Zoologischer Anzeiger 251 (2012) 38–48 Male and female reproductive morphology in the inseminating genus Astroblepus (Ostariophysi: Siluriformes: Astroblepidae) Maria Angélica Spadellaa,∗ , Claudio Oliveirab , Hernán Ortegac , Irani Quagio-Grassiottob , John R. Burnsd a Disciplina Embriologia, Faculdade de Medicina de Marília – FAMEMA, Av. Monte Carmelo, 800, CP 2003, CEP 17.519-030, Marília, São Paulo, Brazil b Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista, CP 510, CEP 18.618-000, Botucatu, São Paulo, Brazil c Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Av. Arenales 1256, Jesús María, AP 14-0434, Lima 14, Peru d Department of Biological Sciences, George Washington University, Washington, DC 20052, USA Received 6 May 2010; received in revised form 11 February 2011; accepted 27 May 2011 Corresponding editor: C. Lueter. Abstract The structure of the reproductive organs of males and females of four species of Astroblepus were studied by light microscopy and TEM. In females, the gonadal and urinary orifices are separated. The urinary pore opens at the tip of a urinary papilla. In males, the testes conform to the anastomosing tubular type. Spermatogenesis is partially cystic, with most spermatid stages taking place within the lumen of the seminiferous tubules. The sperm ducts pass through seminal vesicles. The main sperm ducts fuse and join the urinary duct to form a urogenital duct that passes through to the tip of an long urogenital papilla. A circularly arranged skeletal muscle at the base of the urogenital papilla probably serves as a sphincter. The spermatozoon is an anacrosomal introsperm with an elongate, conical nucleus and an elongate midpiece. The midpiece contains numerous fused mitochondria that form rings within the cytoplasmic collar, but no vesicles were observed. The flagellum has a classic 9 + 2 axoneme with the A-tubules of the peripheral doublets being electron-dense. Two lateral fins on the posterior part of the flagellum have electron-dense termini. Nuclear chromatin is highly condensed, and the two centrioles are oriented perpendicularly to one another and are contained within a nuclear fossa. © 2011 Elsevier GmbH. All rights reserved. Keywords: Intromittent organ; Sperm ultrastructure; Introsperm; Insemination; Partially cystic spermatogenesis 1. Introduction The family Astroblepidae (Siluriformes; Osteichthyes) contains a single genus, Astroblepus, with 54 valid species (Schaefer, 2003; Ferraris, 2007). Phylogenetic studies have established the monophyly of this group (Schaefer, 1990), as ∗ Corresponding author. Tel.: +55 14 3402 1764; fax: +55 14 3413 2594. E-mail address: maspadella@gmail.com (M.A. Spadella). 0044-5231/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2011.05.005 well as its inclusion in the suborder Loricarioidei (Sullivan et al., 2006). Morphological (de Pinna, 1998; Britto, 2003) and molecular (Shimabukuro-Dias et al., 2004; Sullivan et al., 2006) phylogenetic analyses have shown Astroblepidae to be a sister group to the Loricariidae, and these two families together form a sister group to the Scoloplacidae (Sullivan et al., 2006). The reproductive biology of astroblepids is poorly known. Drawings of Astroblepus marmoratus by Johnson (1912) clearly showed an elongate palp-like structure cranial to the M.A. Spadella et al. / Zoologischer Anzeiger 251 (2012) 38–48 39 anal fin, which was not identified. Breder and Rosen (1966) later referred to this structure as an elongate “genital” papilla, while Burgess (1989) suggested that this structure is actually an elongate “urogenital” papilla that apparently functions as an intromittent organ, suggesting the occurrence of insemination in this family. An earlier study of the microscopic structure of the testes, accessory glands and intromittent organs was carried out on several species of Astroblepus (Buitrago-Suárez and Galvis, 1997). In addition, insemination was recently demonstrated in Astroblepus sabalo and Astroblepus chotae (Javonillo et al., 2009). Román-Valencia (2001), in a study on the reproductive ecology of Astroblepus cyclopus, shows that in this species ovaries mature between December and May, fecundity is low and the eggs are small. However, insemination was not documented. The goals of the present study were as follows: corroborate insemination in A. sabalo and A. chotae and provide evidence for insemination in Astroblepus trifasciatus; give more detailed histological analyses of the testes, accessory gland and intromittent organ in the male and of the ovary and the gonopore region in the female; and provide the first description of sperm ultrastructure in an astroblepid, Astroblepus cf. mancoi. 