Early development of zooxanthella-containing eggs of the corals Pocillopora verrucosa and P. eydouxi with special reference to the distribution of zooxanthellae

8 Pages·2000·3.2 MB·

by  M Hirose

Reference: Bwl. Bull. 199: 68-75. (August 2000)
Early Development of Zooxanthella-Containing Eggs
of the Corals Pocillopora verrucosa and P. eydouxi
with Special Reference to the Distribution
of Zooxanthellae
M. HIROSE', R. A. KINZIE III2, AND M. HIDAKA1 *
1 Marine Environmental Science, Department ofChemistry, Biology and Marine Science, University of
the Rviikvus, Nishihara. Okinawa 903-0213, Japan; and 2 Department ofZoology and Hawaii Institute
ofMarine Biologv. University ofHawaii, Honolulu. Hawaii 96822
Abstract. Some hermatypic corals spawn eggs that con- Introduction
tain zooxanthellae. We followed development of zooxan-
Reef-building corals harbor intracellular symbiotic
tvheerlrluac-ocsoantaainndinPg. eeygdgosuxio.f Wtewoalssuochdocspuemceinest,edPocchialnlgoepsorian dSionmotelahgeelrlmaattesy,piczocooxraanltshealclqauei,reinthetihreisrymebnidoondtesrmfarlomctehlelsi.r
the distribution pattern of zooxanthellae during develop- mother colony before fertilization (Kojis and Quinn, 1981;
ment. Oocytes ofboth species took up zooxanthellae 3 to 4 Babcock and Heyward, 1986; Tomascik and Sander, 1987;
days before spawning. At first, zooxanthellae were evenly Yeemin, 1988; Glynn et al., 1991. 1994; Heyward et til..
distributed in oocytes, but they later moved to the hemi- 1987; Kinzie. 1993, 1996; Sierand Olive, 1994; Krugerand
sphere that contained the germinal vesicle. After fertiliza- Schleyer, 1998). It is not known how zooxanthellae are
tion, early cleavage events were holoblastic, progressing by delivered to oocytes and how their distribution relates to
furrow formation. The first cleavage furrow started at the their eventual restriction to the endodermal cells in adults.
hemisphere that contained zooxanthellae. dividing the zoo- Early development of scleractinian corals has been de-
xanthellate complement ofthe zygote about equally into the scribed in various species (e.g.. Szmant-Froelich ct a/..
two blastomeres. The second division divided each blas- 1980, 1985; Babcock and Heyward, 1986; Harrison and
tomere into one zooxanthellae-rich cell and one with few Wallace, 1990). However, early development ofcorals with
zooxanthellae. With continued cell division, blastomeres oocytes containing zooxanthellae has beendescribedonly in
containing zooxanthellae moved into the blastocoel. The the spawning species Montipora effusa (Yeemin, 1988) and
blastocoel disappeared at about 5 h after the first cleavage, M. verntcoxa (Mate et al., 1998) and the brooding species
and the central region of the embryo was filled with cells Porites porites (Tomascik and Sander, 1987).
containing either zooxanthellae or lipid droplets, forming a Although zooxanthellae are generally restricted to the
stereogastrula. Our results suggest that only blastomeres gastrodermis of adult corals, they are at least temporarily
that had been determined to develop into gastrodermal cells observed in the ectoderm of planulae of some corals and
receive zooxanthellae during cleavage. This determination soft corals (Szmant-Froelich, 1985; Benayahu ft al.. 1988;
appears to take place, at the latest, by the second cell Benayahu, 1997; Benayahu and Schleyer. 1998; Sehwarz et
division at the four-cell stage. al.. 1999). This is probably because infection first occurred
in the ectoderm cells ofembryos orearly planulae (Szmant-
Froelich ct al.. 1985) or because dividing cells of these
stages transferred the multiplying symbionts to their daugh-
Received 22 October IWJ; accepted 17 May 2000. ter cells, including presumptive ectoderm cells (Benayahu.
*To whom correspondence should be addressed. E-mail: hidaka 1997; Benayahu and Schleyer, 1998). In these cases, zoo-
@sci.u-ryukyu.ac.jp xanthellae were transferred from ectoderm to endoderm
68
ZOOXANTHELLATE EGGS OF CORALS 69
across the mesoglea before larvae develop into mature plan- Histolog\ and transmission electron microscopy
ulae (Benayahu, 1997; Benayahu and Schleyer. 1998).
