The spermatozoa of three species of Xenotrichulidae (Gastrotricha, Chaetonotida): the two ?d�nne Nebengeisseln? of spermatozoa in Heteroxenotrichula squamosa are peculiar para-acrosomal bodies

Zoomorphology(1995) 115:151-159

9 Springer-Verlag 1995

Marco Ferraguti 9Maria Balsamo 9Elena Fregni

The spermatozoa of three species of Xenotrichulidae (Gastrotricha, Chaetonotida): the two "dfinne Nebengeisseln" of spermatozoa in Heteroxenotrichula squamosa are peculiar para-acrosomal bodies

Accepted: 3 July 1995

Abstract The spermatozoa of xenotrichulid gastrotrichs have been studied with the aim of supplying fnrther characters for the phylogenetic analysis of Gastrotricha and to assess the reported biflagellarity of Heteroxenotrichula squamosa. Three species have been examined, belonging to the two hermaphroditic genera of xenotrichulids. The spermatozoa are filiform cells characterized by a scarcely condensed nucleus followed by a single mitochondrion and a flagellum with large accessory fibers. These show an obliquely striated cortex and a core containing some dense material. In Heteroxenotrichula squamosa and Xenotrichula punctata there is also a simple acrosome flanked by two para-acrosomal bodies which are curious long extracellular structures formed by a pile of electron-dense disks connected by thin threads. Xenotrichula intermedia lacks both acrosome and paraacrosomal bodies. The sperm model of xenotrichulids is very different from that of the Macrodasyida and Chaetonotida so far studied, thus supporting an isolated position of the family. The oblique striation of the tail's accessory fibers is similar in to the one period and inclination of the striated cylinder of macrodasyid gastrotrichs, thus being the only spermatological character shared by the two gastrotrich taxa.

A. Introduction The family Xenotrichulidae (Gastrotricha) only includes marine species and is a clearly distinct group within the order Chaetonotida, which is generally composed of freshwater parthenogenetic species. Phylogenetic relationships of Xenotrichulidae are dubious, even through they are generally considered (Remane 1936; Travis Marco Ferraguti (~) Sezione di Zoologiae Citologia, Dipartimento di Biologia, 26, Via Celoria, 20133 Milano,Italy, Maria Balsamo 9Elena Fregni Dipartimento di BiologiaAnimale, 4, Via Universit~t,41100 Modena, Italy

1983) to be an early, divergent branch of the main evolutionary line of Chaetonotida, close to Macrodasyida and Chaetonotida Multitubulatina (Neodasys), with which they share the marine interstitial habitat and the hermaphroditic sexual condition. Three genera compose the family Xenotrichulidae, namely Draculiciteria, Heteroxenotrichula, and Xenotrichula. Draculiciteria stands out from the other two genera because of its morphology and parthenogenetic reproduction, which is probably secondary (Ruppert 1979). Wilke (1954) observed under the light microscope the spermatozoa of some xenotrichulid species and provided drawings and short descriptions. In the spermatozoa of Heteroxenotrichula squamosa Wilke, 1954, she reported "zwei sehr lange dtinne Nebengeisseln" (which could be translated as "two very long and thin accessory flagella") in addition to a conventional tail. Wilke stated that because of this character these spermatozoa "... gleichen also fast vollkommen gewissen Turbellarienspermien (DendrocoeIum)" [... "were almost perfectly similar to the spermatozoa of some turbellarians (Dendrocoelum)"]. Subsequent authors have interpreted the information by Wilke by taking the "Nebengeisseln" to be two flagella and the tail as a part of the nucleus-containing cell body. From that arose statements such as "... Heteroxenotrichula squarnosa possesses elongate biflagellate sperm" (Hummon and Hummon 1983) and "an unconfirmed report of biflagellate sperm in H. squamosa was given by Witke (1954)" (Ruppert 1991). The other species examined by Wilke, i.e., Xenotrichula beauchampi Levi 1950, X. cornuta Wilke 1954, and X. punctara Wilke, 1954, showed a sperm model without any peculiar differentiation. The spermatozoa of macrodasyid gastrotrichs are characterized by a common general architecture (Ferraguti and Balsamo 1995). The acrosome is, at least in part, corkscrew-shaped and contains complex regular structures, the nucleus is spring-shaped and contains the mitochondria, and the tail has an axoneme surrounded by a striated cylinder (except in Turbanellidae). In a single species, Cephalodasys maximus Remane, 1926, nine ac-

