Classification and Characterization of Ten Hemocytes Types in the Tunicate Halocynthia roretzi(Immunology)

12 Pages·1991·21.3 MB·English

by  Tomoo Sawada

ZOOLOGICAL SCIENCE 8: 939-950(1991) 1991 Zoological Society ofJapan
Classification and Characterization of Ten Hemocytes Types
in the Tunicate Halocynthia roretzi
TOMOO SAWADA1 YOSHIH1SA FUJIKURA1 SUSUMU TOMONAGA2
, ,
and Tetsuo Fukumoto1
{Department ofAnatomy, Yamaguchi University School ofMedicine,
L'hc-city, Yamaguchi 755, and 'School ofAllied Health Sciences,
Yamaguchi University, Uhe-city, Yamaguchi 755, Japan
—
ABSTRACT We propose a standard condition forclassifying living hemocytesofHalocynthia roretzi:
adheringorspreadingonglassinthe presenceofEGTAatpH6.0atroomtemperature(adheringcells).
Accoiing to morphological characteristics revealed by vital staining, autonomous fluorescence and an
effect ofNH4 ions, we classified hemocytes into ten groups; phagocytestype 1 and 2 (pi- and p2-cells),
granularcellstype 1.2.and3(gl-.g2-andg3-cells),vacuolatedcellstype I,2,3and4(vl-,v2-,v3-,and
\4-cells) and lymphoid cells (ly-cells). Two types ofphagocytes, pi- and p2-cells, were the only cells
which actively phagocytize and spread as adhering cells. They were spherical cells with a diameter of
20-40ftm and40-50//m before spreading. Three typesofsmall-granulecontainingcellsandfourtypes
of large-vacuole containing cells were also clearly distinguished not only by their size but also by their
behavior or properties aftervital staining. Ly-cellswere small and spherical and have been referred to
as lymphocytesorhemoblasts. Wecorrelatedsevengroups(pi-,p2-,gl-,g2-,v3-,v4-and ly-cells)with
the formerclassification, andidentifiedforthe first timethreetypesofhemocytes(g3-,vl-andv2-cells).
The relative proportions of various hemocyte types did not agree with earlier reports, and pi-cells,
accounting for 30-45%, were found to be the largest group. These basic analyses of cell types are
essential for future studies of immunodefense properties.
tions.
INTRODUCTION
For the purpose of investigating their functions,
The tunicate Halocynthia roretzi, is an excellent one approach to identifying individual hemocytes
protochordate forresearch dealingwith hemocytes in the living state must be established. However,
and hemolymph. Each individual is large and hemocyteseasily aggregate which makes it difficult
therefore it contains sufficient quantities of to identify individual cells. In addition, the mor-
hemolymph and the animals can be supplied phorological characteristics of hemocytes undergo
throughout the year. Consequently, it isone ofthe substantial change after they adhere on a matrix,
tunicates whose hemolymph has been well investi- after degranulation, or during amoeboid move-
gated biochemically [1-3]. With respect to the ment. The fixation, staining methods and electron
cells, their hemocytes. there is the earlier clas- microscopy also cause deformation. Because of
sification by Ohuye [4] and a later detailed one by this instability, previous classifications were based
Fuke [5]. Turning to function, alloreactivity of upon fixed hemocytes after staining or on hemo-
hemocytes hasalso been studied by Fuke [6, 7] and cytes modified after degranulation, spreading on a
is well known as the "contact reaction". However, matrix or phagocytosis.
these results have not sufficiently established the We considered it essential to classify living
classification of hemocytes and it is important to hemocytes in order to examine their function.
identify and classify them according to their func- Therefore, we searched for: I) the condition in
which hemocytes degranulation was mostk sup-
Accepted April 19. 1991 pressed; 2) when morphological differences among
Received February 14. 1991 hemocytes became maximum. We were able to
940 T. Sawada, Y. Fujikura, et al.
identify each individual hemocyte separately inthe
living state. Finally we confirmed some of the Phagocyticactivityforsheepredbloodcell(SRBC)
studies by Fuke [5], and examined the differences Hemocytes in intact hemolymph were incubated
between her reports and our observations. with sheep redblood cells (SRBC) for5 to 30min.
