an ultrastructural study of spermatozoa of helix aspersa and helix pomatia

/. MoU. Stud. (1989), 55, 389--W4. © The Malacological Society of London 1989

AN ULTRASTRUCTURAL STUDY OF SPERMATOZOA OF HELIXASPERSA AND HELIX POMATIA (GASTROPODA, PULMONATA)

J.M. HEALY** and B.G.M. JAMIESONt'School of Biological Sciences (Zoology, A08), University of Sydney, 2006, NSW Australia

tDepartment of Zoology, University of Queensland, Brisbane, Australia, 4067.(Received 23 May 1988, accepted 17 August 1988)

ABSTRACT

Spermatozoa of the pulmonates Helix aspersa Mullerand H. pomatia Linnaeus are examined in detail usingtransmission electron microscopy (TEM). Importantfeatures such as the acrosome, perinuclear sheath,nucleus and terminal region of the midpiece aredescribed for the first time. Also presented are thefirst ultrastructural observations on spermatozoa fromspermatophores in any pulmonate gastropod (//.aspersa). No morphological differences could befound between sperm taken from spermatophores andthose within the hermaphrodite duct in H. aspersa.Spermatozoa of H. aspersa and H. pomatia snowall the characteristics of euthyneuran spermatozoa,namely: a helically-keeled nucleus; distinctivearrangement of acrosomal components (apical vesicle,acrosomal pedestal), and extremely elongate midpiece(axoneme and glycogen helix enclosed by matrix andparacrystalline layers). The sperm nucleus of bothspecies is short, and the midpiece also forms theterminal portion of the spermatozoon (glycogen pieceabsent). The extraordinary positioning of the acro-some in H. aspersa—reflected backwards from thenuclear apex—is not observed in H. pomatia, thougha perinuclear sheath (possibly another acrosomalcomponent) is present in sperm of both species. Helixspermatozoa are compared with other euthyneuransperm and briefly discussed from the systematic view-point.

INTRODUCTION

Previous studies of Helix aspersa Muller sper-matozoa have demonstrated the internal struc-ture and cytochemistry of the centriolarderivative ('basal body derivative') and variousregions of the midpiece (Anderson & Personne,1967,1969a,b, sec also 1970, 1976; Anderson etal., 1968; Personne & Anderson, 1970; Ritter& Andre\ 1975). These reports have dealtexclusively with sperm taken from the her-maphrodite duct: no ultrastructural observa-

JPreiem address: Department of Zoology, St Lucii, 4067, Brisbane,OLD, AnstraHa

tions have yet been published on spermatophoresperm in H. aspersa or any other euthyneurangastropod (with the exception of two micTO-graphs of Runcina sperm tails: Kress, 1985).Spermatozoa of H. aspersa (and other pul-monates) were examined by Maxwell (1975)using scanning electron microscopy (SEM) andAnderson & Personne (1976) have presented asmall SEM micrograph of the spermatozoon ofan unspecified Helix species (?//. aspersa).Some aspects of Golgi activity (Meek & Brad-bury, 1963; Dauwalder & Whaley, 1975),nuclear maturation (Bloch & Hew, 1960) andmidpiece development (Tahmisian, 1964; seealso Anderson et al., 1968) have been inves-tigated in H. aspersa but the necessity for a fullstudy of spermiogenesis in this important speciesis still apparent. It should also be noted that, atpresent, knowledge of sperm development inboth euthyneuran groups (Pulmonata and Opis-thobranchia) is limited to a few species, of whichonly two or three are known in any detail. Thepurpose of this paper is to demonstrate for thefirst time the morphology of the entire sper-matozoon of H. aspersa (taken from the her-maphrodite duct and spermatophores aftercopulation) with particular emphasis beingplaced on the acrosome and the terminal regionof the spermatozoon. The unusual arrangementof acrosomal components in H. aspersaprompted us to study spermatozoa of anotherHelix species (H. pomatia Linnaeus). Theseresults, though limited to observations on spermfrom the hermaphrodite duct of H. pomatia, arepresented herein.

MATERIALS AND METHODS

Helix aspersa were collected from gardens in Brisbane(southern Queensland, Australia) and Sydney (NewSouth Wales, Australia) between September and Nov-ember (spring months). Animals were seen paired inNovember (Sydney) and from these individuals wereobtained freshly deposited spermatophores. H. poma-

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BFig. 1. Helix aspena: hermaphrodite duct sper-matozoa. A. Longitudinal section (L.S.) of sper-matozoan head showing poorly developed helicalkeels, portion of acrosomal pedestal, and perinuclearsheath (near nuclear apex). B. L.S. complete acro-some and anterior region of nucleus. Note: post-

eriorly-directed posture of apical vesicle andacrosomal pedestal. C. Transverse section (T.S.)through tip of nucleus and acrosomal pedestal. D.Oblique T.S. of acrosomal pedestal, nuclear tip, andperinuclear sheath, showing true profile of the acro-somal pedestal. E. T.S. through apical vesicle, nucleus

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ULTRASTRUCTURE OF HELIX SPERMATOZOA 391

tia from the United Kingdom were obtained throughthe courtesy of Professor R. Chase.

