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Sperm ultrastructure of a member of the black coral family Aphanipathidae: Rhipidipathes reticulata (Anthozoa, Antipatharia)
Tissue and Cell 42 (2010) 391–394
Contents lists available at ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Sperm ultrastructure of a member of the black coral family Aphanipathidae: Rhipidipathes reticulata (Anthozoa, Antipatharia) E. Gaino ∗ , F. Scoccia Dipartimento di Biologia Cellulare e Ambientale, Università degli studi di Perugia, Via Elce di Sotto, 06123 Perugia, Italy
a r t i c l e
i n f o
Article history: Received 26 July 2010 Received in revised form 4 October 2010 Accepted 11 October 2010 Available online 19 November 2010 Keywords: Indonesian coral reef Cnidaria Male gametes Sexual reproduction
a b s t r a c t Fertile male polyps of three colonies of the black coral Rhipidipathes reticulata (Aphanipathidae) from Togian Islands (Indonesia) have been the source of the sperm investigated at ultrastructural level, in order to compare their organization with that of other members belonging to the family Antipathidae and Myriopathidae. The extension of the studies to a representative of the family Aphanipathidae stresses once more the structural similarity of the male gametes in antipatharians. A sketch of the sperm model reports the similarity and differences in the examined taxa. Among the micro-characters, the cup-like body linked to the pericentriolar apparatus, is exclusive of the antipatharians. Other inclusions concern the more common pro-acrosomal vesicles or the acrosomelike structure observed only in Antipathella subpinnata and Myriopathes japonica. Lipid vesicles are occasionally present. A typical inclusion, the electron-dense content of which has a C-shaped configuration, is restricted to Rhipidipathes reticulata and is associated to the cup-like body or to the mitochondrion. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction In black coral, the primary transversal mesenteries interposed between the lateral tentacles of the fertile polyps are involved in the sperm differentiation, which takes place within the spermatic cysts that gradually enlarge filling up the mesentery (Parker et al., 1997; Gaino et al., 2008; Gaino and Scoccia, 2009). Sperm maturation proceeds from the periphery towards the central region where sperm are arranged in row (Gaino et al., 2008; Gaino and Scoccia, 2009; Gaino and Scoccia, in press, 2010). Specimens from the Indonesia and Mediterranean Sea have been the object of our previous investigations carried out with the aim to gather data on the ultrastructural organization of the male gametes in these antipatharians in order to outline their reproductive biology (review in Gaino and Scoccia, in press, 2010). In spite of the different morphologies showed by the colonies of the members belonging to a same family, the fine structure of the male gametes appears to be almost similar with the exception of some details, such as the occurrence of discrete proacrosomal vesicles that, in some genera, as in Myriopathes sp. and Antipathella subpinnata, merge to give rise to an acrosome-like structure. The aim of the present investigation was to add new information on the black coral male gametes by studying the so far unknown
∗ Corresponding author. Tel.: +39 0755855702. E-mail address: [email protected] (E. Gaino). 0040-8166/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2010.10.002
sperm morphology of Rhipidipathes reticulata (ESPER, 1795) (family Aphanipathidae).
2. Material and methods Some polyps from fragments were cut off with scissors from three different colonies of Rhipidipathes reticulata living in Togian Islands (Tomini Gulf, Sulawesi, Indonesia), and placed in sealed containers underwater. The samples were transferred from the sealed containers into Petri dishes and fixed for 12 h in 2.5% glutaraldehyde buffered with filtered sea-water (adjusted to a final pH of 7.5–7.8 with NaOH 0.1 N) and repeatedly rinsed in the same buffer. Underwater photographs coupled with examination of a part of the skeleton allowed taxonomical identification. Five polyps from each of the colonies were processed for transmission electron microscopy investigations. Selected material was post-fixed in 1% osmium tetroxide in an artificial sea-water buffer for 1 h at 4 ◦ C, then repeatedly washed in the same buffer, dehydrated in an incremental alcohol series and propylene oxide, and finally embedded in an Epon–Araldite mixture. Semi-thin sections, 0.5–1.0 mm thick, were cut on a Leica DC 300 F Ultracut (Leica Microsystem AG, Rijswijk, The Netherlands), stained with toluidine blue and observed under a Leica microscope (Leica LSM Holings GmbH, Wetzlar, Germany). Ultrathin sections were cut with the same Ultracut device, collected on copper grids, stained with uranyl acetate and lead citrate, and examined with a Philips EM 208 electron microscope.
