sperm ultra-structure and spermiogenesis in syllis … · introduction the family syllidae is one...

SCIENTIA MARINA 70(4)December 2006, 585-592, Barcelona (Spain)

ISSN: 0214-8358

Sperm ultra-structure and spermiogenesis in Syllis krohni (Polychaeta: Syllidae), with some

observations on its reproductive biology

ELENA LEPORE 1, MARGHERITA SCISCIOLI 1, MARIA MASTRODONATO 1, MIRIAM GHERARDI 1, ADRIANA GIANGRANDE 2 and LUIGI MUSCO 2

1 Dipartimento di Zoologia, Università di Bari, 70125 Bari, Italy. 2 Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, Università di Lecce, 73100 Lecce, Italy.

E-mail: [email protected]

SUMMARY: Syllidae show a wide spectrum of both reproductive strategies and sperm types. Analysis of their reproductivepatterns could drastically change the presently accepted taxonomic hierarchy of the group. To further contribute to theknowledge of Syllidae we have described the sperm ultra-structure and the spermiogenesis of Syllis krohni (Ehlers, 1864).Mature sperm has a cone-shaped acrosome whose distal end is notched by a trough that transversely encircles its anteriorportion. During spermiogenesis, the acrosomal vesicle undergoes modifications leading to its final shape with a posterioropening. The nucleus appears flattened anteriorly and forms a cleft that surrounds the proximal centriole in its distal region.A 9+2 flagellar axoneme is observed. Up to five mitochondria surround the distal centriole. The spermatozoon of S. krohnican be ascribed to the ect-aquasperm type that is typical in species having external fertilisation and appears to be very sim-ilar in appearance to that of the congeneric species thus far investigated. The main difference is in the shape of the acrosome,which is more elongated and as long as the nucleus. Reproduction of syllids seems to be conservative within sub-families,and the sperm morphology can probably help in phylogenetic reconstruction. External fertilisation is a widespread strategywithin the genus Syllis, probably leading to a substantial similarity in sperm morphology being maintained. It is hypothe-sised, however, that within the same sperm type linked to a particularly reproductive strategy, the acrosome ultra-structurecan be indicative of phylogeny.

Keywords: sperm morphology, ultra-structure, Polychaeta, Syllidae, phylogeny.

RESUMEN: ULTRA-ESTRUCTURA DEL ESPERMA Y ESPERMIOGÉNESIS EN SYLLIS KROHNI (POLYCHAETA: SYLLIDAE) CON ALGUNASOBSERVACIONES DE SU BIOLOGÍA REPRODUCTIVA. – La familia Syllidae muestra un amplio espectro de variación tanto de laestrategia reproductiva como del tipo de espermatozoide. La jerarquía taxonómica de la familia actualmente aceptada podríacambiar drásticamente después de una profunda análisis de las modalidades reproductivas. Con el objetivo de dar aún unamayor contribución al conocimiento de la biología de la familia Syllidae, en este trabajo se ha estudiado la ultra-estructuradel espermatozoide y la espermatogénesis de algunos individuos de Syllis krohni Ehlers 1864. El espermatozoide maduropresenta un acrosoma cónico, cuye parte distal está inciso a través de una fisura que transversalmente circunda su parte ante-rior. Durante la espermatogénesis, la vesícula del acrosoma se modifica hasta su forma final en la que es presente de unaapertura posterior. El núcleo es aplanado en su parte anterior y en la región distal presenta una incisión que circunda el cen-tríolo proximal. Se ha observado un axonema flagelar de estructura 9 + 2. Hasta cinco mitocondrias circunda el centríolo dis-tal. El espermatozoide de S. krohni puede ser adscrito al tipo ect-aquasperma que es típico en aquellas especies que tienenuna fecundación externa y que aparece muy similar a las de las especies congenéricas estudiadas hasta el momento. La prin-cipal diferencia se encuentra en la forma del acrosoma que es más largo que en las especies similares y tan largo como elnúcleo. La reproducción de Sílidos parece ser conservativa en el interior de las subfamilias y probablemente la morfologíadel espermatozoide puede ayudar en la reconstrucción filogenético. La fecundación externa es una estrategia muy difundidadentro del género Syllis y esto probablemente lleva a mantener una similitud substancial en la morfología del espermatozoi-de. Se formula la hipótesis, de todas formas, que en el interior del mismo tipo de espermatozoide y en la estrategia repro-ductiva, la ultra-estructura del acrosoma puede ser indicativa de filogenia.