Spermatophores (only H. aspena) and hermaphro-dite ducts (both H. aspena and H. pornand) werediced and fixed in 3% glutaraldehyde (at 0-4°C, in0.1 M phosphate or cacodylate buffer, pH 7.2) forapproximately two hours. Tissue pieces were thenrinsed in buffer, osmicated for 80 minutes (1%osmium tetroxide in 0.1 M phosphate or cacodylatebuffer at (0-4°C), rinsed again, dehydrated through aseries of ethanols (20%-60% at 0-4°C, 70%-100% atroom temperature) and gradually embedded inSpurr's epoxy resin. Thin sections were cut using LKB2128 UM IV Ultrotome and Huxley ultramicrotomescollected on coated or uncoated 200 mesh coppergrids and stained with uranyl acetate and lead citrate.Specimen grids were examined with AEI Corinth 500,Hitachi 300, Siemens Elmiskop I and Philips 300 trans-mission electron microscopes.

RESULTS

Helix aspersa (hermaphrodite ductspermatozoa) Acrosome and nucleus.

The nucleus is approximately 8.1 urn in length,basally invaginated and exhibits one, or possiblyas many as three, poorly developed helical keels(Fig. 1A). An acrosome is present and is com-posed of: (1) an apical vesicle (spherical, 0.2 umin diameter) situated beside the tapered nuclearapex, and (2) a less electron-dense acrosomalpedestal (length 0.33 um, circular in true trans-verse section) which connects the tip of thenucleus to the apical vesicle (Figs. 1B-E; Fig.2A). A third, possibly acrosomal component(composed of a very electron-dense layerembedded in fine granular material) is inti-mately associated with the anterior half of thenucleus and supports the base of the apicalvesicle and acrosomal pedestal (see Figs. 1 A-G; Fig. 2A). This component is here termed theperinuclear sheath. The degree to which thissheath encloses the nucleus varies according tothe level of section—the most complete enclos-ure of nucleus occurring near the nuclear apex

(below level of acrosomal pedestal) and perinuclearsheath. The electron-dense component of the sheathsurrounds the nucleus while the less-electron-densecomponent abuts the apical vesicle. F. T.S. throughnucleus and perinuclear sheath below level of apicalvesicle and acrosomal pedestal—bilateral symmetryof the sheath is obvious. G. T.S. of nucleus andperinuclear sheath below level shown in Fig. IF—notethat the perinudear sheath only surrounds approxi-mately half of the nuclear membrane at this level. Allscales = 0.2 um except: 1A (scale = 1 um), IB(scale = 0.5 um).

as shown in Figs. 1 D-F. Transverse sectionstaken at lower levels of the nucleus (for exampleFig. 1G; for orientation see also Fig. IB), showthe perinuclear sheath lining one half or lessthan one half of the nuclear membrane. Bothtransverse and longitudinal sections of the peri-nuclear sheath (and nucleus) reveal the bilateralsymmetry of this component and of the associ-ated apical vesicle and acrosomal pedestal (seeFigs. 1B-G; Fig. 2A).

Coarse fibres, 'neck' region

Nine coarse fibres accompany the axonemaldoublets throughout most of the midpiece (Figs.2C-F, see also H) and together with the twocentral microtubules of the axoneme, attach tothe centriolar derivative within the basal inva-gination of the nucleus (Fig. 2C). Figure 2Cshows that the coarse fibres are thick (maximumwidth 0.13 um) and periodically banded withinthe sperm 'neck' region (periodicity rangingfrom 400-500 A). A well developed distal access-ory sheath (length approximately 0.37 um,width approximately 0.16|im) surrounds thecentra] pair of axonemal micTOtubules (Figs.2C, D). At the base of the distal accessorysheath, an electron-dense granule is oftenobserved (Fig. 2C arrow) and is also apparent inmicrographs of Euhadra hickonis spermatozoapresented by Takaichi (1975) and Takaichi &Sawada (1973). The purpose or composition ofthis granule in Helix and Euhadra is unknown,but possibly it may act as a connecting linkbetween the axonemal doublets (emergent fromthe coarse fibres) and the distal accessorysheath. From the base of the distal accessorysheath to the nuclear invagination, the axonemaldoublets are individually enveloped (and there-fore gTeatly obscured) by their accompanyingcoarse fibres (see Fig. 2C (longitudinal section),Figs. 2 B, D (transverse section)). The com-posite coarse fibres and enclosed doublets arecontinuous with the cortical region of the cen-triolar derivative (see Fig. 2C). In contrast thecentral pair of axonemal microtubules (which infact appear solid and not tubular in transversesection) attach directly to the core of the cen-triolar derivative (see Fig. 2C).