About0.1% SRBCsuspension (v/v) innormalsea
water was added to the hemolymph, reaching the
MATERIALS AND METHODS
final concentration 0.005-0.01%.
Hemocytes Autonomousfluorescence
Tunicates (Halocynthia roretzi) were collected Hemocytesattachingorspreadingonglassslides
at the Mutsu-bay in northeastern Japan, from in EGTA-solution, in normalseawaterorinintact
April to October, and were transported im- hemolymph were examined under a fluorescence
mediately. Hemocytes were collected within 2 microscope with ultraviolet illumination.
days (fresh-animals) and collected after mainte-
nance in an aquarium for 4-8 weeks without Quantification ofhemoyctes
feeding (4-8 weeks-animals). Hemocytes were Hemocytes were collected in the presence of
collected from the space just beneath epithelium, EGTA, rinsed and resuspended in EGTA-
at a tunic papilla in the upper part of the body, solution. After the pH of hemocyte suspensions
with 1 ml disposable plastic syringes. Harvesting was adjusted to pH6.0, 0.1-0.2ml was added to
was done carefully in the same way and at similar glass slides and incubated for 10-15 min. We
points from each individual. When we collected identified all hemocytes within several different
hemocytes in the presence of ethyleneglycol-bis viewing areas (about 300 cells in total) by phase-
(-aminoethylether) N,N,N',N'-tetraacetic acid contrast microscopy and counted the numbers of
mM
(EGTA), 0.2ml of the solution containing 5 each hemocyte type. Hemocytes in the suspended
EGTA and 0.5 M sodium chloride (EGTA- state, in EGTA-solution at pH5.5, were also
solution, pH7.0 with HC1) was previously drawn counted.
into 1 ml plastic syringes and, with the same
syringe, we collected 0.8 ml of hemolymph which Staining
included numerous hemocytes. Neutral red (0.01% in final concentration) was
added to the hemocyte suspension and vitally
Behavior ofhemocytes on glass slides at different stained hemocytes were observed after 15-30min.
pHs
Hemocytes adheringorspreadingonglassslidesin
Upon centrifuging the mixture of hemolymph the EGTA-solution (pH6.0) were fixed and
and EGTA-solution at 250g for 1 min, hemocytes stained with Wright's blood stain.
were collected and resuspended in 1 ml ofEGTA- NH NH OH
solution. A small quantity (0-20/A) of 0.1 N Effect of 4Cl or 4
sodium hydroxide was added to raise the pH of NH4C1 (5-500mM) or NH4OH (0.1-1%) was
hemocyte suspensions to the range ofpH5.5-8.0. added to the hemocyte suspension in EGTA-
We then added about 100/^l of the hemocyte solution or in intact hemolymph and examined by
suspension on a glass slide and the activity of light microscopy 5-15 min later. Phenol red
hemocytes was viewed as they attached and (0.01%) was added to the hemocyte suspension to
spread. Phenol red (10—30/^l of 0.1% solution in estimate the pH.
distilledwater) was addedtothe hemocytesuspen-
sion to estimate the pH. Hemocytes were then
RESULTS
observed by Nomarski-differential-interference
microscopy (Nomarski-image) and by phase-
contrast microscopy (phase-contrast-image). Adhering and spreading abilities ofhemocytes on
glass slides
Hemocvtes of Tunicate H. roretzi 941
Hemocvtes in intact hemolymph (collected with- ably at pH 5.8-5.9. Although the pH-range and
out EGTA) tended to form small clusters contain- the sequential raising of the pH varied in three
ing 3-10 hemocvtes. They aggregated more inten- eases, the percentages ofspreading cells reached a
sively after placing them on glass slides. In con- maximum in each of three cases at pH 6.0-6.1
trast, hemocvtes in hemolymph containing EGTA (Fie. 2). Most of the spreading hemocvtes under
(at pH 5.0-5.5) did not aggregate and thus each this condition were phagocytes type 1 and 2
individual cell type could be observed (Fig. 1-A). according to the classification to be described later
The addition of EGTA-solution to hemolymph (Fig. 1-B). Above pH 6.5, the percentage of
changed the pH to 5.0, from the original value of spreadingcells tended to decrease slightly whereas
6.0-6.5. No hemocvtes adhered nor were other cell types spread at pH 7.0-8.0.