Palabras clave: morfología espermatozoides, ultra-estructura, Polychaeta, Syllidae, filogenia.

INTRODUCTION

The family Syllidae is one of the most diversewithin Polychaeta (Annelida), numbering about 667species distributed in a large array of habitats, espe-cially in the littoral fringe (see Musco andGiangrande, 2005 and literature cited therein). Thefamily is usually divided into four subfamilies(Syllinae, Eusyllinae, Autolytinae and Exogoninae),but its taxonomic hierarchy is under debate. In fact,several authors (Franke, 1999; Nygren, 1999;Glasby, 2000) have pointed out the ambiguity of thepresent classification and the pressing need for aphylogenetic analysis of the whole family also basedon molecular analysis and reproductive patterns.

From a reproductive point of view a large arrayof strategies have been reported (Franke, 1999; SanMartín, 2003) and a strong link between phylogenyand reproductive modes has been inferred (Franke,1999; Nygren, 1999; San Martín, 2005). After thepioneering studies by Malaquin (1893) and Potts(1911) on the diversity of reproduction modes ofSyllidae, further studies were carried out by Okada(1929, 1937) for the sub-families Syllinae andAutolytinae, and by Schiedges (1979, 1980) andGidholm (1965, 1966) for the sub-familyAutolytinae.

The majority of the syllid species reproduce byepitoky (Malaquin, 1893; Schroeder and Hermans,1975; Franke and Pfannenstiel, 1984; Garwood,1991; Franke, 1999), which may be divided intoepigamy and schizogamy (stolonisation). Inepigamy the whole animal undergoes the modifica-tion into the epitokous form (Wissocq, 1970a, b, c;Daly, 1975; Garwood, 1991). In schizogamy only apart of the individual (usually the posterior one)undergoes the modification into the epitokous form,the so-called stolon, which is the free swimmingstage carrying the germinal products. There are twotypes of schizogamy (Gidholm, 1967; Estapè andSan Martín, 1991; Garwood, 1991): scissiparity(some of the existing segments in the stock aretransformed into a stolon) and gemmiparity (newsegments proliferate and produce stolons).

Epitoky is known in several species of Syllinae,Eusyllinae and Exogoninae. The suppression of epi-toky is often associated with hermaphroditism(Westheide, 1984; Garwood, 1991; Bührmann et al.,1996; Giangrande, 1997; Kuper and Westheide,1997) and external gestation (Mastrodonato et al.,2003). The majority of the species are dioic and usu-

ally lack an evident sexual dimorphism in both atok-ous and epitokous individuals (San Martín, 2003).

Thus, it is not surprising that such a wide spec-trum of reproductive strategies is accompanied by avariety of different sperm types. According toJamieson and Rouse (1989), all the sperm cate-gories, ect-aquasperm, ent-aquasperm and intro-sperm, are encountered within syllids. However,though the family consists of a great number ofspecies, information about spermatogenesis and theultra-structure of their spermatozoa is scarce. In fact,the ultrastructural organisation of spermatozoa hasbeen analysed in Salvatoria (=Grubea) clavata(Franzén, 1974), Syllis pigmentata (as Typosyllispulchra), Syllis sp (as Typosyllis sp) (Jamieson andRouse, 1989; Heacox and Schroeder, 1981),Autolytus sp and Calamyzas amphictenicola(Franzén, 1982), Neopetitia (=Petitia) amphoph-thalma (Bührmann et al., 1996), Sphaerosyllis her-maphrodita (Kuper and Westheide, 1997), Exogonenaidina and E. dispar (Giangrande et al., 2002). SeeLicher (1999) and San Martín (2003) for the resolu-tion of synonymies.