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153 cessory fibers are present around the axoneme (Fischer 1994). Thus, the morphology of xenotrichulid spermatozoa appeared very different from that of the other gastrotrichs. On the other hand, the possession of two flagella is a feature shared with most free-living Platyhelminthes (Hendelberg 1983) and could be used to support the phylogenetic relationship between the two taxa (Remane 1936). We have studied the ultrastructural morphology of the spermatozoa of species belonging to both hermaphroditic genera of the family Xenotrichulidae (Heteroxenotrichula squamosa, XenotrichuIa punctata, and X. intermedia Remane, 1934) with the aim of expanding the set of characters available for phylogenetic studies of gastrotrichs. We focused our attention, in particular, on H. squamosa to check the real nature of its "biflagellate" spermatozoon. Preliminary results of this research have been published in abstract form (Ferraguti et al. 1994).

B. Material and methods The specimens were selected frorn sandy sediments collected in some localities along the coasts of the Tyrrhenian [Marina di Pisa (Pisa), Punta Ala, Mortelliccio (Grosseto)] and Adriatic (Pesaro) Seas by decantation and narcotization with 7% aqueous Mg C12 . Observations on living specimens were carried out by means of a Leitz Dialux microscope equipped with Nomarski optics and phase contrast. Moving spermatozoa within the testes and after isolation were recorded on videotape.

Transmission electron microscopy (TEM) Specimens were fixed in a 0.1 M phosphate buffered (pH 7.3) solution of paraformaldehyde, glutaraldehyde, and picric acid (SPAFG) (Ermak and Eakin 1976), postfixed in 2% aqueous osmium tetroxide, washed in 0.2 M cacodylate buffer, dehydrated in a graded acetone series, prestained en bloc in uranyl acetate in acetone 70%, and embedded in Araldite. Sections were cut with an Ultratome Nova LKB microtome, double stained with aqueous uranyl acetate and lead citrate, carbon coated, and observed under a Jeol 100 XS electron microscope.

Figs. 1-11 Heteroxenotrichula squamosa Fig. 1 Longitudinally sectioned para-acrosomal bodies (p) and acrosome (a) Fig. 2 Cross-section of the animal. The two testes are cut at a level close to the connection Fig. 3 Longitudinal section of nuclear (n) apex and base of the acrosome (a). The para-acrosomal bodies (p) lie on the nuclear region Figs, 4-6 Cross-sections at different levels of the acrosome-nucleus transition Figs. 7, 8 SEM views of the whole spermatozoon (Fig. 8) and detail of the para-acrosomal bodies and the acrosome (a) (Fig. 7) Fig. 9 Cross-section of the main portion of the flagellum Figs. 10, 11 Longitudinal sections of the mitochondrion and basal portion of the flagellum. Note the striated appearance of the accessory fiber cortex (arrow)

Scanning electron microscopy (SEM) The animals were dissected in sea water and the spermatozoa were fixed with the same mixture used for TEM analysis, dehydrated in a graded ethanol series, critical-point dried with CO 2 , coated with gold-palladium and observed under a Philips XL40 or a Cambridge Stereoscan 250 Mk2.

