Histological studies on hermaphroditism, gametogenesis and cyclic changes in the structures of marsupial gills of the introduced asiatic clam, corbicula fluminea, and the Korean clam, corbicula leana.

ABSTRACT The marsh clams, Corbicula fluminea and Corbicula leana, are functional hermaphrodites. They usually appear to be surrounded by numerous spermatozoa in the hermaphroditic follicles. In both species, the follicular ganglia (consisting of the neuronal fiber and neuronal soma-like cells at its periphery) are associated with neurosecretion and the differentiation of complex innervated nerve structures during spermatogenesis and are widely distributed in the follicles in the ripe and spawning stage. Corbicula fluminea and C. leana have two pairs of gills, with the inner-demibranchs acting mainly as marsupia. The non-marsupial demibranchs are not separated, but in the marsupial demibranchs, cyclic changes in the structures of the inner-demibranchs of the gills appear, with the depletion of ripe eggs during incubatory periods and the production of mature and ripe eggs during nonincubatory periods. The reproduction of tripioid C. fluminea and C. leana may occur by parthenogenesis without self-fertilization (or cross-fertilization) by eggs and sperm. The DNA contents of the somatic (gill) and gamete (spermatozoa) cells of C. fluminea are the same. Because reproduction is parthenogenetic, numerous spermatozoa may participate in the activation of the mature eggs and egg cleavage, as a stimulus only for parthenogenesis in the same hermaphroditic follicle or the gonophore.

KEY WORDS: Corbicula fluminea, Corbicula leana, gametogenesis, hermaphroditism. Korea, North American

INTRODUCTION

The freshwater clams Corbicula fluminea and Corbicula leana are hermaphrodites that brood their larvae in the inner-demibranchs (Britton and Morton 1982, Miyazaki 1936). Okamolo and Arimoto (1986) suggested that C. leana reproduce by gynogenesis. Corbicula fluminea and C. leana, on the basis of chromosomal and karyological studies have been reported as being triploid (Okamoto and Arimoto 1986, Park et al. 2000). The triploid condition may closely be related to hermaphroditism.

The Korean clam, C. leana, is one of the commercially important edible clams. However, the North American C. fluminea was introduced from Asia in the 1900s and is now widely distributed throughout the United States (Britton and Morton 1982, McMahon 1982), where it has become a biofouling pest (Mattice 1979, McMahon 1977). There have been a few previous studies of the reproduction of the Corbicula species (Kennedy and van Huekelem 1985, Kraemer 1978, Kraemer 1984, Kraemer et al. 1986. Morton 1982, Williams and McMahon 1986). In addition, gametogenesis and the corresponding morphologic changes in the inner-demibranchs have not been examined. Finally, there is the question whether the reproduction of the two triploid Corbicula species involves self-fertilization, cross-fertilization, or parthenogenesis. The purposes of this study were to understand functional hermaphroditism, gametogenesis, the cyclic changes in the structures of the inner-demibranchs, and the duration of the pediveligers released from the parent clams. The DNA content of the spermatozoa and gill tissues of C. fluminea were also compared.

MATERIALS AND METHODS

Among Corbicula species used for the current study, a total of 366 North American C. fluminea specimens collected from the Bethel Lake Dam, Pitman, Gloucester Country, New Jersey, between July 1995 and December 1996 were examined shortly after being identified. Three hundred fifty-two specimens of C. leana were collected from the Keum River, Korea, between February 2000 and December 2001. The 30 to 40 individuals were collected on a monthly basis fix this study.

The live clams were transported to the laboratory of the Museum of Zoology, University of Michigan, and the Department of Parasitology, Kwandong University, and the shell lengths and heights measured using Vernier calipers and their total weight determined using a balance. Histologic preparations were made to study the sexuality, gametogenesis, and morphologic changes in the structures of the demibranchs by light microscopy. The visceral mass and the gill tissues were subjected to standard histologic procedures (dehydrated in alcohol and embedded in paraffin), and 5-7-[micro]m sections prepared using a rotary microtome. The sections were then mounted onto glass slides, stained with Hansen's hematoxylin-0.5% eosin and Mallory's triple stain, and observed using light microscopy.

