Spermiogenesis in Seison nebaliae (Rotifera, Seisonidea ...Melone1999... · (Rotifera, Seisonidea): further evidence of a rotifer-acanthocephalan relationship* M. Ferraguti, G. Melone

Spermiogenesis in Seison nebaliae(Rotifera, Seisonidea): further evidenceof a rotifer-acanthocephalan relationship*

M. Ferraguti, G. Melone

Abstract. The spermatozoa of Seison nebaliae are filiform cells about 70 µm long with a diameter of 0.6 µm.They have a slightly enlarged head, 2.5 µm long, followed by a long cell body. The flagellum starts from the head,and runs parallel to the cell body, contained in a groove along it. The head contains an acrosome, two large,paired para-acrosomal bodies, the basal body of the flagellum and the anterior thin extremity of the nucleus. Thecell body contains the main portion of the nucleus, a single mitochondrion located in its distal portion, and manyaccessory bodies with different shapes. The flagellum has a 9 + 2 axoneme. The study of spermiogenesis showsthe Golgian origin of the acrosome and the para-acrosomal bodies and reveals some peculiarities: a folding of theperinuclear cisterna is present between the proacrosome and the basal body of the flagellum in early spermatidsand the flagellum runs in a canal inside the spermatid cytoplasm. The basal body migrates anteriorly. These char-acters are shared partly by the Rotifera Monogononta and, to a large extent, by the Acanthocephala studied sofar. Many details of the spermiogenetic process are identical to those of Acanthocephala, thus suggesting that theprocesses in the two taxa are homologous. © 1999 Harcourt Publishers Ltd.

Keywords : Seison, rotifera, spermatozoa, spermiogenesis, phylogeny

Tissue & Cell,1999 31 (4) 428–440© 1999 Harcourt Publishers LtdArticle no. tice.1999.0012

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Introduction

The three classes of the phylum Rotifera, i.e. SeisonMonogononta, and Bdelloidea, are characterized, interby their modalities of reproduction: Bdelloidea are obligparthenogens, Monogonota reproduce mainly by parthgenesis, with rare periods of arrhenothoky with the protion of dwarf haploid males, whereas only in Seisonthere is a bisexual reproduction with regular, contempopresence of males and females. Spermatozoa arepresent, among rotifers, only in species of Monogonand Seisonidea.

Despite the high number of monogonont spe(>1500), their ecological importance, and the key pos

Dipartimento di Biologia, Università di Milano, 26, via Celoria, I-20133,Milano, Italy

Received 3 December 1998Accepted 19 January 1999

Correspondence to: M. Ferraguti. Fax: +39 2 2660 4462; E-mail: [email protected]

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of rotifers in the debate on the phylogeny of lower Metathe morphology of their spermatozoa, at ultrastructlevel, is known only for five species (Melone & Ferrag1998). Even poorer is our knowledge of the ultrastrucand sperm morphology in Seisonidea, a class compostwo species only, belonging to the same genus, Seison. Bothspecies, Seison nebaliae(Grube, 1861) and S. annulatus(Claus, 1876) live epizoic on Nebalia bipes(O. Fabricius1780) (Crustacea, Leptostraca). After the old paperPlate (1888), de Beauchamp (1909), Illgen (1916) Remane (1929–1933), mainly concerned with the anaof the animals, the fine structure of male germ cells studied with the scanning electron microscope only by Ret al. (1993) and with the transmission electron micros

*Since the submission of this paper, a paper has been published (Ahlrichs,WH 1998. Spermatogenesis and ultrastructure of the spermatozoa of S.nebaliae[Syndermata]. Zoomorphology, 118, 255–261). The results presented by Ahlrichs are consistent with the authors’, with some minordiscrepancies. The conclusions reached by the author about sister-grouprelationships of Seisonand Acanthocephala are also similar to those of thepresent paper, and to those of Melone and Ferraguti (1998).

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SPERMIOGENESIS INSEISON NEBALIAE 429

by Ahlrichs (1995a) in his Ph D thesis. A general ultrasttural description is provided in the review by Melone Ferraguti (1998). A poor knowledge of Seisonspermatozoais particularly regrettable for two reasons: first, Seisonmay be the most ancient rotifer taxon (Wallace & Colb1989; Wallace & Snell, 1991) and second the biologmale gametes in Seisonappears particularly complex, wispermatozoa encysted one by one at maturity, then ferred encysted to the female where they ‘unroll’ and trinto the oviduct to the germarium (Ricci et al., 1993).