Eggs and embryos were collected in a microtube and
Montgomery and Kremer (1995) also found that in the
allowed to settle to the bottom. The supernatant was then
larvaeofa scyphozoan,Linucheungiiiculata. the algae were discarded and fixative added. The specimens were fixed in
found mostly in the ectodermal cells, and suggested mech- 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)
anisms by which zooxanthellae could be transferred from containing 3% NaCl for 2 h or more. The specimens were
ezocotTxohadenetrchmoerlatlloasteePnodecgoigdlsel,ropmwohroiafchepyladdniousnlpxaliea.yanadnP.unveevrernucdoissatrriebluetaisoen rdoirsnamstieeddumiinntteahtergorsxaiaddmeeedibnsuetfrhfieeersstoahfmreeaecbetutifomfneeers,faionmrdm1eprohssteo-dnfiixinceed,/(id-nebuh1ty%y-l
ofalgal cells (Hirose et<//., unpubl. data). It is likely that, in glycidyl ether (QY1). and embedded in Spurr's resin. For
these corals, zooxanthellae are not equally delivered to all
tdiavueghetnedrocdeelrlsmbcuetllsg.oImfozroeoxoarntlheesslleaxeclbuescivoemley rtoesptrriecsteudmpt-o wliegrhet smtiacirnoesdcowpiitch o1b%sermveatthioynl,enesecbtliuones-1%0.5a-z1ur/IiIminthi1c%k
borax. Forelectron microscopy, silverto gold sections were
endodermcellsduring the course ofdevelopment, the larvae
donot needtotransferthealgaefromectoderm toendoderm sutnadienredawiJtEhOuLranJyElM-ac2e0ta0t0eEXandelleecatdroncitrmaitceroasncdopoebseatrveadn
as described in the soft corals (Benayahu, 1997).
In the present study, we followed early development of acceleration voltage of 100 kV.
zooxanthellate eggs of the corals P. eydouxi and P. verrn-
cosa. We studied changes in the distribution pattern of Results
zooxanthellae during early development of the corals to Oocytes took up zooxanthellae 3 Idays before spawning
determine mechanisms by which the distribution of zoo- in both species. Zooxanthellae were first distributed evenly
xanthellae becomes localized to the endoderm of planulae. in the ooplasm (Fig. 1A, B), but later. 1-2 days before
spawning, the algae became concentrated in the hemisphere
that contained the germinal vesicle. The other hemisphere
Materials and Methods contained many lipid droplets ofabout the same size as the
zooxanthellae. Although the germinal vesicle was no longer
Branches, 7-12 cm long, were collected from colonies of apparent by the time the eggs were spawned, the zooxan-
Pocillopora verrucosa a few days before the new moon and thellae remained concentrated in the hemisphere ofthe egg
from P. eydouxia few days before the full moon in June and that contained the nucleus (Fig. 1C, D). At spawning, eggs
July 1998. Colonies were collected from reefs at Sesoko of both species were about 140 ju,m in diameter and con-
Island. Okinawa. The branches were placed separately into tained about 130 zooxanthellae (Hirose etai, unpubl. data).
3-1 plastic containers supplied with unfiltered running sea- Cleavage took place by progressive furrow formation
water. The hermaphroditic colonies of P. verrucosa and P. (Fig. IE), at intervals of 30 to 40 min. The first cleavage
eydouxi spawned gametes for about 30 min in the early furrow started at the hemisphere that contained the zooxan-
morning a few days after the new moon, and a few days thellae and the oocyte nucleus, dividing the zooxanthellae
afterthe full moon, respectively (Kinzie, 1993; Hirose etul.. equally into two blastomeres, each with a roughly equal
unpubl. data). Both species first released sperm and then complementofzooxanthellae still concentrated in the hemi-
negatively buoyant eggs. To collect gametes, the supply of sphere containing the nucleus (Fig. IF). At the second
seawater was stopped before the expected spawning time, cleavage, each blastomere was divided into one zooxanthel-
about 0630 h. After sperm had been shed, they were col- lae-rich blastomere and one with few zooxanthellae (Fig.
lected by sucking up seawater from the container in a large 2A). By the 64- to 128-cell stage, some blastomeres in the
plastic pipette. The pipette was rinsed with a diluted hypo- morula contained one or sometimes more zooxanthellae.
