Micron 69 (2015) 43–55

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Spermatogenesis and spermatozoa ultrastructure of two Dipolydora species (Annelida: Spionidae) from the Sea of Japan Vasily I. Radashevsky a,b,∗ , Olga V. Yurchenko a , Sergey A. Tyurin a , Yana N. Alexandrova a a b

A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, 17 Palchevsky Street, Vladivostok 690059, Russia Far Eastern Federal University, 8 Sukhanov Street, Vladivostok 690950, Russia

a r t i c l e

i n f o

Article history: Received 30 September 2014 Received in revised form 9 November 2014 Accepted 11 November 2014 Available online 20 November 2014 Keywords: Polychaete Spermiogenesis Electron microscopy

a b s t r a c t Spermatogenesis and the structure of the spermatozoa of two spionid polychaetes Dipolydora bidentata and Dipolydora carunculata are described by light and transmission electron microscopy. Both species are gonochoristic borers in shells of various molluscs. Proliferation of spermatogonia occurs in paired testes regularly arranged in fertile segments, and the rest of spermatogenesis occurs in the coelomic cavity. Early spermatogenesis occurs quite similarly in the two species but results in formation of tetrads of interconnected spermatids in D. bidentata and octads of spermatids in D. carunculata. Three consecutive stages of spermiogenesis are recognized according to the condensation of chromatin in nucleus: (1) early spermatids with heterogeneous, partly clumped chromatin, (2) middle spermatids with homogeneous, coarsely granular chromatin, and (3) late spermatids with homogeneous fibrillar chromatin. Moreover, late stage of spermatids is further classified into two stages, I and II, according to the position of the acrosome and shape of the nucleus. In late spermatids I, the acrosome is situated in the anterior invagination of the funnel-shaped to oval nucleus, whereas in late spermatids II the acrosome is situated on top of the elongated nucleus. Ultrastructural composition of cells at each stage of spermatogenesis is described and illustrated. The possible process of morphogenesis of organelles during spermato- and spermiogenesis is reconstructed for both species. The proacrosomal vesicle first appears in early spermatids near the Golgi complex and then migrates anteriorly; in the middle spermatids, the acrosome comes to lie in a deep anterior nuclear fossa. In late spermatids I, this fossa evaginates and a posterior fossa develops in the nucleus housing basal body and the anterior part of the axoneme. In late spermatids II, the mitochondria elongate and probably reduce in number due to fusion of some of them. The mature spermatozoa in both species are introsperm with the conical acrosome, subacrosomal plate, long nucleus with short posterior fossa, long midpiece with elongated mitochondria, and long flagellum with 9 × 2 + 2 organization of microtubules. Numerous flat rounded platelets with putative glycogen are present throughout most part of the nucleus and the midpiece. The process of spermatogenesis in D. bidentata and D. carunculata is similar to that in other Dipolydora, Polydora and Pseudopolydora species. Spermatozoa in these polydorin spionids have similar composition and differ mainly in size of the nucleus and the midpiece. Elongated spermatozoa are adapted for transfer in spermatophores and an internal fertilization which is characteristic for brooding species. Diversely modified spermatozoa among spionids may be signs of the diversity of fertilization biology within the Spionidae. The exact places where fertilization occurs in brooding spionids however remains unknown. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Brooding, in various structures produced by females on gamete release, is a rather common kind of parental care of offspring

∗ Corresponding author at: A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, 17 Palchevsky Street, Vladivostok 690059, Russia. Tel.: +7 914 706 1977. E-mail address: [email protected] (V.I. Radashevsky). http://dx.doi.org/10.1016/j.micron.2014.11.004 0968-4328/© 2014 Elsevier Ltd. All rights reserved.

in marine invertebrates (Thorson, 1946). It is usually associated with the occurrence of modified spermatozoa (introsperm) and the mode of fertilization is other than that of gamete release into sea water for external fertilization and where holopelagic development may take place (Franzén, 1956). The polychaetes of the family Spionidae Grube, 1850 demonstrate a great diversity of gametes, methods of reproduction and modes of larval development (reviews by Blake and Arnofsky, 1999; Blake, 2006). The origin and subsequent evolution of these diverse reproductive characteristics remain however uncertain.

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Among spionids (ca. 500 species in 40 genera), Rhynchospio Hartman, 1936 and Streblospio Webster, 1879 brood offspring on the dorsal side of their body (Fonsêca-Genevois and Cazaux, 1987; Radashevsky, 2007a), some Malacoceros Quatrefages, 1843 and Scolelepis Blainville, 1828 brood offspring in mucus masses and cocoons, respectively (Guérin, 1974; Blake and Arnofsky, 1999), while members of the subfamily Spioninae Söderström, 1920 (ca. 230 species in 11 genera) brood their larvae in capsules produced by female’s nephridia. Brooding in Spionidae is usually associated with elongated, filiform or coiled spermatozoa, sperm transfer via spermatophores, sperm storage in various seminal receptacles (Rice, 1978), and mixed development, including lecithotrophy at early stages and planktotrophy at later stages of larval development. Parental care within the Spionidae has likely evolved more than once from the ancestral condition of free spawning and holopelagic larval development (Radashevsky, 2007b). Consequently, modified spermatozoa of different spionid brooders might have evolved independently from short-headed spermatozoa characteristic for free spawning ancestors. In this case, modified spermatozoa in different groups of Spionidae may have different ultrastructural composition. Studies on the ultrastructure of spionid short-headed spermatozoa (ect-aquasperm) were recently overviewed by Radashevsky et al. (2011). Spermiogenesis and fine morphology of spionid modified spermatozoa were described in Polydora ciliata (Johnston, 1838) (Franzén, 1974), Polydora cornuta Bosc, 1802 (Rice, 1981, as Polydora ligni Webster, 1879), Dipolydora socialis (Schmarda, 1861), Polydora websteri Hartman in Loosanoff and Engle, 1943, Streblospio benedicti Webster, 1879 (Rice, 1981), Pseudopolydora sp.1 (Rouse, 1988, as Tripolydora sp.), Polydora neocaeca Williams and Radashevsky, 1999 (Williams, 2000), Scolelepis laonicola2 (Tzetlin, 1985) (Vortsepneva et al., 2006, as Asetocalamyzas laonicola), Boccardiella hamata (Webster, 1879) (Rice, 1992; Reunov et al., 2010), Pseudopolydora paucibranchiata (Okuda, 1937) (Rice, 1992), and Spio setosa Verrill, 1873 (Eckelbarger and Hodgson, 2014). Dipolydora Verrill, 1881 comprises about forty species of polydorin spionids (tribe Polydorini Benham, 1896 sensu Radashevsky, 2012, ca. 170 species in 8 genera, comprising Amphipolydora Blake, 1983, Boccardia Carazzi, 1893, Boccardiella Blake & Kudenov, 1978, Carazziella Blake & Kudenov, 1978, Dipolydora, Polydora Bosc, 1802, Polydorella Augener, 1914, and Pseudopolydora Czerniavsky, 1881) that occupy diverse habitats from the intertidal to deep water and have evolved diverse reproductive strategies. The ultrastructure of gametes has been studied only in D. socialis (Schmarda, 1861) (Rice, 1981) but general morphology of gametes and larval development have been described with light microscopy in a series of species (Blake and Arnofsky, 1999: table 2). Herein, we describe the complete spermatogenesis and ultrastructure of spermatozoa of Dipolydora bidentata (Zachs, 1933) and Dipolydora carunculata (Radashevsky, 1993), common inhabitants in shallow waters of the Sea of Japan. The former species, attaining 80 mm long for 250 chaetigers, is an opportunistic borer in shells of various molluscs, barnacles and also coralline algae. The latter species, attaining 70 mm long for 195 chaetigers, bores in various shells and sponges but also inhabits mud tubes on soft bottom (Radashevsky, 1993; Radashevsky and Pankova, 2013). Males of both species produce spermatophores and pass them to females.

