*Corresponding author.

Invertebrate Reproduction and Development, 51:3 (2008) 119–126 119Balaban, Philadelphia/Rehovot

0168-8170/08/$05.00 © 2008 Balaban

Female gametes of the black coral Cirrhipathes cfr. anguina(Anthozoa, Antipatharia) from the Indonesian Marine Park

of Bunaken

ELDA GAINO* and FRANCESCA SCOCCIADipartimento di Biologia Cellulare e Ambientale, Università degli Studi di Perugia, Via Elce di Sotto, 06123 Perugia, Italy

Tel. +39 075 5855702; Fax: +39 075 5855733; email: gaino@unipg.it

Received 12 December 2007; Accepted 7 January 2009

Summary

Female polyps of Cirrhipathes cfr. anguina were collected in August 2007 from the coral reef ofSiladen Island in the Bunaken Marine Park (North Sulawesi, Indonesia). The primary transversemesenteries, which separate the adjacent lateral tentacles from one another, were filled withoocytes developing within the mesoglea. Cross-sections of the oral region revealed that younggametes were located both at the emergence of the primary transverse mesenteries from thegastroderm lining the coelenterons, and at the point where the mesenteries were linked to thepharynx. By contrast, at a lower level, where the mesenteries gave rise to mesenterial filaments,young gametes were limited to the initial part of the mesenteries. Oocytes were lodged in individualchambers, and were separated from one another by mesogleal elements. On occasion, oocytes werefound in proximity to cells with short microvillous-like processes that interdigitated with thoseemerging from the oocyte surface. Oocytes varied in size according to the stage of the vitello-genetic process, the largest measuring 125 µm in diameter. Their cytoplasm was enriched withinclusions of three main kinds: lipids, yolk and both rod- and ovoid-shaped bodies. These bodieswere characterized by an electron-dense core with a dotted appearance, surrounded by a sequenceof tightly-packed fibrils arranged in parallel fashion. These bodies pushed the oocyte membraneslightly outwards, giving rise to irregular “spiny” protrusions. In addition to the microvillous-likeextensions, oocytes also showed short lobose pseudopodia; both are probably involved in nutrientuptake. No other membrane specializations were observed in the oocytes, the general morphologyof which seemed to be coherent with the assumption that they had reached their final stage ofgrowth. Investigations on the reproduction of the black coral may provide insights into thefeasibility of transplanting fertile colonies into areas where they have been decimated byanthropogenic pressure.

Key words: Sexual reproduction, female polyps, oocyte ultrastructure

Introduction

Little is known about the reproductive biology ofAntipatharia. The lack of information on this topic is

mainly due the bathymetric distribution of the blackcoral, as the great depth at which colonies commonlydevelop makes sampling difficult (Grigg, 1965;

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Opresko, 1972,1976; Grigg and Opresko, 1977; Golbergand Taylor, 1989; Montgomery, 2002).

Shallow colonies offer better opportunities forresearch into sexual reproduction (Grange, 1986; Parkeret al., 1997), and in several groups of Anthozoa, sexratio, gametogenesis and fecundity have been docu-mented (Clark and Dewel, 1974; Chadwick-Furman etal., 2000; Fautin and Mariscal, 1991; Ryland andBabcock, 1991; see review in Fautin, 2002).

Some interesting research into the fecundity offemale colonies was carried out by Parker et al. (1997)on Antipathella fiordensis, a species endemic in theNew Zealand fiords, while Molodtsova (2006) inves-tigated the possible coexistence of dimorphic polypsin the genus Heliopathes: vegetative polyps located onthe stem and lateral pinnules; generative polypsarranged on subpinnules of the anterior row. In two newspecies of the genus Hexapathes, observed that gonad-bearing polyps were found in different areas ofthe colonies from those occupied by polyps withoutgonads.

Among Antipatharia living inside the coral reef ofthe Bunaken Marine Park in Indonesia, coloniesbelonging to the genus Cirrhipathes can be found atdepths as shallow as 5 m (Tazioli et al., 2007), a con-dition that has facilitated investigation of the repro-duction of some of these colonies. The systematics ofthis genus is problematic and the morphology of theskeleton is still under investigation. However, it hasemerged that three undetermined, but probably newspecies, live in sympatry in the same environment(Tazioli et al., 2007).