2. Materials and methods 2.1. Materials For light microscopy, a total of six specimens from three species were used: A. chotae [USNM 121129 (National Museum of Natural History, Smithsonian Institution, USA)]: one adult female (SL 63.2 mm) and one adult male (SL 93.1 mm). A. sabalo (USNM 167877): two adult males (SL 80.0 and 94.9 mm) and one adult female (SL 90.9 mm). A. trifasciatus [LBP 1352 (Laboratório de Biologia e Genética de Peixes, Departamento de Morfologia, Instituto de Biociências, UNESP, Botucatu, São Paulo, Brazil)]: one adult male (SL 71.0 mm), which was collected in the Río Jequetepeque, State of Cajamarca (Peru). These specimens had been fixed in 10% formalin and were subsequently stored in 70–75% ethanol. For transmission electron microscopy (TEM), two adult males specimens were available, which were collected in the Río Chorobamba, Río Ucayali basin, Huancabamba, Pasco (Peru). They were identified as A. cf. mancoi (LBP 3284) and is now also in the fish collection of the Laboratório de Biologia e Genética de Peixes, Botucatu, São Paulo, Brazil. 2.2. Methods For light microscopy, the tissues were dissected, cut into appropriately sized pieces to fit into the molds, dehydrated in an ethanol series, embedded in glycol methacrylate, sec- Fig. 1. Light micrographs of gonads of Astroblepus chotae, stained with toluidine blue. (A) Ovary of a sexually mature female (SL 63.2 mm, USNM 121129), with a yolky-rich oocyte (oc) and with spermatozoa (arrowheads) within the ovarian lumen (ol). (B) Testis of a sexually mature male (SL 93.1 mm, USNM 121129), demonstrating partially cystic spermatogenesis. Only early spermatids (st) with spherical nuclei are located within spermatocysts. Spermatids with ovoid nuclei (ov) have already been released into the lumen (lu) of the seminiferous tubule where the elongation of the nuclei is completed. Abbreviations: el, spermatid with elongate nucleus; sc, primary spermatocytes. Symbol: arrows, walls of spermatocysts formed by Sertoli cells. tioned and stained with either toluidine blue, hematoxylin and eosin, periodic acid-Schiff (PAS) reagent, PAS and hematoxylin, or fast green according Humason (1972) and Neto et al. (2003). For TEM, testis fragments were fixed in 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M Sorensen phosphate buffer, pH 7.4. Tissues were post-fixed in 1% osmium tetroxide for 2 h in the same buffer in the dark, stained in block with aqueous 5% uranyl acetate for 2 h, dehydrated in acetone, embedded in araldite, and sectioned and stained with a saturated solution of uranyl acetate in 50% alcohol, and lead citrate. Electron micrographs were obtained using a Phillips – CM 100 TEM. 40 M.A. Spadella et al. / Zoologischer Anzeiger 251 (2012) 38–48 Fig. 2. Light micrographs of the reproductive organs of a sexually mature male of Astroblepus sabalo (SL 94.9 mm, USNM 167877), stained with toluidine blue. (A) Testis in longitudinal section, with spermatozoa that are formed in the spermatogenic (sg) region of testis and enter a series of sperm ducts (sd). (B) Accessory gland (“seminal vesicle”) in longitudinal section with a main sperm duct containing spermatozoa (sz) passes through the accessory gland (acc), which releases a PAS+ substance (arrowheads). (C and D) Base of intromittent organ. (C) Cranial to the base of the intromittent organ, the main sperm duct (sd), which contains spermatozoa (sz) and a PAS+ substance (arrowhead) from the accessory glands, merges (arrow) with the urinary duct (ud). Abbreviation: sk, skeletal muscle. (D) Immediately cranial to the base of the intromittent organ (bio) circular skeletal muscle (csk) may serve as a sphincter. More caudally, within the intromittent organ proper, skeletal muscle tend to be oriented longitudinally (lsk). Symbol: asterisk, area external to the body of the fish. 