbtleer.II). Again, cell tinobtheepmreifxeeredntcielallcpuhlatguorceyst(oTsaibsleeiItIh)e.rTbhyertehedindonn-ovtaacpupoelaarr
amoebocytes over the granular cells or vice versa (data
Phagocytosis byseparatedphagocytes ofC. intestinalis not shown).
in vitro When the phagocytic amoebocytes were challenged
The in vitro phagocytic ability ofeach ofthe separated with bacteria pre-incubated with LPS-treated CLS, up-
cell types is shown in Table I. Uptake ofthe bacteria P. take was significantly increased from 5.12 to 42.82%
Table I
Phenoloxidaseanilproteaseactivities indifferent bloodcelltypesojCiona intestinalis
216 V. J. SMITH AND C. M. PEDDIE
le !I
m
Phagocytosisin - icrcultures
from i/him<:
aTreat; Percentage phagocytosis
TUNICATE IMMUNITY 217
TableIV
TheI'th'ct<>!I'rc-lrcalnicni c/ Psychrobacter immohilis withfli/lm-nt
ivll/rvi//r sii/vnii/M/i/.v mi uptakehy separatedphagocytes Ironi
Ciona intestinalis
218 V. J. SMITH AND C. M. PEDDIE
is exocytosed from !' ".la cells in a similar way has Johansson, M. VV., and K. Soderhall. 1989. A cell adhesion factor
notbeen determi' ^n LPS-stimulated release of fromcrayfishhaemocyteshasdegranulatingactivitytowardscrayfish
proteasesfror is(ofundefined type)hasbeen Kobagyraasnuhlia,rKc.e,llMs../W.CelJlohBaionls.so1n0,6:an1d79K5.-1S8o0d4er.hall. 1990. The 76kD
reported ! ascidian Halocynthia roretzi by cell-adhesionfactorfromcrayfishhaemocytespromotesencapsulation
Azui Further work is necessary to clarify in vitro. Cell TissueRes 260: 13-18.
the .e solitary ascidian C. intestinalis. Leonard, C.M., N. A. Ratcliffe, and A. F. Rowley. 1985. The roleof
extenttowhich phenoloxidaseand protease prophenoloxidaseactivation in non-selfrecognitionandphagocytosis
5 are linked to other host defense responses in by insectbloodcells.J. insect Pliyxiol. 13: 789-799.
individuals ofC intestinalis is unknown. In crustaceans, Parrinello, N., E. Patricolo, and C. Canicatti. 1977. Tunicate immu-
theproPOsystem hasbeenshown toparticipatein clotting innojbeicotlioogn.y.BoIl.l.TuZnoioclr4e4a:cti3o7n3-o3f81C.iona intestinalis L. to erythrocyte
(Soderhall, 1981). mediate encapsulation (Persson el ai. Persson,M.,A.Vey,andK.Soderhall. 1987. Encapsulationofforeign
1987), generate cell adhesion molecules (Johansson and particlesin vitroby separatedbloodcellsfromcrayfish.Astacuslep-
Soderhall, 1988; Kobayashi et a/.. 1990), and have anti- todactylus. Cell TissueRes. 247:409-415.
microbial activity (Soderhall and Ajaxon, 1982; Soderhall Pestarino,M. 1991. TheneuroendocrineandimmunesystemsinPro-
and Smith, 1986a). While urochordates share with ar- Rafttoosc,hoDr.daAt.e,s.anAdclvE.\Le.iiCroooipmemrw.io1l9.911.: 11P4r-ol1i2f3e.ration oflymphocyte-
thropods a reliance upon such defense strategies for pro- like cells from the solitary tunicate, Slyela clava. in responseto al-
tection against microbial exploitation, more research is logenicstimuli. J. Exp. Zool. 260: 391-400.
necessary to discover the underlying biochemical path- Ratcliffe, N. A., C. M. Leonard, and A. F. Rowley. 1984. Pro-
ways in deuterostomes. phenoloxidaseactivation: nonselfrecognition and cell co-operation
in insect immunity. Science226: 557-559.
Ratcliffe, N. A., A. F. Rowley, S. \V. Fitzgerald, and C. P. Rhodes.
Acknowledgments 1985. Invertebrateimmunity:basicconceptsandrecentadvances.
CupWaerStuhabn-kAqtuhaeCvlaurbio(usBSsApCort1s09d4i)vearns,dUprMinBciSpaMllySpefcrioaml RowlmIneotyr..pRhAeo.vl.Fo.gCyy1t,9o8ld1.i.f9f7e:rTehn1te8i3abl-l3oc5oo0ud.nctesl,lsaonfdthiensveiatrsoqupihratgCoicoytniaciancttesitviintayl.isJ.:
Branch (BSAC 902), for their voluntary assistance with Invenebr. Pathol 37: 91-100.
the collection ofspecimens. We are grateful to Dr. Alan Rowley, A. F. 1982a. Ultrastructuralandcytochemical studiesonthe
Jackson and Mrs. June Chisholm for helpful discussion. blood cells ofthe sea squirt, Ciona intestinalis. I. Stem cells and
The work was supported by grants from SERC, the Nut- amoebocytes. Cell TissueRes 223:403-414.
field Foundation, and the Royal Society. Rowlperyo,beA.X-Fr.a1y98st2ubd.y. /ThMeabrlooBidolc.ellAsssoofcC.iUo.nKa.i6nt2e:st6i0n7al-i6s2:0a.nelectron
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