Ectaquasperm-like parasperm in an internally fertilizing gastropod

Ectaquasperm-like parasperm in an internally fertilizing gastropod

Beatriz C. Winik,1,a Marta Catalan,2 Carlos Gamarra-Luques,3 and Alfredo Castro-Vazquez3

1 Laboratory of Electron Microscopy (INSIBIO-CONICET), National University of Tucuman,

San Miguel de Tucuman City, Argentina2 Miguel Lillo Institute, National University of Tucuman, San Miguel de Tucuman City, Argentina

3 Department of Morphology and Physiology (IHEM-CONICET), National University of Cuyo, Mendoza, Argentina

Abstract. Pomacea canaliculata is an internally fertilizing gastropod that produces, besidesfertilizing sperm (5 eusperm), a large number of unfertile sperm (5parasperm) that have nochromatin, are fusiform, and have three to five flagella. Here, we report that this snail alsoproduces another type of parasperm, which results from a peculiar spermiogenesis includingan anterior cytoplasmic migration. The mature oligopyrene parasperm has: (1) a roundedhead including a partly lysed nucleus, (2) a conical mid-piece with eight large mitochondrialstructures, and (3) a single flagellum (B20mm). These characteristics, although not found inany other gastropod parasperm, are shared with the externally fertilizing ‘‘ectaquasperm’’and with the early spermiogenic stages of internally fertilizing ‘‘introsperm’’ found among theAnnelida and basal Mollusca. There are indications that this sperm type may be produced bya truncation of euspermiogenesis, as proposed by Buckland-Nicks & Scheltema for the ex-pression of ectaquasperm in bilaterian evolution.

Additional key words: caenogastropod parasperm, spermiogenesis, Sertoli cells

Fertilizing sperm (5eusperm) are known to co-ex-ist with non-fertile parasperm in representatives offour invertebrate phyla (Healy 1988; Hodgson 1999)and in a chordate (Hayakawa 2007). Within the classGastropoda, they have been reported for severalprosobranch taxa (Neritimorpha, Skeneidae, andCaenogastropoda; Hodgson 1999) as well as fortwo heterobranches (Lucas 1971). Paraspermaticcells have gained their greatest morphological diver-sity among caenogastropods, and more than one pa-raspermatic type may occur in the same species(Hodgson 1999). These cells may partly or totallylose their chromatin during spermiogenesis, and arethen called ‘‘oligopyrene’’ or ‘‘apyrene’’ parasperm,respectively.

Pomacea canaliculata LAMARCK 1822 (Caeno-gastropoda, Ampullariidae) is a gonochoristic, inter-nally fertilizing snail that produces, besides eusperm(Catalan et al. 1997), a large number of an apyrene,fusiform parasperm. The endpiece of these para-sperm is formed by a bunch of three to five flagella(Winik et al. 2001).

Gamarra-Luques et al. (2006) have mentioned theoccurrence of a second type of parasperm in P. ca-naliculata. We report here that this type of parasperm(an ‘‘oligopyrene’’ one) is a consequence of a peculiarspermiogenesis in which an anterior cytoplasmic mi-gration occurs, and an ephemeral acrosome is formedat the anterior pole. The morphological characteris-tics of the resulting parasperm are unique within gas-tropods, the sperm being similar to the externallyfertilizing ‘‘ectaquasperm’’ (Rouse & Jamieson 1987)found throughout the Radiata and in many diverseBilateria.

Methods

Twelve adult, reproductively active male speci-mens of Pomacea canaliculata were collected duringthe spring and summer seasons in ponds and chan-nels in the Tucuman province, Argentina. Voucherspecimens were deposited in the malacological col-lection of the Fundacion Miguel Lillo, National Uni-versity of Tucuman (FML34#14322). The shells werecracked with scissors and the bodies were carefullyremoved; the testes were cut into thin slices with arazor blade. Slices for light microscopy were fixed inBouin’s fluid and stained with Heindenhain’s iron

Invertebrate Biology ]](]]): 1–9.

r 2009, The Authors

Journal compilation r 2009, The American Microscopical Society, Inc.

