Comparative morphology of zebra (Dreissena polymorpha) and ...

Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: light and electron microscopy

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G.K. Walker, M.G. Black, and C.A. Edwards

Abstract: Adult zebra (Dreissena polymopha) and quagga (Dreissena bugensis) mussels were induced to release large quantities of live spermatozoa by the administration of 5-hydroxytryptamine (serotonin). Sperm were photographed alive using phase-contrast microscopy and were fixed subsequently with glutaraldehyde followed by osmium tetroxide for eventual examination by transmission or scanning electron microscopy. The sperm of both genera are of the ect-aquasperm type. Their overall dimensions and shape allow for easy discrimination at the light and scanning electron microscopy level. Transmission electron microscopy of the cells reveals a barrel-shaped nucleus in zebra mussel sperm and an elongated nucleus in quagga mussel sperm. In both species, an acrosome is cradled in a nuclear fossa. The ultrastructure of the acrosome and axial body, however, is distinctive for each species. The structures of the midpiece are shown, including a unique mitochondria1 "skirt" that includes densely packed parallel cristae and extends in a narrow sheet from the mitochondria. Resume : Des quantitCs importantes de spermatozoi'des vivants ont CtC libCrCes par des Moules zCbrCes (Dreissena polymopha) et des Moules quagga (Dreissena bugensis) traitCes a la 5-hydroxytryptamine (sirotonine). Les spermatozoi'des ont CtC photographiCs vivants au microscope a contraste de phase et ont CtC par la suite fixes au glutaraldihyde, puis traitis au tCtraoxyde d'osmium pour examen au microscope Clectronique, ordinaire ou a balayage. Chez les deux espkces, les spermatozoi'des sont du type ect-aquaspermatozoide. Les dimensions gCnCrales et la forme permettent de diffkrencier facilement les spermatozo'ides au microscope photonique ou au microscope Clectronique balayage. Le microscope Clectronique ordinaire permet de voir le noyau en forme de barillet des spermatozo'ides de la Moule zCbrCe et le noyau allongC des spermatozoi'des de la Moule quagga. Chez les deux espkces, un acrosome repose dans une fosse nucliaire. L'ultrastructure de l'acrosome et du corps axial est cependant distinctive chez les deux espkces. Les structures de la pikce mCdiane sont illustrkes, notamment une a jupe mitochondriale particuliere qui compte une masse compacte de cristae disposCes en parallkle et qui se prolonge en un feuillet Ctroit au-delh de la mitochondrie. [Traduit par la Ridaction] ))

Introduction The present study compares and contrasts the spermatozoa of two closely related bivalves that have been noted to hybridize in the laboratory. We present here the first comparison of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm using light, transmission electron, and scanning electron microscopy. While we expected sperm of these species to have a very similar structure, light and electron microscopy reveal that each has unique features. The sperm of both dreissenids studied here are released freely into the water and, hence, fertilize externally. Franzkn (1956) characterizes cells of this type as "primitive" and states that they are usually small and contain a conical or spherical nucleus capped by a small acrosome. Rouse and Jamieson (1987) prefer the descriptor "ect-aquasperm," to indicate externally fertilizing sperm, in order to eliminate the phylogenetic connotations of "primitive. " While they offer Received June 20, 1995. Accepted October 25, 1995.

G.K. Walker and C.A. Edwards. Department of Biology, Eastern Michigan University, Ypsilanti, MI 48 197, U. S .A. (e-mail: bio , walker@emuvax ,emich. edu) . M.G. Black. National Biological Service, 1451 Green Road, Ann Arbor, MI 48105, U.S.A. Can. J. Zool. 74: 809-815 (1996). Printed in Canada / Imprime au Canada