Knowledge of the reproduction of cnidarians isaccumulating rapidly, although the great plasticity ofthese animals (Boero and Bouillon, 1987, 1989), alongwith their ability to adapt their life-cycle to environ-mental conditions, makes aspects of their reproductionless informative than expected. By contrast, thestructural details of the gametes reflect evolution and arephylogenetically relevant (Fautin, 2002). In this regard,investigation of the male polyps of Cirrhipathes sp.(Gaino et al., 2008), tentatively attributed to Cirrhi-pathes cfr. anguina in the discussion section of thepaper, has shown that sperm have some peculiarultrastructural traits, thereby confirming that gameteshave a set of characteristics that can be useful fortaxonomic and phylogenetic purposes (see review inSchmidt and Zissler, 1979). Additional investigations onthe skeletal structure of Cirrhipathes corroborated thetaxonomic attribution to Cirrhipathes cfr. anguina.

The finding in the female colonies of some fertilepolyps, which are easily distinguishable from non-fecund individuals by the bulge at the base of the lateraltentacles, prompted us to undertake an additional study

on the reproduction of this black coral, with the aim ofinvestigating, mainly at the ultrastructural level, thecharacteristics of the female gametes.

Materials and Methods

Colonies of Cirrhipathes cfr. anguina are mostabundant at depths below 25–30 m. In August 2007, asingle apical fragment (10 cm in length) bearing somelarge polyps was cut from five colonies located alongthe reef of Siladen Island (Sulawesi, Indonesia, Siladen,1E37N37.04O N; 124E48N12.10O E). On the basis of ourpreliminary observations, which had shown that theapical part of these monopodial colonies containedfertile polyps, we carried out our investigation on thesepolyps.

When brought to the surface, each of the fivefragments was subdivided into two portions, each of5 cm in length. One portion was preserved in alcohol at70%, for diagnosis; the other was fixed for 12 h in 2.5%glutaraldehyde buffered with filtered seawater (pHadjusted to 7.5–7.8, with 0.1 N NaOH), for ultra-structural investigation. The samples were repeatedlyrinsed in the same buffer and stored at 4EC before beingtransferred to our laboratory in Italy.

From each part previously fixed for histologicalobservations, two of the largest polyps were treated forobservation by scanning electron microscopy (SEM). Atotal of 10 polyps were observed. For this procedure,these polyps were immersed in liquid nitrogen, fracturedand then dehydrated in an ethanol series. Specimenswere subsequently critical-point dried in a CPD 030Bal-Tec critical-point dryer (Bal-Tec Union, Liechten-stein), mounted on stubs with silver conducting paint,sputter-coated with gold-palladium in an EmitechK550X (Emitech, Ashford, England) sputterer, andobserved with a Philips XL30 electron microscope(Philips, Eindhoven, The Netherlands) at an acceleratingvoltage of 18kV.

For transmission electron microscopy (TEM), twopolyps per portion (10 polyps in total) were post-fixed in1% osmium tetroxide in an artificial seawater buffer for1 h at 4EC. After repeated washing in the same buffer,the polyps were dehydrated in a series of ethanoldilutions and embedded in an Epon-Araldite mixture.Ultrathin sections were cut on a Leica EM UC6 ultracut(Leica Microsystem, Vienna, Austria), collected onformvar-coated copper grids, stained with uranyl acetateand lead citrate, and examined with a Philips EM 208transmission electron microscope (Philips, Eindhoven,The Netherlands).

For light microscopy, 1 µm sections were cut fromthe same blocks as those used for electron microscopy

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by means of a Leica DC 300 F ultracut (Leica Micro-systems, Rijswijk, The Netherlands), stained with 0.5%toluidine blue and observed with a Leica microscope(Leica LMS Holdings, Wetzlar, Germany).

Results

Polyps of Cirrhipathes cfr. anguina were uniformlydistributed along and around the axial skeleton. The sixtentacles encircling the mouth were of two sizes: thefour lateral tentacles were shorter than the two sagittalones, which extended perpendicularly to the skeletalaxis (Fig. 1).