DOI: 10.1111/j.1744-7410.2008.00158.x

a Author for correspondence.

E-mail: [email protected]

i:/BWUS/IVB/158/[email protected]

hematoxylin (Clark 1981). Alternatively, slices fromthe same animals were fixed for transmission electronmicroscopy in a 3% glutaraldehyde solution bufferedwith 0.1molL�1 phosphate buffer, pH 7.4, for 3 h atroom temperature, and the slices were postfixed in1% osmium tetroxide in the same buffer overnight.Tissues were later treated with an aqueous solution of2% uranyl acetate for 40min. Afterwards, they wereserially dehydrated in ethanol, passed through ace-tone, and embedded in Spurr’s resin. Ultrathin silvergray sections were stained with uranyl acetate andlead citrate and examined with a transmission elec-tron microscope. For scanning electron microscopy,testicular slices were fixed and dehydrated in ethanoland acetone as described above. Then, they were crit-ical point dried, fractured, mounted on aluminumstubs, coated with gold, and the exposed surfaceswere examined with a scanning electron microscope.

Results

The spermatogenic epithelium is organized in nestsof germinal cells. The Sertoli cells (somatic cells) areflattened and completely surround the spermatogenicepithelium of each tubule; their large nuclei appear

ovoid in sections and have one or more conspicuousnucleoli (Fig. 1A). Mature eupyrene sperm beforespermiation are organized with their spiral headspointing perpendicular to the outer band of Sertolicells. Nests of the oligopyrene parasperm are less or-derly arranged and are found in the testis with theother sperm lines in all of the studied individuals. Theoligopyrene nests are of about the same size as theeupyrene and apyrene ones, although they occur lessfrequently. It should also be noted that the matureoligopyrene forms are less frequently found than thecorresponding spermatids.

A diagram of oligopyrene paraspermiogenesis inPomacea canaliculata is presented in Fig. 2A–E,while selected micrographs of this process are pre-sented in Figs. 3–5. Oligopyrene paraspermatids(Fig. 3A) show a nucleus whose finely granular chro-matin is beginning to coalesce at several points and,particularly, over the nuclear membrane of the pro-spective posterior pole.

Most cytoplasm of these spermatids is located nearthe posterior nuclear pole (Fig. 3A). An implantationfossa may be seen as an indentation of the nuclearmembrane, while a centriole may be found in thenearby cytoplasm. Conspicuous stacks of Golgi cis-

Fig. 1. A. Light microscopy (iron hematoxylin) of the testes showing portions of three adjacent spermatogenic tubules. A

mature eupyrene nest is seen at the lower left, showing the typical elongated nuclei (white triangles), which are

perpendicularly oriented toward the cytoplasmic band of the Sertoli cell (white arrows), which shows a large nucleus

with two nucleoli. A nest of oligopyrene paraspermatids is seen on the upper center, showing their bowl-shaped nuclei

(black triangles), which are correlative to the posterior chromatin condensation observed in transmission electron

microscopy and that is accompanied first by chromatin dispersion and later by chromatolysis of their anterior nuclear

halves (see Figs. 3, 4); most of the cytoplasm of these oligopyrene paraspermatids is seen at the anterior pole.

Paraspermatids are not orderly arranged with respect to the Sertoli cytoplasmic band. The third tubule (lower right)

probably shows the boundary of a eupyrene and an oligopyrene nest: dark, elongated nuclei of late eupyrene spermatids

(black arrow) are seen among round nuclei (probably bowl-shaped), oligopyrene ones (black triangle). Scale bar5 5mm.

B. Scanning electron microscopy of part of an oligopyrene sperm nest, showing the pear-shaped sperm heads, with their

frequently irregular anterior ends (white triangles) and the conical midpieces (black triangles) giving rise to the single tails.

Scale bar5 5mm.