3. Results 3.2. Testes and accessory glands (“seminal vesicles”) 3.1. Insemination The presence of insemination in the Astroblepus species could be demonstrated by the presence of spermatozoa within the lumen of an ovary of A. chotae (Fig. 1A). No specific sperm-storage structures were evident within the ovary. Instead, spermatozoa were found in the ovarian lumen in the vicinity of developing oocytes. No fertilized oocytes were present. The walls of the ovarian ducts that lead out of each ovary were highly folded, perhaps serving as sperm storage regions in this manner. The cranial portion of each testis of A. sabalo is spermatogenic (Fig. 2A). The germinal epithelium lining the seminiferous tubules contains germ cells ranging from isolated primary spermatogonia to early spermatids, with stages beyond the primary spermatogonia contained within spermatocysts (Fig. 1B). Although all stages of spermatogenesis up to the early spermatid stage take place within spermatocysts (Fig. 1B), the processes of the Sertoli cells that form these spermatocysts soon separate and open releasing the early spermatids into the lumen of the seminiferous M.A. Spadella et al. / Zoologischer Anzeiger 251 (2012) 38–48 41 Fig. 3. Mid-sagittal section through the anal and urogenital region of a sexually mature female Astroblepus sabalo (SL 90.9 mm, USNM 167877), stained toluidine blue. Observe that the wall of the intestine is highly folded. Only one anal opening is founded, but it appears to be more than one due to the angle of the sectioning. Abbreviations: an, anal opening; ovd, oviduct; pl, plug of tissue blocking the gonopore; urd, urinary duct; uro, urinary orifice; urp, urinary papilla. tubules, even though the heads of some spermatids appear to remain associated with the luminal Sertoli cell cytoplasm. Thus, the completion of spermiogenesis, during which the nucleus elongates, takes place within the lumina of the seminiferous tubules. This partially cystic spermatogenesis is clearly shown in Fig. 1B where spermatids with both slightly ovoid as well as greatly elongate nuclei can be seen within the same seminiferous tubule. Sperm packaging does not occur. Spermatozoa flow from the seminiferous tubules into a series of testis ducts lined with a simple cuboidal epithelium (Fig. 2A). These ducts eventually form a large sperm duct that passes through an accessory gland (seminal vesicle) located caudal to each testis (Fig. 2B). At this point the epithelium lining the large sperm duct resembles that of the tubules of the accessory gland (see below). The accessory glands, often referred to as seminal vesicles, are composed of interconnecting tubules whose epithelium releases a PAS+ substance into the sperm ducts (Fig. 2B). The epithelium lining the tubules appears to be simple cuboidal or columnar. However, an accurate description is hindered by the presence of cellular material on the surface, which appears to be sloughing off into the lumen. The sperm ducts of each testis eventually merge before connecting with the urinary duct (Fig. 2C). At the point where the sperm and urinary ducts merge, the opening of the sperm duct appears to be occluded by epithelial cells, so that an open channel is not apparent. 3.3. Female genital region and male intromittent organ In females, the anal, genital and urinary orifices are separate from one another (Fig. 3). The urinary duct opens at the tip of a tapering extension of the body wall, or urinary papilla. In Fig. 3, the genital pore is occluded by what appears to be a plug of epithelial cells. The intromittent organ of male Astroblepus species is an elongate, fleshy palp-like structure located immediately cranial to the anal fin (Fig. 4). It lacks any skeletal elements such as cartilage or bone. Caudal to the merging of the urinary and sperm ducts, the common urogenital duct passes through the center of the intromittent organ (Figs. 5A and 6A) and opens at it tip (Fig. 5B). Immediately cranial to the base of the intromittent organ, circularly arranged skeletal muscle fibers surround the urogenital duct (Fig. 2D). Longitudinally arranged skeletal muscle fibers are seen in the proximal portion of the intromittent organ (Figs. 2D and 5A,D). The distal remainder of the intromittent organ is devoid of skeletal muscle (Fig. 5A). The epithelium covering the intromittent organ is stratified squamous (Figs. 5C,D and 6B) with mucous cells being particularly abundant near the base of the organ (Figs. 5D and 6D). The epithelium lining the urogenital duct of the intromittent organ is simple columnar (Figs. 5C and 6C) and lacks any noticeable surface specializations such as cilia or microvilli. Abundant PAS+ secretion is evident within the lumen of the intromittent organ of the specimen in Fig. 6. 