Tissue and Cell 38 (2006) 1–6

The spermatozoon of a ‘living fossil’:Tubiluchus troglodytes (Priapulida)

Marco Ferraguti∗, Claudio GarbelliDipartimento di Biologia, Universita degli Studi di Milano, 26, via Celoria, I-20133 Milano, Italy

Received 1 April 2005; received in revised form 12 May 2005; accepted 17 May 2005Available online 4 November 2005

Abstract

The spermatozoon ofTubiluchus troglodytes, the first priapulid formally described from the Mediterranean Sea has a head composedof an acrosome and a nucleus. The acrosome is divided in two branches coiled around the nucleus. The nucleus is basally columnar, butapically generates two rods helically coiled one around the other. The midpiece is formed by an axoneme with 27 accessory microtubules,surrounded by three mitochondria. An annulus separates the midpiece from the tail that contains a 9 + 2 axoneme surrounded by nine accessorymicrotubules. The spermatozoon ofT. troglodytes is similar to that of the other two species known from the genus, and completely differentf genusw©

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rom the ‘primitive’ one of the other priapulids. SinceTubiluchus is considered the most basal of the extant priapulids, and the onlyith an internal fertilization, it may be that in priapulids the external fertilization is a derived character.2005 Elsevier Ltd. All rights reserved.

eywords: Spermatozoa; Ultrastructure; Priapulida

. Introduction

Priapulida is a small phylum of psudocoelomate wormseasuring from 0.5 to 20 cm. They live in the marine sedi-ents with a worldwide distribution. They were among theominant forms in the lower-to-middle Cambrian soft sedi-ents, but are now represented by 18 species only, often at

ow densities (Storch et al., 1999b). Tubiluchus troglodytes,he first priapulid formally described from the Mediterraneanauna is a small (less than 2 mm long) meiobentic form,elonging to a genus comprising six species (and probablyany undescribed ones) “from a variety of regions and habi-

ats, including the tropical–subtropical Atlantic Ocean, theed sea, the Philippines area and the Arctic, testifying to

he cosmopolitan nature of the genus” (Todaro and Shirley,003).

Among priapulids the larger species have external fer-ilization and a ‘primitive’ type of sperm (sensu Franzen,956; Afzelius and Ferraguti, 1978), whereas only the smaller

∗ Corresponding author. Tel.: +39 02 5031 4782; fax: +39 02 5031 4781.

species belonging to the genusTubiluchus show internafertilization (Alberti and Storch, 1988) and a peculiar typof sperm. This was described in two species of the geTubiluchus sp. (later identified asTubiluchus philippinensisby Alberti and Storch, 1983, 1988), andTubiluchus coral-licola (Storch and Higgins, 1989) and shows a compleinterdigitation of acrosome and nucleus. The acrosomesists of a small vesicle in apical position from which ttubular extensions arise basally, and helically wind arothe nucleus which is basally column-shaped, but antergenerates two digitiform extensions also helically woand surrounded by the acrosome: “Two lobes of the asomal vacuole and two lobes of the nucleus are wraaround each other to form a cone-shaped corkscrew couration” (Storch and Higgins, 1989). A centriole surroundeby electron-dense material is situated in a centriolar foThe midpiece ofTubiluchus is also unusual in showing 2accessory microtubules surrounding the axoneme. TheTubiluchus species examined, despite their far asidegraphical distribution show a considerable uniformitycharacters, being recognizable only by “some obvious

E-mail address: marco.ferraguti@unimi.it (M. Ferraguti). ferences in dimensions and proportions of acrosome and

040-8166/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.tice.2005.05.001

2 M. Ferraguti, C. Garbelli / Tissue and Cell 38 (2006) 1–6

nucleus, and different number of mitochondria in the mid-piece” (Storch and Higgins, 1989).

T. troglodytes is the first priapulid formally described fromthe Mediterranean Sea, and extends the distribution of thegenus. The description of its spermatozoon could add fur-ther information to the knowledge of one of the eight generaof a phylum considered a textbook example of evolutionarystasis.

2. Materials and methods

The specimens ofT. troglodytes were found by M.A.Todaro in the sandy sediments of the Grotta Piccola del Ciolonear Santa Maria di Leuca, in the Province of Lecce, Italy(details inTodaro and Shirley, 2003). Living priapulids wereextracted by a narcotization–decantation technique using a7% MgCl2 solution in sea water. The males were identifiedwith an optical microscope while still alive and dissected outto expose the testes, which were immediately fixed in a 0.1 Mcacodylate-buffered (pH 7.3) solution of paraformaldehydeand glutaraldehyde in picric acid (Ermak and Eakin, 1976).