2 Winik, Catalan, Gamarra-Luques, & Castro-Vazquez

Invertebrate Biologyvol. ]], no. ]], winter 2009

ternae are found in the posterior cytoplasmic mass.Some ovoid mitochondria and vesicles of the smoothendoplasmic reticulum (SER) are seen in the periph-eral cytoplasm of spermatids.

The nucleus of oligopyrene spermatids remainsround or ovoid (Figs. 3B,C, 4A,B). A condensationof chromatin develops at the posterior nuclear half.Later, a marked chromatolysis occurs, mainly at theanterior nuclear half (Figs. 3C,D, 4A,E). The differ-ence between the two nuclear halves becomes moreevident later in spermiogenesis, because both leafletsof the anterior nuclear membrane become disorga-nized (Fig. 5D) and the anterior nuclear region be-comes full of loosely arranged chromatin fibrils andmyeloid figures (Figs. 3D, 4A–E, 5D). Eventually,chromatin in the posterior half may also become fi-brillar (Fig. 4A–D). Oligopyrene spermatids may beseen lying on the Sertoli cytoplasmic band that limitsthe tubular lumen, and also, some thin Sertoli pro-jections are sometimes observed attached to oligopy-rene spermatids (e.g., Figs. 3C, 4A); however, theseparaspermatids never get wrapped in a ‘‘mantle’’ ofSertoli cell cytoplasm.

Another characteristic feature of oligopyrenespermiogenesis is the anterior migration of cyto-plasm, which includes the Golgi complex. The latterproduces numerous proacrosomal vesicles that con-tribute to the formation of an acrosome in which nobasal plate is formed and in which an amorphous

material is accumulated instead (Figs. 3D, 4A, 5C).Lysosomes, autophagic bodies, rather large vacuoles,and several irregularly flattened cisternae of the SER,as well as some prominent multivesicular bodies

Fig. 2. A diagrammatic representation of oligopyrene

paraspermiogenesis in Pomacea canaliculata (micro-

graphic information related to each figure is indicated in

parentheses). A. An early oligopyrene paraspermatid,

before tail formation, with a subspheroidal nucleus with a

finely dispersed granular chromatin, beginning to coalesce

at some points, and particularly over the prospective

posterior nuclear membrane. An incipient implantation

fossa, with a nearby centriole, is represented at the

posterior nuclear pole; Golgi stacks, ovoid mitochondria,

and both smooth and rough endoplasmic reticula are

represented (Fig. 3A). B. Tail-forming early para-

spermatid with an ovoid nucleus, whose chromatin is

condensed at the posterior nuclear half, while most of the

cytoplasm is migrating forward, including the Golgi stacks

and many vesicles of the smooth endoplasmic reticulum

(SER). Some enlarging mitochondria remain at the

posterior cell pole; a single flagellum originates from a

basal body associated to a thin annulus, and it is

surrounded by a deep and wide flagellar canal. An

electron-dense material occupying the implantation fossa

precludes the visualization of a proximal centriole (if any

exists) (Figs. 3B,C, 4A, 5A,D). C. Oligopyrene para-

spermatid with a nucleus showing a fibrogranular

chromatolysis and degenerative myeloid figures; the

anterior nuclear membrane is becoming disorganized.

The Golgi stacks appear associated to an acrosome with

no basal plate; SER vesicles, lysosomes, and autophagic

bodies are represented at the anterior cytoplasmic mass.

The enlarging mitochondrial structures reduce the flagellar

canal while the annulus is separating from the basal body

(Figs. 3D, 4A, 5B–D). D. Oligopyrene paraspermatid in

which both chromatolysis of the anterior half and the

formation of myeloid figures have progressed; SER

vesicles, lysosomes, and a microvesicular body are

represented at the anterior mass. There are no Golgi

stacks or acrosome; the annulus is represented beyond

the point where the mitochondrial structures and the

axoneme become closer. A shallow flagellar canal

separates the mitochondrial structures from the flagellum

(Figs. 4B,C, 5B–F). E. Mature oligopyrene sperm showing

a nucleus with an electron-dense posterior part, still limited

by the posterior nuclear membrane; the anterior nuclear

membrane has been lost. A fibrillar material (presumably

lysed chromatin fibrils) is shown extending from the

anterior nuclear region and occupying most of the

anterior cell pole; the anterior cytoplasmic mass is

reduced, and the acrosome and the self-digesting

structures are no longer observed. Large mitochondrial

structures are shown forming a conical midpiece around

the flagellar origin (Figs. 1B, 4D–F).

