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Spermatozoal ultrastructure in three species of the genus Uca Leach, 1814 (Crustacea, Brachyura, Ocypodidae)
Micron 39 (2008) 337–343 www.elsevier.com/locate/micron
Spermatozoal ultrastructure in three species of the genus Uca Leach, 1814 (Crustacea, Brachyura, Ocypodidae) A.S. Benetti a, D.C. Santos b,*, M.L. Negreiros-Fransozo a, M.A. Scelzo c a
Departamento de Zoologia—NEBECC, Instituto de Biocieˆncias, Universidade Estadual Paulista (UNESP), Campus de Botucatu, Rubia˜o Ju´nior s/n, 18600-000 Botucatu, SP, Brazil b Departamento de Morfologia, Instituto de Biocieˆncias, Universidade Estadual Paulista (UNESP), Campus de Botucatu, Rubia˜o Ju´nior s/n, 18600-000 Botucatu, SP, Brazil c Departamento de Ciencias Marinas, Universidad Nacional de Mar Del Plata/CONICET, Mar Del Plata, Argentina Received 12 February 2007; received in revised form 5 April 2007; accepted 10 April 2007
Abstract Morphological aspects of spermatozoa in marine animals have been used in recent decades as phylogenetic criteria (spermiotaxonomy). This paper presents ultrastructural descriptions of the spermatozoa from Uca maracoani, U. thayeri, and U. vocator. A small portion of the vas deferens of each species was examined under the transmission and scanning electron microscopy. The ultrastructural analysis showed that each spermatophore consists of a varying number of spermatozoa embedded in a dense fibrillar matrix surrounded by a membrane. The spermatozoa of U. maracoani, U. thayeri, and U. vocator are typical of brachyurans. The voluminous acrosome is characterized by three different layers. The postero-lateral surface of the acrosome is cupped by the reduced cytoplasm, and the anterior surface is covered by the operculum. The perforatorium consists of coiled, helicoidal membranous tubules and is continuous with the cytoplasm. The nucleus is composed by uncondensed chromatin and presents several lateral arms distributed over the entire equatorial plane of the cell. The presence of the apical button is a well defined character among all species of the genus Uca, but in U. thayeri it was not observed. The accessory opercular ring can be found in the three studied species, but in distinct development degree. Two centrioles were detected in U. thayeri and U. vocator, but only one was found in U. maracoani. The presence of centrioles in the mature spermatozoa is the first account for the genus Uca upto-date. Considering the ultrastructure of the spermatozoa of U. maracoani, U. thayeri, and U. vocator, we suggest that these three species partially follow the morphological patterns previously described in other Thoracotremata brachyurans. The absence of the apical button in U. thayeri spermatozoa may represent an evolutionary novelty in the genus Uca. # 2007 Elsevier Ltd. All rights reserved. Keywords: Spermatozoa; Ultrastructure; Crab reproduction; Uca vocator; Uca thayeri; Uca maracoani; Scanning electron microscopy; Transmission electron microscopy
1. Introduction Morphological aspects of spermatozoa in marine animals have been used in recent decades as phylogenetic criteria (Jamieson, 1989a,b, 1990; Jamieson and Tudge, 1990; Medina, 1995). In decapod crustaceans (shrimps, crabs, lobsters, hermit crabs, for instance), the spermatozoa are immobile, aflagellate cells which have an uncondensed nucleus. Brachyuran crabs also have a wide acrosome and radial arms, whose number can
* Corresponding author. Tel.: +55 14 3811 6264; fax: +55 14 3811 6264. E-mail addresses: [email protected] (A.S. Benetti), [email protected] (D.C. Santos). 0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2007.04.004
vary among species (Medina, 1992; Medina and Rodrı´guez, 1992a,b). In some species, the arms seem to be originated from extensions of the nucleus, and in others they seem to be extensions of the cytoplasm (Hinsch, 1986). Recently, morphological studies of spermatozoa ultrastructure in decapods have been widely accepted in phylogenetic and taxonomic approaches, as a tool to solve problems concerning taxonomic distribution and relationships among species (Jamieson, 1989a,b, 1990, 1994; Jamieson and Tudge, 1990; Medina, 1995). Guinot (1977, 1991) and Guinot et al. (1994) classified the Brachyura into three higher groups, Podotremata, Heterotremata and Thoracotremata, taking into account mainly the position of the genital pores. Fiddler crabs of the genus Uca Leach, 1814 belong to the Thoracotremata, in
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which the genital pores are located in thoracic sternite VI in females and sternite VIII in males. In the Thoracotremata, the ambulatory legs have no reproductive function. Spermatozoa morphology has been studied in several species of Uca: U. dussumieri (H. Milne Edwards, 1852) by Jamieson (1991); U. tangeri (Eydoux, 1835) by Medina (1992) and Medina and Rodrı´guez (1992a,b); and Uca pugilator (Bosc, 1802) by Jamieson and Tudge (2000). These investigators found that spermatozoa in Uca have a wide spherical acrosome, a reduced cytoplasm and a filamentous nucleus, from which numerous arms extend. However, further comparative studies are needed, in order to find some general pattern for the genus and distinctive features for each species. The Ocypodidae are represented on the Brazilian coast by the genera Ocypode Weber, 1795, Ucides Rathbun, 1897, with one species each, and by Uca, with 10 species. The species of Uca are U. maracoani (Latreille, 1802–1803), with a narrow front (sensu Crane, 1975); U. thayeri (Rathbun, 1900), with an intermediate front; and U. rapax (Smith, 1870), U. vocator (Herbst, 1804), U. mordax (Smith, 1870), U. leptodactyla Rathbun, 1898, U. uruguayensis (Nobili, 1901), U. cumulanta Crane, 1973, U. burgersi Holthuis, 1967, and U. victoriana von Hagen, 1987, with a wide front (sensu Crane, 1975). Several studies on species of Uca from the Brazilian coast, mainly concern their reproductive biology. These include the reports of Colpo and Negreiros-Fransozo (2003) on U. vocator; Negreiros-Fransozo et al. (2003) and Costa et al. (2006) on U. thayeri; Cardoso and Negreiros-Fransozo (2004) on U. leptodactyla; Benetti and Negreiros-Fransozo (2003, 2004) and Benetti et al. (in press) on U. burgersi; Massunari et al. (2005) on U. maracoani; and Castiglioni and NegreirosFransozo (2006) on U. rapax. None of these studies has included ultrastructural analyses of the reproductive tissues. Considering the usefulness of the spermatozoal ultrastructure in animal taxonomy (Jamieson, 1991; Medina, 1995; Jamieson et al., 1995; Jamieson and Tudge, 2000), this paper presents ultrastructural descriptions of the spermatozoa of U. maracoani, U. thayeri, and U. vocator. We compare features of their spermatozoa with those previously described for other species.