Due to the high diversity in the reproductivemodes of Syllidae, the study of the sperm ultra-structure and its correlation with their reproductivebiology is particularly interesting in order to betterdefine the taxonomic hierarchy within them. Thesperm structure may be useful for the reconstruc-tion of the phylogenetic patterns of the family(Franke, 1999).

To contribute to the knowledge of the diversity ofspermatozoa in the Syllidae family, we studied thesperm ultra-structure of an additional species of thegenus Syllis: S. krohni.

MATERIAL AND METHODS

Target species and study area

Syllis krohni [the original name S. krohnii wasrecently emended by Licher (1999)] is widely dis-tributed in the Atlantic-Mediterranean region andcommonly collected within the algae of the pho-tophilic hard substrata (San Martín, 2003), includingsome human-impacted areas and harbours (Musco,personal observations). During the early reproduc-tive phase the adult specimens undergo schizogamyby scissiparity. The resulting stolon (Chaetosyllistype) is characterised by a typically bilobate head

SCI. MAR., 70(4), December 2006, 585-592. ISSN: 0214-8358

586 • E. LEPORE et al.

(dicerous) with two un-articulated appendages(antennae), four eyes (two ventral and two dorsal),capillary natatory chetae and the non-developmentof the feeding-related organs (San Martín, 2003).

Male specimens of S. krohni were collected inshallow water (0.5-1 m depth) in the harbour of Bari(41°08’01”N, 16°52’06”E, south Adriatic Sea,Apulia, Italy) in May 2005 by scraping the algalsubstrate. The study area suffers from poor hydro-dynamism and wastewater discharge (mainly organ-ic) from both urban and harbour activities, and expe-riences strong annual temperature and salinity vari-ations. The identification of the collected specimens(both atokous and epitokous) was carried out usingLicher (1999) and San Martín (2003) as identifica-tion guides.

Methodology

For transmission electron microscopy investiga-tions, specimens were fixed in a mixture of 2.5%glutaraldehyde, cacodylate buffer (0.4 M) and fil-tered seawater (pH 7.4). Subsequently they werepost-fixed for 1 h in a mixture of 1.0% OsO4 andsea water at 4°C, rinsed in sea water, dehydratedwith a graded series of acetone, and embedded inAraldite (Taab, Aldermaston, England) for section-ing. The ultra-thin sections were cut with an LKBultratome, contrasted with 5% uranyl acetate in50% ethanol and lead citrate in distilled water, andexamined with an EM 109 microscope (Zeiss,Oberkochen, Germany). Semi-thin sections (1 µmthick) were heat-stained with toluidine blue borate(Millonig, 1976).

RESULTS

Morphology of stolons and stocks

The analysed specimens (both stocks and stolons)of S. krohni corresponded well to the morphologicaldescriptions by Licher (1999) and San Martín (2003),apart from the greater dimensions of the stocks exam-ined in the present study (length about 15 mm vs 6.9mm; width about 0.8 mm vs 0.6 mm).

The collected individuals produced one stolonconsisting of ca. 20-30 segments full of gametes.The stolon head bore four eyes and appeared typi-cally bilobate (dicerous) (Fig. 1). The stolon gut wasatrophic and tended to disappear due to compression

by the mass of germinal cells. The nervous gangliawere present but the musculature was reduced. (Fig.2 A). In contrast the gut of the stock was full ofingested material, among which some diatomstogether with chaetae and uncini of a sabellid poly-chaete were easily recognisable.

Some irregularly shaped cells with numerouslong cilia were observed in the epidermis of thestolon (Fig. 2B, C).