a logical argument, the latter term appears to be ingrained in the literature (Eckelbarger et al. 1990; Hodgson et al. 1990; Nicotra and Zappata 1991; Peredo et al. 1990) and may be difficult to supplant. When the sperm fertilize internally, through either copulation or release near the female inhalent siphon, sperm morphology is "modified." The changes are primarily confined to the head region and occasionally include the midpiece. For example, the introsperm (sperm that never contact the water) of several polychaetes have an elongated nucleus and midpiece that contains long, rod-shaped mitochondria (Franzkn 1982). Other digressions from the primitive scheme appear to relate to adaptations to fertilization and brooding behavior (Popham 1974). The longer acrosomal rods and larger acrosomes of teredinid bivalves, which fertilize externally, are contrasted with the smaller acrosomes of brooding teredinids. Popham (1979) further suggests that these differences may be accounted for by the fact that the jelly coat protecting the eggs of the brooding molluscs is potentially thinner than in nonbrooders . Numerous investigators have demonstrated the merit of spermatozoon morphology in evaluating molluscan phylogeny (Popham 1974; Franzen 1983; Healy and Jamieson 1989; Hodgson et al. 1990; Buckland-Nicks and Scheltema 1995). In particular, several studies reveal that closely related species


Can. J. Zool. Vol. 74, 1996

may be distinguished using sperm ultrastructure (Eckelbarger et al. 1990; Healy and Lester 1991; Hodgson et al. 1990; Denson and Wang 1994). This feature captured our interest for several reasons. First, in a research note by Denson and Wang ( 1994), scanning electron microscopy (SEM) revealed differences in the morphology of zebra and quagga mussel spermatozoa confined to the tissues of the visceral mass (gonads). Secondly, since both species have invaded the Great Lakes and co-exist, ultrastructural differentiation of their sperm may be of value in understanding their reproductive biology. And, lastly, since Nichols and Black (1994) revealed that zebra and quagga mussels hybridize regardless of the egg parent, and hybrids have been reared to the D-shell stage in the laboratory, the sperm ultrastructure of these closely related dreissenids takes on additional significance.

Materials and methods Identification of spawning stock Morphological characteristics were used to identify the adult mussels. The features used to identify adult quagga and zebra mussels were those linked by May and Marsden (1992) to genotypic differences. Mussels were identified as quaggas if they were laterally compressed (oval in cross section) and had a rounded ventral surface, a round ridge between the dorsal and ventral surfaces, and the right shell valve larger or more convex than the left. In adults identified as zebra mussels, the shell was triangular in cross section, the ventral surface was flattened with a distinct angular ridge between the dorsal and ventral regions, and the shell valves were equal in size and convexity.

Gamete acquisition The dreissenid adults used to produce gametes in the laboratory were collected from Presque Isle, Pennsylvania (eastern Lake Erie), where zebra and quagga mussels co-exist, and Monroe, Michigan (western Lake Erie), where quagga mussels were not present. Spawning was induced in both types of mussels by exposing them to a 30-min bath of a M solution of the neurotransmitter serotonin (5-hydroxytryptamine) (Ram et al. 1992) and then isolating them in 50-mL containers. Sexually mature mussels released gametes within 0.5-6 h. Once gametes were available, the sperm were prepared for light microscopy and SEM.

Microscopy Light microscopy Live, freshly isolated sperm were photographed using phase-contrast microscopy.

Scanning electron microscopy Freshly isolated active sperm were fixed in cold 2.5 % glutaraldehyde buffered with 0.01 M phosphate buffer for 30 min. Following a buffer wash, the cells were postfixed in buffered 1 % osmium tetroxide for 30 min, immersed in 1% tannic acid for 30 min, and dehydrated in a graded ethanol series. Sperm were either filtered, using 0.45-pm filter paper, following glutaraldehyde fixation or settled onto polished aluminum stubs following ethanol dehydration. The cells were dried using a modification of the hexamethyldisalazane technique (Nation 1983). This modification involved impregnation of sperm with 1% tannic acid, which serves as a mordant, as noted above. Sperm treated with the tannic acid were less likely

to appear collapsed or crenate. Specimens were coated with gold and examined with an AMRay 18201 scanning electron microscope.