For the scanning electron microscopy (SEM) observations, the vas deferens of each species was fixed in 2.5% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.3) for 24 h at 4 8C. Then it was post-fixed in 1% osmium tetroxide solution for 2 h, dehydrated through a graded series of acetone, critical-point-dried with liquid CO2 and sputtered-coated with gold (10 nm thick). The samples were analyzed and photographed with a Transmission Electron Microscope (CM 100, Philips) and Scanning Electron Microscope (Quanta 200, FEI COMPANY). Ultrastructural descriptions of the spermatozoa are made from the innermost to the outermost regions of the cell. The general morphology of brachyuran spermatozoa is illustrated in Fig. 1, which has been drawn from electron micrographs. 3. Results 3.1. General morphology The general morphology of brachyuran spermatozoa was illustrated in Fig. 1, and it was done based in electron micrographs. The spermatozoa of U. maracoani, U. thayeri, and U. vocator are typical of brachyuran in gross morphology, with some generic differences. An acrosome vesicle, forming most of its bulk, with concentric zonation and capped in the apical portion by a dense operculum, is embedded in the posterior region of the nucleous. The acrosome vesicle is centrally penetrated, almost to its apex, by a cylindrical perforatorial column. The nuclear material forms several lateral projections or ‘‘arms’’ (Fig. 1). In all three species of Uca many spermatozoa are contained within thin walled, oval spermatophores of various sizes; the spermatozoa are embedded in a dense fibrillar matrix surrounded by a membrane (an acellular wall) (Figs. 2A and 2B, 3A and 3B, 4A and 4B). All the spermatophores are immersed in an extracellular matrix, the seminal fluids.
2. Material and methods Adult males of U. maracoani, U. thayeri, and U. vocator with developed testicles were obtained, respectively, in mangrove forests at Jabaquara, Parati, state of Rio de Janeiro (238120 10.000 S and 448430 14.100 W); Ubatumirim, Ubatuba, state of Sa˜o Paulo (238200 17.800 S and 448530 2.200 W); and Itamambuca, Ubatuba, state of Sa˜o Paulo (238240 4300 S and 458000 7300 W). For the transmission electron microscopy (TEM) observations, a small portion of the vas deferens of each species was fixed in 2% glutaraldehyde and 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3) for 24 h at 4 8C. Then the material was post-fixed in 1% osmium tetroxide solution for 2 h, dehydrated through a graded series of acetone and embedded in Araldite1. Ultra-thin sections were doublestained with uranyl acetate and lead citrate.
Fig. 1. Schematic representation of mature spermatozoa of Uca with the main structures. Scale bar: 1 mm.
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Fig. 2. Ultrastructure of the vas deferens and of the spermatozoa in U. maracoani. (A and B) Spermatophores in TEM and SEM, respectively. Note the great number of spermatozoa (sp) immersed in an extracellular matrix (M) surrounded by spermatophore membrane (sf). (C and D) Mature spermatozoa in TEM. Note the voluminous acrosome with three different density of layers (1, 2, 3), perforatorium (p), cytoplasm (cy), centriole (c), apical button (ab), operculum (op), nucleus (n), lateral arms (la), accessory opercular ring (aor), mitochondria (m), thickened ring (tr). Scale bar: (A) 10 mm, (B) 20 mm, (C and D) 1 mm.
The spermatozoa are a lens-shaped, aflagellate cell. It has a voluminous acrosome, limited by the acrosomal membrane and characterized by three different layers (varying electron density), in longitudinal section. 3.2. Mature spermatozoa of Uca maracoani (Fig. 2 and Table 1) The innermost region (layer 1) of the acrosome is adjacent to the perforatorium and continuous with the third layer (outermost acrosomal region). The second layer, which is more electronlucent, occupies the intermediate region of the acrosome. These layers are bounded by the acrosomal membrane. The posterolateral surface of the acrosome is bounded by the cytoplasm, and the anterior surface is covered by the subopercular material, forming the operculum region. The zonation of the acrosome is concentric around the perforatorium; it is not ragged, and has a ring on the inner surface of the acrosomal chamber (thickened ring). The operculum is not very wide or thick. It is closed with an apical button. A structure seen on the lateral portion of the operculum is the accessory opercular ring. No periopercular rim was observed. The protuberance of the periopercular material is considered to be absent, because it is covered by the operculum (Fig. 2C and D).
The perforatorium has a rounded anterior end, and extends through most of the acrosomal vesicle length, i.e., preequatorial. This structure consists of coiled, helicoidal membranous tubules. Granular or filamentous material can be seen in the perforatorium base, which is continuous with the cytoplasm (Fig. 2C and D). The nucleus is cup-shaped, and consists of fibrous chromatin surrounding all the lateral, antero-lateral, and posterior surfaces of the acrosomal vesicle. It has several lateral arms, arranged over the entire equatorial plane of the cell, which also consist of fibrous chromatin (Fig. 2C and D). The cytoplasm of U. maracoani is much reduced. It is located between the acrosome and the nucleus. It has a centriole in the perforatorium base, and small membranous mitochondria, located laterally to the acrosome and surrounding the antero-lateral region of the acrosome (Fig. 2C and D). 3.3. Mature spermatozoa of Uca thayeri (Fig. 3 and Table 1) The innermost region of the acrosome (layer 1) and the outermost one (layer 3) are very thin, and the intermediate region (layer 2) is thicker. These layers are bounded by the acrosomal membrane. The postero-lateral surface of the acrosome is
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Fig. 3. Ultrastructure of the vas deferens and of the spermatozoa in Uca thayeri. (A and B) Spermatophores in TEM and SEM, respectively. Note the spermatozoa (sp) immersed in an extracellular matrix (M) surrounded by spermatophore membrane (sf) in TEM photography. In SEM, only the spermatophores (sf) are seen. It was not possible to see the spermatozoa inside them because we could not see a spermatophore with rupture in their membrane, like in the other species analised. (C and D) Mature spermatozoa in TEM. Voluminous acrosome with three different density of layers (1, 2, 3), cytoplasm (cy), centriole (c), apical button (ab), operculum (op), nucleus (n), lateral arms (la), accessory opercular ring (aor), mitochondria (m), thickened ring (tr). Note the perforatorium (p) in a claviform shape. Scale bar: (A) 10 mm, (B) 20 mm, (C) 1 mm, (D) 0.5 mm.
surrounded by the cytoplasm, and its anterior surface is covered by the subopercular material, forming the operculum region. The acrosome present a concentric zonation and the three layers are not ragged (Fig. 3C and D). The apical button is not present, unlike the other two species (Fig. 3C and D). The operculum is moderately thick and discontinuous with the capsule; it is not as wide as in U. maracoani, and the accessory opercular ring seems to be shorter than in the other species. There is no periopercular rim, but there is a thickened ring on the inner surface of the acrosomal capsule. There is no radiate zone or ‘‘xanthid ring’’ (Fig. 3C and D). The perforatorium extends pre-equatorially through the acrosomal vesicle, and the anterior extremity is rounded in a claviform shape. It is filled with coiled membranous tubules. No corrugation was seen on the inner wall of the perforatorium chamber (Fig. 3C and D). There are several lateral arms formed by nuclear material. The nucleus is composed of fibrous chromatin and is continuous with the cytoplasm. The cytoplasm is located between the nucleus and the acrosome; it is much reduced, but apparently thicker than in the other species. There are two centrioles in the region of the
perforatorium base and in the antero-lateral region of the acrosome there are mitochondria (Fig. 3C and D). 3.4. Mature spermatozoa of Uca vocator (Fig. 4 and Table 1) The outermost region of the acrosome (layer 3) is very thin, and the others (layers 1 and 2) are thicker. The postero-lateral surface of the acrosome is surrounded by cytoplasm, and the anterior surface is covered by the subopercular material, consisting the operculum. None of those regions is ragged (Fig. 4C and D). The operculum is apparently narrower than it is in the other two species. It is perforated, with a well-developed apical button. There is no periopercular rim, and the accessory opercular ring is present and seems to be longer than in the other species (Fig. 4C and D). The perforatorium extends throughout most of the acrosomal vesicle length, with its anterior part rounded. It contains coiled, membranous tubules, and is continuous with the cytoplasm. It also has a thickened ring on the inner surface of the acrosomal capsule (Fig. 4C and 4D).