To compare the relative DNA content of the spermatozoa and gill tissue of C. fluminea, cells were isolated on a glass slide by cutting small pieces of gonad and gill tissues in distilled water and air-drying it before fixing with 70% ethanol. The spermatozoa and gill cells from one individual were placed on the slide. The cells were stained with DNA-specific dyes PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindole), and the relative DNA content (fluorescence intensity) per cell estimated by microfluorometry, as in Komaru et al. (1988). The DNA content was assayed at least three times at each of three different concentrations of spermatozoa and gill cells. Twelve individuals were used for these assays. The spermatozoa could easily be distinguished from the other spermatogenic cells due to their elongate and curved morphology.

RESULTS

Sexuality and Functional Hermaphroditism

The gonad of the freshwater clams C. fluminea and C. leana is located between the digestive diverticula and the outer fibromuscular layers, which are compacted by the fibrous connective tissues and muscle fibers. As the gonad matures, it extends to the lowest part of the muscular layers, around the foot. Both Corbicula species were hermaphrodites (monoecious). and gonad consisted of a number of oogenic, spermatogenic, or hermaphroditic follicles. Both female and male germ cells were present in the oogenic, spermatogenic, or hermaphroditic follicles (Figs. 1-20). More specifically, triploid C. fluminea and C. leana showed hermaphroditism, and a group of the ovotestis (Figs. 6, 16, and 20), or intrafollicular embryos, usually appeared to be surrounded by numerous spermatozoa in the hermaphrodilic follicles during egg cleavage (Figs. 8 and 20). The functional hermaphroditism was shown in the hermaphroditic follicles.

[FIGURES 1-20 OMITTED]

Gametogenesis

The oogenesis of the two Corbicula species occurred in the oogenic or hermaphroditic follicles between the digestive and the outer fibromuscular layers and was divided into five stages: oogonial, previtellogenic oocyte, vitellogenic oocyte, and mature oocyte stages. Also, spermatogenesis occurred in the spermatogenic or hermaphroditic follicles, between the digestive diverticula and the outer fibromuscular layers, and was divided into five stages: spermatogonial, primary spermatocyte, secondary spermatocyte, spermatid, and spermatozoon stages. The characteristics of gametogenesis of the two Corbicula species are as follows.

Corbicula fluminea

This species is a hermaphrodite. The gonad consisted of a number of oogenic (Figs. 1, 2, 3, and 4). spermatogenic (Figs. 2, 3, 5, and 7), or hermaphroditic (Figs. 6 and 8) follicles.

Oogenesis Oogenesis occurred in the oogenic and hermaphroditic follicles. The oogenic and hermaphroditic follicles located near the outer muscular layer begin to develop toward the visceral mass. A number of oogonia appeared along the follicular walls (16-18 [micro]m in diameter) and had a round nucleus containing a nucleolus in its center. One nucleolus in the nucleus was distinct in appearance, though the cytoplasm of the oogonium was very poor stained. At this time, a number of undifferentiated mesenchymal tissues and eosinophilic cells were both located near the follicle walls (Fig. 1). The oogonium developed into the previtellogenic oocyte. The previtellogenic oocyte (30-36 [micro]m in diameter) had a round nucleus containing one or more small eosinophilic nucleoli along the nuclear envelope, and the cytoplasm begin to grow in volume. Undifferentiated mesenchymal and eosinophilic granular cells were abundant on the follicular wall. There were widely distributed interfollicular connective tissues near the follicles (Fig. 2). Early vitellogcnic oocytes grew to 130-140 [micro]m in diameter and became eosinophilic oval or pentagonal oocytes in the oogenic or hermaphroditic follicles (Fig. 3). Mature oocytes (about 150 170 [micro]m) had one large nucleolus, 3-4 small nucleoli in the nucleus, and numerous yolk granules in the cytoplasm. At this stage, the vitelline envelope of the mature oocyte was surrounded with a gelatinous substance (Fig. 4).

Spermatogenesis Spermatogenesis occurred between the digestive diverticula and the outer fibromuscular layers. The spermatogonia were 8-9 [micro]m in diameter and contained a large oval nucleus located in the wall of the spermatogenic or hermaphroditic follicles. Undifferentiated mesenchymal cells and eosinophilic cells were located near the spermatogonia and spermatocytes (Fig. 5). The spermatocytes developed into the spermatids. At this time, the spermatids in the center of lumina of the hermaphroditic follicles were occupied with a few oocytes; the interfollicular connective tissues were also widely occupied (Fig. 6). At the early differentiation stage of the spermatid, the shape of the nucleus changed gradually and became slightly elongated and narrow. After spermatogenesis (transformation of the spermatid into the spermatozoon), a number of the spermatozoa formed needle-shaped sperm clusters in the male follicles (Fig. 7). The spermatozoon was approximately 12-13 [micro]m in length, and the sperm head was approximately 1.0 [micro]m in width.