Two masses of germinal elements were located atthe sides of the atrophic gut (Fig. 2A). The stolonscontaining exclusively spermatozoa were orange-coloured, whereas others containing germinal ele-ments at various stages of maturation were whitishin colour. In the coelomic cavity of the latter, sper-matocytes, spermatids and numerous mature spermswere present.


SPERM ULTRA-STRUCTURE IN SYLLIS KROHNI • 587

FIG. 1 – Male stock of Syllis krohni bearing a stolon at the posterior end. Scale bar, 1 mm.

Morphology of the germinal cells

The spermatocytes were irregularly shaped proba-bly due to their closely packed arrangement and someof them were joined by cytoplasmic bridges (Fig. 2D). The nucleus was large and elliptical and containedloose chromatin. The Golgi system, some mitochon-dria and ergastoplasmic cisternae were easily recog-nizable within the cytoplasm (Fig. 2 D). Someproacrosomal granules which give rise to the acro-some were observed in the spermatocytes and in theearly spermatids (Figs 2 D, 3 A). The proacrosomalgranules were variable in number and had an electron-dense peripheral ring, a less dense inner region and anelectron-dense core (Fig. 3 A); the nucleus was stillroundish and contained loose chromatin.

During the process of spermiogenesis the pro-acrosomal granules merged, giving rise to a long,narrow structure that formed a kind of cup beneaththe plasmic membrane (Fig. 3 B). While the acroso-mal vesicle became longer and thinner, it moved tothe anterior pole of the nucleus. In this phase ahomogeneous content defined by an electron-denseperipheral area was visible (Fig. 3 C). In this samearea an expansion was formed, later causing the for-mation of the sub-acrosomal space (Fig. 3 D). Theacrosomal vesicle changed, gradually becomingbell-shaped in the late spermatids (Fig. 4 A). Thesub-acrosomal space of the sperm was filled withgranular material and had a core occupied by fila-mentous substance (Fig. 4 A, B, C, D). In the basalregion of the acrosomal vesicle, closest to the nucle-



FIG. 2 – A, transverse semi-thin section showing the coelomic cavity full of male germinal cells (gc). nervous system (ns); gut (g). Scale bar,60 µm. B, semi-thin section showing a ciliary cell (cc) in the epidermis (e). muscle fibres (mf); male germinal cells (gc). Scale bar, 15 µm.C, ultra-thin section of the epidermis (e) in which a ciliary cell (cc) is visible. Scale bar, 3.5 µm. D, ultra-thin section of spermatocytes.

nucleus (n); ergastoplasmic cisternae (arrows); proacrosomal granules (pg); intercellular bridge (arrowhead). Scale bar, 1.2 µm.

us, it was possible to observe a small opening withan accumulation of electron-dense material (Fig.4A-C). The mature sperms had an elongated cone-shaped acrosome (1.8 µm). The distal end of theacrosome was notched by a trough that transverselyencircled its anterior portion (Fig. 4B). The troughresembled a borderline separating the acrosomal cupfrom the acrosome proper. Another trough was pres-ent at its base (Fig. 4B, C).

The formation of the acrosome occurred simul-taneously with the condensation of the nucleus.The latter began with the irregular accumulation ofchromatin in several areas within the nuclear mem-brane (Fig. 3A-D). The condensation proceededfrom the periphery towards the centre of the nucle-us (Figs 2D; 3A-D); in the mature sperm it

appeared as a cylindrical mass of roughly granularchromatin, subsequently becoming compact andstrongly electron-dense. The nucleus of the maturesperm was cylindrical (barrel-shaped) and meas-ured 1 µm in diameter and 1 µm in length. Itappeared flattened in its anterior part, where itremained in contact with the acrosome. In its distalregion the nucleus formed a cleft that surroundedthe proximal centriole (Fig. 4B, C). The centrioleswere located at the base of the nucleus in the oppo-site position to the acrosome (Fig. 4C). The centri-oles and the flagellum already appeared in the sper-matids. A 9+2 flagellar axoneme was formed bythe distal centriole. Up to five mitochondria sur-rounding the distal centriole were visible at thebase of the nucleus (Fig. 4E).