Midpiece

Some, though very minor, overlap of the nuclearbase and midpiece is visible in longitudinal andtransverse sections of the 'neck' region (see Figs.2C, D). Initially the coarse fibres (with enclosedaxonemal doublets) are surrounded only by

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Fig. 2. Helix aspena: hermaphrodite duct sper-matozoa. A. Longitudinal section (L.S.) acrosomalpedestal and nuclear tip (detail of Fig. 1A)—noteperinudear sheath (this section taken at approxi-mately 90° to the plane of section shown in Fig. IB).B. Transverse section (T.S.) through basal inva-gination of nucleus showing central axonemal micro-tubules, and (arrows) axonemal doublets (faintly

visible) embedded in coarse fibres. C. L.S. sperm'neck' region showing basal invagination of nucleus,centriolar derivative, periodically banded coarse fibres(continuous with cortical portion of centriolar deriva-tive), distal accessory sheath, and also matrix andparacrystalline components of mitochondnal deriva-tive. Note at base of distal accessory sheath, emerg-ence of axonemal doublets from coarse fibres and

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paracrystalline material (Figs. 2C, D). How-ever, further posteriorly, the coarse fibres andemergent axonemal doublets are surrounded byboth paracrystalline and matrix componentsof the mitochondrial derivative (see Figs.2C, E, F). A single, glycogen helix is alsoenclosed by the mitochondrial derivative, but,in the immediate post-nuclear region of the mid-piece, is occupied by tightly-packed mem-branous deposits. These deposits graduallybecome supplanted by glycogen granules in suc-ceeding levels taken through the midpiece (seeFigs. 2E, F, H). The shape of the midpiecechanges considerably throughout its length, suchthat the glycogen helix gradually becomesreduced in extent (compare Figs. 2F, H withFigs. 3A, B) and eventually disappears alto-gether (Figs. 3C, D). The terminal region of themidpiece (the final 2.2 um of the spermatozoon)commences with the loss of the two, centralmicrotubules of the axoneme and is followedsequentially by: (1) the loss of all nine axonemaldoublets (occurring simultaneously), (2) a plugof electron-dense material, (3) a partially occu-pied vesicle, (4) membrane deposits (see Figs.3F, E for successive transverse sections throughthis region) and finally (5) a second, apparentlyempty vesicle. Figure 3G shows in full longi-tudinal section, the complete series of thesestructural changes within the midpiece. Themitochondrial derivative, therefore, forms theposterior extremity of the spermatozoon inHelix aspersa—there being no vestige of eithera glycogen piece or dense ring structure (bothfeatures often occurring in sperm of opis-thobranchs, basommatophoran pulmonates,and in euspermatozoa of all meso- and neo-gastropod prosobranchs).

(arrow) presence of a small, electron dense granule(? function). D. T.S. through sperm 'neck' region.Portions of nucleus and mitochondrial derivative(chiefly paracrystalline material) are visible as arecoarse fibres (with enclosed axonemal doubletsobscured in this section) and distal accessory sheath.E, F. T.S. through midpiece: E. immediately belownucleus (—note membranous deposits partly occu-pying glycogen helix; double matrix layer): F. pos-terior to 2E (—note absence of membranous deposits;single matrix layer; also intra-axonemal (glycogen)granules). G. Paracrystalline and matrix componentsof mitochondrial derivative as seen in oblique section.H. L.S. of midpiece at the same level as 2F. Scales.A,C,D,H (scale = 0.5 um); B,E,F,G (scale =0.2 um).

Helix aspersa (spermatophore spermatozoa)

Spermatozoa from spermatophores (freshlyexchanged during copulation) do not differ mor-phologically from those stored within the her-maphrodite duct. Figs. 4A-C show that theapical vesical and acrosomal pedestal are angu-larly positioned relative to the sperm longi-tudinal axis. The perinuclear sheath is fullyintact and still retains its layered appearance(Figs. 4C-F). Other components of the sper-matozoon, including the nucleus (Figs. 4A-F),midpiece (Figs. 4G, H) and terminal region ofthe cell (Fig. 4H) are as described for her-maphrodite duct spermatozoa.