Spermatozoa are considered as an important sourcharacters for phylogenetic reconstructions (Jamieson 1995) but, on the other hand, our knowledge of psecoelomate sperm is full of gaps. The aim of this paper describe the ultrastructure of the spermatozoa of S. nebaliaeboth at complete maturity and in the encysted formoutline some important steps of spermiogenesis, ancompare the morphological features of seisonidean spethose of the other rotifers and the other lower metaphyla.

Materials and methods

Nebalia bipeswere collected by traps of enrolled fish necontaining dead fishes, and left for 2–3 days on thebottom in Venice lagoon and in Puerto de Andr(Majorca, Spain). Different percentages of collecNebalia bipes were associated with Seison nebaliae:60–80% in Venice lagoon and 10–20% in PuertoAndratx. The rotifers, narcotized with marcaine and geremoved from the host, were examined under a stereomscope. The adult male seisonids, easily recognisable bsize (about 1 mm length) and by the humped trunk (1C), were dissected in sea water with thin tungsten neto isolate mature spermatozoa for in vivo observation omovement and for observations under optical and scanelectron microscopes, after fixation.

Optical microscopyLiving spermatozoa, isolated in sea water, were obseunder a Leitz Dialux phase contrast microscope andsperm movement was recorded on a U-matic videocasCysts, mature spermatozoa and encysted spermatozoaisolated on coverslips coated with poly-1-lysine, fixed wa sucrose-picric acid-paraformaldehyde-glutaraldehsolution (SPAFG: Ermak & Eakin 1976) 1200 mO(Melone & Ferraguti, 1994), stained with 4,6-Diamidinphenylindole (DAPI) solution (2µg/ml) for nuclear identifi-cation and observed under a Leitz DM RB Nomarski inference contrast and fluorescence microscope.

Transmission electron microscopy (TEM)The males of S. nebaliaewere fixed at room temperatuwith 0.1 M cacodylate buffered SPAFG. The worms wleft in the fixative for different times, from 2 h to seve

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days, then washed in 0.1 M cacodylate buffer, postfixecacodylate buffered 1% osmium tetroxide, briefly wasin distilled water, pre-stained for 2 h en bloc in 2% aqueuranyl acetate, dehydrated in a graded ethanol seriesembedded in Spurr resin. Thin sections, obtained witLKB Ultrotome III and V, were stained in lead citracarbon coated, and observed with a Jeol 100 XS elemicroscope.

Scanning electron microscopy (SEM)Whole narcotized animals were fixed with SPAFG,reported in the previous paragraph, then washed in diswater. After the dehydration in a graded ethanol seriesspecimens were critical-point dried with CO2, coated withgold, and observed under a Cambridge Stereoscan 250

The sperm material was isolated on coverslips stained with DAPI. After the observation under the optmicroscope, the coverslips where processed with heethyldisilazane (Melone & Ferraguti, 1994), glued wsilver paint to SEM stubs, coated with gold and obseunder a Cambridge Stereoscan 250 Mk2.

Results

The male genital apparatus of S. nebaliaeis a prominent Ushaped structure almost filling the trunk of the animal. formed by two large paired sac-like testes situated lateto the gut, and connected posteriorly to a commondeferens. This last folds forward, crosses a pear-like odorsal to the stomach, and finally outlets in the cloacal ature, situated at the connection between the neck andof the animals (Remane, 1929–1933). Mature spermacan be found in the testes and in the first tract of thedeferens, whereas the spermiogenesis can be examithe testes. In the pear-like organ the mature spermatozencysted one by one in a process lasting 90 s (Illgen, 1There and in the following tract of the vas deferens we examined the encysted spermatozoa.

Mature spermatozoa (Figs 1–4)The spermatozoon of S. nebaliaeis a thin, filiform cell 70µm long with a fairly constant diameter of approxima0.6µm (Fig. 1A). The diameter is only slightly increased0.8µm at one of the extremities, about 2.5µm long,showing a characteristic hood shape (Figs 1B, D & When observed in sea water the spermatozoa wave acwith the hood anteriorly. The movement is particulaaccentuated in the sperm region opposite to the hFollowing Justine’s convention about Platyhelmint(1995), we consider the hood-shaped slightly enlaportion as the head, and will distinguish it, in the followdescription, from the cell body.