Females store sperm in dorsal seminal receptacles and brood larvae in capsules produced by nephridia and attached to the inner wall of the burrow. Larvae grow 4 chaetigers inside the capsules then hatch and continue development feeding in the plankton until the 18–20-chaetiger stage when they become able to settle and metamorphose (Radashevsky, unpublished). This is one in a series of ultrastructural studies by the authors aiming to describe the diversity of spermatozoa in various spionids to facilitate greater understanding of the pathways of sperm transformation accompanying evolution of parental care in the Spionidae (Reunov et al., 2010; Radashevsky et al., 2010, 2011). 2. Material and methods Dipolydora bidentata and D. carunculata adults were collected in Vostok Bay (part of the larger Peter the Great Bay) of the Sea of Japan, near the Vostok Marine Biological Station (42.8925◦ N, 132.735◦ E) of the A.V. Zhirmunsky Institute of Marine Biology, in April 2011. Worms were extracted from shells of the Yesso scallop Mizuhopecten yessoensis (Jay, 1857) with pliers and sorted under a microscope in the laboratory. Gametes were preliminary examined on semi-squashed preparations from living worms. The gamete-bearing segments of live mature males were cut-off, opened, incubated with acridine orange for 5 min and then rinsed twice in the filtered sea water. Semi-squashed preparations of these segments were examined with a light microscope Carl Zeiss Axio Imager Z2 equipped with a digital camera. The preparations were viewed using differential interference contrast (DIC) and module U1 equipped with an ultraviolet BP 330–380 excitation filter for fluorescent techniques. For the transmission electron microscopy (TEM), the gametebearing segments of mature males were cut-off and fixed for 2 h at 4 ◦ C. The fixative was 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 21 mg/ml NaCl to make the solution isotonic to sea water. Specimens were washed in several changes of buffered sodium cacodylate with added NaCl and then post-fixed in 2% buffered OsO4 for 2 h in a dark room. After dehydration in a graded ethanol series and acetone, they were embedded in Araldite-EMbed812 resin (Araldite-EMbed-812 Embedding Kit, EMS). Semi- and ultra-thin sections were made using a Leica UC6 ultramicrotome. Semi-thin sections were stained with methylene-blue and examined with a Carl Zeiss Axio Imager Z2 light microscope equipped with a digital camera. Ultra-thin sections were stained with 2% alcoholic uranyl acetate and aqueous lead citrate, and then viewed with transmission electron microscopes JEOL JEM 100S and Carl Zeiss Libra 120 at the Far East Centre of Electron Microscopy, Institute of Marine Biology FEB RAS. The sperm cells and their organelles were measured using Carl Zeiss Libra 200 software. The flagella of spermatozoa were measured using a light microscope. The measurements are given in microns, ␮m as mean ± standard deviation (Table 1). Representative adult specimens were fixed in 10% sea-water formalin, rinsed in fresh water, transferred to 70% ethanol, and deposited in the Museum of the Institute of Marine Biology (MIMB) in Vladivostok, Russia. 3. Results 3.1. Early spermatogenesis

1

Rouse (1988) identified the examined worms as Tripolydora sp. These worms were re-examined and re-identified later as Pseudopolydora sp. (Radashevsky, unpublished). 2 Vortsepneva et al. (2006) initially identified the examined worms as Asetocalamyzas laonicola Tzetlin, 1985 (family Calamyzidae) but later recognized them as dwarf males of the spionid Scolelepis laonicola (Tzetlin, 1985) (Vortsepneva et al., 2008).

The paired testes are regularly arranged throughout the midbody region of males, from chaetigers 23–40 to chaetigers 40–140 in D. bidentata, and from chaetigers 20–33 to chaetigers 35–134 in D. carunculata. In both species, each testis is covered by a thin peritoneal epithelium and suspended on a short blindly-ending blood

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Table 1 Dimensions of mature spermatozoa (in microns, ␮m; the number of measurements is given in parentheses after the mean and standard deviation). D. bidentata

Organelle

Acrosome Nucleus Posterior nuclear fossa Midpiece Flagellum

D. carunculata

Length

Width

Length

Width

1.02 ± 0.05 (24) 5.48 ± 0.29 (22) 1.0 ± 0.07 (14) 4.07 ± 0.42 (3) 56 ± 3 (3)