42 M.A. Spadella et al. / Zoologischer Anzeiger 251 (2012) 38–48 3.4. Sperm ultrastructure Fig. 4. Photograph of an intromittent organ of a sexually mature male Astroblepus sabalo (SL 80.0 mm, USNM 167877). (A) Lateral view. (B) Ventral view. Abbreviation: af, anal fin. Symbol: arrow, intromittent organ. The spermatozoon of A. cf. mancoi is an anacrosomal introsperm with an elongate, conical nucleus, a symmetrical midpiece, and a single medial flagellum (Fig. 7A, H, I, J and K). Within the sperm head, the nucleus is 6.0 ␮m long and 0.6 ␮m wide. It is slightly ovoid in transverse section (Fig. 7B). No other organelles are evident in the region of the nucleus. Nuclear chromatin is highly condensed and homogeneous with occasional small electron-lucent areas present (Fig. 7A, B, C, I and J). At the posterior region of the nucleus a deep nuclear fossa (0.7 ␮m long and 0.3 wide) contains the centrioles and the basal segment of the flagellum (Fig. 7A, C, D and J). In a longitudinal section, the shape of the nuclear fossa is an arc with irregular walls (Fig. 7J). The proximal centriole is immediately anterior and perpendicular to the distal one (Fig. 7J). Some electron-dense substance appears to be associated with the distal centriole (Fig. 6J). Posterior to the nucleus, the midpiece is approximately 5.4 ␮m long and 0.6 ␮m wide (Fig. 7A and K). A cytoplasmic canal (4.8 ␮m long and 0.4 ␮m wide), holding the proximal Fig. 5. Longitudinal sections through the intromittent organ of a sexually mature male Astroblepus sabalo (SL 94.9 mm, USNM 167877), stained with toluidine blue. Cranial is to the left. (A) Section through most of the intromittent organ showing a large central duct representing the merged urinary and testicular ducts. (B) Damaged distal tip of the intromittent organ showing the orifice to the exterior (arrowhead). (C) Higher magnification of one side of intromittent organ. (D) Area near the base of the intromittent organ showing a mostly longitudinally oriented skeletal muscle. Abbreviations: bv, blood vessels; dct, dense connective tissue; lct, loose connective tissue; le, luminal epithelium of the duct of the intromittent organ; mc, mucous goblet cells; oe, epidermis of the intromittent organ; sk, skeletal muscle. Symbols: arrowhead, orifice of the intromittent organ; asterisk, lumen of the duct of the intromittent organ. M.A. Spadella et al. / Zoologischer Anzeiger 251 (2012) 38–48 43 Fig. 6. Transverse sections through the intromittent organ of a sexually mature male Astroblepus trifasciatus (SL 71.0 mm, LBP 1352). (A) Section through the whole intromittent organ; stain H&E. (B) Detail of the epidermis; fast green stain. (C) Detail of lumen containing PAS stained substances; PAS stain. (D) Detail of the epidermis showing mucous cells (arrows); PAS stain. Abbreviations: dct, dense connective tissue; lct, loose connective tissue; lu, luminal epithelium; oe, epidermis. Symbols: asterisk, lumen of intromittent organ; arrow, mucous cells. portion of the flagellum, occupies the center of the midpiece (Fig. 7A, E, F, G, J and K). Mitochondria that appear to be fused to one another form rings that occupy the cytoplasmic collar, which surrounds the cytoplasmic canal throughout most of the midpiece (Fig. 7E, J and K). The most posterior region of the cytoplasmic collar is thin and devoid of mitochondria, forming a cytoplasmic sheath (Fig. 7A, F, G and K). Vesicles are not observed within the midpiece. The axoneme of the flagellum exhibits the classic 9 + 2 microtubular pattern, with the A-tubules of the peripheral doublets being electron-dense. The flagellum then becomes free beyond the posterior terminus of the cytoplasmic canal (Fig. 7A and L). Within the posterior portion of the cytoplasmic canal (Fig. 7G), the flagellum begins to develop two lateral fins that are more elongate on the free portion of the flagellum (Fig. 7L, inset). The distal ends of these flagellar fins are dilated and filled with electron-dense material (Fig. 7L, inset). 