Materials

Five species of unionid mussels were collected from three localities in the Kinki and Chugoku regions in Japan; Anodonta woodiana from a creek at Kanmaki, Osaka Prefecture, Inversidens japanensis and Pseudodon omiensis from a creek at Kinomoto, Shiga Prefecture, Inversiunio yanagawensis and Lanceolaria grayana from the Gion Creek, Okayama Prefecture. Sex of all individuals collected was determined by observation of gametes under a light microscope, and 10 to 20 males of each species were used in the experiments.

Spawning induction

Males were reared in freshwater kept at 20°C for twenty-four hr, transferred to 4°C freshwater for two hours, and then returned to freshwater at 20°C. Spawning occurred within one hour after this treatment.

Motility of spermatozoa

Spermatozoa released by thermal stimulation were reared in freshwater of 16°C. Sperm collected by dissecting the gonads were diluted by freshwater or balanced salt solution (BSS : 9.6 mM NaCl, 0.7 mM KCl, 1.7 mM Na₂SO₄, 0.3 mM MgSO₄, 3.2 mM CaCl₂, 7.3 mM NaHCO₃, 0.1 mM NaH₂PO₄, 0.2 mM glucose adjusted to pH 7.3) and maintained at 16°C. The balanced salt solution was prepared after Machii (1992). We observed spermatozoa under a light microscope in every twenty-four hours and their motility was examined. Motility was qualitatively quantified into three categories as follows; very active, active and inactive.

Description

Males discharged spermatozoa as spherical masses (Fig. 1A and 1B). Spermatozoa embedded their heads into the spherical colorless body with their tails extended around the sphere. We termed this sphere as a “sperm sphere”. Although we tried to fix the sperm spheres by 70% ethyl alcohol, Carnoy's fixative (ethyl alcohol : acetic acid = 4:1) and 10% formalin, we failed to fix them. They disintegrated at once in any fixing fluids, and then only the dispersed spermatozoa were observed, which suggests that the spherical body may be composed of a thin, fragile membrane. At that time, the sperm spheres rotated rapidly and showed active movement through the water. But a part of the surface became bare (Fig. 1C), because some spermatozoa detached from the sphere (Fig. 1D). The sperm spheres gradually increased in size with time (Fig. 2). As a result, the sperm spheres moved forward with the bare surface area at the head.

Fig. 1

Spermatozoa of Inversiunio yanagawensis (A, B, C, E) and Inversiunio japanensis (D). MM: membrane, SH: head of spermatozoa, ST: tail of spermatozoa, Scale bar = 50 μm

A Sperm spheres just after discharge

B Schematic representation of a sperm sphere

C Sperm sphere twenty-four hr after discharge

D Sperm sphere twenty-four hr after discharge (Inversiunio japanensis)

E Single spermatozoa obtained from the gonads

Fig. 2

Increase in diameter of sperm spheres. • : Anodonta woodiana, • : Inversidens japanensis, • : Inversiunio yanagawensis, • : Lanceolaria grayana, • : Pseudodon omiensis.

On the other hand, only single spermatozoa were obtained by dissection from the gonads. The spermatozoa in the balanced salt solution also did not form sperm spheres.

Mean diameter of sperm sphere just after discharge was 44.4±3.0 μm in Lanceolaria grayana, 47.9±19.1 μm in Inversiunio yanagawensis, 49.3±6.8 μm in Inversidens japanensis, 53.3±1.8 μm in Pseudodon omiensis and 59.6±8.5 μm in Anodonta woodiana.

Motility

Spermatozoa in the sperm spheres of five species were very active until forty-eight hours after discharge (Table 1). Spermatozoa detached from the sperm sphere and became inactive within a few minutes (Fig. 1E).

Table 1

Change in the motility of spermatozoa after discharge.

In four species except for Inversiunio yanagawensis, no sperm sphere was observed at seventy-two hr. However, in Inversiunio yanagawensis, sperm spheres were active seventy-two hr after discharge. Some spheres had no spermatozoa, and others had active spermatozoa but with very weakly lashing tails.

The motility of spermatozoa extracted from the gonads is shown in Table 2. In balanced salt solution single sperm maintained their motility kept at forty-eight hours while in all species in freshwater, single sperm lost their motility within a few min.

Table 2

Change in the motility of spermatozoa in the water and in the balanced salt solution (BSS) after extraction from gonads.

Sperm sphere

Sperm sphere observed in the present study are the same as the “hollow globular masses” in Lampsilis ventricosa, Lasmigona complanata and Utterbackia ohiensis described by Utterback (1931) and the “spherical masses of sperm” in Anodontoides ferussachianus by Edger (1965). Since all these species belong to the family Unionidae, the use of sperm spheres may be of widespread occurrence in this family. Moreover, sperm spheres were also observed in Margaritifara laevis (Okada and Ishikawa, 1959). This species belongs to the family Margaritiferidae, which suggests that sperm sphere may be common in the superfamily Unionacea.

As sperm spheres have not been found in other freshwater bivalves such as the “Seta clam”, Corbicula sandai and freshwater mytilid mussel, Xenostrobus securis (Ishibashi, unpublished data), they may be unique to Unionacea.

The sperm sphere of unionid mussels is similar to “sperm ball” in the California oyster, Ostrea lurida (Coe, 1932), to the “sperm-discs” in the tubucolous polychaete, Arenicola cristata (Okada, 1941) and to the “disc of mature sperm” in Arenicola mariana (Newell, 1948). But these structures disintegrate immediately when they were released into salt water. On the contrary, sperm spheres moved through the freshwater for a long period. Therefore, sperm spheres appear to play a different role compared with sperm balls and sperm-discs of marine lamellibranchs and polychaetes.

Spermatozoa in the balanced salt solution did not form sperm spheres, which suggest that spheres may form in gonads. We suggested that sperm spheres may form just before spawning in the gonads or gonoducts.

Motility of spermatozoa

In the present study, single spermatozoa dissected from the gonads in freshwater lost their motility within a few minutes, while they were active in the balanced salt solution for forty-eight hr. Further, spermatozoa with their heads embedded in sperm spheres were also active for two days, and sperm spheres increased in size with time. These results suggest that the membrane of the sperm sphere may be semipermeable and the composition of the fluid in the sperm sphere may be similar to the balanced salt solution.

As the concentration of the salt in the balanced salt solution is higher than freshwater, the sperm spheres may protect spermatozoa from the lower osmotic pressure of ambient freshwater and enable them to maintain motility for a long time in order to transport them to females far away from the male.