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E. Gaino, F. Scoccia / Tissue and Cell 42 (2010) 391–394
Fig. 1. The black coral Rhipidipathes reticulata. (a) Part of a colony showing the fan-shaped configuration of the anastomosing branches (arrows); (b) ultrastructural sections of a fertile polyp showing the linear arrangement of the sperm within a spermatic cyst; (c) close up of a sperm showing its fine organization consisting in pro-acrosomal vesicles (arrowhead), nucleus (N), cup-like body (CB) and a single mitochondrion (M). The tail (T) is connected to the distal centriole (double arrow), which in turns is linked to the cup-like body; (d) apical region of the sperm showing the sequence of the pro-acrosomal vesicles (arrowheads); (e), circular profile of the peri-centriolar apparatus whose branched arms connect the distal centriole to the electron-dense granules bordering the perimeter of the cup-like body (CB); (f), tangential section of a region of the cup-like body showing its pattern of electron-dense stripes that, in section, correspond to the electron-dense granules; (g), section of the apical region of an immature
E. Gaino, F. Scoccia / Tissue and Cell 42 (2010) 391–394
393
Fig. 2. Sketch of the sperm model in the examined taxa of antipatharians belonging to Antipathidae, Myriopathidae and Aphanipathidae, focusing the different microcharacters. PAC, pro-acrosomal vesicles; N, nucleus; M, mitochondrion; LD, lipid droplet; CB, cup-like body; T, tail. Note in the frames: the acrosome-like structure of Myriopathes japonica and Antipathella subpinnata; the C-shape inclusion of Rhipidipathes reticulata.
3. Results The colonies of Rhipidipathes reticulata have a typical fan-shaped configuration and the anastomosis of their branches grows in an almost two-dimensional plane (Fig. 1(a)). The polyps are very small and their six tentacles (two sagittal longer than the four lateral ones) are typically grouped together around the mouth and show a rounded tip (Tazioli et al., 2007). Cross-sections of fertile polyps showed that the two transversal primary septa, each separating the lateral tentacles from one another, were involved in spermiogenesis. Male gamete differentiation took place inside the spermatic cysts filling the primary septa. The periphery of each spermatic cyst was delimited by a coat of cells elongated in shape resting on a fibrillar envelope. Inside the spermatic cysts mature sperm were arranged in row and their long tails formed bundles in between (Fig. 1(b)). The close up of the mature sperm (Fig. 1(c)), the head of which measured about 2.5 m, showed electron-dense vesicles located at its apex, and a large and fairly round nucleus (diameter ∼1–1.5 m) apparently flanked by two mitochondria; however, in serial sections, these proved to be two portions of a single mitochondrion partially encircling the nucleus. Some sections revealed that the apical vesicles were placed in sequence along the apical region of the nucleus (Fig. 1(d)). The sperm also included a large, electron-
dense cup-like body of about 800 nm along its long axis and about 500 nm along the shorter one. The perimeter of the cup-like body was delineated by regularly interspaced electron-dense granules (Fig. 1(c)). A thin cytoplasmic sheet was interposed between the mitochondrion and the cup-like body. The two centrioles, proximal and distal, were almost coaxial. The axonema of the tail originated from the distal centriole. The cup like-body was connected to the distal centriole through a complex pericentriolar apparatus consisting of nine arms arranged in a radial pattern, each of them terminally branching into three processes (Fig. 1(e)). The branches were associated with electron-dense granules located along the basal region of the cup-like body (Fig. 1(e)). In tangential sections it emerged that these granules were sections of electron-dense stripes decorating the surface of the cup-like body (Fig. 1(f)). Immature sperm showed peculiar inclusions, ranging from 200 to 250 nm, the section of which showed an electron-dense peripheral region slightly eccentric with respect to its probably lipid inner electron-lucent part. This organization gave them a typical C-shaped morphology (Fig. 1(g)). These inclusions conserved their configuration in mature sperm (Fig. 1(h)), where they were located in the region between the cup-like body and the mitochondrion (Fig. 1(h)) or between the mitochondrion and the nucleus (Fig. 1(i)). On the basis of the analysis carried out on the ultrastructure of the sperm from various families examined so far, a schematic
germ cell showing inclusions whose electron-dense content has a typical C-shaped configuration (arrows); (h), cross section of the inclusion with the typical C-shaped configuration of its content (arrow). Note the location between the sperm mitochondrion (M) and the cup-like body (CB); (i) tangential section of an inclusion with the typical C-shaped content. Note its location between the nucleus (N) and the mithocondrion (M).