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Fig. 4. Ultrastructure of the vas deferens and of the spermatozoa in Uca vocator. (A and B) Spermatophores in TEM and SEM, respectively. Note the spermatozoa (sp) immersed in an extracellular matrix (M) surrounded by spermatophore membrane (sf). (C and D) Mature spermatozoa in TEM. Voluminous acrosome with three different density of layers (1, 2, 3), perforatorium (p), cytoplasm (cy), centriole (c), apical button (ab), operculum (op), nucleus (n), lateral arms (la), accessory opercular ring (aor), mitochondria (m), thickened ring (tr). Scale bar: (A) 5 mm, (B) 10 mm, (C) 1 mm, (D) 0.5 mm.
The nucleus is composed of fibrous chromatin and is continuous with the cytoplasm. There are several lateral arms (with the same nucleus material), distributed over the entire equatorial plane of the cell (Fig. 4C and D). The cytoplasm of U. vocator spermatozoa is reduced, continuous with the nucleus and presents a small extension in the upper portion of the nucleus, near the operculum. In this extension, it was possible to see small membranous and lamellar structures, the mitochondria. There are two centrioles in the base of the perforatorium (Fig. 4C and D). 4. Discussion The spermatozoa of Uca maracoani, U. thayeri, and U. vocator are lens-shaped and aflagellate cells, with a voluminous acrosomal vesicle composed of three layers, with different densities and bounded externally by the acrosomal membrane. This arrangement is similar to other species of Brachyura (Hinsch, 1986, 1988, 1990; Felgenhauer and Abele, 1990; Medina and Rodrı´guez, 1992a,b; Jamieson et al., 1995; Medina, 1995). The absence of the apical button in U. thayeri can means an apomorphy (Jamieson, 1994; Medina and Rodrı´guez, 1992a,b). Uca thayeri shows the greatest structural
difference in the spermatozoa, the absence of the apical button. The other two species are almost indistinguishable, except for small differences in dimensions such as width vs. length of the acrosome (character 1, as in Table 1) and accessory opercular ring (character 9) (Table 1). The spermatozoa of the three species share several characters with other species in the family Ocypodidae (Medina and Rodrı´guez, 1992a,b; Jamieson, 1994). These shared characters are the following: the zonation of the acrosome contents (character 2), which are mainly concentric in the Thoracotremata, according to Jamieson (1994); the operculum is not continuous with the acrosomal capsule (character 5), according to Jamieson (1994) the operculum is continuous only in three species of Raninidae; the operculum is moderately thick (character 6) and not extremely wide (character 7); it has no periopercular rim (character 8) and no true acrosome ray zone (character 11), this last character was presumed to be ‘‘lost’’ in the Thoracotremata (Jamieson, 1994); the outer acrosome zone border is not ragged (character 12); the subacrosomal chamber of the perforatorium (character 16) is extended pre-equatorially; the anterior extremity (head) of the perforatorium (character 17) is rounded and has several lateral arms (character 19) composed only of nuclear material (character 20).
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Table 1 Ultrastructural characters of the spermatozoa and other morphological features of U. maracoani, U. thayeri, and U. vocator (terminology according to Jamieson (1994) and Jamieson et al. (1996)) Characters of spermatozoa
Uca maracoani
Uca thayeri
Uca vocator
1. Acrosome length/width 2. Zonation of the acrosome contents 3. Perforation of operculum
0.77 Concentric Perforated, closed with apical button Absent Discontinuous Moderately thick Not extremely wide Absent Presentb Absent Absent Not ragged Absent Absent Absent Pre-equatorial Amoeboid Absent Several Nuclear only Present Absent Present Absent Absent Absent Absent
0.84 Concentric Imperforatea
0.77 Concentric Perforated, closed with apical button Absent Discontinuous Moderately thick Not extremely wide b Absent Presentb Absent Absent Not ragged Absent Absent Absent Pre-equatorial Amoeboid Absent Several Nuclear only Present Absent Present Absent Absent Absent Absent
4. Opercular projections 5. Continuity of operculum with capsule 6. Thickness of operculum 7. Operculum width 8. Periopercular rim 9. Accessory opercular ring 10. Subopercular protuberance through operculum 11. True acrosome ray zone 12. Outer acrosome zone border 13. Anterolateral pale zone 14. Flangelike peripheral extension of lower acrosome zone 15. ‘‘Xanthid’’ ring 16. Subacrosomal chamber of perforatorium 17. Head of perforatorium 18. Corrugations of wall of perforatorial chamber 19. Lateral arms 20. Composition of lateral arms 21. Centrioles 22. Posterior median process of nucleus 23. Thickened ring 24. Concentric lamellae of acrosome 25. Capsular chambers 26. Capsular projections 27. Capsular flange a b
Absent Discontinuous Moderately thick Not extremely wide Absent Presentb Absent Absent Not ragged Absent Absent Absent Pre-equatorial Amoeboid Absent Several Nuclear only Present Absent Present Absent Absent Absent Absent
Indicates differences among species. Indicates differences in the degree of development among species.
Jamieson (1994) noted that the presence of the apical button (character 3) in Brachyura is an ambiguous feature, because may or may not be present in the spermatozoa. One of the main features not shared among the three species studied here is the absence of this structure in U. thayeri. The absence of the apical button was considered as a loss in a basal condition in the Thoracotremata, which may indicate an apomorphic character of the species (Jamieson et al., 1995). In the Thoracotremata, the absence of an apical button was observed in Macrophthalmus crassipes (H. Milne-Edwards, 1852) (Ocypodidae) by Jamieson (1991), and in Varuna litterata (Fabricius, 1798) (Varunidae) by Jamieson et al. (1996). There is no mention in the literature about a similar loss in the genus Uca. Jamieson (1994) and Jamieson et al. (1995) suggested that a more detailed investigation in Thoracotremata might clarify whether the presence of the button is a basal condition. Jamieson (1990) observed that the true acrosome ray zone (character 11) is well developed in pagurideans and in some crabs of the families Portunidae, Dorippidae, and Xanthidae; however, in the Thoracotremata it may be reduced or not easily recognized. Subsequently, Jamieson et al. (1996) found that this feature is absent in all Thoracotremata; this character is considered as a loss in this group, because only the Heterotremata among Brachyura have a ‘‘true’’ ray zone in the acrosome. Thus, the absence of this structure in U. maracoani, U. thayeri, and U. vocator is considered here as a lost character.