FIG. 3 – Ultra-thin sections. A, spermatocytes (arrow) and early spermatid (arrowhead); nucleus (n); proacrosomal granules (pg); mitochon-dria (m); centriole (c); dictyosomes (d). Scale bar, 0.6 µm. B, early spermatid in which the acrosomal vesicle (av) is formed by the proacro-somal granules (pg) fusion. nucleus (n). Scale bar, 0.4 µm. C, next maturation stage in an early spermatid. nucleus (n); acrosomal vesicle (av).Scale bar, 0.5 µm. D, early spermatids showing the formation of the sub-acrosomal space (ss). nucleus (n); acrosomal vesicle (av).

Scale bar, 0.6 µm.

DISCUSSION

The morphology-based taxonomic examination ofthe herein analysed specimens of S. krohni (shape anddistribution pattern of chaetae, aciculae and cirri, gen-eral morphology and colour pattern) revealed a com-plete correspondence to the available descriptions ofthis species. As for the greater dimensions of thestocks of our specimens, it must be stressed that,though the specimens described by Licher (1999) andSan Martín (2003) were morpho-metrically identical,Licher (1999) clearly states that the specimen that hedescribed was smaller than usual.

An interesting feature observed in the stolon ofthe examined species is the epidermal structure

provided by cilia. Ciliated structures have com-monly been reported in stocks and stolons of syl-lids (Autolytinae), particularly in the prostomialregion (San Martín, 2003), but further studies areneeded to clarify their function. We suggest that,because the brief life of the stolon is exclusivelydevoted to sexual reproduction, the cells withgroups of long cilia observed along its body sur-face may be in some way useful in reproduction,maybe as sensorial organs.

Moreover, the presence of undigested residuals(chaetae and uncini) within the posterior portion ofthe gut of the stock (atokous), possibly belonging toan interstitial form of Jasmineira (Sabellida:Polychaeta), is particularly interesting because S.



FIG. 4 – Ultra-thin sections. A, late spermatids: a bell-shaped acrosome (a) with granular and filamentous sub-acrosomal material (ss) andnucleus (n) with condensed chromatin. Scale bar, 1.12 µm. B, C, spermatozoa: fully formed acrosome (a) with the notch indicating the troughmarking the junction between acrosome and acrosomal cup (arrow); another notch is present near the basal region of the acrosome (arrow-head); sub-acrosomal space (ss); nucleus (n); mitochondria (m); centrioles (c); flagellum (f). Scale bar (B), 0.8 µm; (C), 0.4 µm. D, cross sec-tion at two levels of the acrosome (a) showing the different dimensions of the sub-acrosomal space (ss); flagella (f). Scale bar, 0.35 µm. E,

transverse view of mitochondria (m) surrounding the distal centriole (c). flagella (f). Scale bar, 0.25 µm.

krohni has been previously indicated to be herbivo-rous (Giangrande et al., 2000).

Spermatozoa of S. krohni, which can be ascribedto the ect-aquasperm type (sensu Jamieson andRouse, 1989), appear very similar in morphology tothe thus far investigated species of the Syllis genus.The similarity is related to the general shape of thenucleus, mid-piece and internal structure of theacrosome. The difference is in the dimension of theapical part of the acrosome, which is more devel-oped in S. krohni than in congeneric species, so thewhole structure appears longer than the nucleus.