394
E. Gaino, F. Scoccia / Tissue and Cell 42 (2010) 391–394
diagram was outlined to point out the main differences among these gametes, here including the sperm of Rhipidipathes reticulata for a comparison (Fig. 2). It emerged that the essential differences pertained to some micro-characteristics, such as: the presence of pro-acrosomal vesicles or acrosomal-like structure; occasional lipid droplets; C-shaped inclusions so far observed only in the taxon here investigated.
4. Discussion The lack of information on the reproductive biology of black coral is partially due to the bathymetric distribution of these organisms, which typically grow at high depth (Parker et al., 1997), and partially to the difficulty in distinguishing unfertile colonies from the fertile ones in the natural environment. In fact, no obvious external morphological characteristics exist apart from a slightly enlargement of the polyps due to spermatic cysts differentiation or egg accumulation within the mesentery septa. In this regard, the pigmented stored material filling up the egg cytoplasm can help in distinguishing fertile from non-fertile female polyps. The study of the sperm of Rhipidipathes reticulata (Aphanipathidae) confirms the similarity of the sperm in antipatharians, a feature already stressed in previous investigations on various members of the tropical black coral, such as: Cirrhipathes anguina, C. spiralis, Cirrhipathes sp., Antipathes cfr. simplex, Sticopathes sp. (Antipathidae); Myriopathes japonica, Cupressopathes pumila (Myriopathidae) (Gaino and Scoccia, in press). Also, in the Mediterranean Antipathella subpinnata (Myriopathidae) the sperm organization does not differ significantly from that of the above listed taxa (Gaino and Scoccia, 2010). The common characters are: the single mitochondrion; the cup-like body connected to the complex pericentriolar apparatus, which in turn is linked to the distal centriole; the pro-acrosomal vesiscles. By contrast, Antipathella subpinnata and Myriopathes japonica share an acrosome-like structure instead of the pro-acrosomial vesicles. The occurrence of this kind of sperm vesicles in the apical region is shared with hydrozoans and scyphozoans, thus suggesting to be a primitive acrosome (Hinsch, 1974). The inclusions with a C-shaped electron-dense content observed in Rhipidipathes reticulata seem to be a characteristic of this taxon. These inclusions are already evident in immature sperm and are kept in mature sperm, thus representing a marker useful to trace their origin from the cells bordering the periphery of the spermatic cysts. In contrast with the similar organization observed in antipatharian sperm, the comparison between eight scleractinian corals species identified different types of sperm, mainly on the basis of the nuclear shape (Steiner, 1993). However, a distinction among families seems to be more difficult when they show the same sexual condition because the reproductive pattern prevails on sperm morphology (Steiner and Cortès, 1996). It seems acceptable that antipatharian sperm similarity reflects hybridization, as suggested by the degree of genetic variation observed by Lapian et al. (2007) in several species of Cupressopathes and Myriopathes, and in other Hexacorallia (Diekmann et al., 2001; Frank and Mokady, 2002). Schmidt and Zissler (1979), by comparing the sperm of Anthozoa, reported the cup-like body (named “Ringkörper”) as an apomorphic character exclusive of Antipatharians. This assumption emerged from the observations of specimens belonging to a single genus of the family Antipathidae and was then found by us in other representatives of Antipathidae and Myriopathidae. The study of the sperm morphology in a member of Aphanipathidae confirms that this structure is widespread within the order. Even though there are no data sustaining the actual function of the cup-like body, the connection to the distal centriole through the