observed to spread on glass slides under this Temperature within the range of 15-25°C did
condition. not affect spreading and morphological character-
Rinsed and resuspended in EGTA-solution at istics of hemocytes, except that higher tempera-
pH 5.0-5.5, hemocvtes neither adhered nor tures slighly accelerated their spreading. At 15°C,
spread on glass slides, but exhibited Brownian the number of spreading cells reached a plateau
movement. Raising the pH caused increasing within at least 15 min. When hemocytes were
numbers of hemocvtes to attach whereas some of placed on glassslides for 15 min in EGTA-solution
them spread. In analyzing hemocvtes from several at pH 6.0 at room temperature, all of phagocytes
fresh-animals, the percentage of spreading cells type 1 and 2 spread and all other hemocytes
was low at pH 5.0-5.6 but it increased consider- adhered revealing characteristic features for their
Fig. 1. The hemocytes of Halocynthia
roretzi suspended in the EGTA-
solution at pH5.5 (A: the condition for
cell suspension), and spreading or
adhering on glass slides in the EGTA-
solution at pH6.0 (B: the condition for
adhering cells). The scale bar shows
1(H)/iin. pi. p2: phagocyte type 1 and
2; gl, g2: granularcell type 1 and 2; v2,
v3: vacuolated cell type 2 and 3.
942 T. Sawada, Y. Fujikura, et al.
image revealedthattheywerethickerthanpl-cells
in the fully spread state (Fig. 6-E). They some-
timescontained severalgranularcomponents (Fig.
6-C, D, E). P2-cells were relatively dark in phase-
contrast-image after they spread (Figs. 3-F; 6-C,
D, E), even in a fully spread state.
Granular cells type 1 (gl-cells) 35-40jum in
diameter did not spread but retained the round
shape (Fig. 4-A, E), adhering to glass slides by
means of small pseudopodia; they sometimes
moved actively. In the Nomarski-image they con-
tained many small granules less than 5jum in
pH of the solution diameter (Fig. 4-A), and they were weakly refrac-
Fig. 2. Thepercentageofspreadinghemocytesonglass tile in phase-contrast-microscopy (Fig. 4-E). In a
slidesatdifferentpH. Square (), triangle (A.) and few cases, some of them expanded slightly into
circle (•) indicate the results in the hemocytes of
three different individuals, respectively. Although dark round cells. Granular cells type 2 (g2-cells)
threecaseswereexaminedatdifferentpHandinthe 45-50jum in diameter were large, polygonal cells
different pH range, it was considered that the (Fig. 4-B, C, F, G). Rarely, did they have small
spreading hemocytes reached a maximum at pH protrusions. The Nomarski-image showed that
6.0-6.1 in each case. these cells were filled with numerous small gra-
nules less than 5jum in diameter (Fig. 4-B). In the
cell-types (Fig. 1-B). Afterward, we used this phase-contrast image (Fig. 4-F), theywere slightly
condition (the condition for adhering cells) as the brown anddarkusuallywitharefractileperiphery;
standard to identify orclassifylivinghemocytes: in thesecellshadno pseudopodia nordidtheymove.
the EGTA-solution at pH6.0, placed on glass Granular cells type 3 (g3-cells) were large and
slides for 15 min at room temperature. oval, 80jum in diameter (Fig. 4-D, H), the largest
hemocyte; they did not move. Nomarski-image
Classification of hemocytes at the condition for showed that these cells were filled with numerous
adhering cells small granules less than 5jum in diameter (Fig.