C. Results The spermatozoa of H. squamosa, X. intermedia, and X. punctata were observed in the testes as well as freshly isolated in sea water under a phase contrast microscope. The testes are paired, lateral organs formed by bundles of germ cells in various developmental stages (Fig. 12). These bundles are not surrounded by an epithelium and, in some areas, they are in close contact with free oocytes and the body wall (Figs. 2, 12, 24). The testes join ventrally at the level of the posterior tract of the gut (Fig. 2) where spermatozoa appear scattered and motile. They converge toward the so-called copulatory organ, a glandular structure located ventrally at the level of the pharyngo-intestinal junction. We have been able to observe it under the electron microscope only in X. punctata where it is a narrow duct apparently devoid of any specialized epithelium (Fig. 13). A real male pore is not clearly visible and could be a temporary production as suggested by Ruppert (1979). When observed under the light microscope, the spermatozoa are already actively motile within the testes and look uniflagellate. When freed from the animal, freshly isolated spermatozoa of H. squamosa were seen slowly moving by means of the tail, with the two stiff "Nebengeisseln" passively transported at the anterior extremity. These are parallel, coupled and close together in freshly extracted spermatozoa (Figs. 8,13), but split up shortly after (Fig. 7). X. intermedia does not show any anterior differentiation (Figs. 22, 29, 31). The electron microscope reveals that the "Nebengeisseln" are, both in H. squamosa and X. puntata, thin (approximately 0.2 pm in diameter) anterior processes, 20 pm long (Fig. 8), which lack a surrounding cell m e m brane (Figs. 1, 14, 16) and are formed by a pile of electron-dense disks, each 0.1 pm thick, somewhat connected to one another by thin filaments (Figs. 1, 14, 18). A third, very short process, which is clearly an acrosome (Figs. 3, 4, 16, 17), is also visible between the two longer ones (Fig. 8) that, thus, have been called "para-acrosomal bodies" by Ferraguti et al. (1994) (Fig. 30). These are loosely connected with the anterior portion of the cell body (Figs. 3, 5, 6, 15, 16) but, in the connecting region, the plasma membrane of the spermatozoon is uninterrupted, suggesting an extracellular position of the paraacrosomal structures (Figs. 6, 15). The acrosome, 3.8 gm long in H. squamosa and 2.9 pm long in X. punctata with a diameter of 0.1 gin, contains a small acrosomal vesicle made up of various types of materials of different textures, and surrounded by a large amount of electron-dense material (Figs. 4, 17).

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The acrosome is inserted into a nuclear concavity and is the anteriormost structure of the cell body. No acrosome has been detected in X. intermedia (Figs. 22, 29, 31). The cell body contains, in sequence, the acrosome (except in X. intermedia), the nucleus, a single mitochondrion, and the flagellum (Figs. 10, 19, 30, 31). The nucleus of the three species examined differs both in shape and texture. In H. squamosa (Fig. 10), the nucleus is nearly cylindrical, about 2 ~tm long with a more regular outline (diameter approximately 0.4 ~tm) (Fig. 8). The chromatin is not condensed and shows scattered filaments thickening only at the nuclear tapering extremity (Fig. 6). In X. punctata it is pear-shaped, 1.7-1.9 ~tm long, tapering toward the apex (Fig. 19). The chromatin is incompletely condensed, with thin filaments and scattered dense globules up to 0.18 ~m in diameter (Fig. 19) and the nuclear outline is often irregular (Fig. 12). The nucleus of X. intermedia spermatozoa is much longer (24.5-28.0 tam) and tapers from a basal diameter of 0.27 ~tm to an apical one of 60 nm. Its chromatin is in form of thin filaments (Figs. 28, 29, 31). A single mitochondrion with a conventional aspect (Figs. 30, 31) follows the nucleus. It is 0.6-0.8 ~m long and 0.4 ~ln in diameter in H. squamosa (Fig. 11) and X. punctata (Fig. 19) and more than 4.7 ~tm long with the same diameter in X. intermedia (Fig. 28). No basal body is visible at the base of the flagellum (Figs. 11, 20, 28). The flagellum, the mitochondrion, and, at least in H. squamosa, most of the nucleus have the same diameter (Figs. 8, 22, 23). A thin, annular indentation of the plasma membrane separates the mitochondrion from the flagellum in H. squamosa (Fig. 11) and X. punctata. The mitochondrion is basally inflexed in X. intermedia (Fig 28). The main portion of the flagellum has a conventional 9+2 axoneme surrounded by an outstanding system of nine external accessory structures (Figs. 9, 21, 25). These have the same aspect and position of the accessory fibers present in other sperm models and thus, thereafter, we will call them accessory fibers. Each doublet is connected to two neighboring accessory fibers by evident connections: the one with the closer fiber starts between tubules A and B, and the other with the preceding one starts from the back of B tubule (Fig. 32). The accessory