Helix pomatia

Spermatozoa from only the hermaphrodite ductof this species were examined. The structure ofthe midpiece, nucleus and neck region (Figs.5A-E) is essentially as described for H. aspersa.An important difference lies in the arrangementof the acTOsomal complex. In H. pomatia, theapical vesicle (diameter 0.18 urn) and acrosomalpedestal (length, including pad over nuclear tip,approximately 0.25 um, diameter 0.18 uxn) lieon the nuclear apex (Figs. 5A, B) at only a slightangle to the sperm longitudinal axis (as opposedto the acute angle between nucleus and vesicle-pedestal complex in H. aspersa). The peri-nuclear sheath is present in H. pomatia sper-matozoa (Figs. 5A, B) but lacks the layeredorganization seen in H. aspersa (compare withFigs. 1B-G, Figs. 4C-F). The length of thenucleus is approximately 10 um.

DISCUSSION

Comparison with other euthyneuranspermatozoa

General features of euthyneuran spermatozoa.Euthyneuran spermatozoa—including those ofHelix aspersa and H. pomatia—possess the fol-lowing general characters: (1) an acrosome com-posed of an apical vesicle (usually round) andan acrosomal pedestal (variously shapeddepending on taxon, often columnar), (2) heli-cally-keeled nucleus (either distinct from or par-tially intertwined with the axoneme andmitochondrial derivative), (3) midpiece com-posed of a complex mitochondrial derivative—with one or more incorporated glycogen heli-ces—which surrounds the axoneme and associ-ated coarse fibres, and (4) ordered (usually9+1) rows of intraaxonemal glycogen granules

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throughout midpiece and, if present (absent inHelix), the glycogen piece. While exceptionalcharacter states occur in spermatozoa of someundisputed euthyneurans (for example, theabsence of coarse fibres in spermatozoa of theopisthobranch Tomatina sp.—seeHealy, 1982),the above listed features readily distinguish opis-thobranch and pulmonate spermatozoa from theeuspermatozoa of meso- and neogastropod pro-sobranchs.

'Neck' region and midpiece

The midpiece region of Helix aspersa sper-matozoa has been the subject of previous ultra-structural studies (see Anderson & Personne,1967, 1969a, b, see also 1970, 1976; Andersonetal., 1968; Personne & Anderson, 1970; Ritter& Andr6, 1975). These papers, in conjunctionwith much work on other pulmonate species,have dealt with the mitochondrial derivativeand have helped to establish the presence of aglycogen-filled helix ('helice principale' ofAndr6, 1962) within the derivative. They havealso explored the morphology and biochemistryof the two constituents of the derivative—thematrix material ('helice secondaire' of Andr6,1962) and the paracrystalline material. Ander-son & Personne (1976) have presented a com-prehensive review of the complex enzymatic andenergy pathways occurring within the mito-chondrial derivative of pulmonate spermatozoaand detailed the probable functional relation-

Fig. 3. Helix aspersa: hermaphrodite duct sper-matozoa. A. Transverse section (T.S.) of midpiecebelow level shown in Figs. 2G,H. The glycogen helixand components of the mitochondrial derivative havebeen reduced in extent. B. Longitudinal section (L.S.)through midpiece region shown in 3A. Intraaxonemalgranules are clearly visible. C. T.S. through midpiecefollowing termination of glycogen helix (axoneme/coarse fibre complex surrounded only by mito-chondrial derivative). D. L.S. through midpieceregion shown in 3C. G,E,F. L.S. and T.S. throughterminal region of midpiece. Sequence of structuralchanges is best shown in 3G—note: (1) loss of centralmicTOtubules of axoneme, followed soon after by lossof doublets, (2) an electron-dense plug of materialreplaces axoneme, (3) a vesicle, partly occupied bydense material, (4) membranous deposits, (5) asecond, more extensive vesicle. Note how mito-chondrial derivative continues to the very tip of thespermatozoon. E,F are T.S. through upper (F) andlower (E) levels of the membranous deposit (area 4)shown in 3G.Scales. A,C,E,F (scale = 0.2 um); B,D,G (scale =0.5 urn).