The worms were then sent to Milano were they werewashed in 0.2 M cacodylate buffer for 30 min, postfixed for2 h in a 1% solution of osmium tetroxide in 0.1 M cacody-late buffer, washed repeatedly in distilled water, pre-stainede tiono s ande LKBU ronm

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Fig. 1. Schematic drawing of the spermatozoon ofT. troglodytes.

F 00 except A. (A) Longitudinal section of the anterior portion showing the coiledn the cytoplasm (asterisk) (×37,500). (B) Cross-section of the basal body cut at the levels g the mitochondria surrounding the basal portion of the axoneme. (D) Cross-section oft ranes surrounding the axoneme (arrow). (E) Cross-section of the axoneme at the level oft e annulus. (G) Main portion of the tail. The axoneme is surrounded by single accessorym e disorganization of the axoneme.

n bloc for 2 h in the dark in a 2% filtered aqueous soluf uranyl acetate, dehydrated in a graded ethanol seriembedded in Spurr’s resin. Sections were cut with anltrotome V and observed with a JEOL 100 SX Electicroscope.

. Results

The spermatozoa ofT. troglodytes were examined in thentral portion of testes to which they move at matuFig. 3A). The short (about 28�m long) spermatozoa apperranged with their heads directed towards the peripher

he tails in the centre of the testes. The cells are evidivided into a head, comprising acrosome and nucleus, aiece and a tail (Fig. 1).

The head, about 6�m long, contains basally only tholumn-shaped nucleus, with compact chromatin andounded by a plasma membrane constantly separatedhe chromatin by an electron transparent space (Fig. 2A).he nuclear column generates apically two digitiform exions that coil around each other for 3–4 gyresecoming progressively thinner towards the anterior ext

ty (Figs. 2A and 3B, C). The acrosomal vesicle is t

ig. 2. Ultrastructural features ofT. troglodytes spermatozoon. All×67,5ucleus and acrosome. Some redundant membranes are present inhown in C (arrows). (C) Longitudinal section of the midpiece showinhe midpiece showing three mitochondria. Note the two plasma membhe annulus. (F) Another cross-section at a slightly different level of thicrotubules. (H) Terminal portion of the tails showing the progressiv

M. Ferraguti, C. Garbelli / Tissue and Cell 38 (2006) 1–6 3

4 M. Ferraguti, C. Garbelli / Tissue and Cell 38 (2006) 1–6

Fig. 3. Testis and spermatozoa ofT. troglodytes. (A) Cross-section of an isolated testis. The gonia are arranged at the periphery, and the sperm matures towardthe centre. (B) A nearly sagittal section of the anterior portion. A dense material (subacrosomal material?) is present among the spires of the acrosome (asterisk).(C) A computer-generated model of the anterior portion of the head, showing the relationship between acrosome and nucleus. List of abbreviations: a,acrosome;am, accessory microtubules; an, annulus; ax, axoneme; bb, basal body; G, gonia; m, mitochondria; n, nucleus.

anterior-most portion of the cell. It forms two basal digitiformextensions helically wrapping the nuclear extensions for 3–4gyres each (Fig. 3C). Close to the cell plasma membrane,among the turns of the acrosomal vesicle there is a con-siderable amount of electron-dense periacrosomal material(Fig. 3B). Some redundant membranes are present betweenthe acrosome and the nucleus. The basal portion of thenucleus is conically indented to form an implantation fossahosting the basal body of the flagellum (Fig. 2B and C). Thebasal body inside the fossa does not show a conventionaltriplet structure, but rather looks like a sort of ‘compressed’axoneme with some dense material in the centre, and sur-rounded by accessory microtubules (Fig. 2B). The nucleus isfollowed by a midpiece usually containing three mitochon-dria arranged to delimit a canal that contains the axoneme(Fig. 2C and D). The mitochondria show a usual appear-ance with cristae. The axoneme in this region is delimitedfrom the mitochondria by two plasma membranes close toone another, and shows three accessory microtubules in cor-respondence to each doublet, thus a total of 27 accessorytubules (Fig. 2D). Redundant membranes are also presentaround the mitochondria. The midpiece ends with an elec-

trondense annulus 80 nm thick with a diameter of 0.48�min which 18 of the 27 accessory microtubules seem to termi-nate (Fig. 2E and F). The annulus, surrounds the axoneme andsends nine oblique extensions to reach the axonemal doublets(Fig. 2E). The main portion of the axoneme has the usual 9 + 2structure with nine accessory microtubules arranged betweeneach doublet and the following one (Fig. 2G). There is a longendpiece with a variable number of disordered microtubules(Fig. 2H).