fibers have a peculiar pear-shaped aspect in cross-section and the thinner portion of the pear comes into tight contact with the plasma membrane (Figs. 9, 21, 25, 27). The outline of the accessory fibers is denser than the internal portion which appears largely electron transparent with the exception of the areas toward the doublets and the one adjacent to the thinner extremity. The denser outline of the accessory fibers reveals, in grazing longitudinal sections, its real nature. It is formed by a series of oblique "ribs" with a periodicity of 15-16 nm (Figs. 11, 26). In the regions between two following accessory fibers the flagellar plasma membrane is looser and "falls" toward the axoneme (Fig. 9) thus forming longitudinal furrows visible in the scanning microscope preparations (Fig. 23). In the posterior region of the flagellum the accessory fibers become smaller (Fig.27) and, finally, disappear and, thus, the terminal tract of the tail shows only a conventional 9+2 axoneme.

Figs. 12-21 Xenotrichula punctata Fig. 12 Cross-section of the testes with spermatozoa and spermatids

Figs. 22-29 Xenotrichula intermedia

D. Discussion

The spermatozoa of the three species of Xenotrichulidae are peculiar, not only when compared to the spermatozoa of other Gastrotricha, but also with respect to the sperm models of other animal phyla. The most remarkable feature is the presence in H. squamosa and X. punctata of two para-acrosomal bodies. Of course, such huge and, presumably, energy-costing para-acrosomal bodies can have some function during fertilization. In Xenotrichulidae, as in the other hermaphroditic gastrotrichs, fertilization is most likely to be internal, but it has never been observed. We only know that spermatozoa make their way through a single narrow duct, the so-called copulatory organ. A female pore does not exist (Ruppert 1979). We have observed in X. punctata a few spermatozoa in the narrow "deferent duct", with the para-acrosomal bodies parallelly arranged to surround the acrosome. However, Wilke (1954) failed to observe para-acrosomal bodies in the same species. A possible explanation could reside in their loss during some phase of the fertilization, probably at the passage through the partner's cuticle, which they could facilitate in some way. )

Fig. 13 The copulatory organ contains some spermatozoa (arrow) Figs. 14, 16, 18 Longitudinal sections of the para-acrosomal bodies (p) and the acrosome (a) Fig. 15 Cross-section of the apex of the nucleus (n) with the paraacrosomal bodies (arrowheads) Fig. 17 Cross-section of the acrosome (a) and the para-acrosomal bodies (p) Fig. 19 Detail of testis, a acrosome, m mitochondrion, n nucleus Figs. 20, 21 Cross-sections of the base (Fig. 20) and main portion (Fig. 21) of the flagellum

Figs. 22, 23 SEM view of a sperm apex (Fig. 22) and tail (Fig. 23). Note the furrows on the tail (arrowheads) Fig. 24 Low-power cross-section of the testes (t) surrounding a pre-vitellogenic oocyte Figs. 25, 27 Cross-section of the upper (Fig. 25) and lower (Fig. 27) portion of the flagellum. Note the different appearance of the accessory fibers Fig. 26 Grazing section of the accessory fiber cortex. The oblique striation is visible Fig. 28 Longitudinal section of the mitochondrial/flagellar region. m mitochondrion Fig. 29 Longitudinal section of a nuclear anterior extremity

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Fig. 30 Schematic drawing of Xenotrichula punctata spermatozoon. A acrosome, M mitochondrion, N nucleus, P para-acrosomal body

Fig. 31 Schematic drawing of Xenotrichula intermedia spermatozoon. The plasma membrane has been omitted in the enlargements at right. M mitochondrion, N nucleus

However, it must be stressed that, while we have seen the para-acrosomal bodies in the testicular spermatozoa of X. punctata, we have not seen them in X. intermedia, where the acrosome was also absent. A second feature of xenotrichulid spermatozoa is the incomplete nuclear chromatin condensation. The scattered masses of condensed chromatin present in X. punctata resemble those described in other pseudocoelomates, such as Rotifera (Melone and Ferraguti 1994). The chromatin of macrodasyid spermatozoa as well as that of the other chaetonotids is completely condensed.