The head is a bilaterally symmetric structure contaiapically a conical vesicle (hereafter: acrosome) under wthe flagellum is inserted with its basal body (Figs 2A, B

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Fig. 1 A A single spermatozoon as seen under SEM. The posterior extremity is recognizable for its coiled arrangement. B A head at higher magnifica-tion (compare to Fig. 2 A, B). C Seison nebaliae(m, male; f, female) on its host, Nebalia bipes. D A group of mature spermatozoa. E Encysted sperma-tozoa. F, G The same mature spermatozoon as seen under Nomarski interference contrast microscope (F) and under the fluorescence microscope afterDAPI staining (G). Note that the dye localizes the nucleus all along the cell, with an intenser positivity in the posterior portion. H, I the same microscopicfield containing encysted spermatozoa, as seen under the Nomarski interference contrast microscope (H) and under the fluorescence microscope (I) afterDAPI staining. The whole spermatozoon is highly coiled, thus the encysted sperm is entirely DAPI-positive.

3A). Between the basal body and the acrosome, a struformed by a certain number of tightly packed membraare visible, packed one over the other, thus forming a acteristic structure (Fig. 2C). The membrane surrounthe acrosome is infolded at its base, determining a subsomal space (Fig. 2B, D). Laterally to the flagellar b

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Fig. 2 A–H Head of the spermatozoon in longitudinal (A–C) and cross (D–H) section. A at low magnification the distinction between the head and thecell body is apparent. Note the different shapes of the heads (arrows) when cut with different inclinations. The accessory bodies (arrowheads) are differentin the different sections of the cell body. B In this sperm head there is: an acrosome (a) with a subacrosomal space (asterisk), a paraacrosomal body (p),and the basal body (arrowhead) with the first tract of the flagellum. Dotted lines indicate the level of the cross sections at right. C enlargement of the struc-ture connecting the acrosome and the flagellum. Note the packed plasma membranes (arrowheads). D–H progressively more caudal sections of the head.a, acrosome; p, paraacrosomal bodies; n, nucleus; ab, accessory bodies.

body. In the space between the two tear-shaped vesicextremely thin organelle delimited by two membranevisible (Fig. 2F). Based on DAPI staining we interpret structure as the most apical portion of the nucleus (FigG). The faint reaction to the stain suggests the presencscarce amount of uncondensed chromatin.

The sperm portion posterior to the head is formed bycell body, containing the nucleus and the accessory borunning parallel to the flagellum to which it is tighconnected (Fig. 3B). There is only one basal body iS.nebaliae, continuing in the flagellar axoneme: the babody and the basal portion of the flagellum are immersea dense substance (Fig. 2B, E). Only the basal bocontained in the head’s cytoplasm, but as soon asaxoneme emerges, a flagellum is formed, separate fromain body of the spermatozoon (Figs 1B & 2B, F).

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flagellum runs parallel to the sperm body and netowards its end, contained in a groove (Fig. 3B). flagellar plasma membrane is connected by some weato the plasma membrane of the sperm body (Fig. 2G)flagellar axoneme has a conventional 9+2 structure, inner and outer dynein arms present (Fig. 4C). The plabilateral symmetry of the axoneme always coincides that of the cell body (Fig. 4C–E). Since it is known thatflagellar beat occurs in the plane of bilateral symmetrthe flagellum, we may assume that the movement owhole sperm is planar. Caudally, the axoneme terminafirst with only A tubules of each doublet continuing, thwith a gradual, progressive loss of the microtubules 4F).

The nucleus is nearly as long as the whole spermApically, in the head, it is visible as an extremely flatte


Fig. 3 A schematic drawing illustrating a mature spermatozoon of S. nebaliae. A The head in its tridimensional aspect (left) with the corresponding crosssections at right. B The cell body with some of the characteristic cross sections. Note that the two schemes are at different magnifications. a, acrosome;b, basal body; p, paraacrosomal body; f, flagellum; n, nucleus; ab, accessory bodies.