Another feature observed in these three species is the presence of centrioles (character 21) in the cytoplasm in the base of the perforatorium. Medina and Rodrı´guez (1992a,b) observed centrioles only in the intermediate and final spermatids of U. tangeri, and they reported that this structure is degenerated in the adult spermatozoa. Jamieson (1994) also did not observe centrioles in U. dussumieri and M. crassipes (Thoracotremata, Ocypodidae). However, Hinsch (1986) occasionally found centrioles in mature spermatozoa of Ovalipes ocellatus (Herbst, 1799) (Portunidae) and Libinia emarginata L. (Majidae). Although centrioles are also observed in several species (Hinsch, 1988; Jamieson et al., 1996), such character was excluded from parsimony analyses due to the uncertainty of its occurrence in mature spermatozoa (Jamieson, 1994), without altering their results. A curved line attached to the inner surface of the acrosomal capsule, termed the thickened ring (character 23), was detected in all three species studied. Jamieson (1994) observed that this structure is present only in Heterotremata and Thoracotremata, is reduced in the genus Potamonautes, and is lost in grapsoid crabs (Jamieson, 1991). Such a structure is also present in the other species of the genus Uca that have been studied so far (Jamieson, 1991; Medina and Rodrı´guez, 1992a,b). Tudge et al. (1998) studied the spermatozoal morphology in several representatives of Heterotremata in which the ring occurs: Bythograea thermydron (Williams, 1980); Austinograea
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alayseae (Guinot, 1990); and Segonzacia mesatlantica (Williams, 1980) (Bythograeidae). These authors suggested that this feature is a synapomorphy of Heterotremata. Considering the ultrastructure of the spermatozoa of U. maracoani, U. thayeri, and U. vocator, we suggest that these species partially follow the morphological patterns described for other Thoracotremata brachyurans (Hinsch, 1986, 1988, 1990; Felgenhauer and Abele, 1990; Jamieson, 1991, 1994; Medina, 1992, 1995; Medina and Rodrı´guez, 1992a,b; Jamieson et al., 1995, 1996; Tudge et al., 1998). The absence of the apical button in U. thayeri spermatozoa may represent an evolutionary novelty in the genus Uca. Further studies may clarify whether the presence of the apical button really represents a plesiomorphy in the Thoracotremata. Acknowledgements This work was supported by Brazilian Research Agency CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico). We thanks for Electron Microscopy Center – UNESP, Botucatu, SP, for the technical assistance and Mrs. Janet Reid for English review. References Benetti, A.S., Negreiros-Fransozo, M.L., 2003. Madurez sexual morfolo´gica del cangrejo Uca burgersi Holthuis, 1967 (Brachyura, Ocypodidae) de uma a´rea de manglar subtropical de Brasil. Universidad y Ciencia 19 (37), 9–16. Benetti, A.S., Negreiros-Fransozo, M.L., 2004. Relative growth of fiddler crab Uca burgersi Holthuis, 1967 (Crustacea, Ocypodidae) from two mangroves in the southeastern Brazilian coast. Iheringia, Se´rie Zoologia 94 (1), 67–72. Benetti, A.S., Negreiros-Fransozo, M.L., Costa, T.M., in press. Population and reproductive biology of Uca burgersi Holthuis, 1967 (Crustacea, Brachyura, Ocypodidae) in three subtropical mangroves. Rev. Biol. Tropi. Cardoso, R.C.F., Negreiros-Fransozo, M.L., 2004. A comparison of the allometric growth in Uca leptodactyla (Crustacea: Brachyura: Ocypodidae) from two subtropical estuaries. J. Mar. Biol. Assoc. U.K. 84, 733–735. Castiglioni, D.S., Negreiros-Fransozo, M.L., 2006. Physiological sexual maturity of the fiddler crab Uca rapax (Smith, 1870) (Crustacea, Ocypodidae) from two mangroves in Ubatuba, Brazil. Braz. Arch. Biol. Technol. 49 (2), 239–248. Colpo, K.D., Negreiros-Fransozo, M.L., 2003. Reproductive output of Uca vocator (Herbst, 1804) from three subtropical mangroves in Brazil. Crustaceana 76 (1), 1–11. Costa, T.M., Silva, S.M.J., Negreiros-Fransozo, M.L., 2006. Reproductive pattern comparison of Uca thayeri Rathbun, 1900 and Uca uruguayensis Nobili, 1901 (Crustacea, Decapoda, Ocypodidae). Braz. Arch. Biol. Technol. 49 (1), 117–123. Felgenhauer, B.E., Abele, L.G., 1990. Morphological diversity of decapod spermatozoa. In: Bauer, R.T., Martin, J.W. (Eds.), Crustacean Sexual Biology. pp. 322–341. Guinot, D., 1977. Propositions pour une nouvelle classification des crustace´s decapods brachyoures. Comptes Rendus Hebdomadaires Se´ances l‘Acad. Sci., Paris 285, 1049–1052. Guinot, D., 1991. E´tablissement de la famille des Poupiniidae pour Popinia hirsute gen. nov. sp. nov. de Polune´sie (Crustacea, Decapoda, Brachyura, Homoloidea). Bull. Muse´um Natl. Hist. Nat., section A 12, 577-605. Guinot, D., Jamieson, B.G.M., Richer de Forges, B., 1994. Relationship of Homolidae and Dromiidae: evidence from spermatozoal ultrastructure (Crustacea, Decapoda). Acta Zool. 75, 255–267.
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Hinsch, G.W., 1986. A comparison of sperm morphologies, transfer and sperm mass storage between two species of crab, Ovalipes ocellatus and Libinia emarginata. Int. J. Invert. Reprod. Dev. 10, 79–87. Hinsch, G.W., 1988. Ultrastructure of the sperm and spermatophores of the golden crab Geryon fenneri and a closely related species, the red crab G. quinquedens, from the eastern Gulf of Mexico. J. Crusta. Biol. 8 (3), 340– 345. Hinsch, G.W., 1990. Structure and chemical content of the spermatophores and seminal fluid of reptantian decapods. In: Bauer, R.T., Martin, J.W. (Eds.), Crustacean Sexual Biology. pp. 290–307. Jamieson, B.G.M., 1989a. The ultrastructure of the spermatozoa of four species of xanthid crabs (Crustacea, Brachyura, Xanthidae). J. Submicr. Cytol. 21, 579–584. Jamieson, B.G.M., 1989b. Ultrastructural comparison of the spermatozoa of Ranina ranina (Oxystomata) and of other crabs exemplified by Portunus pelagicus (Brachygnatha) (Crustacea, Brachyura). Zoomorphology 109, 103–111. Jamieson, B.G.M., 1990. The ultrastructure of the spermatozoa of Petalomera lateralis (Gray) (Crustacea, Brachyura, Dromiacea) and its phylogenetic significance. Invert. Reprod. Dev. 17, 39–45. Jamieson, B.G.M., 1991. Ultrastructure and phylogeny of crustacean spermatozoa. Mem. Queensland Museum 31, 109–142. Jamieson, B.G.M., 1994. Phylogeny of the Brachyura with particular reference to the Podotremata: evidence from a review of spermatozoal ultrastructure (Crustacea, Decapoda). Phil. Trans. Roy. Soc. Lond., Ser. B 345, 373–393. Jamieson, B.G.M., Tudge, C.C., 1990. Dorippids are Heterotremata: evidence from ultrastructure of the spermatozoa of Neodorippe astute (Dorippidae) and Portunus pelagicus (Portunidae) Brachyura: Decapoda. Mar. Biol. 106, 347–354. Jamieson, B.G.M., Guinot, D., Richer de Forges, B., 1995. Phylogeny of the Brachyura (Crustacea, Decapoda): evidence from spermatozoal ultrastructure. In: Jamieson, B.G.M., Ausio, J., Justine, J.L. (Eds),. Advances in Spermatozoal Phylogeny and Taxonomy. Memories Museum Natural History 166, 265-283. Jamieson, B.G.M., Guinot, D., Richer de Forges, B., 1996. Contrasting spermatozoal ultrastructure in two thoracotreme crabs, Cardisoma carnifex (Gecarcinidae) and Varunna litterata (Grapsidae) (Crustacea, Brachyura). Invert. Reprod. Dev. 29, 111–126. Jamieson, B.G.M., Tudge, C.C., 2000. Crustacea-Decapoda. In: Jamieson, B.G.M. (Ed.), Reproductive Biology of Invertebrates. Part C, vol. IX. pp. 01–94. Massunari, S., Dissenha, N., Falca˜o, R.C., 2005. Crescimento relativo e destreza dos quelı´podos de Uca maracoani (Latreille) (Crustacea, Decapoda, Ocypodidae) no Baixio Mirim, Baı´a de Guaratuba, Parana´, Bras. Revista Brasileira Zool. 22 (4), 974–983. Medina, A., 1992. Structural modifications of sperm from the fiddler crab Uca tangeri (Decapoda) during