In the partially spawned follicle, an embryo uncleavaging, surrounded by a number of sperm, appeared in the hermaphroditic follicle (Figs. 8 and 9), indicating that C. fluminea is a functional hermaphrodite. Male reproductive tissue was less common than female tissue. At this time, the gonoduct was located near the spermatogenic follicle, and the follicular ganglia (consisting of the neuronal fiber and neuronal soma-like cells at its periphery), associated with neurosecretion and the differentiation of complex innervated nerve structures during spermatogenesis, were widely distributed in the follicles during the ripe and spawning stages (Figs. 10 and 11).

Cyclic Changes in Structures of the Inner-Demibranchs A pair of the inner demibranchs acted mainly as marsupium. Incubated ripe eggs and D-shaped veliger larvae were rarely found within a pair of the outer-demibranchs. The non-marsupial demibranchs were not separated and had no secondary septa and tripartite marsupial structure. In this case, the outer-membranes of the demibranchs were slightly thicker (Fig. 12A). In the case of the marsupial demibranchs, the morphologic and structural changes in the inner-demibranchs of the gills showed a distinct seasonal alternation or periodicity in relation to depletion of ripe eggs during the incubatory periods, with the production of mature and ripe eggs during the nonincubatory periods. In the late nonincubatory period, during gametogenesis, or before depletion of the ripe gametes, the tripartite marsupial structures completely disappeared, and the epithelial cells lining the inner-lamellar spaces of the inner-demibranchs became gradually thicker (Fig. 12B). During the early incubatory period of the inner demibranchs (removal of ripe gametes or early developing embryos from the gametogenic follicles to the gills), a number of ripe eggs and D-shaped veliger larvae were filled in thickened inter-lamellar spaces of the inner-demibranchs (Fig. 12C). In the late incubatory period (gonads in the partially spawned stage), a number of incubated D shaped veliger or pediveliger larvae were found in the inter-lamellar spaces of the inner-demibrauchs. Thereafter, as the marsupial demibranchs began to be separated by the secondary septa, the secondary septa produce secondary water tubes (Fig. 12D). Consequently, in the early nonincubatory period, the tripartite marsupial organization that was formed by the secondary septa and water tubes appeared in the inner-demibranches (Fig. 12E). At this time, the inter-lamellar spaces of the tripartite marsupial demibranchs were vacant, as a number of the D shaped veligers in the marsupial gills were released.

Some characteristics of the relationships between the structural changes of the inner-demibranchs in the marsupial gills and gametogenesis in the gonads were found: most gonads became de generate and appeared to be depleted of female and male gametes during the incubatory and larval release periods. Therefore, inhibition of gametogenesis might occur during the incubatory periods (especially during the mid-summer and early autumn incubation and D-shaped veliger larvae release),

Corbicula leana

This species is a triploid (Okamoto and Arimoto 1986) and a hermaphrodite. The gonad consisted of a number of the oogenic (Figs. 13 and 15) and spermatogenic (Figs. 17, 18, and 19) or hermaphroditic (Fig. 20) follicles. The spermatogenic and oogenic or hermaphroditic follicles were distributed among the interfollicular connective tissues. The characteristics of gametogenesis of this species were as follows.

Oogenesis Oogenesis occurred in the oogenic follicles. Many oogonia (15-18 [micro]m) propagated along the follicle wall near the mesenchymal tissues. The oogonia had one large basophilic nucleus and light basophilic cytoplasm (Fig. 13). The oogonia developed into the previtellogenic oocytes; at the beginning of cytoplasmic growth, each oocyte (ranging in diameter from 20 to 40 [micro]m) had an egg-stalk and was attached to the follicle walls of the oogenic follicle near the spermatogenic follicle, which contained a number of spermatids. In the early vitellogenic oocytes (50-70 [micro]m), the cytoplasm was markedly stained with eosin (Fig. 14). The vitellogenic oocytes (80-100 [micro]m) and the late vitellogenic oocyte (110-130 [micro]m) were located in the center of the lumina of the follicle, and numerous eosinophilic yolk granules in the cytoplasm were filled (Fig. 15). The mature oocytes (more than 140-160 [micro]m) had 4-7 nucleoli in the nucleus, and many yolk granules in the cytoplasm were filled. The mature oocytes were surrounded by a gelatinous substance. Several mature oocytes were found in the lumen of the hermaphroditic follicles and contained numerous spermatozoa (Fig. 16).