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Fig. 4 A–F Cell body of a spermatozoon in longitudinal (A, B) and cross (C–F) sections. A, B two different tracts of the cell body showing the differentshapes of the accessory bodies (arrowheads). C–F Progressively more distal sections showing the different aspect of the accessory bodies (ab), of thenucleus (n) and the flagellum. In the more distal sections (E, F) the unique, long mitochondrion is visible and is accompanied by two symmetric c-shapedbodies (arrowheads).

vesicle containing almost no chromatin (Fig. 2F–H). Mposteriorly, in the cell body, nuclear sections becomeshaped, with the long portion of the Y in the planesymmetry of the spermatozoon (Fig. 4C, D). The lportion of the Y becomes progressively longer in mcaudal sections, so as the nucleus reaches the sperm membrane opposite to the flagellum. More caudally,nucleus progressively assumes a triangular shape in section, with the top of the triangle toward the axonemethe base toward the mitochondrion (Fig. 4E, F). In region the chromatin is particularly condensed (Fig. 1G

A single, long mitochondrion is present in the posteportion of the cell body, flattened against the plamembrane (Fig. 4E, F). It is accompanied by two bilatesymmetric columnar structures placed one at each side4E, F). These structures are C-shaped in cross section.origin and function are unknown.

Finally, for nearly its whole length, the spermatozshows a considerable amount of vesicles (hereafter: asory bodies), starting already in the head (Fig. 2G, H),continuing to the mitochondrial region (Fig. 4): theaccessory bodies are always paired structures situa

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each side of the nucleus, and varies characteristicatheir shape and number in the different regions of the matozoon (compare Fig. 4A and B). However, due tofact that the spermatozoa were studied with TEM intestes and in the deferent ducts, where they are extrebent, we could not observe longitudinal sections sufficielong enough to measure the relative length of the tractsdifferent accessory bodies.

Encysted spermatozoa (Figs 1 & 5)Mature spermatozoa of S. nebaliaebecome encysted in thpear-like organ, one at a time through a very rapid prostarting from the sperm’s caudal extremity. The encystmconsists of a rolling-up of mature spermatozoa that aresurrounded with two different sheaths, an inner and an one (Fig. 5A, B). The inner one, approximately 35 nm thand with a characteristic crystalline periodicity of 9 ntightly involves the sperm (Fig. 5B), whereas the outer thinner, is more loose fitting but also shows a someperiodical appearance. The encysted spermatozoa areshaped structures about 8µm long and 1.5µm in diamete(Figs 1E & 5A). All the different parts of the spermatozo

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Fig. 5 The encysted spermatozoa and their fate. A Longitudinal section of an encysted sperm. Even if the fixation is poor probably for the presence ofthe dense sheaths (arrowhead), some parts of the mature spermatozoa, like the flagellum (asterisk) and the accessory bodies (arrows) are recognizable. Ontop of the encysted sperm, the cup-shaped appendage is visible. B A detail seen at higher magnification shows the crystalline appearance of the innersheath. C At the level of the cup-shaped appendage, the outer sheath is interrupted (arrowhead) D A cross sectioned cup-shaped appendage shows at leastthree types of filaments connecting it to the encysted sperm. E A male S. nebaliaewith a ‘necklace’ of encysted sperm (arrow) around the neck. F Thetrunk of a female S. nebaliaeshows a sperm ‘unrolled’ with its typical appearance (arrow) into the ovary. m, muscle; o, oocyte.

are recognizable in the encysted form, although deforand poorly fixed because of the tight packaging (Figs 51H–I). The average volume of the encysted sperm csponds to that of the mature spermatozoa before enment. Evident longitudinal spiral grooves mark the exteof the encysted sperm when seen under SEM (Fig. 1E)

Each one of the encysted sperm has a characteristicshaped appendage at one of the two extremities (Figs 5A, C). The cup is formed by a large amount of electdense spherules of different sizes, close to one anothe5C, D). The cup-shaped appendage is connected tencysted sperm through an extremely complex array ofments of at least three different morphologies, size,disposition (Fig. 5C, D). The more external layer of fments is formed by large filaments, 25–28 nm in diamwith a periodicity of 29–31 nm, superficially resemblcollagen fibrils (Fig. 5D). The array of filaments evidenconnects the ‘cup’ to the encysted sperm, passing thrthe outer sheath, to end against the inner one (Fig.Once formed, the encysted spermatozoa are probably elated, since they are found as a sort of ‘necklace’ arounneck of the male (Fig. 5E). At mating they are passed tfemale in which only ‘unrolled’ spermatozoa can be fo(Fig. 5F).