Transmission electron microscopy Small segments of the testis were excised from zebra and quagga mussels and fixed in cold 2.5 % glutaraldehyde in 0.01 M phosphate buffer for 1 h. After washing in buffer, the tissue was postfixed in 1 % osmium tetroxide for 1 h, dehydrated in a graded ethanol series, and infiltrated and embedded in Spurr's medium. Thick sections were cut for orientation and stained with toluidine blue. Thin sections were stained with uranyl acetate followed by lead citrate. Sections were examined with a Philips 301 transmission electron microscope.

Results Cell morphology The sperm of D. polymorpha and D. bugensis are both of the ect-aquasperm type and are composed of three regions: (i) a head consisting of an acrosome and nucleus, (ii) a midpiece containing mitochondria and basal bodies, and (iii) a flagellum. The primary difference in morphology, which can be seen at the level of light microscopy (Figs. 1, 2) and is clearly seen with SEM (Figs. 3, 4), is the contrast in cell size and shape. Zebra mussel sperm are consistently shorter (4.0 pm) and straighter than quagga mussel sperm, which appear significantly longer (4.6 pm) and are obviously curved. The slimmer (1.2 pm) quagga mussel sperm taper to a pointed acrosomal tip, whereas the wider (1.5 pm) zebra mussel sperm form a blunter tipped acrosome. Ten sperm from each species were measured from the tip of the acrosome to the distal end of the midpiece, using SEM to arrive at the averages noted. Acrosome Both species have conical acrosomes that taper in diameter from a broad base, which sits in a nuclear fossa (Figs. 5, 6, 7, 8). While the acrosome of zebra mussel sperm is slightly shorter and wider than that of quagga mussel sperm, both occupy nearly 25 % of the length of the sperm head. The acrosome of quagga mussel sperm is divided into an outer thin, electron-dense cortex, which enlarges at the nuclear fossa, and an inner, less electron-dense region composed of most of the acrosomal material. A deeply invaginated core of electron-lucent material holds a well-defined electron-dense acrosomal rod that projects from the expanded electrondense cortical material at the base, through the center of the acrosome, and extends to the tip (Fig. 8). The acrosome of zebra mussel sperm is distinguished from that of quagga mussel sperm by a thicker, less electron-dense cortical region surrounding a slightly less dense zone. The diffuse, poorly defined hcrosomal rod is composed of numerous subunits (Fig. 7). Midpiece The midpiece, in every case noted, contains four welldefined spherical mitochondria (Figs. 9, 11) and, typically a proximal and a distal centriole oriented approximately at right angles to one another, each containing nine triplet sets of microtubules. The posterior segment of the nucleus is indented by the mitochondria and proximal centriole (Figs. 5, 6, 10). Numerous examples of unique mitochondria1 "skirt"

Walker et al

Fig. 1. Phase-contrast micrograph showing the bullet-shaped heads of zebra mussel sperm, with clearly defined acrosomes and midpieces. Tails extend in excess of 1 0 the ~ head length. x 1600. Fig. 2. Phase-contrast micrograph showing quagga mussel


sperm. The sperm can be differentiated from zebra mussel sperm by the slightly longer, tapered, curved head. x 1600.

Fig. 3. Scanning electron micrograph showing the zebra mussel sperm head in detail. Note the acrosome (with an indented tip) and the four mitochondria of the midpiece. x 1750. Fig. 4. Scanning electron micrograph showing the typical slightly curved sperm of the quagga mussel. The arrow shows the mitochondria1 "skirt."

structures were seen in sections of zebra mussel sperm (Fig. 12), and similar structures were seen in sections of quagga mussel sperm (Fig. 6). These structures extend in a narrow sheet from the mitochondria. They appear to include densely packed parallel cristae, the outer membrane of the mitochondria, and the sperm cell membrane (Fig. 12). Indications of a centriolar anchoring apparatus are evident in several sections (Figs. 5, 6, 10) and four of these centriolar projections can be seen with SEM (Fig. 11). A collar extends

x 1650.

from the centriolar attachment region below the mitochondria (Figs. 5, 6, and 10).