In the fecund polyps of Cirrhipathes cfr. anguina,the lateral tentacles bulged markedly in their basalregion (Fig. 2a), owing to the presence of oocytes,which were easily detectable in transparency on accountof their orange colour. By contrast, the morphology ofthe sagittal tentacles, which were longer than the lateralones, remained unchanged.

Cross-sections that included the oral region showedthe presence of numerous thin, short mesenteries sup-porting the pharynx, and primary transverse mesenteriesinterposed between the lateral tentacles. These primarymesenteries were markedly convoluted along theirlength and full of oocytes (Fig. 2b). Oocytes varied insize from 30 µm to 125 µm, according to their phase ofmaturation.

The connection of the primary mesenteries to thepharynx was very thin, as evident in fractured specimensobserved by means of scanning electron microscopy.However, these mesenteries dramatically enlarged toinclude maturing oocytes (Fig. 2c), which werearranged very close to one another (Fig. 2d).

Fig. 1. Morphology of the polyps of Cirrhipathes cfr.anguina. Note the arrangement of the lateral (1–4) andsagittal (5,6) tentacles encircling the mouth (M). (Photo:Massimo Boyer/edge-of-reef.com).

Magnification of histological sections of the oralregion of the polyp provided evidence that oocytegrowth mainly proceeded from the initial regions of themesentery towards its inner core (Fig. 2e). Younggametes were located in the mesoglea of the mesentery,

Fig. 2. Female polyps of Cirrhipathes cfr. anguina. a: Gravidpolyp showing one of the lateral tentacles enlarged by thegrowing oocytes, which are faintly visible in transparency(arrow). b: Histological cross-section of the oral region. Thecentral pharynx (Ph), which is supported by short mesen-teries, is flanked by the sagittal (5,6) and lateral (1–4)tentacles. Note that the primary transverse mesenteries,which separate the lateral tentacles 1 and 3 from 2 and 4, aremarkedly convoluted owing to the presence of developingoocytes. c: SEM view of a fractured fecund polyp showingthe primary transverse mesentery (Ms), which dramaticallyenlarges after emerging from the gastroderm that lines thepharynx (Ph). The convolution of the mesentery is due to thepresence of the maturing oocytes within the mesoglea.d: SEM view of a fractured fecund polyp showing thearrangement of the oocytes (Oo) within the mesoglea of alateral primary transverse mesentery. Gd, gastroderm; Ph,pharynx. e: Histological cross-section showing a lateralprimary transverse mesentery (arrows) interposed betweentwo adjacent lateral tentacles. Note the young gametes (YG)located in the regions where the mesentery originates fromthe gastroderm (Gd).

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Fig. 3. Female polyps of Cirrhipathes cfr. anguina. a: Histo-logical cross-section of a lower level of the pharynx (Ph).The convolutions of the mesentery represent the mesenterialfilaments (MF). Young oocytes (arrows) tend to gatherwithin the mesoglea proximal to the origin of the mesenteryfrom the gastroderm (Gd) lining the body cavity. b: SEMview of a fractured fecund polyp showing growing oocytes(Oo) within the mesoglea. Note that the oocytes areseparated from one another by an individual mesoglealenvelope (arrows). c, SEM view of a fractured oocyte whosecytoplasm is filled with inclusions that push the nucleus (N)aside.

both at the point where the mesentery emerged from thegastroderm lining the body wall and at the point whereit joined the pharynx. By contrast, cross-sections inter-secting the primary mesenteries at a lower level showedimmature germ cells limited to the region of themesentery opposite to the body wall; in the regionproximal to the pharynx, no gametes were present, andmesentery convolutions gave rise to the mesenterialfilaments (Fig. 3a).

Mesogleal elements separated the growing oocytesfrom one another (Fig. 3b). The level of oocyte maturitywas marked by the amount of stored material thatpushed the nucleus slightly aside (Fig. 3c).