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Spermiogenesis (Figs 6 & 7)The process of spermatogenesis in S. nebaliaeis extremelycomplex and still incompletely understood. We hsucceeded to follow only some spermiogenetic evwhich will be described and discussed for their possphylogenetic implications. The spermatogenesis occuSeisonidea, as in many other invertebrate species, in i.e. isogenic groups of germ cells undergoing some synnous divisions not followed by cytodieresis. The cyststhus formed by a central, anucleate, cytoplasmic m(cytophore) to which a certain number of germ cellconnected. We were able to count the number of spermattached to each cytophore in only a few cases: we co32 cells in all cases but one, which contained 64 cells. Inot been possible to determine at what stage of spermiesis the germ cells lose their connection to the cytopho

The process of spermatogenesis occurs in the testwhich the cysts in the different developmental stagesfound, without any apparent order, and tightly packed. explains why it has not been possible to follow and studentire process of sperm production. Apparently the procespermatogenesis begins in the centre of the testes angresses centrifugally, so late spermatids and mature sptozoa are found free in the more external portion of the te

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Fig. 6 Early stages of spermiogenesis (compare to the scheme in Fig. 7 H–K). A The proacrosome makes its first appearance in close connection with afolding of the perinuclear membrane. The basal body of the flagellum (arrowhead) is attached to the same folding. B The flagellum encircles the nucleus.A complex series of vesicles is present in the proacrosome area (asterisk). C Chromatin condensation begins in the area close to the flagellar canal. D Anenlargement of the proacrosome region showing the likely confluence of vesicles in the proacrosome (asterisk). E The multiple vesicle visible also in Fig.6 B is of probable Golgian origin. a, proacrosome; n, nucleus; m, mitochondria.

In the earliest stage we could identify, the spermatidroundish cell with a large nucleus showing pores inperinuclear cistern in certain areas, and chromatin alrcondensed in an area close to the basal body (Fig. 6A)

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at least in its basal portion, within a cytoplasmic canal.canal is surrounded by the cell plasma membrane aadjacent to the nucleus (Fig. 6A, B). In the nuclear rewhere the basal body is attached, the perinuclear cisforms a fold: the basal body is attached to one side whereas a small electrondense vesicle (the proacrosomalways attached to its other side (Figs 6A & 7I). Some mchondria are also present in the cytoplasm: curiouslleast one is always connected to the nucleus in a regiofrom the basal body.

The next event in spermiogenesis is the ‘deepeninthe flagellar canal which, at the end of the procsurrounds more than half of the nucleus (Figs 6B, C & 7JThis process is probably not accompanied by a rotatiothe nucleus, so as the fold of the perinuclear cisternsomewhat ‘stretched’ and the area of condensed chromoves backward with respect to the basal body (Fig. 7I

A Golgi apparatus is often present in the spermatid cplasm in close proximity with electrondense cytoplasvesicles of different shapes (Fig. 6E). Some of the veswill fuse with the proacrosome (Fig. 6D), whereas two oassume progressively a fibrous aspect and will becomparaacrosomal bodies (Fig. 7A, B).

At a following stage the nucleus elongates, at least distal, more condensed portion, always parallel to ancontact with, the flagellum, whereas in the more proxiportion it remains uncondensed with apparent nuclear p(Fig. 7C, D). Only at the end of spermiogenesis this poof the nucleus flattens against the flagellum, and the worganelle is now a U-shaped cylinder.

A fusion of the cell plasma membrane with those offlagellar canal makes the flagellum free, but always in ccontact with the cell body (Fig. 7E–G). The fusion stfrom the apical portion of the flagellum, recognizable bythin nuclear profile associated (Fig. 7F). The last eventhis unusual process of spermiogenesis consists straightening of the formerly U-shaped nucleus. Tprocess pushes the sperm head externally to the cytopat this final spermiogenetic stage, thus, the cysts connesperm tails, whereas the nuclei stand outside.