We classified hemocytes under the conditions 4-D). They contained highly refractile granules in
for adhering cells into ten groups: two types of the phase-contrast-image.
phagocytes, three types of granular cells, four Vacuolated cells type 1 (vl-cells) were small
types ofvacuolated cells and lymphoid cells (Figs. cells 30-35jum in diameter (Fig. 4-C, G). They
3,4). containedonlyone (rarelytwo)prominentvacuole
Phagocytes type 1 (pl-cells) were 80-100jum in 10-15jum in diameter. Hyaline cytoplasm was
diameter and they spread over wide distances observed in these cells, and they occasionally
(Figs. 3-A, E; 5). They did not move or they spread but exhibited no obvious movement.
moved so slowly that we only observed motion by Vacuolated cells type 2 (v2-cells) were round or
intense observation. The Nomarski-image re- polygonal cells, 30-40jum in diameter (Fig. 3-B,
vealed that they spread as thin, flat sheets (Fig. F), without any pseudopodia. In the Nomarski-
3-A). These sheets were transparent in the phase- image they were filled with several vacuoles 5-8
contrast image (Figs. 3-E; 5-C, D) and usually jum in diameter (Fig. 3-B). In the phase-contrast-
containedseveral darkgranules (Fig. 3-E). Phago- image, theywereweaklyrefractilewitharelatively
cytes type 2 (p2-cells), 60-100 jum in diameter, darkcentralarea. Theywere transparentandtheir
were also spreading cells (Figs. 3-B, F; 6), mod- contour was indistinct when observed by light
ifying their shapes frequently by amoeboid move- microscopy.
ment: either rounding up, spreadng extensively or Vacuolated cells type 3 (v3-cells) 35-45jum in
sending out long pseudopodia. The Nomarski- diameter contained several large vacuoles which
Eiemocytes of Tunicate H. roretzi 943
Fig. 3. The five bemocyte t\pesatthecondition foradheringcells, in Nomarski-differential-intcrference-microscopy
(A-D) and in phase-contrast-microscopy (E-G). E-G show the same cells as A-C, respectively. D showed the
slightly spread type v4 cells when the cover slip was slightly pressed. The scale bar shows 10//m. pi, p2:
phagocyte type 1 and 2: \2. v3, v4: vacuolated cell type 2, 3 and 4.
occupied most of the cytoplasm. In both the vacuoleschanged shape when the entire cell-shape
phase-contrast- and in the Nomarski-image. they was altered during movement. Vacuolated cells
were tightly refractile (Figs. 3-C, D. G; 4-C, G). type 4 (v4-cells) 35-50/im in diameter were also
The diameters oftheir cytoplasmic vacuoles varied filled with many highly refractile vacuoles both in
from 10 to 30pan. A hyaline cytoplasm was hardly phase-contrast- and in Nomarski-images (Fig. J-B,
distinct by phase-contrast microscopy. These cells C. D. F, G). The vacuolesclosely resembled those
sometimes moved activelv and the refractile ofv3-cells while the size of the vacuoles in v4-cells
944 T. Sawada, Y. Fujikura, et al.
Fig. 4. Thesix hemocytetypes attheconditionforadheringcells, inNomarski-differential-interferencemicroscopy
(A-D) and in phase-contrast microscopy (E-H). E-H show the same cells as A-D, respectively. The scale bar
shows 10fjtm. gl, g2, g3: granular cell type 1, 2 and 3; vl, v3: vacuolated cell type 1 and 3; ly: lymphoid cell.
wasrelativelyuniformwithintherangeof8-10jum v4-cells to spread.
in diameter (Fig. 3-C, D). V4-cells contained Lymphoid cells (ly-cells) were the smallest and
morevacuoles than v3-cells, andv4-cells had high- themostsphericalcells 12-20jumindiameter(Fig.
