Ž fully induced in many fish species and used for examining sex-determination Avtalion
. Ž
. and Don, 1990 and gene–centromere recombination Arai et al., 1991 , as well as
Ž production of inbred line, mono-sexual broods or clones Naruse et al., 1985; Tabata and
Gorie, 1988; Taniguchi et al., 1988; Fujino, 1989; Fujioka, 1993; Kobayashi et al., .
1994 . In molluscs, however, studies on gynogenesis have been still preliminary. Although
Ž induction of gynogenetic diploids has been reported for Haliotis discus hannai Fujino
. Ž
. Ž
. et al., 1990 , Crassostrea gigas Guo et al., 1993 , Mytilus edulis Fairbrother, 1994
Ž .
and Mytilis galloproÕincialis Scarpa et al., 1994 , practical procedures for induction of gynogenetic diploid have not been established for molluscs as for fishes. One of the
plausible reasons for this is considered to be the decreasing fertilization rate with Ž
. increasing ultraviolet UV irradiation time of sperm that has been observed in all these
studies. Since the sperms of fish species do not lose their fertilizing ability at a dosage of Ž
UV irradiation for genetic inactivation Onozato and Yamaha, 1983; Tabata et al., 1986; .
Ž .
Taniguchi et al., 1986 , Kijima 1992 supposed that UV irradiation affects not only the Ž
. sperm genome, but also the acrosome structure which is absent in teleost fishes in the
Pacific abalone. However, the possibility has not been evidenced. The Japanese scallop, Patinopecten yessoensis, is one of the most commercially
important bivalves in Japan. Numerous studies have been performed on propagation of Ž
. the scallop Yamamoto, 1964; Kanno and Sato, 1980; Nagasaki, 1999 , but few studies
have been undertaken on gynogenesis or polyploidy. In the present study, the effect of UV irradiation on sperm morphology was examined
Ž .
by scanning electron microscopy SEM , and various durations of UV irradiation of sperm were also examined to determine the optimum conditions for induction of
gynogenesis in the Japanese scallop, P. yessoensis.
2. Materials and methods
2.1. Gametes Ž
. Specimens of sexually matured P. yessoensis shell length, 12.5 0.6 cm were
collected in late March and early April at Onagawa Bay, Miyagi Prefecture, Japan. They were separated into males and females and kept in running seawater tanks until used.
Spawning of eggs and sperms was induced by raising water temperature from 98C to Ž
. 148C and injecting 0.5 ml of 1 mM serotonin-creatinine sulfate Wako, Japan into the
gonad. Discharged eggs were collected by suction and rinsed in filtered seawater several times. Sperm suspension was prepared at a concentration of 1 = 10
7
spermrml by dilution with filtered seawater.
2.2. UV irradiation of sperm and insemination Two milliliters of sperm suspension were spread on a 9.0-cm diameter plastic petri
Ž .
dish Nunclon dish; Nalge Nunc, Denmark . The dish was placed on a shaker 25 cm Ž
. below an 18 W UV germicidal light TOSHIBA GL15, 254 nm; Toshiba, Japan that
provided an UV intensity of 720 mW cm
y2
s
y1
as measured by a digital radiometer Ž
. DRC-100X; Spectronics, USA . The sperm was exposed to the UV light for either 0
Ž .
control , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70 or 90 s. During the UV irradiation, the sperm suspension was shaken at approximately 0.5 cyclers.
Ž
4
. At completion of irradiation, 10 ml of egg suspension 1 = 10 eggrml was added
to each petri dish, mixed and finally transferred to a beaker for culture at 118C. The fertilization and development rates were calculated by counting the cleaved eggs and the
D-shaped larvae at 8 h and day 4 after insemination, respectively. The experiment was repeated three times separately using gametes from different spawning.
2.3. Ploidy determination The relative value of DNA content in larval cells was determined by DNA microfluo-
Ž .
rometry in accordance with Komaru et al. 1988 . Samples of veliger larvae were collected from each group on day 3 postinsemination, subjected to a hypotonic treatment
Ž with 0.075 M KCl solution, fixed by cold Carnoy’s solution methanol:acetic acid, 3:1,
. vrv and stored in y208C. Larvae were dissociated in 50 acetic acid by aspirating
Ž .
with a pipette. A sample of the cell suspension and control cells diploid veliger cells were placed on a warmed glass slide, air-dried and stained with 4
X
,6-diamidino-2-phenyl- Ž
. indole dihydrochloride DAPI . The fluorescence intensity of nuclei was measured with
a Nikon P1 photometer.
Fig. 1. Relationships between UV irradiation duration and the rates of fertilization and development of D-shaped larvae in the scallop, P. yessoensis, estimated at 8 h and day 4 post-insemination, respectively.
2.4. SEM of spermatozoa The sperm suspension UV-irradiated for either 0, 30, 60, 90 or 120 s was placed on a
Ž
2
. small piece of slide glass approximately 5 = 5 mm
coated with 0.1 poly-
L
-lysine Ž
. Ž
. Ž
Wako , prefixed with 1 paraformaldehyde Wako and 2.5 glutaraldehyde EM Sci., .
Ž .
USA in 0.2 M phosphate buffer pH 7.4 for 2 h at room temperature, washed three Ž
. times in phosphate buffered saline PBS, pH 7.2, 0.85 NaCl , and then postfixed in
Ž .
2 Osmic acid solution Wako for 1 h at 48C. After washing three times with PBS, the Ž
. samples were dehydrated in ethanol 50–100 , freeze-dried in t-butyl alcohol using an
Ž .
HITACHI ES2030 freeze dryer Hitachi, Japan , attached to larger metal discs, sputter- coated with Platinum–Paladium using an HITACHI E-1030 ion sputter, and observed
with an HITACHI S-4200 scanning electron microscope.
Fig. 2. Histogram of fluorescence intensity from veliger larvae cells of the scallop, P. yessoensis by UV irradiation for 10, 20, 30, 40, 50 and 60 s. The mean fluorescence intensity values from diploid control cells in
10–60 s irradiation groups were 47.73.1, 48.13.5, 46.93.9, 48.53.5, 48.93.0, and 48.73.2 FU, respectively. The data in experiments 1, 2 and 3 were combined.
3. Results