Facing oocytes (Fig. 4a), in the region proximal tothe border of the mesentery, were separated from oneanother by vacuolated cells that delimited the mesenteryand penetrated inwards, running close to the oocytesurface (Fig. 4b). A thin fibrillar matrix surrounded thegrowing oocytes and included the short, irregular,microvillous-like projections emerging from the oocyteperiphery (Fig. 4c). On occasion, mesogleal cellsapproached the oocyte surface, and the space in betweenwas filled with microvillous-like projections protrudingfrom both cell types (Fig. 4d), which interdigitated inthis narrow space (Fig. 4d inset).

In some inner mesenterial regions, mesogleal cellshad long cytoplasmic extensions, which formed an inter-cellular network (Fig. 4e), and, on occasion, reached thecells located close to the oocyte surface (Fig. 4f).

While microvillous-like projections were usuallyseen when oocytes were close to other cell types, shortlobose pseudopodia bulged from the oocyte where itssurface was delimited by a fibrillar coat (Fig. 5a).Lobose pseudopodia usually lacked inclusions (Fig. 5b),which instead filled the oocyte cytoplasm. Oocyteinclusions consisted of at least three kinds of material:lipids, appearing as electron-lucent vesicles; proteinyolk, appearing as electron-dense granules of varyingsize and shape; rod- and ovoid-shaped bodies (Fig. 5a).These bodies tended to accumulate just below theoocyte membrane (Fig. 5c) and were characterized by acentral electron-dense core, surrounded by a sequenceof tightly-packed fibrils arranged in parallel fashion(Fig. 5d). These bodies were also found in the lobosepseudopodia (Fig. 5e) and on occasion pushed the cellmembrane outwards, thereby forming spiny protrusions(Fig. 5f).

Discussion

The Antipatharia studied so far are dioecious but,owing to the absence of gonads in the polyps, noobvious external morphological differences allow thesex to be distinguished (Parker et al., 1997). In thereproductive period, the general shape of the fertile malepolyps does not differ from that of the unfertile ones,whereas gravid females are larger than non-gravid ones,owing to the presence of the growing oocytes (Shick,1991; Chadwick-Furman et al., 2000). In particular, inthe black coral Antipathella fiordensis, it has beenobserved that the polyp tends to change in colour, afeature that reflects changes in reproductive conditions(Parker et al., 1997).

Underwater inspection of Cirrhipathes cfr. anguinacolonies enables gravid females to be easily dis-tinguished owing to the bulging of the lateral tentaclesof the polyp.

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Fig. 4 TEM images of female polyps of Cirrhipathes cfr. anguina. a: Adjacent oocytes (Oo) separated by vacuolated cellspenetrating inwards (arrow) from the mesenterial border. b: Detail of a vacuolated penetrating cell that runs close to thesurface of adjacent oocytes (Oo). c: Irregular microvillous-like projections (arrows), which penetrate the fibrillar matrix (FM)separating the oocyte (Oo) from the mesogleal cells (MC). d: Interdigitation via microvillous-like projections (arrows)emerging from both oocyte (Oo) and mesogleal cell (MC) surfaces. Note the detail of the interdigitation (inset). e: Mesoglealcells situated in proximity to the oocyte (Oo) have long cytoplasmic extensions (arrows), which form an intercellular network.f: Mesogleal cells with long, thin cytoplasmic extensions (arrows) proximal to another mesogleal cell (MC), which is locatedclose to the oocyte (Oo) surface.

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Fig. 5. TEM images of the oocyte of Cirrhipathes cfr. anguina. a: Peripheral region of the oocyte displaying lobosepseudopodia (arrows) and diversification of the stored material consisting of electron-lucent lipid vesicles (Ve), electron-denseprotein yolk granules (YG) and both rod- and ovoid-shaped bodies (Bd). b: Detail of a lobose pseudopodium devoid ofinclusions. c: rod- and ovoid-shaped bodies (Bd) close to the egg surface. d: Magnification of an ovoid-shaped body showingits central core (CC) surrounded by tightly-packed fibrils arranged in parallel fashion (F). e: An ovoid-shaped body within alobose pseudopodium of an oocyte (arrow). f: An ovoid-shaped body (arrow) pushes the oocyte membrane outwards, givingthe oocyte surface a spiny appearance.