0.3 ± 0.03 (25) 0.64 ± 0.04 (24) 0.3 ± 0.03 (14) 1.5 ± 0.27 (20) 0.3

1.05 ± 0.08 (12) 4.6 ± 0.13 (12) 1.11 ± 0.07 (14) 6.1 ± 0.30 (5) 55 ± 3 (3)

0.34 ± 0.02 (16) 0.76 ± 0.07 (17) 0.34 ± 0.02 (14) 1.7 ± 0.32 (15) 0.3

Fig. 1. Male internal anatomy in Dipolydora carunculata. (A) Semi-thin cross-section through a fertile segment. (B) Part of testis associated with a genital blood vessel. (C) Sperm cells at various stages of development free floating in the coelom. Scale bars: (A)—100 ␮m; (B and C)—20 ␮m. bv, genital blood vessel; gut, midgut; dm, band of dorsal longitudinal muscles; sc, spermatocytes; sd, spermatids; sg, spermatogonia; sz, spermatozoa.

vessel in the anterior ventro-lateral part of the coelom (Fig. 1A and B). The genital vessels rise up from the segmental blood vessels interconnecting main dorsal and ventral vessels. Of sperm cells, testes contained only proliferating spermatogonia. The coelom of the fertile segments of mature D. bidentata males was filled with diads of spermatogonia, spermatocytes I and spermatocytes II, tetrads of spermatids, and individual spermatozoa (Fig. 2). In D. carunculata males, coelom contained individual and diads of spermatogonia, diads of spermatocytes I, octads of spermatids, and individual spermatozoa (Figs. 1C and 2); rarely observed were “diads” of partially divided spermatocytes with four and eight nuclei (Fig. 2C). Although during spermato- and spermiogenesis sperm cells remained interconnected in different groups in the two species, they had similar fine composition. Generalized descriptions of the sperm stages presented below therefore relate equally to both of them. Differences between some details in the species are noted where appropriate.

3.1.1. Spermatogonia Spermatogonial cells in the testes (Fig. 1B) appear similar to those in the coelom (Fig. 1C) and are characterized by the presence of single dense nucleolus. Rarely, in the case of D. carunculata, mitotic figures were observed in spermatogonia in the coelom. The spermatogonia in this species are about 9 ␮m in diameter, each containing a large nucleus about 5.8 ␮m in diameter (ca. 27% of the total cell volume), and a nucleolus 1.5 ␮m in diameter. The nuclear double membrane contains numerous small pores. Clumps of coarse chromatin are scattered in the nucleoplasm (Fig. 3A). Golgi bodies (Fig. 3B), cisterns of endoplasmic reticulum

(Fig. 3C), electron-lucent vesicles, nuage-material (Fig. 3D) and small mitochondria are present in the cytoplasm. 3.1.2. Spermatocytes The spermatocytes differ from the spermatogonia mainly by the absence of nucleoli. In spermatocytes, the nuclei are of similar diameter but the cytoplasm is reduced comparing to the spermatogonia. Consequently, nuclei occupy a greater part of cell in the spermatocytes than in the spermatogonia. In D. carunculata, the spermatocytes I are about 8 ␮m in diameter, with a nucleus about 6 ␮m in diameter (ca. 42% of the total cell volume). The chromatin is finely granular and homogenous with distinct synaptonemal complexes in the prophase of the first meiotic division (Fig. 3E and F). Golgi bodies (Fig. 3G) and cisterns of endoplasmic reticulum (Fig. 3I) are well developed in the cytoplasm. A centriole is situated near the Golgi complex (Fig. 3G). The flagellum is not yet developed. Numerous small electron-dense granules of nuage-like material are gathered in clumps (Fig. 3H) on either side of the cytoplasmic bridge. Numerous electron-lucent vesicles are scattered all over the cytoplasm of spermatocytes in the beginning (Fig. 3E and I) and in the end (Fig. 3J and K) of cytokinesis. The vesicles are usually spherical to oval, up to 0.3 ␮m in diameter (Fig. 3I and J). Elongated vesicles appeared along division line of the dividing spermatocytes (Fig. 3K). 3.2. Spermiogenesis The spermatids resulted from the meiotic divisions of spermatocytes are interconnected by cytoplasmic bridges in 4- and 8-cell

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Fig. 2. Coelomic sperm cells in D. bidentata and D. carunculata observed in life with differential interference contrast (DIC) and fluorescent techniques after incubating with acridine orange (AO). (A) Spermatogonia. (B) Spermatocytes I. (C) Spermatocytes II. (D) Spermatids. (E) Spermatozoa. Scale bar (same for all figures): 10 ␮m.

groups in D. bidentata and D. carunculata, respectively (Fig. 2). Early spermatids are smaller than the spermatocytes and have smaller single nuclei. In D. carunculata, the spermatids are about 4.5 ␮m in diameter, with nuclei about 3.5 ␮m in diameter (ca. 47% of the total cell volume). Cells in each group synchronously pass through a continuous series of cytoplasmic changes and split apart in the late stage of spermiogenesis. Changes leading to the formation of mature spermatozoa are similar in the two species. The main visible changes include: condensation of chromatin, transformation of shape of the nucleus and of the mitochondria. The condensation of chromatin is used here as a marker to distinguish three consecutive stages of spermiogenesis viz, early, middle and late. In early spermatids, the chromatin is heterogeneous, partly clumped (Figs. 4A and 8A). In middle spermatids, the chromatin is homogeneous and coarsely granular (Figs. 4B and C and 8B), whereas in late spermatids the chromatin is homogeneous and fibrillar (Figs. 4D–K and 8C and D). Moreover, late stage of spermatids may be further classified into two stages, I and II, according to the position of the acrosome and

shape of the nucleus. In late spermatids I, the acrosome is situated in the anterior invagination of the funnel-shaped to oval nucleus (Figs. 4D–G and 8C), whereas in late spermatids II the acrosome is situated on top of the elongated nucleus (Figs. 4H–K and 8D). Ultrastructural composition of spermatids at each stage is described below. The possible process of morphogenesis and the behavior of organelles during spermiogenesis are reconstructed in Section 4. 3.2.1. Early spermatids Early spermatids with heterogeneous clumped chromatin have spherical to slightly oval nuclei without invaginations (Figs. 4A and 8A). Prospective polarity of the cells is determined by the localization of the Golgi complex and nearby aggregation of mitochondria. These organelles are usually situated on a side opposite to the bridge interconnecting cells in a group. In some spermatids, however, the intercellular bridge occurs on a side oblique to perpendicular to the axis formed by the nucleus and the Golgi complex (see those bridges in middle spermatids on Fig. 4B and C). An