pericentriolar apparatus is coherent with the hypothesis that it is involved in tail movement (Gaino et al., 2008). In cnidarian sperm, the presence of a striated pericentriolar process complex associated with the distal centriole has been frequently observed (Hinsch, 1974; see references in Kleve and Clark, 1980; Corbelli et al., 2003). Structural observations of the filamentous nature of the pericentriolar processes in sperm of an hydrozoan, together with the presence of actin, corroborate its contractile function, thus participating in tail movement and facilitating the directional motility in response to egg-released chemoattractants. The connection of the pericentriolar apparatus to the cup-like body, restricted to antipatharians, is very unusual and needs to be better investigated in order to understand if it has an actual role in sperm motility. Investigations on the sperm organization of antipatharians can be included in a broader context aimed to know how sexual reproduction can contribute to assure biological diversity, thus offering to these inhabitants of the barrier reefs better opportunity to respond to the environmental stresses in this fragile ecosystem. Also, according to Harrison and Wallace (1990), knowledge of reproductive biology represents a prerequisite for ecological studies and for directing policy intervention for the conservation of the coral communities. Acknowledgements We are pleased to thank Dr. Massimo Boyer for his helpful collaboration in sampling the specimens and for the underwater photos. We express our gratitude to Dr. Marzia Bo for the confirmation of the specific attribution of the specimens. References Corbelli, A., Avian, A., Marotta, M., Ferraguti, M., 2003. The spermatozoon of Carybdea marsupialis (Cubozoa, Cnidaria). Invertebr. Reprod. Dev. 43, 95–104. Diekmann, O.E., Bak, R.P.M., Stam, W.T., Olsen, J.L., 2001. Molecular genetic evidence for probable reticulate speciation in the coral genus Madracis from a Caribbean fringing reef slope. Mar. Biol. 139, 221–233. Frank, U., Mokady, O., 2002. Coral biodiversity and evolution: recent molecular contributions. Can. J. Zool. 80, 1723–1734. Gaino, E., Scoccia, F., 2009. Release of sperm clusters in spheres by the black coral Cupressopathes pumila (Anthozoa, Antipatharia). Coral Reefs 28, 851–857. Gaino, E., Scoccia, F., in press. Ultrastructural investigation of the sexual reproduction of some members of the black coral: a review. In: Méndez-Vilas, A., Díaz, J. (Eds.), Microscopy: Science, Technology, Applications and Education. Gaino, E., Scoccia, F., 2010. Gamete spawning in Antipathella subpinnata (Anthozoa, Antipatharia): a structural and ultrastructural investigation. Zoomorphology, doi:10.1007/s00435-010-0112-x. Gaino, E., Bo, M., Boyer, M., Scoccia, F., 2008. Sperm morphology in the black coral Cirrhipathes sp. (Anthozoa, Antipatharia). Invertebr. Biol. 45, 249–258. Harrison, P.L., Wallace, C.C., 1990. Reproduction, dispersal and recruitment of scleractinian corals. In: Dubinsky, Z. (Ed.), Ecosystems of the World: Coral Reefs, vol. 25. Elsevier, Amsterdam, the Netherlands, p. 133-207. Hinsch, G.W., 1974. Comparative ultrastructure of cnidarians sperm. Am. Zool. 14, 457–465. Kleve, M.G., Clark Jr., W.H., 1980. Association of actin with sperm centrioles: isolation of centriolar complexes and immunofluorescent localization of actin. J. Cell Biol. 86, 87–95. Lapian, H.F.N., Barucca, M., Bavestrello, G., Biscotti, M.A., Bo, M., Canapa, A., Tazioli, S., Olmo, E., 2007. A systematic study of some Black Corals species (Antipatharia, Hexacorallia) based on rDNA internal transcribed spacers sequences. Mar. Biol. 151, 785–792. Parker, N.R., Mladenov, P.V., Grange, K.R., 1997. Reproductive biology of the antipatharian black coral Antipathes fiordensis in Doubtful Sound, Fiordland, New Zealand. Mar. Biol. 130, 11–22. Schmidt, H., Zissler, D., 1979. Die Spermien der Anthozoen und ihre phylogenetische Bedeutung. Zoologica 129, 1–98. Steiner, S.C.C., 1993. Comparative ultrastructural studies on scleractinian spermatozoa (Cnidaria Anthozoa). Zoomorphology 113, 129–136. Steiner, S.C.C., Cortès, J., 1996. Spermatozoan ultrastructure of scleractinian corals from eastern Pacific: Pocilloporidae and Agariciidae. Coral Reefs 15, 143–147. Tazioli, S., Bo, M., Boyer, M., Rotinsulu, H., Bavestrello, G., 2007. Ecological observations of some common antipatharian corals in the marine Park of Bunaken (North Sulawesi Indonesia). Zool. Studies 46, 227–241.