Ectaquasperm-like parasperm 3


Fig. 3. Transmission electron microscopy. A. Early oligopyrene paraspermatid showing nuclear condensations (white

arrows), an incipient implantation fossa with a nearby centriole (c), a posteriorly located and conspicuousGolgi complex (g),

some ovoid mitochondria (m), and vesicles of the smooth endoplasmic reticulum (SER, black arrows). B. Early oligopyrene

paraspermatid: a Golgi body (g) occurs in a latero-anterior cytoplasmic mass together with SER vesicles (black arrows); the

posterior nuclear half shows a condensed granular chromatin. A Sertoli cell projection is seen loose in the nearby

extracellular space (black triangle). C. Early oligopyrene paraspermatid is lying over the cytoplasmic band of a Sertoli

cell (S) together with two adjacent sister spermatids (on the right part of the micrograph); the Golgi body is in a latero-

anterior position; the anterior cytoplasmic masses of the paraspermatids show vesicles of SER (black arrows) and some

lysosomes (white triangles). The initial portion of the flagellum (f) is seen close to the wide flagellar canal (�) of the centralparaspermatid, and it is surrounded by the growing mitochondrial structures (m); a Sertoli cell projection (black triangle) is

seen in the space between two spermatids. The Sertoli cell (S) shows many ovoid mitochondria and small SER vesicles;

beyond the Sertoli cell, a myoid cell is seen (M). D. An oligopyrene paraspermatid showing nuclear chromatolysis and

degenerative myeloid figures (white triangles) in the anterior nuclear pole; the Golgi body (g) is associated with an acrosome

with no basal plate (a); enlarging mitochondrial structures are seen at the posterior cell pole (m). All scale bars5 1mm.



Fig. 4. A. An oligopyrene paraspermatid showing a nucleus with lysed chromatin and a disorganized anterior nuclear

membrane (white triangle). An electron-dense material fills the implantation fossa (white arrow); it also shows an

anteriorly located Golgi body (g), some nearby proacrosomal vesicles (black arrow), and some massive mitochondrial

structures (m) at the posterior end. Thin Sertoli cell projection/s is/are seen close to one side of this oligopyrene

paraspermatid (black triangles). B. A late oligopyrene paraspermatid, with a shallow flagellar canal separating the

flagellum (f) from the surrounding mitochondrial structures (m), showing chromatolysis and a disorganized anterior

nuclear pole (white arrow) with myeloid figures (white triangle) and a resorbing anterior cytoplasmic mass, with no Golgi

stacks nor acrosome, but numerous smooth endoplasmic reticulum (SER) vesicles (black arrows) and a large

multivesicular body (black triangle). C. A late oligopyrene spermatid with lysed chromatin and an electron-dense

material in the implantation fossa (white arrow); remnants of the anterior cytoplasmic mass show numerous SER vesicles

(black arrows). Between the mitochondrial structures (m), the flagellum (f) emerges surrounded by the annulus (white

triangles); the flagellar canal is disappearing. The nucleus shows chromatolysis. D. A late oligopyrene spermatid showing

the chromatolysed nucleus, the basal body (bb), and the large mitochondrial structures (m); the implantation fossa is

delimited by a condensed posterior nuclear membrane (white triangle). E. Mature oligopyrene sperm showing a

condensed posterior nuclear half (white arrows), still delimited by the posterior nuclear membrane (black triangles),

while fibrils extending from the disorganized anterior nuclear half occupy most of the anterior cell pole (black arrow); the

annulus appears posterior to the mitochondrial structures (white triangle). F. Transverse section through the midpiece of

an oligopyrene sperm, showing the flagellum (f) and the eight radially arranged mitochondrial structures (m).