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Fig. 3. Ultrastructure of early sperm cells in D. carunculata. (A–D) Spermatogonium. (A) General view of a spermatogonium with heterogeneous chromatic and single electron-dense nucleolus inside nucleus. (B) Golgi bodies and double nuclear membrane. (C) Endoplasmic reticulum. (D) Nuage-like material. (E–H) Spermatocytes I. (E) Spermatocytes I interconnected by a cytoplasmic bridge, with synaptonemal complexes inside nucleus and nuage-like material in cytoplasm. (F) Higher magnification of a fragment of nucleus with synaptonemal complexes. (G) Fragment of cytoplasm with Golgi bodies and centriole. (H) Fragment of cytoplasm with nuage-like material. (I) Putative spermatocyte II. (J and K) Successive stages of spermatocyte division with large number of electron-lucent vesicles. Scale bars: (A, E and I–K)—2 ␮m; (B, C and G)—1 ␮m; (F and H)—0.5 ␮m. c, centriole; ch, chromatin; er, endoplasmic reticulum; Gb, Golgi bodies; m, mitochondria; n, nucleus; nm, nuage-like material; nu, nucleolus; arrows—synaptonemal complexes; arrowheads—electron-lucent vesicles.

electron-dense proacrosomal vesicle can hardly be distinguished from other products of the Golgi complex when it is situated nearby the complex. A small electron-dense vesicle appears on a lateral side of the nucleus. An axoneme is not yet developed at this stage. 3.2.2. Middle spermatids Middle spermatids with homogeneous coarsely granular chromatin have a shallow invagination in the anterior end of nucleus, housing a spherical to oval acrosomal vesicle with homogeneous

electron-dense contents (Figs. 4B and C and 8B). Electron-dense and electron-lucent inclusions are scattered all over the cytoplasm. 3.2.3. Late spermatids Late spermatids with homogeneous fibrillar chromatin exhibit the highest diversity of the shape of nucleus and shape and position of acrosome and mitochondria. Late spermatids I show various degrees of the development and transformation of the anterior nuclear fossa which, in initial and

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Fig. 4. Ultrastructure of late sperm cells in D. carunculata. (A) A group of early spermatids, showing spherical nuclei with partly clumped chromatin; 5 cells (of 8 in total) interconnected by cytoplasmic bridges (not in the plane of section). (B and C) Middle spermatids. (B) Longitudinal section, showing oval nucleus with coarsely granular chromatin and small anterior fossa housing spherical homogeneous electron-dense acrosomal vesicle. (C) Longitudinal section, showing nucleus with homogeneous coarsely granular chromatin and short anterior fossa housing oval acrosomal vesicle; mitochondria located in basal part of cell. (D–G) Late spermatids I. (D) Longitudinal section, showing nucleus with fibrillar chromatin and deep conical anterior fossa housing elongated acrosome vesicle. (E) Longitudinal section through basal part of a spermatid, showing distal and proximal centrioles with pericentriolar complex. (F) Transverse section through basal part of a spermatid, showing cisterns of endoplasmic reticulum and 8–9 spherical mitochondria arranged in a circle near posterior end of nucleus. (G) Longitudinal section of a spermatid, showing ovoid nucleus with fibrillar chromatin and partly reduced anterior fossa housing acrosome vesicle. (H–K) Late spermatids II. (H) Longitudinal section of a spermatid, showing narrow, slightly elongated nucleus with fibrillar chromatin and greatly reduced anterior fossa, an acrosome with electron-lucent inner compartments, and platelets in periphery of cell. (I) Longitudinal section through basal part of a spermatid, showing posterior nuclear fossa housing a basal body, and mitochondria around anterior part of axoneme. (J) Transverse section through basal part of a spermatid, showing eleven mitochondria tightly packed in a circle around anterior part of axoneme. (K) Longitudinal section of a spermatid, showing elongated nucleus with homogeneous electron-dense chromatin and without anterior fossa, acrosome on top of the nucleus, and spherical mitochondria. Scale bars: (A)—3 ␮m; (B–I and K)—1 ␮m; (J)—0.5 ␮m. av, acrosomal vesicle; ax, axoneme; bb, basal body; dc, distal centriole; er, endoplasmic reticulum; Gb, Golgi bodies; m, mitochondria; n, nucleus; pc, proximal centriole; pcc, pericentriolar complex; arrows—electron-dense inclusions; arrowheads—electron-lucent inclusions; asterisk—intercellular bridge.

final stages, may appear quite similar to each other. The length and the complexity of the acrosomal vesicle, length of the anterior fossa and shape of the nucleus can be used as markers of the consecutive stages of cell development (Figs. 4D–G and 8C). Spermatids

with deepest anterior fossa (up to 1.8 ␮m in both species) extending through the greater part of the nucleus have a long acrosomal vesicle occupying the entire length of the fossa. In longitudinal sections of this stage, the nucleus appears bilobed or funnel-shaped,

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Fig. 5. Ultrastructure of spermatozoa in D. carunculata (A and C) and D. bidentata (B, D–L). (A and B) Longitudinal sections through spermatozoa. (C and D) Longitudinal sections through acrosomes. (E) Transverse section through acrosome. (F) Longitudinal section, showing interstitial plate between acrosome and nucleus. (G) Transverse section through anterior part of nucleus. (H) Transverse section through nucleus with asymmetrically situated platelets. (I) Transverse section through middle part of nucleus surrounded by platelets. (J) Transverse section through basal part of nucleus showing centriole inside posterior nuclear depression and a sheath of platelets. (K) Longitudinal section along nuclear surface showing tight mosaic arrangement of rounded platelets. (L) Transverse section through platelets showing their heterogeneous structure. Scale bars: (A and B)—2 ␮m; (I, J and K)—0.5 ␮m; (C, D, F–H and L)—0.2 ␮m; (E)—0.1 ␮m. a, acrosome; ax, axoneme; bb, basal body; c, centriole; mp, midpiece; n, nucleus; p, platelets; arrow—interstitial plate.