early stages of fertilization. J. Crustacean Biol. 12 (4), 610–614. Medina, A. 1995. Spermatozoal ultrastructure in Dendobranchiata (Crustacea, Decapoda): Taxonomic and phylogenetic considerations. In: Jamieson, B.G.M., Ausio, J., Justine, J.L., (Eds). Advances in Spermatozoal Phylogeny and Taxonomy. Memoires Museum Natural History 166, 231–242. Medina, A., Rodrı´guez, A., 1992a. Spermiogenesis and sperm structure in the crab Uca tangeri (Crustacea, Brachyura), with special reference to the acrosome differentiation. Zoomorphology 111, 161–165. Medina, A., Rodrı´guez, A., 1992b. Structural changes in sperm from the fiddler crab Uca tangeri (Crustacea, Brachyura), during the acrosome reaction. Mol. Reprod. Dev. 33, 195–201. Negreiros-Fransozo, M.L., Colpo, K.D., Costa, T.M., 2003. Allometric growth in the fiddler crab Uca thayeri (Brachyura, Ocypodidae) from a subtropical mangrove. J. Crustacean Biol. 23 (2), 273–279. Tudge, C.C., Jamieson, B.G.M., Segonzac, M., Guinot, D., 1998. Spermatozoal ultrastructure in three species of hydrothermal vent crabs, in the genera Bythograea, Austinograea and Segonzacia (Decapoda, Brachyura, Bythograeidae). Invert. Reprod. Dev. 34 (1), 13–23.
Spermatozoal ultrastructure in three species of the genus Uca Leach, 1814 (Crustacea, Brachyura, Ocypodidae) A.S. Benetti a, D.C. Santos b,*, M.L. Negreiros-Fransozo a, M.A. Scelzo c a
Departamento de Zoologia—NEBECC, Instituto de Biocieˆncias, Universidade Estadual Paulista (UNESP), Campus de Botucatu, Rubia˜o Ju´nior s/n, 18600-000 Botucatu, SP, Brazil b Departamento de Morfologia, Instituto de Biocieˆncias, Universidade Estadual Paulista (UNESP), Campus de Botucatu, Rubia˜o Ju´nior s/n, 18600-000 Botucatu, SP, Brazil c Departamento de Ciencias Marinas, Universidad Nacional de Mar Del Plata/CONICET, Mar Del Plata, Argentina Received 12 February 2007; received in revised form 5 April 2007; accepted 10 April 2007
Abstract Morphological aspects of spermatozoa in marine animals have been used in recent decades as phylogenetic criteria (spermiotaxonomy). This paper presents ultrastructural descriptions of the spermatozoa from Uca maracoani, U. thayeri, and U. vocator. A small portion of the vas deferens of each species was examined under the transmission and scanning electron microscopy. The ultrastructural analysis showed that each spermatophore consists of a varying number of spermatozoa embedded in a dense fibrillar matrix surrounded by a membrane. The spermatozoa of U. maracoani, U. thayeri, and U. vocator are typical of brachyurans. The voluminous acrosome is characterized by three different layers. The postero-lateral surface of the acrosome is cupped by the reduced cytoplasm, and the anterior surface is covered by the operculum. The perforatorium consists of coiled, helicoidal membranous tubules and is continuous with the cytoplasm. The nucleus is composed by uncondensed chromatin and presents several lateral arms distributed over the entire equatorial plane of the cell. The presence of the apical button is a well defined character among all species of the genus Uca, but in U. thayeri it was not observed. The accessory opercular ring can be found in the three studied species, but in distinct development degree. Two centrioles were detected in U. thayeri and U. vocator, but only one was found in U. maracoani. The presence of centrioles in the mature spermatozoa is the first account for the genus Uca upto-date. Considering the ultrastructure of the spermatozoa of U. maracoani, U. thayeri, and U. vocator, we suggest that these three species partially follow the morphological patterns previously described in other Thoracotremata brachyurans. The absence of the apical button in U. thayeri spermatozoa may represent an evolutionary novelty in the genus Uca. # 2007 Elsevier Ltd. All rights reserved. Keywords: Spermatozoa; Ultrastructure; Crab reproduction; Uca vocator; Uca thayeri; Uca maracoani; Scanning electron microscopy; Transmission electron microscopy
1. Introduction Morphological aspects of spermatozoa in marine animals have been used in recent decades as phylogenetic criteria (Jamieson, 1989a,b, 1990; Jamieson and Tudge, 1990; Medina, 1995). In decapod crustaceans (shrimps, crabs, lobsters, hermit crabs, for instance), the spermatozoa are immobile, aflagellate cells which have an uncondensed nucleus. Brachyuran crabs also have a wide acrosome and radial arms, whose number can
* Corresponding author. Tel.: +55 14 3811 6264; fax: +55 14 3811 6264. E-mail addresses: [email protected] (A.S. Benetti), [email protected] (D.C. Santos). 0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2007.04.004
vary among species (Medina, 1992; Medina and Rodrı´guez, 1992a,b). In some species, the arms seem to be originated from extensions of the nucleus, and in others they seem to be extensions of the cytoplasm (Hinsch, 1986). Recently, morphological studies of spermatozoa ultrastructure in decapods have been widely accepted in phylogenetic and taxonomic approaches, as a tool to solve problems concerning taxonomic distribution and relationships among species (Jamieson, 1989a,b, 1990, 1994; Jamieson and Tudge, 1990; Medina, 1995). Guinot (1977, 1991) and Guinot et al. (1994) classified the Brachyura into three higher groups, Podotremata, Heterotremata and Thoracotremata, taking into account mainly the position of the genital pores. Fiddler crabs of the genus Uca Leach, 1814 belong to the Thoracotremata, in
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which the genital pores are located in thoracic sternite VI in females and sternite VIII in males. In the Thoracotremata, the ambulatory legs have no reproductive function. Spermatozoa morphology has been studied in several species of Uca: U. dussumieri (H. Milne Edwards, 1852) by Jamieson (1991); U. tangeri (Eydoux, 1835) by Medina (1992) and Medina and Rodrı´guez (1992a,b); and Uca pugilator (Bosc, 1802) by Jamieson and Tudge (2000). These investigators found that spermatozoa in Uca have a wide spherical acrosome, a reduced cytoplasm and a filamentous nucleus, from which numerous arms extend. However, further comparative studies are needed, in order to find some general pattern for the genus and distinctive features for each species. The Ocypodidae are represented on the Brazilian coast by the genera Ocypode Weber, 1795, Ucides Rathbun, 1897, with one species each, and by Uca, with 10 species. The species of Uca are U. maracoani (Latreille, 1802–1803), with a narrow front (sensu Crane, 1975); U. thayeri (Rathbun, 1900), with an intermediate front; and U. rapax (Smith, 1870), U. vocator (Herbst, 1804), U. mordax (Smith, 1870), U. leptodactyla Rathbun, 1898, U. uruguayensis (Nobili, 1901), U. cumulanta Crane, 1973, U. burgersi Holthuis, 1967, and U. victoriana von Hagen, 1987, with a wide front (sensu Crane, 1975). Several studies on species of Uca from the Brazilian coast, mainly concern their reproductive biology. These include the reports of Colpo and Negreiros-Fransozo (2003) on U. vocator; Negreiros-Fransozo et al. (2003) and Costa et al. (2006) on U. thayeri; Cardoso and Negreiros-Fransozo (2004) on U. leptodactyla; Benetti and Negreiros-Fransozo (2003, 2004) and Benetti et al. (in press) on U. burgersi; Massunari et al. (2005) on U. maracoani; and Castiglioni and NegreirosFransozo (2006) on U. rapax. None of these studies has included ultrastructural analyses of the reproductive tissues. Considering the usefulness of the spermatozoal ultrastructure in animal taxonomy (Jamieson, 1991; Medina, 1995; Jamieson et al., 1995; Jamieson and Tudge, 2000), this paper presents ultrastructural descriptions of the spermatozoa of U. maracoani, U. thayeri, and U. vocator. We compare features of their spermatozoa with those previously described for other species.