Contents lists available at ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Sperm ultrastructure of a member of the black coral family Aphanipathidae: Rhipidipathes reticulata (Anthozoa, Antipatharia) E. Gaino ∗ , F. Scoccia Dipartimento di Biologia Cellulare e Ambientale, Università degli studi di Perugia, Via Elce di Sotto, 06123 Perugia, Italy
a r t i c l e
i n f o
Article history: Received 26 July 2010 Received in revised form 4 October 2010 Accepted 11 October 2010 Available online 19 November 2010 Keywords: Indonesian coral reef Cnidaria Male gametes Sexual reproduction
a b s t r a c t Fertile male polyps of three colonies of the black coral Rhipidipathes reticulata (Aphanipathidae) from Togian Islands (Indonesia) have been the source of the sperm investigated at ultrastructural level, in order to compare their organization with that of other members belonging to the family Antipathidae and Myriopathidae. The extension of the studies to a representative of the family Aphanipathidae stresses once more the structural similarity of the male gametes in antipatharians. A sketch of the sperm model reports the similarity and differences in the examined taxa. Among the micro-characters, the cup-like body linked to the pericentriolar apparatus, is exclusive of the antipatharians. Other inclusions concern the more common pro-acrosomal vesicles or the acrosomelike structure observed only in Antipathella subpinnata and Myriopathes japonica. Lipid vesicles are occasionally present. A typical inclusion, the electron-dense content of which has a C-shaped configuration, is restricted to Rhipidipathes reticulata and is associated to the cup-like body or to the mitochondrion. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction In black coral, the primary transversal mesenteries interposed between the lateral tentacles of the fertile polyps are involved in the sperm differentiation, which takes place within the spermatic cysts that gradually enlarge filling up the mesentery (Parker et al., 1997; Gaino et al., 2008; Gaino and Scoccia, 2009). Sperm maturation proceeds from the periphery towards the central region where sperm are arranged in row (Gaino et al., 2008; Gaino and Scoccia, 2009; Gaino and Scoccia, in press, 2010). Specimens from the Indonesia and Mediterranean Sea have been the object of our previous investigations carried out with the aim to gather data on the ultrastructural organization of the male gametes in these antipatharians in order to outline their reproductive biology (review in Gaino and Scoccia, in press, 2010). In spite of the different morphologies showed by the colonies of the members belonging to a same family, the fine structure of the male gametes appears to be almost similar with the exception of some details, such as the occurrence of discrete proacrosomal vesicles that, in some genera, as in Myriopathes sp. and Antipathella subpinnata, merge to give rise to an acrosome-like structure. The aim of the present investigation was to add new information on the black coral male gametes by studying the so far unknown
∗ Corresponding author. Tel.: +39 0755855702. E-mail address: [email protected] (E. Gaino). 0040-8166/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2010.10.002
sperm morphology of Rhipidipathes reticulata (ESPER, 1795) (family Aphanipathidae).
2. Material and methods Some polyps from fragments were cut off with scissors from three different colonies of Rhipidipathes reticulata living in Togian Islands (Tomini Gulf, Sulawesi, Indonesia), and placed in sealed containers underwater. The samples were transferred from the sealed containers into Petri dishes and fixed for 12 h in 2.5% glutaraldehyde buffered with filtered sea-water (adjusted to a final pH of 7.5–7.8 with NaOH 0.1 N) and repeatedly rinsed in the same buffer. Underwater photographs coupled with examination of a part of the skeleton allowed taxonomical identification. Five polyps from each of the colonies were processed for transmission electron microscopy investigations. Selected material was post-fixed in 1% osmium tetroxide in an artificial sea-water buffer for 1 h at 4 ◦ C, then repeatedly washed in the same buffer, dehydrated in an incremental alcohol series and propylene oxide, and finally embedded in an Epon–Araldite mixture. Semi-thin sections, 0.5–1.0 mm thick, were cut on a Leica DC 300 F Ultracut (Leica Microsystem AG, Rijswijk, The Netherlands), stained with toluidine blue and observed under a Leica microscope (Leica LSM Holings GmbH, Wetzlar, Germany). Ultrathin sections were cut with the same Ultracut device, collected on copper grids, stained with uranyl acetate and lead citrate, and examined with a Philips EM 208 electron microscope.