These last spermiogenetic stages are accompanied complex morphogenesis of the dense bodies. The cheshaped dense bodies become visible just before thestraightening of the sperm (Fig. 7G). However, the wprocess it is at present poorly understood, and will behere described in detail.

Discussion

S. nebaliaesperm and biology of fertilizationAccording to Franzén (1956) the spermatozoa with rounhead, simple anterior acrosome, midpiece with round mchondria, and posterior flagellum are related with extefertilization; all other spermatozoa, with different shaand organization, are related with internal fertilizati

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Among Rotifera, species belonging to Seisonidea Monogononta have internal fertilization and a modified tof spermatozoon. Nevertheless the different sperm pain the two rotifer taxa are possibly related to differmodality of sperm-egg interaction. In particular, for Seison,Ahlrichs (1995a) says: ‘Das Spermium legt sich längsan die Eizelle an und dringt dann auf ganzer Breite inein’, which means: ‘The spermatozoon lays down onegg for its whole length, then enters the egg laterally’. perhaps explain the rich equipment of the Seisonspermatozoon in dense bodies that, possibly, are involved infertilization. On the other hand, also the Monogononta smatozoa have dense bodies of different shapes ancannot exclude that they could be involved in the fertiltion.

Male seisonids lack a penis and this condition influethe sperm transmission to the female. The spermatozSeisonhave to get in contact with the sea water duringpassage from the male to the female genital openings. opinion, the encystment of the mature spermatozoa cexplained as an adaptive strategy to avoid an osmotic sand also to obtain more compact gametes easy to ‘haMale seisonids, in fact, collect the encysted spermatosqueezed from the cloaca, with the head, being the completely retracted (Ricci et al., 1993). The adhesiothe encysted spermatozoa to the head-neck region, male, is possibly due to a sticky reaction of the substacontained in the cup region of the spermatozoa. The tmission of the encysted spermatozoa to the female happen through the rubbing of the male head on the fecloaca. The large amount of sperm (thousands) produca male Seisonis possibly related with this peculiar matisystem during which a number of spermatozoa can be

Comparison with the other rotifer sperm modelsThe five monogonont sperm models known (MeloneFerraguti, 1998) are characterized by being elongate cewhich there is an anterior, flagellar portion, and a postone, the cell body, where the nucleus and the mitochoare found. The axoneme is a conventional 9+2 one in wthe inner dynein arms only are present. It is located inthe sperm cell, starts anteriorly in the centriole, and tenates in the posterior region of the cell body. The mitocdria are situated in the cell body posterior to the nucThere are various accessory vesicles in the different reof the spermatozoon. Their shape and content varies different species. Nuclear chromatin is irregulacondensed forming dense clumps.

The general architecture of the S. nebaliaespermatozoois similar to that of Monogononta: the centriole is antethe mitochondrion is posterior, the chromatin is irregulcondensed, there are many and different accessory veall along the cell. However, there are many important diences: in S. nebaliaethe nucleus runs, flattened, alonearly the whole cell (even if chromatin is complecondensed only in the posterior portion of it).


Fig. 7 Later stages of spermiogenesis A The two paraacrosomal bodies appear at first as vesicles filled by some fibrillar material. The nucleus, at thisstage, is already an extremely thin tread lining the flagellar canal. B At a later stage the proacrosome is enlarged and the two paraacrosomal bodies arealready in their definitive place. C The nucleus starts elongating, and the chromatin condenses mainly in its posterior region. The basal body (not includedin the plane of the section) is at right. The dotted line indicates the level of section shown in D. D A cross section of a spermatid at the level indicated in C.At this stage the flagellum (arrows) is U-shaped and still contained in a cytoplasmic canal. E–G Three sections showing how the flagellum becomesprogressively free: in E the flagellum is contained in the cytoplasmic canal (as in D); in F only the proximal portion is free, whereas in G the flagellum isfree at both sides. The ‘straightening’ of the spermatid is a later event. H–K Four schematic drawings illustrating our ideas on the successive spermio-genetic stages: the stage depicted in H is only hypotetical. a, proacrosome; f, flagellum; g, Golgi apparatus; n, nucleus; p, paraacrosomal bodies.