For the scanning electron microscopy (SEM) observations, the vas deferens of each species was fixed in 2.5% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.3) for 24 h at 4 8C. Then it was post-fixed in 1% osmium tetroxide solution for 2 h, dehydrated through a graded series of acetone, critical-point-dried with liquid CO2 and sputtered-coated with gold (10 nm thick). The samples were analyzed and photographed with a Transmission Electron Microscope (CM 100, Philips) and Scanning Electron Microscope (Quanta 200, FEI COMPANY). Ultrastructural descriptions of the spermatozoa are made from the innermost to the outermost regions of the cell. The general morphology of brachyuran spermatozoa is illustrated in Fig. 1, which has been drawn from electron micrographs. 3. Results 3.1. General morphology The general morphology of brachyuran spermatozoa was illustrated in Fig. 1, and it was done based in electron micrographs. The spermatozoa of U. maracoani, U. thayeri, and U. vocator are typical of brachyuran in gross morphology, with some generic differences. An acrosome vesicle, forming most of its bulk, with concentric zonation and capped in the apical portion by a dense operculum, is embedded in the posterior region of the nucleous. The acrosome vesicle is centrally penetrated, almost to its apex, by a cylindrical perforatorial column. The nuclear material forms several lateral projections or ‘‘arms’’ (Fig. 1). In all three species of Uca many spermatozoa are contained within thin walled, oval spermatophores of various sizes; the spermatozoa are embedded in a dense fibrillar matrix surrounded by a membrane (an acellular wall) (Figs. 2A and 2B, 3A and 3B, 4A and 4B). All the spermatophores are immersed in an extracellular matrix, the seminal fluids.
2. Material and methods Adult males of U. maracoani, U. thayeri, and U. vocator with developed testicles were obtained, respectively, in mangrove forests at Jabaquara, Parati, state of Rio de Janeiro (238120 10.000 S and 448430 14.100 W); Ubatumirim, Ubatuba, state of Sa˜o Paulo (238200 17.800 S and 448530 2.200 W); and Itamambuca, Ubatuba, state of Sa˜o Paulo (238240 4300 S and 458000 7300 W). For the transmission electron microscopy (TEM) observations, a small portion of the vas deferens of each species was fixed in 2% glutaraldehyde and 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3) for 24 h at 4 8C. Then the material was post-fixed in 1% osmium tetroxide solution for 2 h, dehydrated through a graded series of acetone and embedded in Araldite1. Ultra-thin sections were doublestained with uranyl acetate and lead citrate.
Fig. 1. Schematic representation of mature spermatozoa of Uca with the main structures. Scale bar: 1 mm.
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Fig. 2. Ultrastructure of the vas deferens and of the spermatozoa in U. maracoani. (A and B) Spermatophores in TEM and SEM, respectively. Note the great number of spermatozoa (sp) immersed in an extracellular matrix (M) surrounded by spermatophore membrane (sf). (C and D) Mature spermatozoa in TEM. Note the voluminous acrosome with three different density of layers (1, 2, 3), perforatorium (p), cytoplasm (cy), centriole (c), apical button (ab), operculum (op), nucleus (n), lateral arms (la), accessory opercular ring (aor), mitochondria (m), thickened ring (tr). Scale bar: (A) 10 mm, (B) 20 mm, (C and D) 1 mm.
The spermatozoa are a lens-shaped, aflagellate cell. It has a voluminous acrosome, limited by the acrosomal membrane and characterized by three different layers (varying electron density), in longitudinal section. 3.2. Mature spermatozoa of Uca maracoani (Fig. 2 and Table 1) The innermost region (layer 1) of the acrosome is adjacent to the perforatorium and continuous with the third layer (outermost acrosomal region). The second layer, which is more electronlucent, occupies the intermediate region of the acrosome. These layers are bounded by the acrosomal membrane. The posterolateral surface of the acrosome is bounded by the cytoplasm, and the anterior surface is covered by the subopercular material, forming the operculum region. The zonation of the acrosome is concentric around the perforatorium; it is not ragged, and has a ring on the inner surface of the acrosomal chamber (thickened ring). The operculum is not very wide or thick. It is closed with an apical button. A structure seen on the lateral portion of the operculum is the accessory opercular ring. No periopercular rim was observed. The protuberance of the periopercular material is considered to be absent, because it is covered by the operculum (Fig. 2C and D).
The perforatorium has a rounded anterior end, and extends through most of the acrosomal vesicle length, i.e., preequatorial. This structure consists of coiled, helicoidal membranous tubules. Granular or filamentous material can be seen in the perforatorium base, which is continuous with the cytoplasm (Fig. 2C and D). The nucleus is cup-shaped, and consists of fibrous chromatin surrounding all the lateral, antero-lateral, and posterior surfaces of the acrosomal vesicle. It has several lateral arms, arranged over the entire equatorial plane of the cell, which also consist of fibrous chromatin (Fig. 2C and D). The cytoplasm of U. maracoani is much reduced. It is located between the acrosome and the nucleus. It has a centriole in the perforatorium base, and small membranous mitochondria, located laterally to the acrosome and surrounding the antero-lateral region of the acrosome (Fig. 2C and D). 3.3. Mature spermatozoa of Uca thayeri (Fig. 3 and Table 1) The innermost region of the acrosome (layer 1) and the outermost one (layer 3) are very thin, and the intermediate region (layer 2) is thicker. These layers are bounded by the acrosomal membrane. The postero-lateral surface of the acrosome is
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Fig. 3. Ultrastructure of the vas deferens and of the spermatozoa in Uca thayeri. (A and B) Spermatophores in TEM and SEM, respectively. Note the spermatozoa (sp) immersed in an extracellular matrix (M) surrounded by spermatophore membrane (sf) in TEM photography. In SEM, only the spermatophores (sf) are seen. It was not possible to see the spermatozoa inside them because we could not see a spermatophore with rupture in their membrane, like in the other species analised. (C and D) Mature spermatozoa in TEM. Voluminous acrosome with three different density of layers (1, 2, 3), cytoplasm (cy), centriole (c), apical button (ab), operculum (op), nucleus (n), lateral arms (la), accessory opercular ring (aor), mitochondria (m), thickened ring (tr). Note the perforatorium (p) in a claviform shape. Scale bar: (A) 10 mm, (B) 20 mm, (C) 1 mm, (D) 0.5 mm.