4. Discussion

The spermatozoon ofT. troglodytes is considerably similarto that of the other two species of the genus so far studied,and is characterized only by the different dimensions of thevarious portions (Table 1). The fact that the measures of thenucleus and the number of mitochondria ofT. troglodytes aremore similar to those ofT. corallicola, a Caribbean species,than to those ofT. philippinensis, from the Pacific is paralleledby a similarity between the conventional characters of the twospecies (Todaro and Shirley, 2003).

M. Ferraguti, C. Garbelli / Tissue and Cell 38 (2006) 1–6 5

Table 1Sperm characters and geographic distribution of the threeTubiluchus species spermatologically known

Species Number of mitochondria Cork-screw-shaped portionof the nucleus (�m)

Straight portion ofthe nucleus (�m)

Geographic distribution

T. philippinensis 4 (rarely 5) 4.5 3.4 PacificT. corallicola 3 (rarely 4) 3.5 2.2 Atlantic (Mediterranean?)T. troglodytes 3 (rarely 4) 3.5 2.2 Mediterranean

The spermatozoa of the genusTubiluchus show manypeculiar features, starting from the coiling of the two rods ofthe acrosome vesicle around the twisted nucleus. This condi-tion is perhaps shared in the animal kingdom only with somemacrodasyid gastrotrichs belonging to the family Thaumas-todermatidae (Balsamo et al., 1999) in which, however, aconnection between the vesicle coiling around the nucleusand the acrosome has never been demonstrated.

The shape of the nucleus, i.e. a cylinder of condensedchromatin anteriorly dividing in two portions coiled one overthe other is unique and typical of the genus. The presence of adouble sheath of plasma membranes around the basal portionof the axoneme, a feature common betweenT. troglodytes andT. corallicola (Fig. 6 inStorch and Higgins, 1989) deservessome comments. Apparently, in mature spermatozoa thesemembranes are not continuous with the plasma membrane,but terminate at the annulus. It may reasonably be supposedthat, as suggested by some images of spermiogenesis ofT.philippinensis published byAlberti and Storch (1983)theaxoneme is located inside a cytoplasmic canal in the firststeps of spermiogenesis, then the connection between thecanal and the exterior is closed by the growth of the annulus.

The structure of the tail, with a proximal portion show-ing 27 accessory tubules of the axoneme is again peculiar. Ifmicrotubules as accessory structures of the axoneme is a fea-ture present in, and typical of, other higher level taxa, sucha s( elsw aetesa ft nb th thes threei ne int 27m thet lese rob-a alsos ots liket

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and confirmed by Lemburg,Tubiluchus turned out to be themore basal taxon among the extant priapulids, and forms,with Meiopriapulus fijiensis, also belonging to the meio-fauna, the Tubiluchidae. The Tubiluchidae form the sistergroup of all the extant priapulids. All known fossil priapulidsare macrobenthic. Thus, or there are other macrobenthicforms not yet discovered, which could link the extant mac-robenthic forms to the fossil ones, confining the Tubiluchidaeto a lateral position, or the meiobentic life style is the primitiveone for priapulids, and their fossils have not been preserved,and the macrobenthic forms are derived. If this is correct, thenthe internal fertilization, typical of the meiobenthic forms,is the plesiomorphic condition for priapulids, and the pecu-liar spermatozoon ofTubiluchus species is the plesiomorphicsperm model for priapulids. This ‘reversion’ of the usualsequence, with the suggestion of the internal fertilization(with modified spermatozoon) as the plesiomorphic condi-tion of an evolutionary line has already been suggested inother animal groups, like sabellid polychaetes (Rouse andFitzhugh, 1994).

Acknowledgements

We want to warmly thank M.A. Todaro who collected andfi grantf

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s insects (Dallai and Afzelius, 1999) and onychophoranStorch et al., 1999a), we are aware of only two sperm modith 27 accessory microtubules in the tail, both in polychpecies:Hrabeiella periglandulata (Rota and Lupetti, 1997)ndQuesta sp. (Jamieson, 1983). The similarity of the tail o

his last species to that ofTubiluchus species is striking. Ioth sperm models accessory tubules are arranged wiame geometry and number: 27 in the basal portion (n correspondence with each axonemal doublet) and nihe distal portion; in both sperm models the region withicrotubules is that where mitochondria also surround

ail; in both sperm models the region with 27 microtubunds in an annulus. This similarity, although striking, is pbly homoplasic, thus lacks a phylogenetic value; this isupported by the fact that inQuesta sp. the axoneme is nurrounded, in its basal portion, by plasma membraneshose ofTubiluchus.

Tubiluchus is the only priapulid genus with internal fertzation. This is probably due to the meiobenthic life stylehe genus. Consequently, it is also the only priapulid gith a modified sperm modelsensu Franzen (1956). How-ver, in the phylogenetic analysis published byWillis in 1998,

xed the specimens. This research was supported by arom MURST (Rome) to a COFIN project.

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