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E. Gaino, F. Scoccia / Tissue and Cell 42 (2010) 391–394
Fig. 1. The black coral Rhipidipathes reticulata. (a) Part of a colony showing the fan-shaped configuration of the anastomosing branches (arrows); (b) ultrastructural sections of a fertile polyp showing the linear arrangement of the sperm within a spermatic cyst; (c) close up of a sperm showing its fine organization consisting in pro-acrosomal vesicles (arrowhead), nucleus (N), cup-like body (CB) and a single mitochondrion (M). The tail (T) is connected to the distal centriole (double arrow), which in turns is linked to the cup-like body; (d) apical region of the sperm showing the sequence of the pro-acrosomal vesicles (arrowheads); (e), circular profile of the peri-centriolar apparatus whose branched arms connect the distal centriole to the electron-dense granules bordering the perimeter of the cup-like body (CB); (f), tangential section of a region of the cup-like body showing its pattern of electron-dense stripes that, in section, correspond to the electron-dense granules; (g), section of the apical region of an immature
E. Gaino, F. Scoccia / Tissue and Cell 42 (2010) 391–394
393
Fig. 2. Sketch of the sperm model in the examined taxa of antipatharians belonging to Antipathidae, Myriopathidae and Aphanipathidae, focusing the different microcharacters. PAC, pro-acrosomal vesicles; N, nucleus; M, mitochondrion; LD, lipid droplet; CB, cup-like body; T, tail. Note in the frames: the acrosome-like structure of Myriopathes japonica and Antipathella subpinnata; the C-shape inclusion of Rhipidipathes reticulata.
3. Results The colonies of Rhipidipathes reticulata have a typical fan-shaped configuration and the anastomosis of their branches grows in an almost two-dimensional plane (Fig. 1(a)). The polyps are very small and their six tentacles (two sagittal longer than the four lateral ones) are typically grouped together around the mouth and show a rounded tip (Tazioli et al., 2007). Cross-sections of fertile polyps showed that the two transversal primary septa, each separating the lateral tentacles from one another, were involved in spermiogenesis. Male gamete differentiation took place inside the spermatic cysts filling the primary septa. The periphery of each spermatic cyst was delimited by a coat of cells elongated in shape resting on a fibrillar envelope. Inside the spermatic cysts mature sperm were arranged in row and their long tails formed bundles in between (Fig. 1(b)). The close up of the mature sperm (Fig. 1(c)), the head of which measured about 2.5 m, showed electron-dense vesicles located at its apex, and a large and fairly round nucleus (diameter ∼1–1.5 m) apparently flanked by two mitochondria; however, in serial sections, these proved to be two portions of a single mitochondrion partially encircling the nucleus. Some sections revealed that the apical vesicles were placed in sequence along the apical region of the nucleus (Fig. 1(d)). The sperm also included a large, electron-
dense cup-like body of about 800 nm along its long axis and about 500 nm along the shorter one. The perimeter of the cup-like body was delineated by regularly interspaced electron-dense granules (Fig. 1(c)). A thin cytoplasmic sheet was interposed between the mitochondrion and the cup-like body. The two centrioles, proximal and distal, were almost coaxial. The axonema of the tail originated from the distal centriole. The cup like-body was connected to the distal centriole through a complex pericentriolar apparatus consisting of nine arms arranged in a radial pattern, each of them terminally branching into three processes (Fig. 1(e)). The branches were associated with electron-dense granules located along the basal region of the cup-like body (Fig. 1(e)). In tangential sections it emerged that these granules were sections of electron-dense stripes decorating the surface of the cup-like body (Fig. 1(f)). Immature sperm showed peculiar inclusions, ranging from 200 to 250 nm, the section of which showed an electron-dense peripheral region slightly eccentric with respect to its probably lipid inner electron-lucent part. This organization gave them a typical C-shaped morphology (Fig. 1(g)). These inclusions conserved their configuration in mature sperm (Fig. 1(h)), where they were located in the region between the cup-like body and the mitochondrion (Fig. 1(h)) or between the mitochondrion and the nucleus (Fig. 1(i)). On the basis of the analysis carried out on the ultrastructure of the sperm from various families examined so far, a schematic
germ cell showing inclusions whose electron-dense content has a typical C-shaped configuration (arrows); (h), cross section of the inclusion with the typical C-shaped configuration of its content (arrow). Note the location between the sperm mitochondrion (M) and the cup-like body (CB); (i) tangential section of an inclusion with the typical C-shaped content. Note its location between the nucleus (N) and the mithocondrion (M).