(Figs. 4B,C, 5B,E,F), develop at the anterior cyto-plasmic mass. Later, neither the Golgi body nor theacrosome is found. Even later, in the mature oligopy-rene sperm, the remnant of the anterior mass, is only

occupied by a fibrillar material that is continuouswith that of the lysed anterior nuclear pole (Fig. 4E).

While these processes are taking place, the implan-tation fossa becomes deeper and is occupied by an

Fig. 5. A. Early oligopyrene paraspermatid showing the annulus (black triangles) attached to the basal body (bb) by a

moderately electron-dense material, and the origin of the flagellum (f) surrounded by a wide and deep flagellar canal. A

portion of the nucleus (n) and some mitochondria (m) are also seen. B. Interspermatid junction showing several smooth

endoplasmic reticulum (SER) vesicles (white triangles) in the vicinity, an electron-dense structure with a double

membrane (probably an autophagosome, white arrows), and typical membrane condensations (black triangles) at the

junction connecting adjacent spermatids.C.Acrosome free in the anterior pole of an oligopyrene paraspermatid, showing

a bell-shaped cap (black triangles) full of an amorphous material (white triangles) and no basal plate; part of a Golgi stack

(g) is seen. D. Anterior nuclear pole of an oligopyrene paraspermatid, showing vesicles of the Golgi body (g), and the

discontinuous nuclear membrane (black triangles); degenerative myeloid figures (white triangles) are also seen. E. SER

vesicles (black triangles), lysosomes (black arrows), and autophagic figures (white triangles) in the antero-lateral

cytoplasmic mass of an oligopyrene paraspermatid, which was still bearing an acrosome (same as Fig. 3D). F. A

multivesicular body (white arrow), some SER profiles (white triangles), and a lysosome (black arrow) in the anterior

cytoplasmic mass of a late oligopyrene paraspermatid.



electron-dense material that may preclude the visu-alization of the proximal centriole if it exists(Fig. 4A,C); the basal body is attached to an annu-lus, which is later freed and progresses posteriorly(Figs. 4C, 5A) around the single axoneme (Figs. 3C,4B–F, 5A). Some mitochondria remain caudal andenlarge, becoming eight radially arranged structuresaround the initial part of the 912 flagellum(Figs. 3C,D, 4A–F, 5A). These mitochondrial struc-tures progressively occupy the space anterior to theannulus, so that the flagellar canal disappears and ashort, conical midpiece is finally formed (Figs. 1B,4D,E). There is no glycogen accumulation in oligopy-rene spermatids. Also, as a result of the anteriorcytoplasmic migration, the mitochondrial structuresaround the initial flagellar portion are left covered byonly a thin cytoplasmic layer (Fig. 4D–F). No resid-ual cytoplasmic droplet is formed at the caudal pole.The terminal piece consists of only the axoneme andthe surrounding plasma membrane.

Finally, the mature oligopyrene parasperm hasan approximately pear-shaped body B3.5mm longand 3mm wide (Fig. 1B) because of the conical mid-piece formed by mitochondrial structures. The singletail isB20–30mm long.

Discussion

Eusperm in Pomacea canaliculata (Catalan et al.1997) result from a typical spermatogenic process andthe mature form resembles the basic ‘‘introsperm’’type of Rouse & Jamieson (1987), which comprises:(1) a conical acrosome; (2) an elongated nucleus; (3) aposterior invagination, which encloses the basal body;(4) a set of mitochondria-derived structures arrangedas a sheath around the axoneme, and that form anelongated midpiece; and (5) an endpiece composed ofan initial glycogen-rich region and a single flagellum.Males of P. canaliculata also generate a fusiform pa-rasperm, which terminates in a bunch of three to fiveflagella (Winik et al. 2001). The latter loses its chro-matin completely during spermiogenesis, although itsgeneral shape and spermiogenetic process resemble,with some differences, the fusiform apyrene andoligopyrene parasperm of other gastropods (Buck-land-Nicks 1998; Hodgson 1999).