er motility than v3-cells. However, it was some- 4-A, E), appearing as dark spheres in the phase-
times difficult to distinguish v4-cells from v3-cells contrastimage. They rarelyspread onglass slides,
in the condition for adhering cells. The identifica- and had no particular characteristics other than
tion of v3- and v4-cells was easy after we slightly their small size.
pressed the cover slips which could make the
Hemocvtes of Tunicate H. roretzi 945
Fig 5. A, B, C: The change of the shape
of phagocyte type 1 with spreading;
*%* round(A),slightlyspread(B)andcom-
pletelyspreadasasheet (C). Thescale
barshows 10//m. D: The syncytium as
a large sheet formed by the fusion of
spread phagocyte type 1. The scale bar
shows 100fim.
^
9
0m
Fio. 6. A. B. C: The change of the shape ofa phagocyte type 2 cell With spreading; round (A). slightly long (B) and
spread as a dark sheet (C). D. E: Phagocyte type 1 and type 2 spread on a glass slide Note the difference
between two types of cells, in darkness in phase-contrast miCTOSCOp) (I)) and in thickness m NOniarski
differential-interference microscopy (E). The scale bar shows L0/<fll.
946 T. Sawada, Y. Fujikura, et al.
in red-violet. On the other hand, g3-, pi-, p2- and
Identification of hemocytes suspended in EGTA- v2-cells required 5-10min and they stained
solution orange-red. Neither gl- and g2-cells nor ly-cells
Following the observation of pi- and p2-cells stained.
from adhering through spreading on glass slides, Autonomousfluorescence underthe illumination
we identified both cell types in the non-spreading of UV-rays: Intensive and autonomous blue
state in cell suspensions. fluorescence was exhibited by vl- and v3-cells.
In EGTA-solution pl-cells were small cells 20- The brightness of the fluorescence increased dur-
40jum in diameterwith irregularprotrusions (Figs. ing first 15-30sec, then weakened and the color
1-A; 5), whereas they were small spherical cells changed into whitish blue. Slightly weak fluoresc-
with frill-like or spine-like pseudopodia in intact ence was exhibited by v4-cells, but the fluoresc-
hemolymph. Most of them had small, refractile ence was faster in diminishing than that ofvl- and
vacuoles which often disappeared when they v3-cells. After 2-5 min of illumination, pi- and
spread by adherence. p2-cells began to exhibit blue fluorescence.
In EGTA-solution p2-cells were relatively large Phagocytosis: Only pi- and p2-cells showed
cells 40-50jum in diameter, spherical (Fig. 6) or active phagocytosis against SRBCs, engulfing
often with irregularly protruding pseudopodia more than 2 and up to 5-7 SRBCs. Little activity
(Fig. 1-A). It was sometimes difficult to disting- of phagocytosis was observed in v4-cells.
uish p2-cells from gl-cells in suspension, and the Effect of NH4Cl or NH4OH: When NH4C1
neutral red-staining was useful to identify p2-cells; (pH5.0) or NH4OH (pH8.0) was added to the
p2-cells contained cytoplasmic granules which hemocyte suspension, the highly refractile cyto-
were stained with neutral red while gl-cells were plasmic vacuoles of vl-, v3- and v4-cells changed
not stained. intoorange-brownspheresbothinEGTA-solution
The identification ofothereight hemocyte types and in intact hemolymph, which we refer to the
in suspension was possible using the same charac- ammonium-effect (Fig. 7). The ammonium-effect
teristics as those which adhered (Fig. 1). Other occurred more quickly in the presence of higher
than the ten hemocyte types, we found non-motile concentrations of NH4C1 or NH4OH. When the
cells having cilia-like projections in suspension final pH of the hemocyte suspension was shifted
(Fig. 1-A an arrow). The projections were within the range of5.0-8.0 after adding NH4C1 or
absorbed into cell-body after adherence when the NH4OH, the ammonium-effect occurred similarly
pHwasraised. Theywerecountedasv2-cellsafter throughout that range.