chlorite solution and then with seawater to avoid contami- while others contained none (Fig. 2B). As cleavage pro-
nation of gametes. Released eggs were collected by pipette ceeded, a blastocoel formed and blastomeres with zooxan-
from the bottom of the container and placed in a plastic thellae moved into this space (Fig. 2C, D). The outer layer
beaker. Eggs were fertilized by mixing released gametes of the blastula consisted of relatively large blastomeres
(100-300 ml suspension each) from twoorthree colonies in containing no zooxanthellae. At about 5 h after fertilization,
a plastic beaker. Filtered (0.45 /u,m) seawater was added to blastomeres containing zooxanthellae, lipid droplets, or
the beaker to make the final volume to 1 or 2 I. Fertilized both filled the blastocoel, resulting in a stereogastrula (Fig.
eggs were kept in the seawater at a room temperature 2E, F). Gastrulation appeared to occur by delamination
28-30C. Eggs and embryos were sampled and observed rather than by invagination.
under a light microscope at intervals offrom 30 min to I h, Blastomeres in the outer layer had microvilli on their
and photomicrographs were taken with a microscope outer surface and characteristic granulesjust below the cell
equipped with an epifluorescent system (Nikon Microphot). membrane (Fig. 3A). Blastomeres in the outer layer were
70 M. HIROSE ET AL
Figure 1. Early developmentofPocillopora vermcosa: from unfertilizedegg totwo-cell stage. (A)Oocyte
isolated from the gonad. Zooxanthellae are distributed evenly in the cytoplasm. The germinal vesicle is at the
centerofthe oocyte. (B) Oocyte viewed underepifluorescence (BV excitation). The red fluorescence is due to
algal chlorophyll. Cytoplasm of the oocyte exhibits blue-green autofluorescence. (C) Spawned egg. Zooxan-
thellae are mainly located in the right hemisphere and lipiddroplets in the left hemisphere. (D)The same egg.
observed underepifluorescence (BV excitation). (E) Firstcleaving stage. Cleavage furrow (arrow) starts at the
hemisphere that contains the /.ooxanthellae. (F)Two-cell stage. Zooxanthellae are dividedequally intothe two
hl.isii'ineres. Bars = 100 fim.
connected to each other by the contact junctions near the those from neighboring blastomeres were often intermin-
apical surface (Fig. 3B). In other regions of the interface, gled (Fig. 3C). When blastomeres in the outer layer con-
blastomeres were only loosely attached to each other or tained Zooxanthellae, the /ooxanthellae were usually at the
were separated by extracellular space. Villi-like cellular lower or lateral margin of the blastomere. Zooxanthellae at
processes were observed in the extracellular space, and the lateral margin of the blastomere bulszed into the extra-
ZOOXANTHELLATE EGGS OF CORALS 71
B
Figure2. EarlydevelopmentofPocilloptmi verrucosa: four-cellstagetogastrula. (A)Four-cell stage.The
secondcleavageplane wasnormal tothe firstcleavageplane, thusdividingtheblastomereintoazooxanthellae-
rich blastomere and a lipid-droplet-rich blastomere. (B) Morula-stage embryo. Blastomeres are round; some
contain asinglezooxanthellae. (Cl Blastula. Zooxanthellaearestill restrictedtoone hemisphere. (D)Sectionof
a blastula. Blastomeres containing one or more zooxanthellae (arrow) and those containing lipid droplets are
located in the blastocoel (be), while the surface layer is composed of larger blastomeres with no algae. (E)
Gastrula. Thecenterofthe gastrula appears dark due to accumulation ofzooxanthella-containing blastomeres.
(F)Sectionofagastrula. Blastomerescontaining zooxanthellaeand thosecontaining lipiddroplets fill the inner
space ofthe gastrula. forming a stereogastrula. Bars = 100 /urn.