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Fig. 6. Ultrastructure of spermatozoa in D. bidentata. (A) Longitudinal sections through two spermatozoa showing basal part of nucleus with posterior fossa housing basal body, long midpiece with electron-lucent platelets, elongated mitochondria and axoneme, and flagellum. (B–F) Transverse sections through midpiece (successive sections towards most posterior part of midpiece). (B) Platelets and seven mitochondria. (C) Platelets and four mitochondria. (D) Only electron-lucent platelets without mitochondria. (E) Satellite fibers of anchoring apparatus directed from axoneme to cell membrane. (F) Terminal part of midpiece with cytoplasmic annulus surrounding a flagellum. (G) Longitudinal section through terminal part of midpiece showing satellite fibers of anchoring apparatus. Scale bars: (A–D)—0.5 ␮m; (E–G)—0.2 ␮m. an, annulus; ax, axoneme; bb, basal body; f, flagellum; n, nucleus; p, platelets; sf, satellite fibers.

with a deep conical fossa in the middle (Fig. 4D). The posterior part of the nucleus is blunt or has a shallow depression. Two centrioles, distal and proximal are present in the middle of cell posterior to the nucleus (Fig. 4E). Elements of the pericentriolar complex and axoneme first appear in cells at this stage (Fig. 4D and E). In both

species, eight to eleven spherical mitochondria are arranged in a circle around the anterior part of the axoneme, close to the posterior end of the nucleus (Fig. 4F). Spermatids with an ovoid nucleus have the anterior fossa shorter than in the previous stage, and the acrosomal vesicle partly extends out of fossa (Fig. 4G).

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midpiece, platelets gradually becoming larger, fewer, electronlucent and appearing as vacuoles (Figs. 5A and B and 6B–D). The midpiece is long and slightly wider than the nucleus. The mitochondria are elongated and twisted around the axoneme (Fig. 6A), absent in the most posterior part of the midpiece (Fig. 6D). A maximum of seven mitochondria appear in transverse sections of both species (Fig. 6B) but there may be more in total because each mitochondrion is likely to extend only a part of the length of the midpiece (Fig. 6C). On the posterior edge of the midpiece, the cell membrane forms a short collar, the annulus, surrounding the anterior part of the flagellum (Fig. 6A and F). The most posterior part of the midpiece contains an anchoring apparatus comprising nine electron-dense satellite fibers arranged in a circle and each extending from the axoneme towards the cell membrane (Fig. 6E and G). In both species, the axoneme has a typical 9 × 2 + 2 arrangement of the microtubules.

4. Discussion 4.1. Reconstruction of spermatogenesis

Fig. 7. Schematic reconstruction of the spermatogenesis explaining possible way of formation of the tetrads of spermatids in D. bidentata and the octads of spermatids in D. carunculata.

Late spermatids II show various degrees of narrowing and elongation of nucleus and cytoplasm. The anterior fossa is reduced (Fig. 4H), but another invagination housing the basal body appears in the posterior part of the nucleus (Figs. 4I and 8D). The acrosome is situated on top of the nucleus and appears completely developed (Fig. 4H and K). Initially spherical (Fig. 4H), the cytoplasm eventually appears drop-like, with the posterior end expanded and situated progressively in a more posterior part of the cell (Fig. 4K). Initially spherical (about 0.5 ␮m in diameter in both species) and tightly packed in a circle posterior to the nucleus (Fig. 4I), the mitochondria appear progressively elongated in advanced stages of late spermatids. Platelets first appear at this stage in the periphery of cell. In spermatids with greatly elongated nucleus, the chromatin is tightly packed, thus the fibers can hardly be distinguished (Fig. 4K). At the end of spermiogenesis, the cytoplasm appears as narrow elongated midpiece characteristic for mature spermatozoa. 3.3. Mature spermatozoa Spermatozoa of D. carunculata and D. bidentata are similar in general morphology and composition to each other but slightly differ in the length of principal organelles and the midpiece (Table 1) (Fig. 8E). The acrosome is conical, with the most anterior part homogeneous electron-dense and the rest subdivided by thin walls into electron-lucent compartments (Fig. 5C–E). The nucleus is elongated and slightly conical, with the anterior part narrow and chromatin homogeneous electron-dense (Fig. 5A and B). A thin interstitial plate is present on top of the nucleus, separating it from the posterior part of the acrosome (Fig. 5F). An implantation fossa is present in the posterior part of the nucleus housing the basal body and the anterior part of the axoneme (Figs. 5A and B and 6A). The cell membrane envelops tightly the acrosome and the anterior third of the nucleus (Fig. 5G), whereas farther posterior a space between the membrane and the nucleus is tightly packed with flat rounded platelets forming a sheath around the nucleus (Fig. 5H–K). Each platelet is covered by a membrane and has an electron-dense structure on the periphery and electron-lucent contents in the center (Fig. 5L). In the

The complete course of spermatogenesis in D. bidentata and D. carunculata can be summarized as follow (Fig. 7). During the reproductive period, spermatogonia proliferate steadily by the mitotic divisions in the testis and are released continuously into the coelom. We observed rare individual coelomic spermatogonia in D. carunculata and assume that in D. bidentata, as in many other annelids, the spermatogonia are also released into the coelom individually. In both species, the spermatogonia in the coelom probably undergo one mitotic division before differentiation into spermatocytes. In D. carunculata, the resulted cells remain interconnected by intercellular bridges in diads and differentiate into spermatocytes I. The following meiotic divisions in this species are quick and also incomplete resulting in duplications of the nuclei without cytokinesis, thus the spermatids form a syncytium. Consequently, “diads” of spermatocytes II with four nuclei are immediately followed by the formation of “diads” of spermatids with eight nuclei. These “diads” of spermatocytes II and “diads” of spermatids are quick stages and rarely observed in the coelom. The following incomplete division of the cytoplasm results in the formation of octads of spermatids interconnected by thin cytoplasmic bridges. These octads are usually observed in the coelom in this species. In D. bidentata, one of the first divisions of sperm cells in the coelom is complete but whether the complete cytokinesis occurs during division of spermatogonia or spermatocytes I remains uncertain. Individual spermatogonia and spermatocytes I were not observed in the coelom in this species, but frequently observed diads of these cells may be equally interpreted either as final incomplete cytokinesis or transient condition towards the complete division of cells. It is quite plausible, however, that the complete cytokinesis in this species occurs during division of the coelomic spermatogonia which then differentiate into individual spermatocytes I. These individual cells may be quick stages and therefore were not observed in the coelom. The two meiotic divisions of the spermatocytes are incomplete and result in the formation of tetrads of spermatids which are usually observed in the coelom in this species. Both in D. bidentata and D. carunculata, very late spermatids separate, float freely in the coelomic fluid and differentiate into individual spermatozoa. The mature spermatozoa are sorted by the nephridial funnel and packed in the middle part of the nephridium in spermatophores which are released through the nephridiopore at spawning. Spermatozoa of both species described herein have a structure similar to that of other polydorin spionids with elongated sperm,