Spermatogenesis The testes were composed of a number of spermatogenic and hermaphroditic follicles, including oogenic tissues. The spermatogonia, which were distributed along the follicular wall, were approximately 9 [micro]m in diameter and had relatively little cytoplasm. The spermatogonia developed into the primary spermatocytes, and the primary spermatocytes developed into the secondary spermatocytes. The chromatin in the nucleus of the spermatocyte became gradually more concentrated, while the volume of the cytoplasm of the secondary spermatocyte gradually became smaller. The secondary spermatocytes developed into spermatids, which were darkly stained with hematoxylin, and distributed in the center of the lumina of the spermatogenic follicle. Stratified layers of the spermatogonia, spermatocytes, and spermatids formed in the male follicle (Fig. 17). The spermatids developed into spermatozoa in the spermatogenic or hermaphroditic follicles, containing a few ovotestis on the oogenic tissue (Fig. 18). A number of needle-shaped sperm clusters, formed by numerous spermatozoa, filled the spermatid follicle. The spermatozoon was approximately 12 [micro]m in length, and the head was approximately 1.2 [micro]m in width. During spermatogenesis, follicular ganglia (consisting of neuronal fiber and neuronal soma-like cells) were widely distributed in the spermatogenic follicle, but gradually disappeared (Fig. 19). In the spawning period, the embryo-like body, surrounded by a number of sperm clusters, appeared in the hermaphroditic follicle (Fig. 20).

Relative DNA Content of Spermatozoa and Somatic (Gill) Cells

In C. fluminea, the relative content of the DNA in the spermatozoa was identical to that in the gill cells (Fig. 21). Komaru et al. (1997) reported that C. leana is a triploidy species, and from microfluorometric analyses, the sperm and somatic cells were shown to have the same DNA content. They suggested that meiosis I or II may be abortive in spermatogenesis, with only one equal division resulting in nonreductional spermatozoa.

[FIGURE 21 OMITTED]

DISCUSSION

In general, hermaphroditism is seen more frequently in freshwater than marine mollusks (Van der Schalie 1970). Corbicula species can be categorized into three major groups, based on their reproductive characters and ecologies (Miyazaki 1936). Species belonging to Group 1 are monoecious, viviparous, and incubatory. They have nonswimming planktonic veliger larvae and live in freshwater, The species belonging to Group 2 are dioecious, oviparous, nonincubatory, and also live in freshwater regions. The species belonging to Group 3 are dioecious and oviparous. They do not incubate their young, have free-swimming planktotrophic larvae, and live in brackish waters. In this study, C. fluminea and C. leana were confirmed to be hermaphroditic species (Group 1). According to our histologic observation, the two Corbicula species were hermaphroditic throughout their lifetime, with no sex reversal. According to Coe's report (Coe 1943), cited by Heard (1975), hermaphroditic conditions in pelecypods were divided into four categories, according to the sequence of reproductive events: (1) functional hermaphroditism (eggs and sperm produced simultaneously), which can be subdivided into two groups: (i) normal (typically in monoecious species) and (ii) accidental or development (typically in dioecious species): (2) consecutive sexuality (single sex-reversal, usually protandrous), (3) rhythmical sexuality (>1 sex-reversal, usually protandrous); and (4) alternative sexuality (adults function seasonally as separate sexes). Monoecious species can be grouped as male (predominance of testicular tissue; animal not gravid) and female (ovarian tissue slightly or greatly exceeding quantity of testicular tissue; animal may became gravid) hermaphrodites. In the current study, C. leana and C. fluminea were found to be functional hermaphrodities.