surrounded by the cytoplasm, and its anterior surface is covered by the subopercular material, forming the operculum region. The acrosome present a concentric zonation and the three layers are not ragged (Fig. 3C and D). The apical button is not present, unlike the other two species (Fig. 3C and D). The operculum is moderately thick and discontinuous with the capsule; it is not as wide as in U. maracoani, and the accessory opercular ring seems to be shorter than in the other species. There is no periopercular rim, but there is a thickened ring on the inner surface of the acrosomal capsule. There is no radiate zone or ‘‘xanthid ring’’ (Fig. 3C and D). The perforatorium extends pre-equatorially through the acrosomal vesicle, and the anterior extremity is rounded in a claviform shape. It is filled with coiled membranous tubules. No corrugation was seen on the inner wall of the perforatorium chamber (Fig. 3C and D). There are several lateral arms formed by nuclear material. The nucleus is composed of fibrous chromatin and is continuous with the cytoplasm. The cytoplasm is located between the nucleus and the acrosome; it is much reduced, but apparently thicker than in the other species. There are two centrioles in the region of the
perforatorium base and in the antero-lateral region of the acrosome there are mitochondria (Fig. 3C and D). 3.4. Mature spermatozoa of Uca vocator (Fig. 4 and Table 1) The outermost region of the acrosome (layer 3) is very thin, and the others (layers 1 and 2) are thicker. The postero-lateral surface of the acrosome is surrounded by cytoplasm, and the anterior surface is covered by the subopercular material, consisting the operculum. None of those regions is ragged (Fig. 4C and D). The operculum is apparently narrower than it is in the other two species. It is perforated, with a well-developed apical button. There is no periopercular rim, and the accessory opercular ring is present and seems to be longer than in the other species (Fig. 4C and D). The perforatorium extends throughout most of the acrosomal vesicle length, with its anterior part rounded. It contains coiled, membranous tubules, and is continuous with the cytoplasm. It also has a thickened ring on the inner surface of the acrosomal capsule (Fig. 4C and 4D).
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Fig. 4. Ultrastructure of the vas deferens and of the spermatozoa in Uca vocator. (A and B) Spermatophores in TEM and SEM, respectively. Note the spermatozoa (sp) immersed in an extracellular matrix (M) surrounded by spermatophore membrane (sf). (C and D) Mature spermatozoa in TEM. Voluminous acrosome with three different density of layers (1, 2, 3), perforatorium (p), cytoplasm (cy), centriole (c), apical button (ab), operculum (op), nucleus (n), lateral arms (la), accessory opercular ring (aor), mitochondria (m), thickened ring (tr). Scale bar: (A) 5 mm, (B) 10 mm, (C) 1 mm, (D) 0.5 mm.
The nucleus is composed of fibrous chromatin and is continuous with the cytoplasm. There are several lateral arms (with the same nucleus material), distributed over the entire equatorial plane of the cell (Fig. 4C and D). The cytoplasm of U. vocator spermatozoa is reduced, continuous with the nucleus and presents a small extension in the upper portion of the nucleus, near the operculum. In this extension, it was possible to see small membranous and lamellar structures, the mitochondria. There are two centrioles in the base of the perforatorium (Fig. 4C and D). 4. Discussion The spermatozoa of Uca maracoani, U. thayeri, and U. vocator are lens-shaped and aflagellate cells, with a voluminous acrosomal vesicle composed of three layers, with different densities and bounded externally by the acrosomal membrane. This arrangement is similar to other species of Brachyura (Hinsch, 1986, 1988, 1990; Felgenhauer and Abele, 1990; Medina and Rodrı´guez, 1992a,b; Jamieson et al., 1995; Medina, 1995). The absence of the apical button in U. thayeri can means an apomorphy (Jamieson, 1994; Medina and Rodrı´guez, 1992a,b). Uca thayeri shows the greatest structural
difference in the spermatozoa, the absence of the apical button. The other two species are almost indistinguishable, except for small differences in dimensions such as width vs. length of the acrosome (character 1, as in Table 1) and accessory opercular ring (character 9) (Table 1). The spermatozoa of the three species share several characters with other species in the family Ocypodidae (Medina and Rodrı´guez, 1992a,b; Jamieson, 1994). These shared characters are the following: the zonation of the acrosome contents (character 2), which are mainly concentric in the Thoracotremata, according to Jamieson (1994); the operculum is not continuous with the acrosomal capsule (character 5), according to Jamieson (1994) the operculum is continuous only in three species of Raninidae; the operculum is moderately thick (character 6) and not extremely wide (character 7); it has no periopercular rim (character 8) and no true acrosome ray zone (character 11), this last character was presumed to be ‘‘lost’’ in the Thoracotremata (Jamieson, 1994); the outer acrosome zone border is not ragged (character 12); the subacrosomal chamber of the perforatorium (character 16) is extended pre-equatorially; the anterior extremity (head) of the perforatorium (character 17) is rounded and has several lateral arms (character 19) composed only of nuclear material (character 20).
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Table 1 Ultrastructural characters of the spermatozoa and other morphological features of U. maracoani, U. thayeri, and U. vocator (terminology according to Jamieson (1994) and Jamieson et al. (1996)) Characters of spermatozoa
Uca maracoani
Uca thayeri
Uca vocator
1. Acrosome length/width 2. Zonation of the acrosome contents 3. Perforation of operculum
0.77 Concentric Perforated, closed with apical button Absent Discontinuous Moderately thick Not extremely wide Absent Presentb Absent Absent Not ragged Absent Absent Absent Pre-equatorial Amoeboid Absent Several Nuclear only Present Absent Present Absent Absent Absent Absent
0.84 Concentric Imperforatea
0.77 Concentric Perforated, closed with apical button Absent Discontinuous Moderately thick Not extremely wide b Absent Presentb Absent Absent Not ragged Absent Absent Absent Pre-equatorial Amoeboid Absent Several Nuclear only Present Absent Present Absent Absent Absent Absent
4. Opercular projections 5. Continuity of operculum with capsule 6. Thickness of operculum 7. Operculum width 8. Periopercular rim 9. Accessory opercular ring 10. Subopercular protuberance through operculum 11. True acrosome ray zone 12. Outer acrosome zone border 13. Anterolateral pale zone 14. Flangelike peripheral extension of lower acrosome zone 15. ‘‘Xanthid’’ ring 16. Subacrosomal chamber of perforatorium 17. Head of perforatorium 18. Corrugations of wall of perforatorial chamber 19. Lateral arms 20. Composition of lateral arms 21. Centrioles 22. Posterior median process of nucleus 23. Thickened ring 24. Concentric lamellae of acrosome 25. Capsular chambers 26. Capsular projections 27. Capsular flange a b
Absent Discontinuous Moderately thick Not extremely wide Absent Presentb Absent Absent Not ragged Absent Absent Absent Pre-equatorial Amoeboid Absent Several Nuclear only Present Absent Present Absent Absent Absent Absent
Indicates differences among species. Indicates differences in the degree of development among species.