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Fig. 8. Schematic reconstruction of spermiogenesis in Dipolydora carunculata and D. bidentata. (A) Early spermatid. (B) Middle spermatid. (C) Late spermatid I. (D) Late spermatid II. (E) Mature spermatozoon. a, acrosome; an, annulus; av, acrosomal vesicle; bb, basal body; c, centriole; f, flagellum; Gb, Golgi bodies; ib, intercellular bridge; ip, interstitial plate; m, mitochondria; n, nucleus; p, platelets; sf, satellite fibers.

but differ from them and one from another mainly in size of the nucleus and the midpiece (Table 1). 4.2. Spermatid aggregations Early stages of spermatogenesis in polychaetes are difficult to differentiate and the processes leading to the formation of spermatids have rarely been described (Olive, 1983; Sawada, 1984; Rice, 1992; Rouse, 2006). Numerous mitotic divisions (proliferation) of spermatogonia in the testes usually result in complete separation of the spermatogonial cells. The spermatogonia released into the coelomic cavity may also undergo one or more mitotic divisions but remain interconnected via cytoplasmic bridges. The number of these divisions varies according to the species. The subsequent meiotic divisions are usually incomplete, and the developing spermatids always remain interconnected via cytoplasmic bridges leading to the production of groups, clusters of cells referred to as tetrads, octads, morulae, rosettes, mulberries, raspberries, platelets, or spheres according to the number of aggregated cells,

shape of the aggregation and taste of the beholder. A cytophore, a central mass of residual cytoplasm, is usually present when numerous spermatids are joined in a large cluster or syncytium. The number of spermatids in a cluster depends on the number of previous incomplete divisions of spermatogonia and spermatocytes and is species specific. These numbers were used as characters in phylogenetic analyses of sabellid polychaetes (Rouse and Fitzhugh, 1994; Fitzhugh and Rouse, 1999), and also as taxonomic characters to distinguish two sibling species of spionids, Polydora calcarea (Templeton, 1836) and P. manchenkoi Radashevsky and Pankova, 2006 (Radashevsky and Pankova, 2006). Spionids demonstrate a great variety of spermatid aggregations which has not yet been analyzed in the literature. The free spawners with short-headed spermatozoa have spermatids interconnected in tetrads, e.g., Aonides oxycephala (Sars, 1862) (Radashevsky et al., 2011), Laonice spp. (Radashevsky and Lana, 2009), Marenzelleria viridis (Verrill, 1873) (Bochert, 1996), Prionospio patagonica Augener, 1923 (Radashevsky et al., 2006), Apoprionospio spp., Dispio spp., Malacoceros spp., Paraprionospio

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spp., Prionospio spp., Scolelepis spp., Spiophanes spp. (Radashevsky, unpublished), whereas brooders with elongated spermatozoa have spermatids joined in tetrads or more numerous aggregations. Among the brooders, tetrads of spermatids have been reported in D. bidentata (present study), Polydora rickettsi Woodwick, 1961 (Radashevsky and Cárdenas, 2004), S. setosa Verrill, 1873 (Simon, 1967), and S. benedicti (Rice, 1981). Octads of spermatids have been reported in D. carunculata (present study), D. socialis and P. websteri (Rice, 1981), P. ciliata (Franzén, 1956), P. cornuta (Radashevsky, 2005), P. neocaeca (Williams, 2000), Polydora triglanda Radashevsky & Hsieh, 2000 (Radashevsky and Hsieh, 2000a), Pseudopolydora diopatra Hsieh, 1992 (Radashevsky and Hsieh, 2000b), and P. paucibranchiata (Rice, 1992). The details of the cytokinesis leading to the formation of spermatid aggregations have been suggested only in D. bidentata and D. carunculata via fluorescent technique after incubating of the live sperm cells with acridine orange. The multicellular (more than 8 cells) aggregations of spermatids may result from more than one incomplete mitotic divisions of the coelomic spermatogonia followed by incomplete meiotic divisions of the spermatocytes I and II. The clusters of probably 32 spermatids in B. hamata may result from three incomplete mitotic divisions of the coelomic spermatogonia (Reunov et al., 2010), whereas the 128-cell clusters in a Rhynchospio sp. from Taiwan result from five spermatogonial divisions (Radashevsky, unpublished). ‘Masses framboisées’ of spermatids (each composed of 64 or 128 cells) are also present in Boccardiella ligerica (Ferronnière, 1898) (Rullier, 1960: fig. 15). The number of spermatids in a cluster varies within some genera (tetrads and octads in Dipolydora and Polydora, probably different numbers in Boccardiella species) but appears to be fixed within other genera of Spionidae (tetrads of spermatids in free spawners, octads of spermatids in brooding Pseudopolydora). The cytophore in annelids was suggested to serve as a means of synchronizing differentiation of the attached sperm cells (Sawada, 1984). No cytophore is observed, but the cytoplasmic bridges are present in the spionids with tetrad and octad clusters of spermatids. Probably 32-cell aggregations of spermatids in B. hamata are joined by a small central cytoplasmic mass referred to as cytophore by Reunov et al. (2010). Spermatids in larger clusters in spionids are probably also attached to a small central cytoplasmic mass but not in massive cytophore as in some sabellids and terebellids. Remarkably, strong synchronization of spermatogenesis in an individual and within the whole population is typical for the free-spawning spionids with spermatids joined in tetrads (e.g., Aonides spp., Apoprionospio spp., Dispio spp., Laonice spp., Malacoceros spp., Paraprionospio spp., Prionospio spp., Scolelepis spp., Spiophanes spp., Radashevsky, unpublished), whereas the brooders tend to be less synchronized and may simultaneously have all stages of spermatogenesis present in the coelom during the entire reproductive period (Rice, 1981; Rouse, 1988; present study). The asynchronous spermatogenesis and multicellular aggregations of spermatids might have evolved with the evolution of brooding and sperm storage from the freespawning within the Spionidae. The adaptive significance of various spermatid aggregations within the family remains unknown. 4.3. Morphogenesis of organelles 4.3.1. Acrosome formation As a product of Golgi complex, proacrosomal vesicles first appear nearby the complex in early spermatids. Early vesicles, however, can hardly be distinguished from other secretion of the complex. In the two examined Dipolydora species, an acrosomal vesicle first appears clearly in middle spermatids, when it migrates to the prospective anterior end of the cell. In late spermatids I, the vesicle comes to lie in the depression temporary formed at the anterior part of the nucleus. As the depression deepens and becomes a