72 M. HIROSE ET AL.
Figure3. Electron micrographsofanearly gastrulaofPot-illo/rum vt'iriicuxti. (A) Blastomeres inthe outer
layerofanembryo. (B) Highermagnificationoftheboxedarea in (A), showingcontactjunction neartheapical
surface (arrow). (C) Blustomere in the outer layer containing zooxanthellae. Zooxanthellae bulge into the
neighboringblastomere. (D)Twozooxanthellae inacellularprocess, whichappears tobe stillconnectedtothe
ouier layer hlastomere. (E) Zooxanthellae surrounded by a small amount ofhost cytoplasm. (F) Lipid droplet
surrounded by a small amount ofcytoplasm, be = blastocoel. bl = blastomere. cp = cytoplasm, li = lipid
droplet, mv = microvilli. zo = zooxanthella. Bars = 5 /J.m except in (Bl, where bar = 1 /nm.
cellular space and sometimes into neighboring blastomeres blastocoel. In such cases, a constriction was often observed
(Fig. 3C). Similarly, /ooxunthellae or lipid droplets located between thecentral cytoplasmand the protrusion containing
at the lower margin of the hlastomercs bulged into the a /ooxanthella or a lipid droplet (Fig. 3D). Most zooxan-
ZOOXANTHELLATE EGGS OF CORALS 73
Figure4. PlanulaofPocillnpora verrucosa 24hafterfertilization. (A) Photomicrographofa fixedplanula
taken under differential interference optics. The planula is completely ciliated at this stage. (B) Histological
sectionofaplanula. Zooxanthellae and lipiddroplets are in the endodermal cells. (C-D) Photomicrographs of
tmheesobgoldeyaw(aalrlroowfhaeapdl)a.nuTlhaeteakcetnoduenrdmercoonislisitmsmeorfscioolnu.mTnhaerceecltlso,decr=m aencdtoednedrmo,degrm=agraestcrleoavralsycusleaprarcaatvietdy,bymth=e
mouth opening, n = endoderm. Bars = 100 ;xm in (A) and (B), 20 fj.m in (C) and (D).
thellae and lipid droplets in the blastocoel were surrounded cock and Heyward, 1986; Harrison and Wallace, 1990), this
by a small amount ofcytoplasm and appeared to be free is the first report describing the processes by which zoo-
that is, detached from blastomeres in the outer layer (Fig. xanthellae become restricted to the endoderm during the
3E, F). course of embryogenesis. In the two Pocillopora species
Spherical embryos with a smooth surface as shown in studied, regions ofegg cytoplasm are differentiated and cell
Figure 2E and F were observed 6 h after fertilization. fates are apparently decided early in development, possibly
Ciliated larvae started to swim 8 h after fertilization. The before fertilization. Zooxanthellae moved towardthe animal
embryos became elliptical and swam spirally by 9-10 h pole 1-2 days before spawning. The first cleavage appor-
after fertilization. An oral pore was formed by invagination tioned zooxanthellae more or less equally between the first
ofthe epidermis, and a gastrovascularcavity was formed as two blastomeres. At the second cleavage, however, two of
gastrodermal cells became organized 24 h after fertilization the four blastomeres received almost all the zooxanthellae,
(Fig. 4A, B). At this stage the ectodermal layer the plan- while the other two had few or none. This uneven distribu-
ula's epidermis consisted ofcharacteristic columnarcells, tion of zooxanthellae persisted until the zygotes developed
and the epidermis and gastrodermis were separated by dis- into gastrulae.
tinct mesoglea (Fig. 4C, D). Embryos at this stage were As cleavage progressed, relatively large blastomeres
typical planulae. Generally, only gastrodermal cells con- without zooxanthellae came to occupy the outerlayerofthe
tained zooxanthellae, though a few zooxanthellae were ob- embryo as the blastocoel opened. Later, blastomeres con-
served in ectoderm of some planulae. Planulae 48 h old taining zooxanthellae or lipid droplets detached from the
possessed some nematocysts. outerlayeranddropped intothe blastocoel until it was filled
with blastomeres containing zooxanthellae and lipid drop-
Discussion
lets. In these two species of Pocillopora, gastrulation may
Although early development of scleractinians has been occur due to delamination rather than invagination, result-
described (e.g., Szmant-Froelich et al., 1980, 1985; Bab- ing in a stereogastrula. Gastrulation through delamination
74 M. HIROSE ET AL
has been suggested forAstrangiadanae (Szmant-Froelich el 1997; Benayahu and Schleyer, 1998). Montgomery and
til.. 1980). Faviafragum (Szmant-Froelich ela!., 1985), and Kremer (1995) suggested that ectoderm cells infected by
Montipora rerrucosa (Mate el at., 1998). zooxanthellae may migrate to the endoderm of planulae.