However, the oligopyrene parasperm in P. canal-iculata has: (1) a rounded head, which includes apartly lysed subspheroidal nucleus; (2) a conical midpiece, with eight large and radially arranged mi-tochondrial structures; and (3) a single, long, flagel-lar endpiece, with no glycogen-storing structures atits origin (present study). Some of these characteris-

tics, although not found in any other gastropod pa-rasperm (Healy 1988), make it similar to thebilaterian ‘‘ectaquasperm,’’ which conforms to a ba-sic plan including a subspheroidal nucleus, a mi-tochondrial midpiece, and a single flagellum (Rouse& Jamieson 1987).

It should also be noted that these ectaquaspermcharacteristics, except for the transient occurrence ofan acrosomal structure, make the oligopyrene para-sperm similar to the round-headed sperm ofRadiata, which Franzen (1956) and Jamieson(1987) have considered either as the ‘‘primitivesperm’’ or the ‘‘plesiosperm,’’ and have led to thewidespread opinion that ectaquasperm of Radiataare the direct ancestors of bilaterian ectaquasperm.This has been contested, however, by Buckland-Nicks & Scheltema (1995), who proposed that theoccurrence of internal fertilization and of introspermwas a condition present in early Bilateria. Such a hy-pothesis on the ancient origin of introsperm impliesthat the occurrence of ectaquasperm in so many,quite unrelated bilaterian taxa is a case of conver-gent evolution.

Buckland-Nicks & Scheltema (1995) have furtherproposed that this multiple, secondary expression ofthe round-headed plesiosperm formmay be the resultof the precocious, ‘‘progenetic’’ development of sper-matids to maturity. They based their proposition onthe fact that early spermatids in digeneans closely re-semble the mature sperm of more recent schistosomes(Justine 1991) and on the observation that the plesio-sperm form is always expressed as an intermediatestage in the development of the introsperm foundamong the Annelida (Franzen 1956; Jamieson 1987)and basal Mollusca (Franzen 1955; Buckland-Nicks& Scheltema 1995). They have further proposed thatthe progenetic maturation of secondarily expressedectaquasperm was the result of truncation of thespermiogenetic process by some communication fail-ure between germ cells and the somatic Sertoli cells(Buckland-Nicks & Scheltema 1995). At that time,these interpretations were based mainly on Abe’s(1987) and Onoda & Djakiew’s (1993) findings inchordates, regarding the involvement of signal com-munication between these cells for the achievementof the final maturation of sperm. More recently, mo-lecular research has shown that the expression of anintercellular junction protein (connexin 43) by Sertolicells is essential for an adequate germ cell nursery(Wang et al. 2006; Brehm et al. 2007; Sridharan et al.2007). Hence, it may be relevant that contact ofoligopyrene paraspermatids with some thin Sertolicells’ projections in P. canaliculata is only occasion-ally observed, and that these spermatids never get



wrapped in the cytoplasmic ‘‘mantle’’ of Sertoli cells,as spermatids of the other sperm lines do (Buckland-Nicks & Chia 1986a; Catalan et al. 1997; Winik et al.2001). Hence, development into an ectaquasperm-like form may be the result of a progenetic trunca-tion of euspermiogenesis, caused by the failure of the‘‘normal’’ germ cell nursery by Sertoli cells.

This (presumably) secondary appearance of an ect-aquasperm in an internally fertilizing gastropod is,however, incomplete, because a process of nuclearlysis occurs during spermiogenesis, equivalent to thatoccurring in the paraspermiogenesis of very differentbilaterian forms (Hodgson 1999).