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prominent anterior nuclear fossa, the acrosomal vesicle elongates and keeps its position at the bottom of fossa. When the fossa reaches its maximum, the vesicle seems to attain its maximal length as well. During the following evagination of the anterior nuclear fossa, the vesicle moves forward and occupies the definitive position on top of the nucleus. This final move is accompanied by the development of internal compartments in the acrosome. When on top of the nucleus in late spermatids II, the acrosome appears to be completely developed. The acrosomes in various polydorins have similar composition. An electron-transparent vesicle was present around the tip of the acrosome in late spermatids of P. cornuta (Rice, 1981: fig. 6). This vesicle persisted throughout most of the development of the acrosome but disappeared in mature spermatozoa. No vesicle appears around the acrosome in spermatids of other examined polydorins. 4.3.2. Nucleus transformation and condensation of chromatin Both in D. bidentata and D. carunculata, the nucleus remains spherical to slightly oval in spermatogonia, spermatocytes and then until the middle spermatid stage. Through all these stages, chromatin becomes more heterogeneous and clumpy, reaching a maximum in early spermatids. The nucleolus disintegrates during the transformation of spermatogonia into spermatocytes and does not appear in sperm cells after that. The most drastic changes in the structure of the nuclei occur in the middle and late spermatids. Condensation of the clumpy chromatin into homogenous granular chromatin of middle spermatids is accompanied by arrival of the acrosomal vesicle in the apical position and the beginning of invagination of the anterior part of the nucleus. In late spermatids I, the chromatin condenses further to homogenous fibrillar and the anterior nuclear invagination (fossa) deepens to its maximum and then evaginates and disappears completely. Simultaneously, another invagination appears and deepens in the posterior part of the nucleus. In late spermatids II, the posterior fossa extends about 1 ␮m of the length of the nucleus and houses the basal body and the anterior part of the axoneme. The final transformation of the nucleus includes its narrowing and elongation. Chromatin becomes completely condensed so the fibrils can hardly be distinguished and the nucleus on sections appears homogenously black. In the end of spermiogenesis, a thin interstitial plate (subacrosomal plate, nuclear plate or cap) appears between the acrosome and the nucleus. The origin and function of the plate remain unknown. The anterior nuclear invagination (acrosomal depression or fossa) housing the acrosome appears during the middle spermatids stage and disappears in late spermatids II in Dipolydora, Polydora and Pseudopolydora species (Rice, 1981; Rouse, 1988; Williams, 2000; present study). Such a transformation does not occur in B. hamata (Reunov et al., 2010) (although the four genera are closely related members of the tribe Polydorini), and in S. benedicti (Rice, 1981) (remarkably, both B. hamata and S. benedicti have filiform mature spermatozoa). A deep anterior nuclear fossa houses the maturating acrosome in late spermatids and retains in mature spermatozoa of S. laonicola (Vortsepneva et al., 2006) and S. setosa (Eckelbarger and Hodgson, 2014). The phylogenetic and adaptive significance of the temporary development of anterior nuclear fossa in some polydorins remains unknown. The posterior nuclear invagination (centriolar depression or implantation fossa) housing the basal body and the axoneme is quite common in elongated spermatozoa of annelids. Among spionids, a short implantation fossa extends 0.8–1.2 ␮m of the nucleus in the polydorins D. bidentata, D. carunculata, D. socialis, P. ciliata, P. neocaeca, P. websteri, P. paucibranchiata (Franzén, 1974; Rice, 1981, 1992; Williams, 2000; present study), and also in S. laonicola and S. benedicti (Rice, 1981; Vortsepneva et al., 2006). A long fossa extends the entire length of the nucleus in P. cornuta, Pseudopolydora sp., and B. hamata (Rice, 1981, 1992; Rouse, 1988; Reunov et al., 2010),