Jamieson (1994) noted that the presence of the apical button (character 3) in Brachyura is an ambiguous feature, because may or may not be present in the spermatozoa. One of the main features not shared among the three species studied here is the absence of this structure in U. thayeri. The absence of the apical button was considered as a loss in a basal condition in the Thoracotremata, which may indicate an apomorphic character of the species (Jamieson et al., 1995). In the Thoracotremata, the absence of an apical button was observed in Macrophthalmus crassipes (H. Milne-Edwards, 1852) (Ocypodidae) by Jamieson (1991), and in Varuna litterata (Fabricius, 1798) (Varunidae) by Jamieson et al. (1996). There is no mention in the literature about a similar loss in the genus Uca. Jamieson (1994) and Jamieson et al. (1995) suggested that a more detailed investigation in Thoracotremata might clarify whether the presence of the button is a basal condition. Jamieson (1990) observed that the true acrosome ray zone (character 11) is well developed in pagurideans and in some crabs of the families Portunidae, Dorippidae, and Xanthidae; however, in the Thoracotremata it may be reduced or not easily recognized. Subsequently, Jamieson et al. (1996) found that this feature is absent in all Thoracotremata; this character is considered as a loss in this group, because only the Heterotremata among Brachyura have a ‘‘true’’ ray zone in the acrosome. Thus, the absence of this structure in U. maracoani, U. thayeri, and U. vocator is considered here as a lost character.
Another feature observed in these three species is the presence of centrioles (character 21) in the cytoplasm in the base of the perforatorium. Medina and Rodrı´guez (1992a,b) observed centrioles only in the intermediate and final spermatids of U. tangeri, and they reported that this structure is degenerated in the adult spermatozoa. Jamieson (1994) also did not observe centrioles in U. dussumieri and M. crassipes (Thoracotremata, Ocypodidae). However, Hinsch (1986) occasionally found centrioles in mature spermatozoa of Ovalipes ocellatus (Herbst, 1799) (Portunidae) and Libinia emarginata L. (Majidae). Although centrioles are also observed in several species (Hinsch, 1988; Jamieson et al., 1996), such character was excluded from parsimony analyses due to the uncertainty of its occurrence in mature spermatozoa (Jamieson, 1994), without altering their results. A curved line attached to the inner surface of the acrosomal capsule, termed the thickened ring (character 23), was detected in all three species studied. Jamieson (1994) observed that this structure is present only in Heterotremata and Thoracotremata, is reduced in the genus Potamonautes, and is lost in grapsoid crabs (Jamieson, 1991). Such a structure is also present in the other species of the genus Uca that have been studied so far (Jamieson, 1991; Medina and Rodrı´guez, 1992a,b). Tudge et al. (1998) studied the spermatozoal morphology in several representatives of Heterotremata in which the ring occurs: Bythograea thermydron (Williams, 1980); Austinograea
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alayseae (Guinot, 1990); and Segonzacia mesatlantica (Williams, 1980) (Bythograeidae). These authors suggested that this feature is a synapomorphy of Heterotremata. Considering the ultrastructure of the spermatozoa of U. maracoani, U. thayeri, and U. vocator, we suggest that these species partially follow the morphological patterns described for other Thoracotremata brachyurans (Hinsch, 1986, 1988, 1990; Felgenhauer and Abele, 1990; Jamieson, 1991, 1994; Medina, 1992, 1995; Medina and Rodrı´guez, 1992a,b; Jamieson et al., 1995, 1996; Tudge et al., 1998). The absence of the apical button in U. thayeri spermatozoa may represent an evolutionary novelty in the genus Uca. Further studies may clarify whether the presence of the apical button really represents a plesiomorphy in the Thoracotremata. Acknowledgements This work was supported by Brazilian Research Agency CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico). We thanks for Electron Microscopy Center – UNESP, Botucatu, SP, for the technical assistance and Mrs. Janet Reid for English review. References Benetti, A.S., Negreiros-Fransozo, M.L., 2003. Madurez sexual morfolo´gica del cangrejo Uca burgersi Holthuis, 1967 (Brachyura, Ocypodidae) de uma a´rea de manglar subtropical de Brasil. Universidad y Ciencia 19 (37), 9–16. Benetti, A.S., Negreiros-Fransozo, M.L., 2004. Relative growth of fiddler crab Uca burgersi Holthuis, 1967 (Crustacea, Ocypodidae) from two mangroves in the southeastern Brazilian coast. Iheringia, Se´rie Zoologia 94 (1), 67–72. Benetti, A.S., Negreiros-Fransozo, M.L., Costa, T.M., in press. Population and reproductive biology of Uca burgersi Holthuis, 1967 (Crustacea, Brachyura, Ocypodidae) in three subtropical mangroves. Rev. Biol. Tropi. Cardoso, R.C.F., Negreiros-Fransozo, M.L., 2004. A comparison of the allometric growth in Uca leptodactyla (Crustacea: Brachyura: Ocypodidae) from two subtropical estuaries. J. Mar. Biol. Assoc. U.K. 84, 733–735. Castiglioni, D.S., Negreiros-Fransozo, M.L., 2006. Physiological sexual maturity of the fiddler crab Uca rapax (Smith, 1870) (Crustacea, Ocypodidae) from two mangroves in Ubatuba, Brazil. Braz. Arch. Biol. Technol. 49 (2), 239–248. Colpo, K.D., Negreiros-Fransozo, M.L., 2003. Reproductive output of Uca vocator (Herbst, 1804) from three subtropical mangroves in Brazil. Crustaceana 76 (1), 1–11. Costa, T.M., Silva, S.M.J., Negreiros-Fransozo, M.L., 2006. Reproductive pattern comparison of Uca thayeri Rathbun, 1900 and Uca uruguayensis Nobili, 1901 (Crustacea, Decapoda, Ocypodidae). Braz. Arch. Biol. Technol. 49 (1), 117–123. Felgenhauer, B.E., Abele, L.G., 1990. Morphological diversity of decapod spermatozoa. In: Bauer, R.T., Martin, J.W. (Eds.), Crustacean Sexual Biology. pp. 322–341. Guinot, D., 1977. Propositions pour une nouvelle classification des crustace´s decapods brachyoures. Comptes Rendus Hebdomadaires Se´ances l‘Acad. Sci., Paris 285, 1049–1052. Guinot, D., 1991. E´tablissement de la famille des Poupiniidae pour Popinia hirsute gen. nov. sp. nov. de Polune´sie (Crustacea, Decapoda, Brachyura, Homoloidea). Bull. Muse´um Natl. Hist. Nat., section A 12, 577-605. Guinot, D., Jamieson, B.G.M., Richer de Forges, B., 1994. Relationship of Homolidae and Dromiidae: evidence from spermatozoal ultrastructure (Crustacea, Decapoda). Acta Zool. 75, 255–267.
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