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Monogononta the nucleus is a lobated structure contaonly in the cell body. In S. nebaliaethere is an acrosomabsent in all Monogononta. The flagellum is, except forbasal body, external to the cell and contained in a grooS. nebaliae, whereas the axoneme is located inside thein Monogononta. The axoneme has both inner and dynein arms in S. nebaliae, but only the inner ones Monogononta. There is only one, long mitochondrion iS.nebaliae, whereas four to eight are present in MonogonoThe extremely unusual fact that the flagellum and nucleus are rolled up in S. nebaliaelate spermatids to bunrolled only in the final stages has been reported also monogonont Brachionus plicatilis(see Melone & Ferragut1994)

Comparison to other metazoan phylaThe spermatozoa of the other lower metazoan phyla ttionally called ‘aschelminths’ or ‘pseudocoelomat(Ruppert, 1991) varies greatly in their structure: a contional ‘primitive’ (Franzén, 1956) spermatozoon is presonly among Priapulida, in Priapulus caudatus(Afzelius &Ferraguti, 1978): in the same phylum there is a transitioa ‘modified’ (Franzén, 1956) sperm model in species ogenus Tubiluchus, connected to a peculiar fertilizatiobiology (Alberti & Storch, 1988). Nematoda (Foor, 19and Nematomorpha (Schmidt-Rhaesa, 1997) have afllate spermatozoa characterised by the presence of vvesicular bodies. The spermatozoa of Gastrotricha belothe modified type, with some difference in membersChaetonotida and Macrodasyida (Balsamo et al., 1998)spermatozoa of xenotrichulid Chaetonotida show mfeatures primitive for the phylum, including scarccondensed chromatin and a simple acrosome striksimilar to that of S. nebaliae. Whereas the spermatozoaLoricifera and Cycliophora are virtually unknown, thoseKinorhyncha show a considerable number of different vcles (Adrianov & Malakov, 1991), some of them derivfrom the transformation of mitochondria (Nyholm Nyholm, 1982). There is no acrosome, the nucleus is atread with the same length of the cell body, the flagelluexternal to the cell body, but parallel and tightly conneto it. At least in a region, the nucleus runs parallel toregion connecting the cell body to the flagellum. The smatozoa of the Acanthocephala (Carcupino & Dez1995) has an anterior centriole, a flagellum externamost of its length to the cell body, but parallel to it contained within a groove. The flagellum has an axonwith both inner and outer dynein arms (Marchand & Ma1978; Figs 3A & 4B). They lack mitochondria and nucleus loses its envelope during spermiogenesis: nuchromatin becomes thus a lamina flattened againsplasma membrane of the spermatozoon not surroundan envelope. Only a small portion of the perinuclear cistremains visible in the mature spermatozoon againsplasma membrane at the level of the flagellar gr(Whitfield, 1971; Marchand & Mattei, 1978). Th

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Acanthocephala have no visible acrosome, but a sGolgian vesicle is located inside the basal body early ispermiogenesis (Marchand & Mattei, 1977; CarcupinDezfuli, 1995). The sperm contacts the eggs exactly this centriolar derivative (Marchand & Mattei, 1977). Madense vesicles are present in the sperm cytoplasm.

Within the complex panorama of the aschelminth spmodels, there is no doubt that the most important simties with rotifers are to be found among acanthoceph(Melone & Ferraguti, 1994; 1998; Ahlrichs, 1995b).particular, the anterior flagellar insertion has been inpreted by Ahlrichs (1995a; 1995b) as a synapomobetween Rotifera and Acanthocephala, whereas the ence of dense bodies along the sperm was interpreted same author as a synapomorphy between Acanthoceand Seisonidea. These and other, somatic, charactersused to strengten the hypothesis that Acanthocephala asister group of Seisonidea (Ahlrichs, 1995b). Howevermust not forget that an anterior flagellar insertion anposterior nuclear position characterize nearly all plhelminthes (Watson & Rohde, 1995; Justine, 19whereas dense vesicles are present in the cytoplasm ofother ‘aschelminths’ (see above) and ‘turbellarian’ plhelminthes as well. Thus, characters interpreted as symorphies may well have been inherited by some reancestor.