ships between the two components of the deriva-tive, the incorporated glycogen helix (or helices)and the coarse fibres. They propose that thefollowing metabolic function(s) can be ascribedto specific midpiece components: matrixmaterial—Krebs tricarboxylic acid cycle; gly-cogen helix (or helices)—phosphorylase,dehydrogenase activity; axoneme, coarse fibre-axonemal doublet interface, surface of cen-triolar derivative—sites of Mg++ activatedATPase; paracrystalline material—cytochromeoxidase activity. Workers who have examinedin detail the architecture of paracrystallinematerial in pulmonate spermatozoa generallyagree that it is a continuous, three dimensionallatticework composed of proteinaceous, hollowrodlets or granules (80-90 A in diameter)(Andre, 1962; Ritter & Andre, 1975; Anderson& Personne, 1976; Reger & Fitzgerald, 1982;Reger et al., 1982). Such an interpretationapplies equally well to paracrystalline materialof opisthobranch spermatozoa (for example seeHealy, 1982; Healy & Willan, 1984) since itappears, morphologically at least, indistinguish-able from that occurring in pulmonate sperm.Paracrystalline material has also been dem-onstrated within the midpieces of certainprosobranch euspermatozoa (Cochlostomamontanum—Selmi & Giusti, 1980; Stenothyrasp.—Healy, 1983a; Cypraea errones—Healy,1986a but in all prosobranch cases the arrange-ment and form of this material differs substanti-ally from that observed in sperm ofeuthyneurans. Ritter & Andre (1975) found thatthe 'crystal' (=paracrystalline material) of Helixaspersa spermatozoa, together with its con-tained cytochromes, resists even prolonged tryp-sin treatment, but is readily digested by pepsin.Their observation that cytochrome content ofthe paracrystalline material increases during theperiod of trypsin treatment (10 min to 12 h)seems to correlate well with the fact that trypsincan induce motility in otherwise immotile her-maphrodite duct spermatozoa of Helix aspersa(see Anderson & Personne, 1967). Anderson& Personne (1976) have even suggested thattrypsin may directly stimulate ATPase activitywithin the axoneme/coarse fibre complex. How-ever trypsin treatment does not apparently elicitmovement in hermaphrodite duct spermatozoaof another stylommatophoran Anguispira alter-nata (Atkinson, 1982). It is likely that otherchemical factoid—intrinsic or extrinsic—maygovern sperm motility, or at least, determinethe degree of response of pulmonate sperm tostimulants such as trypsin.

While corroborating the work of other

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Fig. 4. Helix aspersa: spermatophore spermatozoa. A. Longitudinal section (L.S. slightly oblique)showing angularly tilted apical vesicle and acrosomal pedestal near nuclear apex. B. L.S. of acrosome.C. Transverse section (T.S.) near nuclear apex showing nucleus, perinudear sheath and laterallypositioned apical vesicle. D,E. T.S. through anterior portion of nucleus and (layered) perinudearsheath. F. T.S. through nucleus «nd base of perinudear sheath. G. T.S. through anterior portion ofmidpiece. H. T.S. through posterior (left) and near-terminal (right) regions of the midpiece.Scales. A-H (scale = 0.2 urn).

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Fig. 5. Helix pomatia: hermaphrodite duct spermatozoa. A. Longitudinal section (L.S.) acrosomalcomplex (apical vesicle, acrosomal pedestal, perinuclear sheath) and anterior portion of nucleus. B.L.S. apical vesicle and acrosomal pedestal positioned at apex of nucleus. C. L.S. neck region ofspermatozoon showing distal accessory sheath, centriolar derivative, coarse fibres (banded), nudeus andcomponents of the mitochondrial derivative. D. Detail of paracrystalline component of mitochondria!derivative. E. Transverse sections through anterior region of midpiece and posterior region of midpiece.Scales. A.C.E (scale = 0.5 um); B,D (scale = 0.2 urn).

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authors who have studied the midpiece of Helixaspera spermatozoa, our results have in additionshown that the axoneme terminates abruptly,and symmetrically, within the midpiece (2.2 jimfrom the posterior extremity of the sperma-tozoon), and is replaced by electron-densematerial, two chamber-like vesicles and closelypacked membranes. The same appears to betrue of H. pomatia though we have not observedlongitudinal sections of the terminal region inthat species. The terminal region of Euhadrahickonis spermatozoa (see Takaichi, 1975, Figs.12-15) is similar to that of H. aspersa, as in fact,are other features of the midpiece and neckregion (including the presence of an electron-dense granule near the distal accessory sheath—see Takaichi, 1975; Takaichi & Sawada, 1973;Dan & Takaichi, 1979). The glycogen pieceobserved in spermatozoa of many opistho-branchs and basommatophoran (s.l) pulmon-ates (Anderson & Personne, 1970; Ohsako,1971; Thompson, 1973; Kitajima & Paraense,1976; Ackerson & Koehler, 1977; Maxwell,1980; HeaJy, 1982, 1983b; Healy & Willan,1984) and in all investigated meso- and neoga-stropod euspermatozoa (see Healy, 1983c forreferences), does not occur in spermatozoa ofHelix aspersa, H. pomatia, Euhadra hickonis,or, as far as is known, in any stylommatophoranpulmonate. In some opisthobranchs, the gly-cogen piece is short and may show partialdegeneration or (rarely) total absence of theaxoneme (Healy & Willan, 1984; Healy 1984).Such a trend towards regression of the glycogenpiece in euthyneuran spermatozoa may be dueto the fact that substantial glycogen depositsare enclosed by the mitochondria! derivative,thereby reducing the need perhaps for a gly-cogen piece (Healy & Willan, 1984).