4. Discussion Insemination, namely the introduction of spermatozoa into the female reproductive tract (Burns et al., 1997), had been suggested previously to occur in astroblepids (Breder and Rosen, 1966), but was only recently demonstrated histologically by the presence of spermatozoa in the ovaries of A. sabalo and A. chotae (Javonillo et al., 2009) and in the present study of these two species and A. trifasciatus. The only other catfish families with species known to be inseminating are Scoloplacidae (Burns et al., 2000; Spadella et al., 2008; Javonillo et al., 2009) and Auchenipteridae (von Ihering, 1937; Loir et al., 1989; Burns et al., 2000). Although spermatozoa were shown to be present within astroblepid ovaries, i.e., insemination, no fertilized oocytes have been observed. Therefore, the exact timing of fertilization, i.e., the union of sperm and egg, is still unknown for these species. Given that in some teleosts insemination can actually be followed by external fertilization, as in the process of internal gametic association found in some species of Cottidae (Abe and Munehara, 2009), the term “insemination” rather than “internal fertilization” best describes the current data until the time of fertilization is clearly established in a given inseminating species. Other examples of the inseminating species among Teleostei, are found in Cyprinodontiformes, in the families Poeciliidae (Grier, 1975; Kobayashi and Iwamatsu, 2002), 44 M.A. Spadella et al. / Zoologischer Anzeiger 251 (2012) 38–48 Fig. 7. Transmission electron micrographs of spermatozoa from caudal testis ducts of Astroblepus cf. mancoi (SL 95.0 mm, LBP 3284). (A) Longitudinal section through most of the spermatozoon. (B–G) Series of successively more caudal transverse sections through a spermatozoon. (B) The mid-region of a nucleus. (C) Cranial region of the nucleus fossa at the level of the basal body. (D) Caudal region of the nuclear fossa showing the flagellum within the cytoplasmic canal. (E) The midpiece region with mitochondria, showing the flagellum within the cytoplasmic canal. (F) The cytoplasmic sheath with the flagellum within the cytoplasmic canal. (G) Caudal region of the cytoplasmic sheath; the flagellum within the cytoplasmic canal has short fins. (H) Spermatozoa within testicular ducts are not arranged into packets. (I) Longitudinal section through the nucleus showing a curved anterior tip (arrow). (J) Longitudinal section through the posterior part of the nucleus showing the arrangement of centrioles within the nuclear fossa. (K) Longitudinal section through the elongate midpiece showing the cytoplasmic sheath at its posterior extremity. (L) Longitudinal sections through flagella; inset: transverse sections through flagella showing the two flagellar fins, each with an expanded terminus (arrowhead) containing electron-dense material. Abbreviations: bb, basal body; cc, cytoplasmic canal; cs, cytoplasmic sheath; dc, distal centriole; f, flagellum; m, mitochondrion; n, nucleus; pc, proximal centriole; s, spermatozoon. Symbols: arrow, curved anterior tip of a nucleus; arrowhead, expanded terminus of a flagellar fin. M.A. Spadella et al. / Zoologischer Anzeiger 251 (2012) 38–48 Anablepidae, and Jenynsiidae (Grier et al., 1981); in Scorpaeniformes, in several species of the family Scorpaenidae (Jamieson, 1991; Muñoz et al., 2002), and in Characiformes, in some species of the Characidae subfamilies: Cheirodontinae (Burns et al., 1997), Glandulocaudinae and Stevardiinae (Burns et al., 1995; Burns et al., 1998; Azevedo et al., 2000; Pecio et al., 2005; Burns and Weitzman, 2005). The testis of astroblepids conforms to the anastomosing tubular type (Grier, 1993; Grier and Uribe Aranzábal, 2009), with primary spermatogonia with no apparent pattern throughout the seminiferous tubules rather than being restricted to the testis periphery as in the Atherinomorpha. Spermatogenesis in astroblepids is partially cystic, where the later stages of spermiogenesis occur in the lumina of the seminiferous