staining them with neutral red. Thevacuolesofvl-, v3-andv4-cellsalsolostthe
autonomous fluorescence after the ammonium-
Other characteristics ofhemocytes effect. All hemocytes including pi- and p2-cells
NH
Behavior on glass slides in intact hemoly- did not spread in the presence of 4C1 or
mph: Most ofpl-cells spread and fused together
to form a large syncytium, while they never fused
in the EGTA-solution. Many ofgl-cells extended
long rod-shaped pseudopodia, forming clusters
together with the same cell types and at the same
time discharging cytoplasmic granules. Some-
times, g2- and g3-cells moved with a small hyaline
A - B
cytoplasmiccapattheirtiportailwithoutapparent
degranulation nor extensive change in shape.
uUsiusahleldy,frvo4m-cve3l-lcselslsp.read and were easily disting- Fig. Ca7.d;di3tT0ihomeninovfaac1fu0toelmratMtehdeNcaHedld4liCt1tiyo(pnAe);.31cTmhihanen,givBan;cgu5oumlpeionninatnhAde
Vitalstaining with neutral red: Within 2-3 min changedintotwosphericalgranulesin C. Thescale
ofthestaining, vl-, v3- andv4-cellsstained rapidly bar shows 10jum.
)
Hemocytes of Tunicate //. roretzi 947
Table 1. Hemocyte composition of tunicate Halocynthia roretzi at different
conditions. Each cell type was counted at pH 5.5 (cell suspensions) and at
pH 5.9-6.2 (adhering cells).
case 1 case case 3
type of hemocytes Sus* Ad* Sus Ad Sus Ad
(5.5) (6.2) (5.5) (5.9) (5.5) (6.0)
(%) (%) (%) (%) (%) (%)
pl-CCll 34.6 30.3 34.9 33.2 35.4 28.9
p2-cell 7.2 6.6 7.3 6.3 11.6 9.6
gl-cell 13.7 18.8 23.2 18.cS 15.0 12.4
g2-ceII 1.4 1.4 0.6 1.9 1.9 1.9
g3-cell 0.3
vl-cell 1.4 1.0 1.0 1.9 0.9 0.9
\2-celI 10.6 10.1 6.0 5.6 10.7 5.3
v3-ceU 27.7 24.4 22.2 26.0 21.9 33.5
v4-cell 2.4 3.5 1.6 3.4 0.9 6.2
ly-cell 1.0 3.8 3.2 2.8 1.6 1.6
number of total 292 287 315 319 319 322
cell counted
See text for the abbreviation of hemocytes types.
* Sus: cell suspensions (pH)
Ad: adhering cells (pH)
NH.OH. The composition of hemocytes in fresh-animals
Wrights blood stain offixed hemocytes: After and4-8 weeks-animals: We analyzed the relative
fixation with methanol, we clearly identified only proportions of various hemocyte types in five
gl- and g2-cells because oftheirsizes, round shape fresh-animals within 2 days after they were col-
and frequency. The cytoplasm of both gl- and lected and in eight 4-8 weeks-animals (Table 2).
g2-cells stained dark-blue or violet, definitely in-
dicating that those cells have basophiliccytoplasm. Table2. Hemocyte composition of tunicate
Halocynthia roretzi, in freshly collected animals
All other cell types appeared to have acidophilic and in others kept in an aquarium for 4-8 weeks.