Only a single glycogen helix occurs in sper-matozoa of Helix aspersa, H. pomatia and otherstylommatophorans (herein; Grasse et al., 1956;Andre, 1962; Personne & Andre, 1964; Ander-son & Personne, 1969a, b; Anderson et al., 1968;Yasuzumi et al., 1974; Ritter & Andre, 1975;Takaichi & Sawada, 1973; Takaichi, 1975; Dan& Takaichi, 1979; Shileiko & Danilova, 1979;Odiete, 1982; Atkinson, 1982; Reger & Fitz-gerald, 1982) and this is also the case for mostopisthobranchs (see Holman, 1972; Thompson& Bebbington, 1969; Healy, 1982; Healy &Willan, 1984; Healy, 1984) and at least somebasommatophoran (s.l.) pulmonates (Salinator,Siphonaria—Healy, 1983b; Onchidium—Healy, 1986b). Multiple helices (ranging fromtwo to three, possibly as many as four in somespecies) have been noted in sperm of some

basommatophorans (s.l.) (lymnaeids, planor-bids, ellobiids—Anderson & Personne, 1970;Ohsako, 1971; Kitajima & Paraense, 1976;Healy, 1983b; see also Thompson, 1973) andcertain cephalaspid opisthobranchs (Thompson,1973—Acteon; Healy, 1984—Bullina). Thepossibility that some stylommatophoran sper-matozoa may possess more than a single gly-cogen helix within the midpiece remains to beexplored.

Finally, some comment should be made con-cerning the internal structure of the nine coarsefibres and their relationship with the axonemaldoublets and centriolar derivative. In Helixaspersa, and H. pomatia (herein; see alsoAnderson & Personne, 1967): (1) the doubletsare enveloped by the coarse fibres between thebase of the distal accessory sheath and the cen-triolar derivative, and (2) the coarse fibres (withenclosed axonemal doublets) fuse to the cortexof the centriolar derivative. Precisely the sameorganization has been described in Euhadrahickonis (Takaichi, 1975; Dan & Takaichi, 1979)and other stylommatophorans (see Atkinson,1982; micrographs of Maxwell, 1976; Yasuzumiet al., 1974) some basommatophorans (s.l.)(Kitajima & Paraense, 1976; Healy, 1983b) andsome opisthobranchs (nudibranchs—Healy,1984). In some opisthobranchs however, thedoublets either remain distinct from the coarsefibres or are only incompletely enclosed by them(Healy, 1984). Marked obscuring of the dou-blets by associated coarse fibres often makes itdifficult to establish the exact spatial relationshipbetween these two components in some species.Coarse fibres of all euthyneuran spermatozoaare periodically banded—the principal variablesbeing the periodicity (ranging from 200 A inAnguispira alternata (Atkinson, 1982), to 400-500 A in Helix aspersa and H. pomatia (Ander-son & Personne, 1967; herein) and as great as750 A in the lymnaeid Radix japonica (Ohsako,1971)), width (variable depending on speciesand level of section), and persistence of period-icity into the midpiece region (according toMaxwell, 1976, banding in coarse fibres ofDiscus rotundatus extends from the neck regioninto the midpiece for a distance of up to 30 |im).The distal accessory sheath of Helix aspersa andH. pomatia spermatozoa is also observed inother stylommatophorans {Euhadra hickonis—Takaichi, 1975, Dan & Takaichi, 1973, Takaichi& Sawada, 1973; Discus rotundatus—Maxwell,1976; Umax flavus—Yasuzumi et al., 1974;Bentosites bloomfieldi—Healy, 1984) and insome basommatophoran (s.l.) species (Biom-phalaria glabrata—Kitajima & Paraense, 1976;

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Salinator fragilis, S. solida—Healy, 1983b). It isreminiscent of the inner dense cylinderdescribed for the spermatozoal axoneme of thepolychaete Questa by Jamieson (1983). Nodefinite function for this structure has beendetermined, but it is likely that is performs somerole in determining motility of the axial complexand does not, as suggested by Yasuzumi et al.(1974), represent the 'chromatoid body'.