tubules rather than within spermatocysts. Classic semicystic spermatogenesis, in which all stages beyond the primary spermatocyte occur outside of spermatocysts, has recently been shown to occur in the siluriform families Cetopsidae, Asprenidae, and Nematogenyidae (Spadella et al., 2006a), Ariidae (Ortiz, 2008; Burns et al., 2009), and some species of Doradidae (Ortiz, 2008). It is also known to occur in the more distantly related teleost orders Scorpaeniformes (Mattei et al., 1993; Muñoz et al., 2002; Hernández et al., 2005), Lophiiformes (Yoneda et al., 1998) and the perciform family Blenniidae (Lahnsteiner et al., 1990). The accessory glands located caudal to each testis can be classified as seminal vesicles based on the definition of Lahnsteiner and Patzner (2009), given that they are lobe-like glands that empty into the sperm ducts. The brief description of the astroblepid seminal vesicle presented by BuitragoSuárez and Galvis (1997) agrees with that in the present study. The secretion may be released through an apocrine mechanism as has been reported for the seminal vesicle of Zosterisessor ophiocephalus (Gobiidae) (Lahnsteiner and Patzner, 2009). The secretion forms part of the final seminal secretion (semen or milt) released through the intromittent organ. The function of the mucoid PAS+ secretion of the astroblepid seminal vesicles is unknown. Testicular secretions are known to inhibit sperm motility while in the testis in Alburnus alburnus and Leuciscus cephalus (Lahnsteiner et al., 1994), Perca fluviatilis (Lahnsteiner et al., 1995) and Scaphirhynchus platorhynchus and Polyodon spathula (Cosson et al., 2000), and may, therefore, also serve that same function during sperm storage in the ovary. In addition, seminal fluids often contain abundant nutrients that may serve as energy substrates for the spermatozoa both in the testis (Lahnsteiner et al., 1994, 1995) and within the ovary during sperm storage (Muñoz et al., 2002). Finally, the seminal vesicle secretion of an inseminating auchenipterid catfish, Trachycorystes striatulus, was reported to function as a plug that is inserted into the oviduct during insemination (von Ihering, 1937). Seminal vesicles are present in a number of externally fertilizing siluriforms (Lahnsteiner and Patzner, 2009), as well as in the inseminating family Auchenipteridae (von Ihering, 1937; Loir et al., 1989; Meisner et al., 2000; Burns et al., 2002). In Loricariidae, the sister taxon 45 of Astroblepidae, the seminal vesicles were “poorly defined” in three loricariid genera, Hypostomus, Pseudancistrus and Harttia (Loir et al., 1989), while in Ancistrus, the seminal vesicles were absent (Mansour and Lahnsteiner, 2003). In spite of seminal vesicles being absent in species from two loricariid subfamilies, Neoplecostominae and Hypoptopomatinae, abundant secretion was present in the lumina of the seminiferous tubules (Spadella, 2007) and had perhaps been released by Sertoli cells. Thus, the presence of a luminal secretion does not necessarily indicate the presence of seminal vesicles. Given that the Loricariidae is comprised of 683 recognized species (Reis et al., 2003), further studies are necessary to determine the status of the seminal vesicle in this speciose taxon. Seminal vesicles are absent in the inseminating Scoloplacidae (Spadella et al., 2008), the sister taxon of Astroblepidae and Loricariidae, but this absence could relate in some manner to the miniaturization of these fishes. The results presented here clearly demonstrate that the astroblepid intromittent organ is an elongate urogenital papilla. In the female, the genital and urinary ducts exit the body through separate openings. However in the male, the sperm duct merges with the urinary duct and the combined urogenital duct passes through to the tip of the intromittent organ. The epithelium lining the urogenital duct more closely resembles the lining the urinary duct than that of the sperm duct. Therefore, the intromittent organ may have developed through elongation of the urinary papilla, which is still present in the female. Although the astroblepid intromittent organ is separated from the anal fin, the second, third and fourth fin rays of the anal fin are modified in males (BuitragoSuárez and