Kraemer and Lott (1977) reported that although the largest Corbicula species exceeded 20 mm in size, they consistently showed a clear predominance of ovarian over testicular development. Corbicula fluminea had greater volumes of oogenic than spermatogenic tissue. The oogenic follicles were larger, with a clear predominance and well-developed oocytes. In particular, in the case of triploid species, this phenomenon might occur because of parthenogenetic reproduction. The results of this study on Corbicula species support the previous findings by Kraemer and Lott (1977). Okamoto and Arimoto (1986) proposed that triploid species might closely be related to hermaphroditism. With reference to hermaphroditism and fertilization, there have been some reports that C. fluminea carry out both self-fertilization (Kraemer 1978, Kraemer et al. 1986) and cross-fertilization (Kraemer 1978). In this study, some intrafollicular embryos were found in the course of egg cleavage in the hermaphroditic follicles of C. fluminea (Fig. 8). At a glance, some intermingling of a number of spermatozoa and a few oocytes in the same follicle suggest that self-fertilization may occur in the hermaphroditic follicle. However C. fluminea is a triploid species (Park et al. 2000), and its DNA content in the nucleus of the somatic cell (gill) is the same as that in the gamete cell (sperm). Komaru et al. (1997) reported that C. fluminea was a diploid species and produced nonreductional spermatozoa. If C. fluminea sexually reproduces itself, the DNA content in the nucleus of the somatic cell. and the number of the homozygote genes, would increase in volume and chromosome numbers, respectively, due to egg and sperm fertilization. Therefore, reproduction of this species may occur by parthenogenesis, without self-or cross-fertilization of eggs and sperm. The DNA contents in the nuclei of the somatic and gamete cells of C. fluminea were the same. In the case of parthenogenesis, it is assumed that numerous spermatozoa may participate in activation of the mature eggs, and the eggs cleavage as a stimulus only for parthenogenesis in the same hermaphroditic follicle or the gonopore. Komaru et al. (1998) reported that cytologic observations and DNA microfluorometry of the hermaphrodite freshwater triploid clam C. leana revealed androgenetic development.

Bivalves have two pairs of gills. In the case of Corbicula species, two pairs of gills act as the marsupia, but the pair of inner-demibranchs mainly play an important role as the marsupia: in general, a pair of the outer demibranchs occasionally act as the marsupia. Differences in the structural and morphologic changes between the non marsupial and marsupial demibranchs are very clear. Heard (1975) described that in Anodonta, the non-marsupial demibranchs did not form the secondary sepia, and a comparatively large number of filaments were distributed between the in nor membranes of the primary septa, whereas marsupial demibranchs were divided by the secondary septa that produced the secondary water tubes, resulting in tripartite marsupial organization. However, Kwon and Park (1985) stated that after the glochidia had been released, the secondary water tube was continuously present in Lanceolaria acrorhyncha. In this study the patterns reported by Heard (1975) were found in C. fluminea. In the late nonincubatory period, during gametogenesis or before spawning, the structures of the tripartite marsupial inner-demibranchs disappeared, while the inner-membranes of the marsupial gills were vacant and became very thick. In the early incubatory period, during movement of ripe gametes or early developing embryos in the gametogenic follicles ti inner-demibranchs. a number of ripe eggs or D-shaped veliger larvae filled the thickened epithelial cells lining the inter-lamellar spaces of the inner demibranchs. In the late incubatory period, the late pediveligers or straight-hinged juveniles (approximately 230-240 [micro]m in size), which had grown in the marsupial gills for approximately 7-10 days, began to shed into the environmental water. At this time, the primary water tubes began to separate, and the secondary water septa formed in the marsupial gills. Consequently, in the early nonincubatory period, the tripartite marsupial organization appeared in the inner-demibranches of the gills. In our histologic study, the two Corbicula species were found to be hermaphrodites. with specified demibranchs of the gills. Particularly, gametogenesis inhibition occurred in the gametogenic follicles during the gills incubatory period whereas active gametogenesis occurred during the nonincubatory period. The structural hypertrophy of the epithelial cells lining the inter-lamellar spaces was especially changed in relation to the nonincubatory and incubatory periods of the inner-demibranchs. Therefore, the results of this study suggest that the larvae may receive nourishment from the hypertrophied epithelial cells that line the inter-lamellar spaces of the inner-demibranchs of the adult clam. Thus, the morphologic changes in the structure of the marsupial gills, during the incubatory and nonincubatory periods, with gametogenesis and spawning patterns, may occur cyclically or have a certain short-term periodicity. In conclusion, C. fluminea and C. leana are functional hermaphrodites, with the spermatozoa in the triploidy Corbicula species showing an identical DNA content to the somatic tissue of the parent.

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GAB-MAN PARK (1), * AND EE-YUNG CHUNG (2) (1) Department of Parasitology Kwandong University College of Medicine, Gangneung, Gangwon-do 210-701, Korea; (2) School of Marine Life Science, College of Marine Science and Technology Kunsan National University, Kunsan, Chollabuk-do 573-701, Korea

* Corresponding author. E-mail: [e-mail]