Acrosome and nucleus

Perhaps the most interesting aspect of Helixaspersa and H. pomatia spermatozoa is the formof the acrosome and its positioning at the nuclearapex. Bloch & Hew (1960) recorded a markeddifference in acid lability of nuclear histonesbetween spermatozoa from the hermaphroditeduct and those from spermatophores in H.aspersa. We have observed no significant ultra-structural differences between spermatozoataken from these two sources in H. aspersa—the apical vesicle and acrosomal pedestal con-tinue to be angularly tilted, and associated witha perinuclear sheath within the spermatophore.The perinuclear sheath appears, therefore, tobe a real feature of spermatozoa in H. aspersaand H. pomatia. Elsewhere within the Gastro-poda, this structure has been recorded in thestylommatophoran Bentosites bloomfieldi(Healy, 1984; possibly also Anguispira alter-nata—Atkinson, 1982, and Agriolimax reti-culatus—Bayne, 1970) and evidently inVaginulus borellianus (see micrographs of Lanza& Quattrini, 1964).

The orientation of the acrosomal elementsin H. aspersa may represent only an extremesituation within the Pulmonata, where 'grades'of tilting of the apical vesicle and acrosomalpedestal in relation to the longitudinal axis ofthe nucleus occur. A slightly angular orientationof the apical vesicle and acrosomal pedestal(relative to the nuclear tip) has been noted byTakaichi & Dan (1977) for spermatozoa ofEuhadra hickonis and this is also the case inHelix pomatia (herein) and Bentosites bloom-fieldi (Healy, 1984). In contrast, the acrosomalpedestal and apical vesicle of late spennatids ofstylommatophoran species examined by Healy& Jamieson (unpublished data—Pedinogyrahayii, Varohadra curtisiana, Xanthomelonpachystylum), despite obvious signs of imma-turity, are aligned in the longitudinal axis of thespermatid, and no equivalent of the perinuclearsheath can be identified.

Although we have studied spermatozoa frommature spermatophores, there is no guarantee

that the angular positioning of the Helix aspersaacrosome may not, in some way, alter prior tocontact with the egg. Certainly an analysis ofsperm-egg interaction would clarify this, andhelp determine what role the perinuclear sheathmay play in fertilization.

The form of the sperm nucleus in Helixaspersa and H. pomatia—short, helically keeled,with a shallow basal imagination—occurswidely within the Euthyneura (? in all pulmon-ates) and almost certainly represents the primi-tive form for the ancestral euthyneuranspermatozoon. In contrast, sperm nuclei ofsome opisthobranchs (aplysiids—Thompson &Bebbington, 1969; Thompson, 1973; Kubo &Ishikawa, 1981; certain notaspids—Healy &Willan, 1984; the saccoglossan Elysia australis—Healy, 1984) take the form of an elongate cordwhich spirals around the proximal portion ofthe mitochondrial derivative and axial complex.Such a condition is here considered advancedand may have been independently acquired inthese groups of opisthobranchs. The length ofthe spermatozoan head (that is, acrosome plusnucleus) of Helix aspersa, as determined in thepresent study is 8.1 um—appreciably longerthan the SEM results of Maxwell (1975) for thisspecies (sperm head length 5.5 ± 0.2 um), butreasonably close to the measurement of Ander-son & Personne (1967) for typsin-treated sper-matozoa (7.4 um). Grasse et al., (1956) give alength of 7.5 um for the spermatozoan head ofH. pomatia, while Thompson (1973) found thisregion of the spermatozoa of H. pomatia tomeasure 12 um (including 2 |im acrosome). Ourown observations on H. pomatia indicate a headlength of approximately 10 um. It seems possiblethat sperm head length may be variable in bothH. aspersa and H. pomatia or that significantdifferences may exist between widespread popu-lations. Further research on this aspect seemswarranted. Table 1 summarizes comparativesperm morphology of Helix and other pul-monates.

Systematic significance of stylommatophoranspermatozoa

Apart from those studies dealing with the cyto-chemistry of pulmonate spermatozoa whichhave been briefly reviewed above, very littleinterest has been shown in comparative spermmorphology of the Stylommatophora. The orderis large (over 50 families, approaching 15,000species—see Boss, 1982) and diverse. Under-standably, any comprehensive review of styl-ommatophoran spermatozoa would be onerous.

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Tab

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. Com

paris

on

of

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p.)

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(Rad

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nic

a)(L

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ity

500A

400

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1 gl

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en h

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elic

es

2 gl

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ogen

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coge

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t

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pres

ent

pres

ent

7 ab

sent

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eren

ce

Hea

ly,

1986

b

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ly,

1983

b

Hea

ly,

1983

b

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1983

b

Ohs

ako,

197

1H

ealy

, 19

83b

Klta

jima

&P

arae

nse

, 197

6

Shile

iko

&

5 BO O o

Danilo

va,

1979

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AR

ION

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EA

(An

gu

isp

ira

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rnat

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ly,

1984

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pape

rs)

This

pap

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Shi

leik

o &

Dan

ilova

, 19

79

to m 3 8

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402 J.M. HEALY & B.G.M. JAMIESONIt would, however, provide an opportunity toassess relationships within the order (fromsuborder to generic levels) and between thesepulmonates, the Basommatophora (s.l.) andthe Opisthobranchia.