However, the acrosomal internal organisation,particularly the presence of a fibrillar electron densematerial in the sub-acrosomal space, can be astronger indication of similarity. The basal invagina-tion of the acrosomal vesicle forming the sub-acro-somal space in Syllis is a common feature in poly-chaete sperms (Jamieson and Rouse, 1989; Rice,1992). In S. krohni as well as in the ect-aquaspermof Syllis sp and S. pigmentata (Heacox andSchroeder, 1981; Jamieson and Rouse, 1989), only asmall opening is retained between the sub-acroso-mal space and the underlying nucleus, and the sub-acrosomal material within the enclosed cavity is dif-ferentiated as an ellipsoidal with a number of elec-tron-dense thick filaments.

The general morphology of the sperm of S.krohni is in accordance with the existence of exter-nal fertilisation, as expected from the presence ofswarming stolons. This reproductive strategy is con-sidered as the most ancestral (sensu Franke, 1999)reproductive mode in Syllidae.

From a phylogenetic point of view, variousauthors consider the general reproduction pattern ofsyllids to be conservative within sub-families(Garwood, 1991; Franke, 1999; Nygren, 1999). Inthis case the sperm morphology can also probablyhelp in the phylogenetic reconstruction. However,accepting phylogenetic constraint as the sole struc-tural designer of sperms may be dangerous. It ishighly probable that ecology and ethology play asimilarly important role. This could be seen in thesub-family Exogoninae: the species belonging toSalvatoria and Exogone so far examined are verysimilar in sperm morphology (ent-aquasperm), andthey also have similar reproductive behaviour(external brooders). By contrast, sperm of speciesbelonging to the same genus may be different mostprobably due to the different colonised habitat andreproductive behaviour. The difference in the sperm

morphology between Sphaerosyllis hystrix (dioicand usually epibiotic, having a typical intro-sperm)and S. hermaphrodita (hermaphroditic and intersti-tial, having an extremely modified intro-sperm) fitsthis last case (Franzén, 1956; Kuper and Westheide,1997; Giangrande et al., 2000).

In Syllidae, the ect-aquasperm type has exclu-sively been observed in the three Syllis speciesexamined so far. It can be hypothesised that as exter-nal fertilisation is a more widespread strategy with-in the Syllis genus, this can lead to maintaining asubstantial similarity in sperm morphology. It wouldbe interesting therefore to examine the few speciesof the genus that have been reported as brooders(e.g. S. vivipara) and other Syllinae belonging to theclosely related genera.

Sperm morphology can be indicative of phyloge-ny within species having the same sperm type. Anexample occurs within the family Sabellidae, wherethe similarity of the acrosome within ect-aquasperms of different species can be indicative ofa shared phylogeny (Patti et al., 2003). Even thoughthe presently limited available data prevent any gen-eralisation, the substantial similarity between acro-somal internal features observed in sperms withinthe genus Syllis seems to confirm this hypothesis.

Although the internal structure of the acrosomecan be indicative of affinity among congenericspecies sharing a common reproductive strategy, thedifference in the acrosomal morphology (acrosomalelongation) observed here could be indicative of phy-logeny within the Syllis genus. According to the mor-phology-based classification of Licher (1999), S. pig-mentata and S. krohni belong to different groups(complexes) within the same genus; in anticipation ofa deeper cladistic analysis of the whole family, ourobservations are not in contrast with Licher’s hypoth-esis. As already pointed out by Nygren (1999) for theambiguous taxonomic position of the genusPionosyllis within the sub-family Eusyllinae, theanalysis of the reproductive patterns in Syllidae couldcontribute to a drastic change in the presently accept-ed taxonomic hierarchy of the family.

ACKNOWLEDGEMENTS

The work of the authors A. Giangrande and L.Musco was carried out within the MARBEFNetwork of Excellence “Marine Biodiversity andEcosystem Functioning”, which is funded in the



Community’s Sixth Framework Programme (con-tract no. GOCE-CT-2003-505446). The above-men-tioned authors also wish to thank Marta Chirac forher kind help.

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Received April 11, 2006. Accepted June 27, 2006.Scient. ed.: M.P. Olivar.Published online October 23, 2006.



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