cytoplasm except for a group of small cells whose
cell-type was not defined. type of fresh animals 4-8 weeks-animals
hemocytes (5 animals) (8 animals)
The composition ofhemocytes {percentage ofeach {%) {%)
cell type in total hemocytes) pi-cell 34.1+5.4* 33.8± 10.3*
The composition of hemocytes analyzed in the p2-ccll 9.4±2.9 9.01 2.1
conditionforadheringcells andin theconditionfor gl-cell 15.8±3.3 17.7 + 4.3
cell suspension: We added 0.1-0.2 ml of hemo- g2-cell 1.4±0.4 1.31 0.5
cyte suspension (pH 5.5) on glass slides and g3-cell 0.2±0.3 HI 0.1
counted the number of each type first in cell vl-cell 1.0 | 0.5 1.1 I 0.8
suspensions. Then, we raised the pH of the same v2-ccll 5.8 | 2.8 7.3 | 2.7
hemocyte suspension to 5.8-6.2. added 0.1-0.2 ml v3-cell 24.8 • 6 23.2 | 7.2
on glass slides and then counted adhering cells v4-cell 4.211.4 4.6 • 2.4
(Tablet). From three different individuals, the ly-cell 3.4 • 1 \ L.9 • 2
relative proportions of various hemocyte types did See text for the abbreviation of hcmoc\tcs types.
not change significantlywith changes in conditions. * average • SI
948 T. Sawada, Y. Fujikura, et al.
The analysis was done on adhering cells and the pension did not differ significantly. Therefore, we
composition of hemocytes in the 4-8 weeks- considered that a classification in the cell suspen-
animals was essentially equal to that in fresh- sions most nearly correlated with the classification
animals. for adhering cells. Thus, adherence of hemocytes
on slidesdidnotinduce anyspecificlossofparticu-
lar hemocyte groups. Because the classification of
DISCUSSION
adhering cells was mainly based on morphological
We propose a standard condition for classifying characteristics of living cells, easily observed in
living hemocytes of Halocynthia roretzi, and we light microscopy, it may be useful forinvestigating
have classified them into ten groups (two types of hemocyte functions.
We
phagocytes, three types of granular cells, four alsodescribedthe othercharacteristics, such
types of vacuolated cells and lymphoid cells). It asautonomousfluorescenceortheeffectofammo-
was also possible to classify hemocytes in suspen- nium on hemocytes (Table 3). Autonomous
sion, although vital staining was sometimes neces- fluorescence hasnotbeen systematicallyemployed
sary. Therelativeproportionsofvarioushemocyte in classifying hemocytes, but some investigators
types analyzed as adhering cells and cells in sus- have made significant observations (personal com-
Table3. Characteristics of each hemocyte type in tunicate Halocynthia roretzi
hteympoecyotfe cell size phtaogsoisc^y- svtiatianli3n)g ammefofnecitusm- u\oq•a afultuoorneosmcoeuncse5) vgraacnuuolloeerss ((GV)) (soptrshleeiarddeirngeglmaasorsnk6))
pl-cell 80-100//m ++++ orange +* one or some +
G
(20-40) small (syncytium)
p2-cell 60-100 ++++ orange +* several +
(40-50) small G (active
movement)
gl-cell 35-40 many small G —
<5//m (rod-shape
extension)
g2-cell 45-50 many small G —
<5/im
g3-cell 80 orange + many small G —
<5fxm (largest cell)
vl-cell 30-35 red-violet + one V
10-15/urn
v2-cell 30-40 orange - many V
5-8/urn
v3-cell 35-45 red-violet + many large V
10-30fim (refractile)
v4-cell 35-50 red-violet + many V
8-10jum (refractile)
ly-cell 12-20
See text for the abbreviation of hemocytes types.
1) Cell-size: adhering cells on glass slides (cell suspension).
2) Only the phagocytic activity against sheep red blood cells (SRBC) was examined.
+: at least some of them could phagocytize SRBC, ++++: they phagocytized many SRBCs actively.
3) Vital staining with neutral red.
4) Staining of cytoplasm; a: acidophilic, b: basophilic, ?: uncertain.
5) Fluorescence under the UV-illumination; ?: uncertain.
*: Fluorescence gradually appeared after 2-5min of illumination.
6) The ability to spread on slide glasses at the condition for adhering cells.