In this paper we have presented structuralinformation for the two most economicallyimportant species of Helix (H. pomatia—typespecies for the genus, prized as a delicacy, andH. aspersa—a widespread pest species). Theresults have shown numerous similaritiesbetween the two species (nucleus, midpiece)and one major difference (position of the acro-some—? possibly reflecting current subgenericassignments of H. pomatia and H. aspersa). Itis hoped that these data will provide the impetusfor a wide-ranging survey of helicid sper-matozoa.

On a more general level, spermatozoa of H.aspersa and H. pomatia (Helicidae (Helicinae),Helicoidea) are similar to those of otheradequately studied stylommatophorans{Euhadra hickonis—Bradybaenidae, Heli-coidea; Anguispira alternata—Punctidae,Arionoidea; Trichia hispida—Helicidae(Hygromiinae) Helicoidea; Succinea putris—Succineidae, Succinoidea). Varying amounts ofdata are available for other species thoughusually with no reference to acrosomal mor-phology. A perinuclear sheath similar to that ofHelix pomatia, though less structured than thatof H. aspersa is also observed in Bentositesbloomfieldi (Camaenidae, Helicoidea—Healy,1984). Bentosites bloomfieldi and Euhadrahickonis (see—Takaichi & Dan, 1977) both haveacrosomes only slightly tilted relative to thesperm longitudinal axis, as in Helix pomatia.The laterally-displaced acrosome of Helixaspersa has, as far as we are aware, no equivalentin any studied gastropod species. Interestingly,neither perinuclear sheath nor tilting of the acro-some are evident in late spermatids of the cama-enids Xanthomelon pachystylum or Varohadracurtisiana (Healy & Jamieson, unpublisheddata) or the helicid Trichia hispida (Hygro-miinae, Helicoidea—see Shileiko & Danilova,1979). Clearly more information is required onspermatozoa of the Helicoidea before any usefuldiscussion of systematics within the group canbe attempted.

Absence of a glycogen piece in spermatozoaof all investigated stylommatophoran speciessuggests that this may even be an ordinal charac-ter (in all studied opisthobranchs, a glycogenpiece, albeit sometimes very poorly developed,is still present—see Healy & Willan, 1984).Spermatozoa of basommatophoran (s.l.) pul-

monates Salinator fragilis and S. solida (seeHealy, 1983b) approach the form of styl-ommatophoran spermatozoa (especially Suc-cinea putris Succinoidea—see Shileiko &Danilova, 1979) more closely than do otherbasommatophorans, and it is pertinent that inSalinator, the axoneme terminates within themidpiece, leaving a glycogen piece composedsolely of the plasma membrane and glycogengranules surrounding a central lumen. Thisarrangement, though neither proving nor refut-ing an Amphiboloidea-Stylommatophora link,provides a hypothetical intermediate towardsthe stylommatophoran condition (absence ofglycogen piece).

ACKNOWLEDGEMENTS

The authors are grateful to Professor R. Chase(Department of Biology, McGill University) forproviding material of Helix pomatia, and withinthe Zoology Department, University of Cam-bridge, to Professor G. Hom and Dr B. Guptafor facilities given to B.J. Thanks also are dueto Mrs L. Daddow (Zoology Department, Uni-versity of Queensland), Mr G. Rouse (now ofthe Zoology Department, University of Sydney)and the electron microscope units at the Uni-versity of Cambridge, University of Sydney andUniversity of Queensland for their combinedtechnical assistance. Professor D. T. Andersonhelped in collecting some of the spermatophoresof Helix aspersa used in this study. Valuablecriticisms of an earlier draft of this paper wereprovided by Drs M. Bishop (University of Cam-bridge) and R. C. Willan (Zoology Department,University of Queensland). Mrs H. Sanchez delRio is thanked for her skilful typing of themanuscript. Financial support for this projectwas provided by a Commonwealth PostgraduateAward, Farrand Postdoctoral Research Fel-lowship and the Joyce W. Vickery ScientificResearch Fund (J.H.) and Australian ResearchGrants Committee funding (B.J.).

ABBREVIATIONS USED IN FIGURES

ap acrosomal pedestalav apical vesicle (of acrosome)cd centriolar derivativecf coarse fibresdas distal accessory sheathgh glycogen helix (of midpiece)ma matrix material of mitochondria] derivativen nucleus

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p paracrystalline material of mitochondrialderivative

pns perinuclear sheath (? acrosomalcomponent)

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an ultrastructural study of spermatozoa of helix aspersa and helix pomatia

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