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Like many other colonial animals, black coral arebranched or monopodial, and are considered to bemodular organisms. In modular organisms, the peri-pheral modules are typically younger, non-reproductiveand specialized for defence (Hall and Hughes, 1996).This pattern contrasts with the condition observed in themonopodial black coral, in which the fertile polyps areinstead located in the apical region of the colonies.

In Cirrhipathes cfr. anguina, gametogenetic areasare limited to the mesenteries that divide the adjacentlateral tentacles; this confirms the involvement of thesestructures in sexual reproduction, in agreement withprevious observations that spermatocyst differentiationoccurs exclusively inside these mesenteries (Brook,1889; Gaino et al., 2008).

In Anthozoa, germ cells originate in the gastrodermof the primary mesenteries, from which they move to themesoglea for their ensuing differentiation (Fautin andMariscal, 1991). In Cirrhipathes cfr. anguina, earlyoocytes are preferentially observed at the point wherethe primary mesentery connects with both the pharynxand the body wall, whereas, at a lower level, they arelimited to the region without mesenterial filaments.Consequently, mature oocytes coexist with the youngerones, thereby suggesting that egg spawning may occurrepeatedly in successive waves.

Yolk accumulates in the growing oocytes in the formof different kinds of inclusions. The microvillousprojections and the lobose pseuodopodia emerging fromthe oocyte surface may be involved in the uptake ofnutrients from the mesoglea. Indeed, Antipatharia lacktrophonema, a structure involved in nutrition, which inother Anthozoa is associated with gametes undergoingvitellogenesis (Schmidt and Schäfer, 1980). As in otherAnthozoa, yolk granules predominate, and are dispersedamong inclusions with a fibrillar border that delimits adark core, as already described in Antipatharia (Schmidtand Schäfer, 1980). In Endomyarian oocytes, theseinclusions, which are defined as rod-shaped, may berelated to peculiar cytospines that strengthen the oocytesurface (Dewel and Clark, 1974). As reported by(Schmidt and Schäfer, 1980), in Cirrhipathes cfr.anguina, too, these inclusions, which originated in theooplasm migrate to the periphery. The outermost surfaceof the oocytes of the Cirrhipathes cfr. anguina so farinvestigated has no actual cytospines, but the rod- orovoid-shaped inclusions that push the cell membraneslightly outwards could represent similar structures.

It is reasonable to suppose that the describedorganisation of the oocytes concerns mature elementsready to be released. Indeed, no more advanced phasesof differentiation were observed, in spite of numerousobservations carried out at the ultrastructural level.

We have no data on the completion of meioticdivision before spawning, as no polar bodies wereobserved. It is worth mentioning that in a Japanese seaanemone, the release of a polar body was observed justbefore external fertilisation carried out in experimentalconditions (Fukui, 1991).

No developing embryos or larvae were found insidethe polyps. This corroborates the assumption that fertili-zation occurs externally, a process that leads to plank-tonic development of larvae, which is by far the mostcommon mode of reproduction in tropical corals follow-ing broadcast spawning of gametes (Harrison et al.,1984; Babcock et al., 1986; Ryland and Babcock, 1991;Harrison and Wallace, 1990; Chadwick-Furman et al.,2000; Miller and Mundy, 2003).

Reef habitats display various level of degradation asa result of anthropogenic pressure. In addition to yield-ing biological data, investigation of the reproduction ofCirrhipathes cfr. anguina, albeit in a restricted area,may provide insights into how such pressure can bewithstood (Rinkevich, 1995). In this regard, trans-planting gravid colonies, or rearing larvae and collectingthem on artificial substrates before transplantation ontothe reef, could help to replenish black coral stocks inareas where they have been decimated (Romero, 1997;Montgomery, 2002; Petersen et al., 2006). The ultimateaim is to safeguard the biodiversity of the coral reefs,which are unique marine ecosystems that should bepreserved for future generations.

Acknowledgements

We thank Prof. Giorgio Bavestrello and Dr. MarziaBo of the University of Ancona for their helpful supportin sampling and determination of the black coralcolonies investigated in the present study. This researchwas carried out within the framework of the cooperationagreement between the Faculty of Fishery and MarineSciences of the Sam Ratulangi University, Manado,Università Politecnica delle Marche and Universitàdegli Studi di Perugia.

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