Galvis, 1997). The possible role of the anal fin during insemination is still unknown at this time. BuitragoSuárez and Galvis (1997) provide a single transverse section through what is labeled as the “copulatory organ” of an unidentified astroblepid showing dense “fibroelastic tissue” within its wall. Similar tissue was not seen in the intromittent organs of the present study. It has not yet been established whether, the ducts through the intromittent organs of the inseminating Auchenipteridae represent urogenital ducts or solely genital ducts. Elongate “genital” papillae were reported in both males and females of the inseminating Scoloplacidae (Spadella et al., 2008). A urogenital duct passes through the intromittent organ, which is also an elongate urogenital papilla, of Peruvian populations of characid fishes ascribed to the genus Monotocheirodon (Burns and Weitzman, 2006). These fishes also have bands of circularly arranged skeletal muscle surrounding the duct at the base of the organ, possibly serving as a voluntary sphincter for the release of spermatozoa during insemination (Burns and Weitzman, 2006). The gonoducts of both a male and female A. sabalo in the present study were occluded by similar plugs of cells. In the male this occurred internally at the point where the urinary and sperm ducts merge (Fig. 2C), whereas in the female the opening to the exterior was occluded (Fig. 3). Although both specimens had achieved sexual maturity, they appeared to be 46 M.A. Spadella et al. / Zoologischer Anzeiger 251 (2012) 38–48 reproductively inactive as evidenced by the lack of abundant PAS+ secretion within the tubules of their seminal vesicles (Fig. 2B) by an empty urogenital duct within the intromittent organ of the male (Fig. 4A), and by very few spermatozoa within the ovary of the female. Occluded gonoducts have also been reported in a female specimen of the inseminating catfish Scoloplax distolothrix (Javonillo et al., 2009), a female specimen of the inseminating characid Brittanichthys axelrodi (Javonillo et al., 2007), and viviparous females in the families Poeciliidae (Weishaupt, 1925; Peters and Mäder, 1964) and Anablepidae (Weyenbergh, 1875; Burns and Flores, 1981). Although the function of duct closure is unknown, blockage of the opening to the ovarian duct may prevent insemination by additional males, suggesting a possible role in sperm competition. It would be interesting to know if a subsequent opening of occluded ducts during active reproductive periods involves the secretion of hydrolytic enzymes, as has been demonstrated for the rupture of the ovarian follicle in Oryzias latipes (Beloniformes) during ovulation (Ogiwara et al., 2005). The spermatozoon of Astroblepus can be classified as an anacrosomal introsperm based on the absence of an acrosome and the presence of an elongate nucleus and midpiece (Jamieson, 1991). Astroblepidae species share many sperm characters with species of Loricariidae (Mansour and Lahnsteiner, 2003; Spadella, 2007) reflecting the sister relationship of these two families based on molecular data (Sullivan et al., 2006). Astroblepidae species also share additional characters with species of Scoloplacidae (Spadella et al., 2006b), which is hypothesized to be a sister group to the group formed by Astroblepidae and Loricariidae (Sullivan et al., 2006). These additional characters shared by Astroblepidae and Scoloplacidae species, that include mitochondria forming an internal ring surrounding the cytoplasmic canal in the midpiece, a conical nucleus with curved tip and two lateral flagellar fins, may relate more to sperm modifications associated with the reproductive mode of insemination seen in these two groups and likely to have evolved in convergence. Acknowledgments We would like to thank Mário C.C. de Pinna, Gino Asteaga Koo, Denis Orihuela Preising, and Edgardo Castro Belapatiño for their help during the collection expedition, and the E.M. Laboratory of Instituto de Biociências de Botucatu-Universidade Estadual Paulista, São Paulo, Brazil for allowing the use of their facilities. This research was supported by the Brazilian agencies Fundação de Apoio à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico. 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