394
E. Gaino, F. Scoccia / Tissue and Cell 42 (2010) 391–394
diagram was outlined to point out the main differences among these gametes, here including the sperm of Rhipidipathes reticulata for a comparison (Fig. 2). It emerged that the essential differences pertained to some micro-characteristics, such as: the presence of pro-acrosomal vesicles or acrosomal-like structure; occasional lipid droplets; C-shaped inclusions so far observed only in the taxon here investigated.
4. Discussion The lack of information on the reproductive biology of black coral is partially due to the bathymetric distribution of these organisms, which typically grow at high depth (Parker et al., 1997), and partially to the difficulty in distinguishing unfertile colonies from the fertile ones in the natural environment. In fact, no obvious external morphological characteristics exist apart from a slightly enlargement of the polyps due to spermatic cysts differentiation or egg accumulation within the mesentery septa. In this regard, the pigmented stored material filling up the egg cytoplasm can help in distinguishing fertile from non-fertile female polyps. The study of the sperm of Rhipidipathes reticulata (Aphanipathidae) confirms the similarity of the sperm in antipatharians, a feature already stressed in previous investigations on various members of the tropical black coral, such as: Cirrhipathes anguina, C. spiralis, Cirrhipathes sp., Antipathes cfr. simplex, Sticopathes sp. (Antipathidae); Myriopathes japonica, Cupressopathes pumila (Myriopathidae) (Gaino and Scoccia, in press). Also, in the Mediterranean Antipathella subpinnata (Myriopathidae) the sperm organization does not differ significantly from that of the above listed taxa (Gaino and Scoccia, 2010). The common characters are: the single mitochondrion; the cup-like body connected to the complex pericentriolar apparatus, which in turn is linked to the distal centriole; the pro-acrosomal vesiscles. By contrast, Antipathella subpinnata and Myriopathes japonica share an acrosome-like structure instead of the pro-acrosomial vesicles. The occurrence of this kind of sperm vesicles in the apical region is shared with hydrozoans and scyphozoans, thus suggesting to be a primitive acrosome (Hinsch, 1974). The inclusions with a C-shaped electron-dense content observed in Rhipidipathes reticulata seem to be a characteristic of this taxon. These inclusions are already evident in immature sperm and are kept in mature sperm, thus representing a marker useful to trace their origin from the cells bordering the periphery of the spermatic cysts. In contrast with the similar organization observed in antipatharian sperm, the comparison between eight scleractinian corals species identified different types of sperm, mainly on the basis of the nuclear shape (Steiner, 1993). However, a distinction among families seems to be more difficult when they show the same sexual condition because the reproductive pattern prevails on sperm morphology (Steiner and Cortès, 1996). It seems acceptable that antipatharian sperm similarity reflects hybridization, as suggested by the degree of genetic variation observed by Lapian et al. (2007) in several species of Cupressopathes and Myriopathes, and in other Hexacorallia (Diekmann et al., 2001; Frank and Mokady, 2002). Schmidt and Zissler (1979), by comparing the sperm of Anthozoa, reported the cup-like body (named “Ringkörper”) as an apomorphic character exclusive of Antipatharians. This assumption emerged from the observations of specimens belonging to a single genus of the family Antipathidae and was then found by us in other representatives of Antipathidae and Myriopathidae. The study of the sperm morphology in a member of Aphanipathidae confirms that this structure is widespread within the order. Even though there are no data sustaining the actual function of the cup-like body, the connection to the distal centriole through the