Interestingly, among Platyhelminthes there are models of spermiogenesis: in the first, found in m‘turbellarians’, the basal bodies migrate centrifugally frthe cytophore (Watson & Rhode, 1995), whereas insecond, the so-called proximodistal fusion typical of parasitic Pletyhelminthes, the basal bodies lie close tcytophore, whereas the nucleus and the mitochomigrate centrifugally (Justine, 1991; 1995). If we acceporientation of the platyhelminth sperm proposed by Ju(1991; 1995), i.e. we consider ‘anterior’ the sperm porcontaining the basal body, then it becomes obvious thatwo spermiogenetic patterns are opposed to one anotthe polarity of cell formation. In S. nebaliae, the orientationof spermatids is the same as that of the non-parPlatyhelminthes.

Stronger elements for stating that those character sshared by Seisonidea and Acanthocephala may indesynapomorphies come from their peculiar spermiogenThe orientation and morphogenetic movements of flagellum in S. nebaliaeand in Acanthocephala are idetical: the formation of a cytoplasmic canal in which flagellum is located in the early spermiogenesis is paralby a similar structure in different acanthocephalan spe(flagellar cleft in Whitfield, 1971; gouttière nucléaire Marchand & Mattei, 1976; cytoplasmic crack in ZhaoLiu, 1992). (It is worth recalling that in early spermatidsthe monogonont Asplanchna brightwellithe flagellum isalso seen ‘… occurring in deep cytoplasmic infoldings[Koehler & Birky, 1966]). The anterior migration of tcentriole occurs in both phyla, as well as the associati

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the centriole to a Golgi-derived vesicle and to a fold ofperinuclear cisterna.

However, while in Acantocephala the centriole continto migrate forward of to the rupture of the perinuccisterna fold, leaving in mature sperm only portions ofperinuclear cisterna intact, in Seisonidea the connebetween centriole and nucleus probably remains anrepresented by the pack of membranes always seenrating the acrosome and the basal body and bymembranes seen in the sperm head close to the flagroove. Interestingly, in the posterior region of the Seisoncell body, the chromatin is highly condensed and assthe shape of a small electrondense triangular prism clothe mitochondrion. The nuclear envelope, on the contremains in close contact with the flagellar groove, as doremnants of nuclear envelope in Acanthocephala Whitfield, 1971). However, while in Acanthocephala Golgi-derived proacrosome is progressively reduced very small vesicle placed inside the basal body as a srelict acrosome, in S. nebaliaea real acrosome is visible mature spermatozoa. To summarize, some features S.nebaliaemature spermatozoa are reminiscent of spergenetic stages of Acanthocephala (compare Fig. 7H–the present paper to Fig. 17 in Marchand & Mattei, 197

Sperm morphology and, even more, the spermiogepattern, support the assumption of a sister-group positithe Acanthocephala with respect to the Seisonidea, alsuggested on the basis of somatic, spermiologic (Ahlr1995a; 1995b), and molecular data (Mark Welch, perscommunication on sequences of genes encoding a pro

To conclude, we wish to consider the ‘problem’ collagen. Collagen is reported as absent in Rot(Clément, 1993), while it is abundant in AcanthocephThe presence, around Seisonencysted spermatozoa, of filments morphologically resembling collagen suggest investigations on the genes encoding collagen in Athocephala and Seisonidea are highly needed.

ACKNOWLEDGEMENTS

We would like to thank Claudia Ricci for her contributioncollecting living seisonids, David Mark Welch fproviding unpublished molecular data. Alessio PlebaniRoberta Zolio carried out most of TEM sectioning. Tresearch has been supported by a grant from MU(Rome) under the National Research Project ‘Biology Evolution of the Recognition and Interaction of the Animcells’.

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AbstractKeywordsIntroductionMaterials and methodsOptical microscopyTransmission electron microscopy (TEM)Scanning electron microscopy (SEM)

ResultsMature spermatozoa (Figs 1–4)Figure-1Figure-2Figure-3Figure-4Encysted spermatozoa (Figs 1 & 5)Figure-5Spermiogenesis (Figs 6 & 7)Figure-6

DiscussionS. nebaliaesperm and biology of fertilizationComparison with the other rotifer sperm modelsFigure-7Comparison to other metazoan phyla

AcknowledgementsReferences

Documents

Spermiogenesis in Seison nebaliae (Rotifera, Seisonidea ...Melone1999... · (Rotifera, Seisonidea): further evidence of a rotifer-acanthocephalan relationship* M. Ferraguti, G. Melone