Two shell color types of the exotic bivalve Corbicula fluminea were collected in Kyoto city, Japan. DNA microfluorometry revealed that both types were diploids with non-reductional spermatozoa. Maternal chromosomes were found to be extruded as two polar bodies at the first meiosis, and the second meiosis could not be observed. Only the male pronucleus was present in the egg cytoplasm and became metaphase chromosomes at the first mitosis. The present study indicates that the diploid C. fluminea in Japan has the same mode of androgenetic reproduction as the triploid C. leana.
INTRODUCTION
Corbicula fluminea (Müller 1774) is the most widely-distributed Asian clam species and is highly invasive and known also from North America, South America, Europe and Australia (Britton and Morton, 1986). C. fluminea reproduces by self-fertilization and broods its young in the inner demibranchs (Kraemer, 1978; Kraemer, 1983; Britton and Morton, 1986). Their spermatozoa have two flagella (Konishi et al., 1998).
Komaru and Konishi (1999) found three different shell color types of C. fluminea at the same location in Taiwan. They were composed of diploids and triploids with nonreductional spermatozoa (Komaru and Konishi, 1999).
In North America, it was voluntarily introduced from Asia and subsequently spread to many major river basins in North America. At present C. fluminea has become a major “pest” of industrial and domestic water supply systems (Britton and Morton, 1986; Balcom, 1994). C. fluminea was also introduced into Japan (Masuda and Habe, 1988). According to Nakai and Matsuda (2000), Corbicula leana (Prime, 1864) is an indigenous species to Japan, has been replaced by C. fluminea in some regions of Japan.
C. leana is a freshwater, hermaphrodite and triploid in Japan (Miyazaki, 1936; Habe, 1977; Ikematsu and Yamane, 1977; Okamoto and Arimoto 1986). Komaru et al. (1997) and Konishi et al. (1998) revealed that C. leana produces non-reductional spermatozoa with two flagella, which have the same DNA content as the somatic cells. C. leana reproduces by androgenesis (Komaru et al., 1997; Komaru et al., 2000) in which all the maternal chromosomes of eggs are extruded as two polar bodies at the first meiosis. After extrusion, only the male pronucleus forms the metaphase chromosomes at the first cleavage. It is indicated that C. leana is close relation to C. fluminea because they produces nonreductional spermatozoa with two flagella. However, little is known about egg developmental mode of C. fluminea.
In the present study, we examined the meiosis and fertilization of zygotes in C. fluminea by using DAPI staining for fluorescence microcopy.
MATERIALS AND METHODS
Animals
Two color types of Corbicula fluminea were collected from the same point of Shishigatani creek, Kitashirakawa, Kyoto city, Kyoto Prefecture, Japan. 23 mature samples were collected and used in present study in June 1999. This creek is a part of the Lake BiwaYodo River system. We defined the two color types (n=23), as green (n=17) and pink (n=6). Triploid Corbicula leana samples were collected from the Amano River in Kitatawara, Ikoma city, Nara Prefecture, Japan, for determination of the ploidy of C. fluminea. Samples were collected in June 1999. The samples used in this study are deposited in the Lake Biwa Museum, Oroshimo, Kusatsu, Shiga Prefecture, Japan (green type: LBM-1300008873, pink type: LBM-1300008872).
DNA Microfluorometry
Ploidy of the somatic cells was determined as follows: gill or mantle cells of C. fluminea (green: n=17, pink: n=6 and C. leana n=5) were fixed with Carnoy's fixation and a cell suspension was prepared on a glass slide in 50% acetic acid with a scalpel and air-dried. Three or four samples of C. fluminea and triploid C. leana as a control were spread on the same slide. The slides were stained with the DNA-specific dye DAPI, and the relative DNA content per cell was measured by microfluorometry (Komaru et al., 1998). Ploidy of the spermatozoa was examined from the gonad and somatic cells from the gill or mantle of the same animal which were smeared and placed on the same slide. These slides were stained with DAPI using the above-mentioned method.
Spawning induction
Spawning was induced by rising the water temperature from 20°C to 27°C. Spawned eggs of the pink type were collected and mixed from three individuals, while those of green type were collected from one individual.
Observations of chromosome behavior in eggs with DAPI staining
Spawned eggs were incubated at 27°C. Both color types were kept in different aquarium. The eggs were released into the inner demibranch or into the water from the exhalant siphon. Spawning eggs were fixed with 99% cold ethanol at every 10 or 15 min after spawning.
Fixed eggs were rinsed with 0.2M phosphate buffer (pH 7.5) three times and stained with DAPI (0.25 μg/ml). The chromosome behavior was observed using fluorescence microscope (Nikon ECLIPSE E600-Y-FL).
RESULTS
Shell color
The samples consisted of two distinct shell color types. Green type: External surface was light or dark green, and inside was deep purple (Fig. 1A). Pink type: External surface was yellow or light brown, and inside was white, although the umbone areas are pink and was characterized by a purple flash along with the anterior and posterior lateral teeth (Fig. 1B).
Ploidy
The DNA content of the somatic cells is shown in Table 1. The mean relative fluorescence intensity of the green type of the C. fluminea compared to the triploid C. leana was 0.58 to 0.69. This showed that all the clams of the green type (sample Nos. 1–17) of C. fluminea were diploids. The mean relative fluorescence intensity of the pink type of C. fluminea compare to the triploid C. leana was 0.69–0.70, indicating those (sample Nos. 18–23) of pink type to be diploids.