pericentriolar apparatus is coherent with the hypothesis that it is involved in tail movement (Gaino et al., 2008). In cnidarian sperm, the presence of a striated pericentriolar process complex associated with the distal centriole has been frequently observed (Hinsch, 1974; see references in Kleve and Clark, 1980; Corbelli et al., 2003). Structural observations of the filamentous nature of the pericentriolar processes in sperm of an hydrozoan, together with the presence of actin, corroborate its contractile function, thus participating in tail movement and facilitating the directional motility in response to egg-released chemoattractants. The connection of the pericentriolar apparatus to the cup-like body, restricted to antipatharians, is very unusual and needs to be better investigated in order to understand if it has an actual role in sperm motility. Investigations on the sperm organization of antipatharians can be included in a broader context aimed to know how sexual reproduction can contribute to assure biological diversity, thus offering to these inhabitants of the barrier reefs better opportunity to respond to the environmental stresses in this fragile ecosystem. Also, according to Harrison and Wallace (1990), knowledge of reproductive biology represents a prerequisite for ecological studies and for directing policy intervention for the conservation of the coral communities. Acknowledgements We are pleased to thank Dr. Massimo Boyer for his helpful collaboration in sampling the specimens and for the underwater photos. We express our gratitude to Dr. Marzia Bo for the confirmation of the specific attribution of the specimens. References Corbelli, A., Avian, A., Marotta, M., Ferraguti, M., 2003. The spermatozoon of Carybdea marsupialis (Cubozoa, Cnidaria). Invertebr. Reprod. Dev. 43, 95–104. Diekmann, O.E., Bak, R.P.M., Stam, W.T., Olsen, J.L., 2001. Molecular genetic evidence for probable reticulate speciation in the coral genus Madracis from a Caribbean fringing reef slope. Mar. Biol. 139, 221–233. Frank, U., Mokady, O., 2002. Coral biodiversity and evolution: recent molecular contributions. Can. J. Zool. 80, 1723–1734. Gaino, E., Scoccia, F., 2009. Release of sperm clusters in spheres by the black coral Cupressopathes pumila (Anthozoa, Antipatharia). Coral Reefs 28, 851–857. Gaino, E., Scoccia, F., in press. Ultrastructural investigation of the sexual reproduction of some members of the black coral: a review. In: Méndez-Vilas, A., Díaz, J. (Eds.), Microscopy: Science, Technology, Applications and Education. Gaino, E., Scoccia, F., 2010. Gamete spawning in Antipathella subpinnata (Anthozoa, Antipatharia): a structural and ultrastructural investigation. Zoomorphology, doi:10.1007/s00435-010-0112-x. Gaino, E., Bo, M., Boyer, M., Scoccia, F., 2008. Sperm morphology in the black coral Cirrhipathes sp. (Anthozoa, Antipatharia). Invertebr. Biol. 45, 249–258. Harrison, P.L., Wallace, C.C., 1990. Reproduction, dispersal and recruitment of scleractinian corals. In: Dubinsky, Z. (Ed.), Ecosystems of the World: Coral Reefs, vol. 25. Elsevier, Amsterdam, the Netherlands, p. 133-207. Hinsch, G.W., 1974. Comparative ultrastructure of cnidarians sperm. Am. Zool. 14, 457–465. Kleve, M.G., Clark Jr., W.H., 1980. Association of actin with sperm centrioles: isolation of centriolar complexes and immunofluorescent localization of actin. J. Cell Biol. 86, 87–95. Lapian, H.F.N., Barucca, M., Bavestrello, G., Biscotti, M.A., Bo, M., Canapa, A., Tazioli, S., Olmo, E., 2007. A systematic study of some Black Corals species (Antipatharia, Hexacorallia) based on rDNA internal transcribed spacers sequences. Mar. Biol. 151, 785–792. Parker, N.R., Mladenov, P.V., Grange, K.R., 1997. Reproductive biology of the antipatharian black coral Antipathes fiordensis in Doubtful Sound, Fiordland, New Zealand. Mar. Biol. 130, 11–22. Schmidt, H., Zissler, D., 1979. Die Spermien der Anthozoen und ihre phylogenetische Bedeutung. Zoologica 129, 1–98. Steiner, S.C.C., 1993. Comparative ultrastructural studies on scleractinian spermatozoa (Cnidaria Anthozoa). Zoomorphology 113, 129–136. Steiner, S.C.C., Cortès, J., 1996. Spermatozoan ultrastructure of scleractinian corals from eastern Pacific: Pocilloporidae and Agariciidae. Coral Reefs 15, 143–147. Tazioli, S., Bo, M., Boyer, M., Rotinsulu, H., Bavestrello, G., 2007. Ecological observations of some common antipatharian corals in the marine Park of Bunaken (North Sulawesi Indonesia). Zool. Studies 46, 227–241.