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thus the basal body in spermatozoa is situated anteriorly almost beneath the acrosome. The axoneme extending the entire length of the nucleus may increase motility and penetrative ability of the sperm (Westheide, 1984). It is likely that long fossa evolved several times within various groups during evolution of Spionidae. Fibrous (fibrillar) chromatin was described in late spermatids of polydorins by Franzén (1974), Rouse (1988), Williams (2000) and Reunov et al. (2010) and also in the present study, while Rice (1981: fig. 5), Rice (1992: fig. 20) illustrated lamellar chromatin in P. websteri and P. paucibranchiata and reported fibrous chromatin in S. benedicti. The overall process of chromatin condensation or at least the shape of the condensed chromatin in late spermatids may provide relevant characters for phylogenetic inferences (Rice, 1992) and therefore require further examination. 4.3.3. Midpiece formation Essential transformation of the cytoplasm leading to the formation of midpiece occurs in late spermatids II when the acrosome arrives at the definitive position and the nucleus undergoes elongation. Almost spherical in previous stages, the cytoplasm in late spermatids II of both Dipolydora species stretches out along the longitudinal axis of the cell and appears drop-like with the posterior part expanded. As the posterior part of the cytoplasm glides backwards along the nucleus and then axoneme, its anterior part narrows and tightly envelopes the nucleus and then forms the midpiece. Formation of the definitive midpiece terminates when the cytoplasm is completely stretched out. Stretching and gliding of the drop-like cytoplasm along the nucleus and axoneme appears to be typical for elongated and filiform spermatozoa of spionids. The length of the midpiece is likely defined by the volume of cytoplasm which appears to be fixed and species-specific. 4.3.4. Cytoplasm organelles and inclusions A manchette of microtubules is often present during spermiogenesis in annelids with elongated sperm. These microtubules appear in spermatids as they enter nuclear elongation and are usually absent from the mature spermatozoa. They likely play a role in movement of cytoplasmic organelles and elongating of the nucleus (reviewed by Rice, 1992). Among spionids, transient appearance of microtubules has been demonstrated in P. paucibranchiata, S. benedicti (Rice, 1981, 1992), and Pseudopolydora sp. (Rouse, 1988). Microtubular manchette were not however observed in Dipolydora spp. (Rice, 1981; present study), Polydora spp. (Franzén, 1974; Rice, 1981; Williams, 2000), B. hamata (Rice, 1992; Reunov et al., 2010), S. laonicola (Vortsepneva et al., 2006), and S. setosa (Eckelbarger and Hodgson, 2014). A centriole first appears near the Golgi complex in spermatocytes I (Fig. 3G), and distal and proximal centrioles and an axoneme are present in late spermatids I in both examined Dipolydora species. A basal body in late spermatids II is likely formed by the fusion of the two centrioles (Figs. 4I and 6A), same as it was shown in other Dipolydora and Polydora species (Rice, 1981). In all the examined spionids, the flagellar axoneme has the typical 9 × 2 + 2 arrangement of microtubules. A pericentriolar complex first appears in late spermatids I, comprising an anterior set of satellite fibers associated with the proximal centriole, and a posterior set of fibers associated with the distal centriole (Fig. 4D and E). The anterior set of satellite fibers is probably used to draw the basal body and the axoneme into the posterior nuclear fossa. The posterior set of satellite fibers (also called anchoring apparatus, ring centriole) is associated with a cytoplasmic collar (annulus) situated in the posterior part of the midpiece, and probably used to add strength and rigidity to the sperm. Similar anchoring apparatus and annulus are also present in the posterior part of the midpiece in other Dipolydora, Polydora and Pseudopolydora species (Franzén, 1974; Rice, 1981, 1992; Rouse,

1988; Williams, 2000) with the elongated midpiece. However, an annulus is present but anchoring apparatus was not observed in filiform spermatozoa with short midpiece for example in B. hamata and S. benedicti (Rice, 1981; Reunov et al., 2010). Neither anchoring apparatus nor annulus appear in the spermatozoa with elongated midpiece of S. laonicola (Vortsepneva et al., 2006). Up to eleven spherical mitochondria are tightly arranged in a circle around the axoneme near the posterior end of the nucleus in late spermatids I in both Dipolydora species examined in the present study. In late spermatids II, synchronously with stretching of cytoplasm and formation of the midpiece, the mitochondria elongate and slightly twist along the axoneme. Fewer mitochondria on transverse sections of spermatozoa (up to seven) may be due to either their shifted arrangement or fusion of some of them. Similar morphogenesis of mitochondria occurs in other Dipolydora, Polydora and Pseudopolydora species with elongated midpiece (Franzén, 1974; Rice, 1981, 1992; Rouse, 1988; Williams, 2000). Six long and apparently straight mitochondria are present in elongated spermatozoa of S. laonicola (Vortsepneva et al., 2006). Filiform spermatozoa of S. benedicti and coiled spermatozoa of S. setosa have short midpiece with up to 8 and 10 spherical mitochondria, respectively (Rice, 1981; Eckelbarger and Hodgson, 2014). Filiform spermatozoa of B. hamata have short midpiece with four oval mitochondria (Reunov et al., 2010). Four to five spherical mitochondria are typically present in short-headed spermatozoa of free-spawning spionids (Franzén and Rice, 1988; Rouse, 1988; Bochert, 1996; Radashevsky et al., 2010, 2011). Golgi complex and endoplasmic reticulum are well developed in spermatogonia and succeeding stages until late spermatids in both examined Dipolydora species. Beside acrosome, products of their secretion apparently include membrane-bounded platelets. The platelets increase in number towards the end of spermiogenesis. Until late spermatids they are scattered in the cytoplasm but in the end of spermiogenesis they appear orderly arranged inside the cell. In spermatozoa, flat rounded electron-dense platelets are clamped between cell membrane and the nucleus, whereas the vacuole-like electron-lucent platelets are lined along cell membrane in the midpiece. The observed gradation of shape and electron density of the vacuoles into platelets and the kind of their arrangement inside cell appear as evidence of maturating transformation of the same structure. The electron-dense platelets are also present in other polydorins with elongated and filiform spermatozoa (Franzén, 1974; Rice, 1981, 1992; Rouse, 1988; Williams, 2000; Reunov et al., 2010), and also in S. benedicti (Rice, 1981). Rice (1981) has postulated that these platelets are energy storage organelles facilitating prolong sperm storage in female seminal receptacles, and later confirmed the inside presence of glycogen and other complex carbohydrate inclusions (Rice, 1992). No storage membrane-bounded organelles appear in elongated spermatozoa of S. laonicola (Vortsepneva et al., 2006). 5. Conclusions Spermatogenesis in D. bidentata and D. carunculata occurs similar to that in other Dipolydora, Polydora and Pseudopolydora species. Spermatozoa in these polydorin spionids have similar composition and differ mainly in size of the nucleus and the midpiece. These spermatozoa with elongated nucleus and midpiece are adapted for transfer in spermatophores and an internal fertilization which are characteristic for brooding polydorins. Spermatozoa with filiform nucleus and short midpiece, as in Boccardiella and Streblospio species, have probably evolved in relation with other ways of internal fertilization. Diverse modified spermatozoa in Rhynchospio, Scolelepis and Spio species may also be signs of the diversity of fertilization biology among spionids. The exact places of fertilization remain however unknown in all of these species.

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