Titlyanov etal. (1996, 1998) observed degraded zooxan- Benayahu (1997) and Benayahu and Schleyer (1998) ob-
thellae in planulae as well as in adult polyps of hermatypic served that, in the soft corals they studied, zooxanthellae
corals and suggested that digestion ofzooxanthellae occurs pass through temporarily opened gaps in the mesoglea to-
both in planulae and in adult polyps. We saw no such wards the endoderm. Throughout the process, each zooxan-
degraded zooxanthellae in the surface layer of embryos or thella resides within a vacuole in the detached ectodermal
early planulae. If zooxanthellae are not digested during cytoplasm. However, we did not observe such a transfer of
early development, they must be transferred from blas- zooxanthellae fromectodermtoendoderm in planulae ofthe
tomeres that are determined to develop into symbiont-free two Pocillopora species. In these corals, zooxanthellae ap-
ectodermal cells to blastomeres that are fated to develop peared to be transferred more or less exclusively to blas-
into algae-bearing endodermal cells. This ontogenetic redis- tomeres that were fated to develop into endodermal cells.
tribution of algae might occur in several ways. One possi- This suggests that determination of presumptive endoderm
bility is that zooxanthellae move basally within blastomeres cells and specificity of zooxanthellae towards presumptive
so that subsequent horizontal cell division results in surface endoderm cells occur earlier in the two Pocillopuni species
ectodermal cells and centrally located endodermal cells that than in the soft corals studied.
contain zooxanthellae. Our observations suggest that zoo- We describedchanges in the distributionofzooxanthellae
xanthellae, along with small amounts of cytoplasm, were during early development as well as during final maturation
separated fromsurfacecells and dropped intotheblastocoel. of oocytes in the corals Pocillopora verrncosa and P. ey-
This process is similar to the "pinching off suggested for douxi. Zooxanthellae moved to the hemisphere of the oo-
the transfer mechanism of zooxanthellae from follicle cells cyte that contained the germinal vesicle 1 to 2 days before
to oocytes and from ectoderm to endoderm in some soft spawning. Zooxanthellae moved to the lateral or basal mar-
corals (Benayahu, 1997; Benayahu et al., 1992; Benayahu gins ofthe surface blastomeres andbulged intoextracellular
and Schleyer, 1998). However, the small "blastomeres" spaces or into the blastocoel. Blastomeres containing zoo-
containing zooxanthellae (Fig. 3E, F) could also be pro- xanthellae or lipid droplets along with a small amount of
duced by unequal division rather than by pinching off. If cytoplasm were produced, probably by unequal mitotic di-
this were the case, there should be animal nuclei in these vision, and then dropped into the blastocoel and became
structures. These basally derived cells would then develop endodermal cells. It is not clear whether the presence of
into gastrodermal cells. Another possibility is that ectoder- zooxanthellae affects development of the blastomere or if
mal cells expel zooxanthellae by exocytosis and adjacent the fate of a blastomere is determined by the nature or
endodermal cells take them up by phagocytosis. The obser- quantity of its cytoplasm. Further study is necessary to
vation that zooxanthellae within vacuoles ofthe blastomere understand how zooxanthellae move to a region ofoocytes
in the outer layer often protruded to the intercellular space, and to certain areas of blastomeres.
bulging into the neighboring cell, suggests that this possi-
bility cannot be ruled out. Acknowledgments
gasFteruwlaiefaornythzeoeoxcatnotdheerlmlaoefwplearneulfaoeunodftihnetthewoouPtoecrillalyoeproroaf We thank the staffofSesoko Station. Tropical Biosphere
sfpoeucnidesinsttuhdeiede.ctoHdoewremveorf,eazroloyxapnltahneulllaaee oafresosmoemetcoirmaelss RtheissesatrucdhyCweanstedro,neU.niYv.erNsoiztaywoaf,tNh.e TRaykuakhyaussh,i,wahnedreW.parDtiaohf
(Faviafragum: Szmant-Froelich el al.. 1985; Fiingiu scii- Permata kindly helped us. This study was partly supported
taria: Schwarz ct til.. 1999), soft corals (Xeiiiu iimbellata: by the Grant-in-Aid for Scientific Research No. 08644216
and 11694223 from the Ministry of Education, Science,
Benayahu et al., 1988; Litophytoii tirhoreiim: Benayahu et
al.. 1992; Benayahu. 1997; Ant/ieliu glauca: Benayahu and Sports and Culture. Japan, and by the Sasagawa Scientific
Schleyer. 1998), and the scyphozoan Linuche unguiculata Research Grant from the Japan Science Society.
(Montgomery and Kremer, 1995). It has been suggested
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