Tail The flagella of all zebra mussel sperm observed extended from the distal centriole and contained the typical 9 + 2 structure of microtubules. While the quagga mussel flagella do show 9 + 2 structures, sections also revealed that a variety of modified transitional doublet flagellae (Fig. 6, inset)

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Fig. 5. Transmission electron micrograph of a zebra mussel sperm head. Note the typical cylindrical nucleus capped by a conical acrosome and the collar extending from the distal region of the distal centriole. x 27 200. Fig. 6. Transmission electron micrograph of a quagga mussel sperm head, illustrating the slightly curved sperm head, the collar region of the mitochondria1 midpiece, and an unusual double flagellar structure (arrowheads), which was noted in several of our quagga mussel sperm sections but never found in zebra mussel sperm sections. x 29 550. Inset: Microtubule arrangement of the double-flagellar structure. x 45 600.

are common. Sections of these structures always occur below the midpiece.

Discussion Both quagga and zebra mussels have proved easiest to identify in their adult form. Recently, Nichols and Black (1994) detailed characteristics useful in distinguishing these two closely related species at the larval stage. Denson and Wang (1994) further revealed differences between quagga and zebra mussel sperm using SEM. That study, however, used

sperm contained within the gonads (visceral masses) of the mussels. We examined extruded sperm for the SEM segment of this study to determine morphological effects associated with activation. Sections of the gonads were used for transmission electron microscopy to maximize regular sperm orientation within ducts and to avoid the inducing artifacts by centrifuging extruded sperm. These dreissenid mussels are unusual among freshwater mollusks, adopting the primarily marine strategy of external fertilization with a planktonic larval stage. The sperm, eggs, and veligers of these mussels have successfully adapted to the

Walker et al.


Fig. 7. Transmission electron micrograph showing zebra mussel acrosomes in oblique (left) and longitudinal (right) section. The diffuse axosomal rod visible in the longitudinal section comprises numerous discrete subunits, as revealed in the oblique section. x 24050. Fig. 8. Longitudinal section of a quagga mussel acrosome, showing a typical dense, well-defined acrosomal rod, in contrast to the more diffuse version noted in the zebra mussel. x 35 100.

Fig. 9. Cross section of the midpiece of a zebra mussel sperm. Note the cross section of the distal centriole and the densely packed cristae. x 47 700. Fig. 10. Transmission electron micrograph of the midpiece of a zebra mussel sperm. Note the cross section of the proximal centriole. x 41 000.

osmotic stresses associated with a freshwater environment. This examination of sperm structure confirms the correlation with the biology of fertilization. The benefit of the characteristic, slightly curved quagga mussel sperm head in movement and fertilization remains to be studied; however, a curved sperm head has been noted in other mussels, including Venus striatula (Ockelmann 1964), Ruditapes decussatus (Pochon-Masson and Gharagozlou 1970), Mysella bidentata (Ockelmann 1978), and Callista chione (Nicotra and Zappata 1991). Contained within the midpiece, the centriolar apparatus reveals indications of the anchoring structures commonly described for other invertebrate sperm. For example, elaborate structures such as nine centriolar projections arborize into two radii

and form the anchoring apparatus for Priapulus sp. sperm (Afzelius 1979). This intricate fabric of fibers is thought to secure the base and, in sperm with highly structured, centriolar branching processes, perhaps control the motion and direction of the gamete (Bozzo et al. 1993). The distal centriole of the dreissenids appears to be stabilized by a dense pericentriolar apparatus that extends toward the surrounding mitochondria and elaborates distally into the folded plasma membrane region, termed the collar. FranzCn (1983) reports four mitochondria for the midpiece of mature sperm of D. polymorpha, and mitochondria consistently numbered four in the scores of mature sperm examined for each species in this study. Collar structures associated with the sperm have been identified in other

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Fig. 11. Scanning electron micrograph of the midpiece of a quagga mussel sperm. Note the four centriolar attachment structures extending longitudinally along the distal centriole. x 32 450. Fig. 12. Typical representations of zebra mussel mitochondrial skirts. The mitochondrial extensions appear to involve elaborations of the cristae. X 35 200.