A third feature of xenotrichulid spermatozoa, i.e., the existence of a single mitochondrion between nucleus and flagellum, is again a feature not shared with other gastrotrichs and can be regarded as an autapomorphy of Xenotrichulidae. A single mitochondrion is also interposed between nucleus and flagellum in hirudineans, but we consider this feature to be a homoplasy. A fourth relevant character of xenotrichulid spermatozoa is the presence of large accessory fibers in the tail. Accessory fibers are somewhat linked to internal fertilization, especially, but not only, in terrestrial animals and

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Fig. 32 An artist view of the accessory fibers structure in xenotrichulid spermatozoa in the proximal (top) and distal (bottom) part of the flagellum. Two following doublets are represented. For simplicity, arms and links connecting the doublets, as well as most of the plasma membrane have been omitted. D flagellar doublet, PM plasma membrane have been "invented" many times independently in the different lineages acquiring internal fertilization (Baccetti 1985). Thus, one can expect that they differ in the various groups. Xenotrichulid gastrotrichs are not an exception, showing again a peculiar and complex model which, however, appears uniform in the species observed, suggesting a basic plan for the family. Fischer (1994) has shown short accessory fibers in a single macrodasyid species (CephaIodasys maximus), but they appear different from the xenotrichulid ones. In the Xenotrichulidae, in fact, the accessory fibers have a dense outline somewhat similar to a plasma membrane and an in-

ner electron-transparent core with one or two dense areas. There are two evident connections between fibers and axonemal doublets: one starting from the A/B tubule boundary is directed toward the corresponding accessory fiber and the other link starts from the back of B tubule and is directed toward the preceding accessory fiber. The connections between the doublets and the fibers and between these last and the plasma membrane of the xenotrichulids may have a linking function of the plasma membrane of the accessory fibers and the axonemal machinery. Connections with a similar geometry are present in the sperm accessory fibers of other phyla (Dallai and Afzelius 1993) and, even more importantly, seem to be also present in the macrodasyid Cephalodasys maximus (see Fischer 1994). The spelan model of Chaetonotida Xenotrichulidae that comes out from our observations is very different from that of Macrodasyida. The only evident feature in common seems to be the striation in the cortex of the xenotrichulid accessory fibers which is similar in period and inclination to that of the striated cylinder surrounding the axoneme of the macrodasyid spermatozoa (Ferraguti and Balsamo 1994). This character is possibly homologous in the two orders. The short accessory fibers observed by Fischer (1994) in Cephalodasys maximus remain an isolated observation within the Macrodasyida. The spermatozoon of Chaetonotida Multitubulatina, known through a single published micrograph of Neodasys sp. (see Ruppert 1991), is again different from that of xenotrichulids in that there is a large and simple acrosome, a condensed nucleus, and a simple 9+2 flagellum. Among the numerous parthenogenetic Chaetonotida Pancitubulatina, the aberrant and probably relictual spermatozoa described in several species are nothing but simple rods of condensed chromatin (Hummon 1984). On the other hand, the morphology of xenotrichulid spermatozoa shows in both genera a number of consistent characters, such as the para-acrosomal bodies (with the exception of X. intermedia), the scarcely condensed nucleus, the single mitochondrion between nucleus and flagellum, and the peculiar accessory fibers in the tail. Thus, the family is spermatologically uniform: in particular the single mitochondrion and the accessory fibers of the axoneme, shared by the three species examined, can be considered autapomorphies. Thus, sperm morphology supports the phylogenetic relationships of Xenotrichulidae suggested by the analysis of other morphological features. They appear to be a monophyletic taxon included within the Chaetonotida, but clearly distinct from all the other families of the Pancitubulatina (Rieger and Rieger 1977; Ruppert 1982; Travis 1983). Acknowledgements We want to thank G. Alberti for his help in the understanding of the German literature, G. Melone for the SEM pictures, L. Valenti for the drawings, G. Barozzi and E. Ghelfi for their technical assistance, G. Manicardi for fluorescence checks, and G. Bernardini for the helpful discussions. This research has been supported by a grant from the Ministry and University and Scientific Research to M.F. (40% project Cell Interactions) and to M.B. (40% project Animal Populations of the Western Mediterranean Sea).

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