An intriguing characteristic of oligopyrene para-spermiogenesis in P. canaliculata is that the posteriorlylocated cytoplasm of oligopyrene paraspermatocytesmigrates forward (including the Golgi complex) duringspermiogenesis, i.e., contrariwise to the migration oc-curring during euspermiogenesis in this and other spe-cies, but somehow similar to the migration ofcytoplasm observed during euspermiogenesis in theaplacophoran Epimenia australis, a basal mollusc(Buckland-Nicks & Scheltema 1995).

In such primitive gastropods as the Neritimorpha,and in basal caenogastropods such as the Cerithio-idea (Buckland-Nicks & Chia 1986b; Buckland-Nicks & Hodgson 2005), the acrosome is formed inthe posterior cytoplasmic mass and it attaches to theplasma membrane to migrate forward, accompaniedby the Golgi complex. In contrast, acrosome forma-tion during oligopyrene paraspermiogenesis in P. ca-naliculata does not begin at the posterior pole, butwhen the Golgi body is half-way in its anterior mi-gration. Also, the rudimentary acrosome formed inP. canaliculata does not develop a basal plate, and itnever gets attached to the plasma membrane, as oc-curs in the species mentioned above (Buckland-Nicks& Chia 1986b; Buckland-Nicks & Hodgson 2005).Coincident with this anterior migration, cytoplasmicdigestion that normally occurs at the posterior poleof euspermatids instead occurs at the anterior poleof oligopyrene paraspermatids, as noted by theoccurrence of lysosomes, autophagic bodies, andmultivesicular bodies. Apparently, the rudimentaryacrosome is also lost with most of the anterior cyto-plasmic mass, because we have not seen any in theapical cytoplasmic remnants of late oligopyrene sper-matids and spermatozoa.

Much has been elaborated on the functional roleof parasperm, particularly in the context of paternityassurance (Buckland-Nicks 1998; Till-Bottraud et al.2005). In the case of P. canaliculata, it has beenshown (Yusa 2004) that continuous mating with asecond male displaces sperm deposited by a former

one. In principle, both eusperm and parasperm arelikely to participate in displacing rival sperm; how-ever, oligopyrene parasperm have not been observedtogether with eusperm and apyrene parasperm in theseminal receptacle of females of P. canaliculata col-lected during the spring/summer season (Catalan2008). However, it would be premature to suggestthat oligopyrene parasperm do not have a role insperm competition on the sole basis of the latter ob-servation, because we do not know what factorswould affect the ascent and interaction of spermthrough the intricate oviduct in P. canaliculata(Thiengo et al. 1993; Catalan et al. 2002) beforethey reach the seminal receptacle.

Among these many factors, on the male part, it isunknown whether sperm are transferred continu-ously during the protracted copulations observed(9–20h: Albrecht et al. 1996; Burela & Martın2007) or whether they are transferred in a single orin several repeated quanta. It is also unknownwhether the proportions of eusperm and paraspermtransferred are always the same or whether theychange between successive copulations (or betweenthe possibly repeated quanta within a single copula-tory episode). And finally, it should be kept in mindthat the female structures may also have a role in de-termining sperm availability at the fertilization site(as discussed by Paterson et al. 2001) and that thesestructures, which are particularly complicated in thegenus Pomacea (Thiengo et al. 1993; Catalan et al.2002), might differentially affect the ascent of thedifferent sperm types.

In summary, (1) an oligopyrene paraspermwithmor-phological similarities to ectaquasperm is produced byreproductively active males of P. canaliculata, an inter-nally fertilizing gastropod; (2) oligopyrene paraspermapparently result from a truncated spermiogenesis,which includes an anterior migration of the posteriorcytoplasm; and (3) the oligopyrene parasperm thatreach the fertilization site (if any) are much less numer-ous than eusperm and apyrene parasperm.

Acknowledgments. This work was supported by grantsfrom CONICET, FONCYT, and from the NationalUniversities of Tucuman and Cuyo (Argentina).

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Ectaquasperm-like parasperm in an internally fertilizing gastropod

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