bivalves such as Choromytilus merionalis and Mytilus galloprovincialis (Hodgson and Bernard 1986) as well as the mollusc Chaetoderma sp. (Buckland-Nicks and Chia 1989). In the case of Chaetoderma sp., a micrograph and interpretive drawing show the collar as a "plasma membrane folded back around the annulus" (Buckland-Nicks and Chia 1989). Hodgson and Bernard (1986) similarly suggest that the sperm tail "originates from the distal centriole and is surrounded at its base by a collar of thickened plasma membrane." A unique and elaborate skirt is observed in quagga and zebra mussel sperm. While neither a collar nor a skirt is seen in Franzkn's (1983) micrographs of D. polymorpha, he does note that "fixation does not permit any observation" of centriolar attachment structures. Perhaps fixation also prevented observation of the collar and skirt structures. We, too, had problems with fixation until we dramatically diluted our buffer to 0.01 M . These structures may be sensitive to fixation at the higher molarities used by both Franzkn (1983) and Denson and Wang (1994). The integration of mitochondrial cristae into the skirt may reflect a mechanism for increasing the surface area of the cristae, since these appear to be tightly packed into the spherical mitochondria. The skirt may also provide a structure to guide the sperm. The selective pressures involved in maintaining this structure are not understood. The unusual incomplete "doublet" flagella observed in quagga mussel sperm probably represent an aberrancy, since it is unlikely these sperm would manifest significant motility. The recognition that hybrids of quagga and zebra mussels have been reared to the D-shell stage in the laboratory (Nichols and Black 1994) suggested to us that the sperm may be quite similar in morphology. However, the overall sizes and shapes differ substantially, and these criteria can certainly be used to distinguish the sperm of the two species. Ultrastructural differences in acrosomal morphology, such as axial rod structure and cortical densities, further serve to distinguish these cells from one another. However, these

slight differences in acrosomal substructure are clearly not enough to thwart hybridization. Cross fertilization has been noted for other bivalves, between, for example, species of Crassostrea (Galtsoff and Smith 1932) and species of Mytilus (Lubet et al. 1984), in spite of differing acrosomal morphologies in the latter species (Hodgson and Bernard 1986).

Acknowledgments This is Contribution No. 921 of the National Biological Service, 145 1 Green Road, Ann Arbor, MI 48 105, U.S .A. We gratefully acknowledge the assistance of Dr. John Lynn, Department of Biology, Louisiana State University, and the comments of Drs. Douglas Wilcox, S. J. Nichols, National Biological Service, Ann Arbor, Michigan, and an anonymous reviewer. References Afzelius, B.A. 1979. Sperm function in relation to phylogeny in lower Metazoa. In The spermatozoon. Edited by D. W. Fawcett and J.M. Bedford. Urban and Schwarzenberg, Baltimore. pp. 243-251. Bozzo, M.G., Ribes, E., Sagrista, E., Poquet, M., and Durfort, M. 1993. Fine structure of the spermatozoa of Crassostrea gigas (Mollusca, Bivalvia). Mol. Reprod. Dev. 34: 206-21 1. Buckland-Nicks, J., and Chia, F. 1989. Spermiogenesis in Chaetoderma sp. (Aplacophora). J . Exp. Zool . 252: 308 - 3 17. Buckland-Nicks, J., and Scheltema, A. 1995. Was internal fertilization an innovation of early Bilateria? Evidence from sperm structure of a mollusc. Proc. R. Soc. Lond. B Biol. Sci. 261: 11-18. Denson, D.R., and Wang S.Y. 1994. Morphological differences between zebra and quagga mussel spermatozoa. Am. Malacol. Bull. 11: 79-81. Eckelbarger, K.J., Bieler, R., and Mikkelsen, P.M. 1990. Ultrastructure of sperm development and mature sperm morphology in three species of commensal bivalves (Mollusca: Galeommatoidea). J. Morphol . 205: 63 - 75.


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