Table 1
The relative DNA contents of somatic cells in C. fluminea compared to those of C. leana based on microfluorometry.
Non-reductional spermatozoa
The mean DNA content of sperm and somatic cells of the two color types of C. fluminea is shown in Table 2. The relative DNA content of spermatozoa of both types was almost identical to those of the somatic cells. The relative fluorescence intensity from both types ranged from 0.98 to1.06. The ploidy of the spermatozoa of both the green and pink was the same as that of their somatic cells.
Table 2
The relative DNA contents of spermatozoa and somatic cells (gill or mantle) in diploid C. fluminea.
Egg size
The mean diameter of eggs from the green type just after spawning was 115.7±3.9 μm (n=105) and the pink type eggs was 127.9±4.9 μm (n=132). On the other hand, the egg diameter of triploid C. leana was 158.2±4.4 μm (n=98). Between the green and pink types, the egg diameter was not statistically different (t-test, p>0.5), however, the egg diameter of the two types of diploid C. fluminea was significantly smaller (t-test, p<0.01) than that of the triploid C. leana.
Process of androgenesis
Green type: Fig. 2 summarizes the process of andro-genesis in the eggs. At 10 min after spawning, 20.7% of the eggs were unfertilized (Figs. 2A and 3) and 77.8% were fertilized and at the M-I stage (Figs. 2B and 3). At 20 min, most eggs were fertilized (87.9%). At 30 min, 81.5% of the eggs were at the anaphase of the first meiosis (A–I), and the maternal chromosomes were divided into two groups (Figs. 2C and 3). At 45 min, two polar bodies were formed in 75.3% of the eggs, but the female pronucleus was not formed (Figs. 2D and 3). All meiotic chromosomes were extruded as two polar bodies and only one male pronucleus was present in the egg cytoplasm. At 75 min, the male pro-nucleus became the metaphase chromosomes of the first cleavage (Figs. 2F and 3), and at 90 min, 66.3% of the eggs were at the anaphase of the first cleavage.
Fig. 2
DAPI images from the unfertilized egg stage to metaphase of the first cleavage in C. fluminea eggs. A: unfertilized at 10 min. B: fertilized at 20 min. C: anaphase of first meiosis at 30 min. D: polar body formation at 45 min. E: male pronucleus expanded at 60 min. F: metaphase of first cleavage at 75 min. Arrows indicate the polar body, arrow heads indicate the male pronucleus. Scale bar: 50 μm
Fig. 3
Chromosome behavior from metaphase of first meiosis to two cell stages in the androgenetic C. fluminea eggs (green type).
Pink type: The eggs of the pink type also showed androgenetic development as shown in Fig. 4. All the maternal chromosomes were also extruded as two polar bodies. Thus, no female pronucleus was formed in the egg cytoplasm.
DISCUSSION
The present study showed that hermaphroditic freshwater diploid clam C. fluminea reproduces by androgenesis. The process was follows; androgenetic diploid C. fluminea were hermaprodite and produced non-reductional spermatozoa and its spermatozoa have two flagella. All the maternal chromosomes were extruded as two first polar bodies at the first meiosis. Only one male pronucleus was present in the egg cytoplasm and became the metaphase chromosomes at the first mitosis.
Komaru and Konishi (1999) and Qiu et al. (2001) reported that hermaprodite C. fluminea in Taiwan and China has various shell color types. Komaru and Konishi (1999) suggested that in C. fluminea from Taiwan, the shell color types might have different genetic background. C. fluminea consists of diploids, triploids and tetraploids and they produce non-reductional spermatozoa with two flagella in respective ploidy (Qiu et al., 2001). Diploid, triploid and tetraploid C. fluminea in Taiwan and China may reproduce androgenesis because they were produced non-reductional spermatozoa such as C. fluminea in Japan.
Ishibashi et al. (2002) demonstrated that in androgenetic C. leana eggs treated with Cytocalasin D (CD) to inhibit polar body formation, the second meiosis occurred. Second meiosis proceeded normally in CD treated eggs when maternal chromosomes and centrosomes existed in egg cytoplasm, and typical meiosis system still proceeded. It is suggested that androgenetic form may have appeared from the meiotic form.
Ishibashi et al. (2002) showed the possibility that an androgenetic triploid form may originate from the meiotic form in Corbicula. Triploid C. fluminea may elevate the ploidy level by accidental formation of the female haploid pronucleus (Komaru et al., 2001). So Diploid androgenetic form may arise from the meiotic form. Ploidy may have been elevated to the triploid levels. It is likely that the polyploid C. fluminea may have originated from the diploid androgenetic C. fluminea.
Acknowledgments
We especially thank Prof. Takaki Kondo of the Division of Natural Sciences, Osaka Kyouiku University for his critical comments on the manuscript and we wish to express our sincere thanks to Mr. Toru Koyama and Mr. Shinsuke Hatanaka, Kinki University Faculty of Agriculture for providing the C. leana specimens. This study was supported by a grant from the Narishige Zoological Science Award and a grant for research from the Ministry of Education, Science, Sports and Culture, Japan (No.13660183).
