3. Results
3.1. Effects of UV irradiation dosage on the rates of fertilization and deÕelopment Relationships between the duration of UV irradiation and the rates of fertilization and
development of D-shaped larvae are shown in Fig. 1. In experiments 1, 2 and 3, the fertilization rate for controls was more than 90, but was apparently reduced with
increasing exposure time, falling to 70–80 by 30 s and to approximately 45–60 by 60 s irradiation. As UV irradiation duration increased, the developmental rate of
D-shaped larvae decreased sharply and became 0 by 30 s irradiation in each of the three experiments, at the same time more and more eggs developed into abnormal larvae and
never reached D-larval stage.
3.2. Microfluorometry Fig. 2 shows the distribution of fluorescence intensity from veliger larvae cells of P.
yessoensis by UV irradiation for 10, 20, 30, 40, 50 and 60 s. For the 10- and 20-s irradiation groups, the fluorescence intensity modal values of veliger larvae cells from
eggs inseminated with UV-irradiated sperm were still observed at 40–55 FU, the same as those of diploid control cells, and cells having lower fluorescence intensity value
apparently increased. For the 30- and 40-s irradiation groups, in spite of a small number of cells showing the diploid control values, the mode at around 20–27 FU, which was
estimated to be the haploid values, was clearly seen. For the 50- and 60-s irradiation groups, the mode at around 20–27 FU became more distinct and almost no cells
exhibited the diploid control values, and the mean fluorescence intensity values of larvae cells were, respectively, 25.2 5.4 and 24.5 5.6 FU, which were approximately half
Ž .
that of diploid control cells 48.9 3.0 and 48.7 3.2 FU , indicating that haploid gynogenesis was successfully induced.
Ž . Fig. 3. SEM micrographs of normal spermatozoa of the scallop, P. yessoensis. a Whole spermatozoa
Ž .
Ž . bar s 4 mm . Arrowhead indicates a immature spermatozoon with a rounded head; b the nucleus region of a
Ž .
spermatozoon bar s1 mm . N, Nucleus; A, Acrosome; M, Mitochondria; F, Flagellum.
3.3. Effect of UV irradiation on sperm morphology The structure of normal untreated sperm was observed by SEM for comparison with
Ž .
the irradiated sperm Fig. 3a,b . The mature spermatozoon of P. yessoensis was
3.9 0.2 mm long when measured from the tip of the acrosome to the distal end of the Ž
. mid-piece n s 30 . It was composed of a cone-shaped acrosome, an elongated nucleus,
Ž .
a short mid-piece and a single flagellum Fig. 3b . The nucleus was conical in form, Ž
. 3.0 0.2 mm long and 1.3 0.1 mm wide n s 30 . The acrosome measured 0.5 0.1
Ž .
mm in length and 0.5 0.0 mm in maximum diameter n s 5 . The midpiece showed the typical primitive arrangement with mitochondrial spheres around a centriolar appara-
Ž .
tus. The flagellum had a total length of 50.2 2.9 mm n s 10 . In addition, the
immature sperm showing a rounded head and a flagellum, but without an acrosome, was also seen in the discharged sperms of the scallop.
Next, the sperms that have been irradiated for 0, 30, 60, 90 and 120 s were examined. Based on the changes in their morphology, the UV-irradiated mature sperms were
Ž . Ž .
Fig. 4. SEM micrographs of UV-irradiated spermatozoa of P. yessoensis bar s1 mm . a A 30-s irradiated Ž .
spermatozoon characteristic of group A, in which no morphological changes could be observed; b a 30-s Ž .
irradiated spermatozoon characteristic of group B, in which the flagellum was lost; c a 60-s irradiated Ž .
spermatozoon characteristic of group C, in which the acrosome was abnormal; d
a 120-s irradiated spermatozoon characteristic of group D, in which the acrosome and flagellum were abnormal.
Table 1 Ž
. Distribution of spermatozoa from various UV irradiation doses, classified into groups A–D based on the
changes in morphology as assessed by SEM Figures in parentheses are number of spermatozoa observed.
Ž . UV irradiation duration s
30 60
90 120
Ž . Ž
. Ž
. Ž
. Ž .
Ž . Group A
98 51 64 36
29 16 4 2
0 0 Ž .
Ž . Ž .
Ž . Ž .
Ž . Group B
2 1 4 2
4 2 2 1
0 0 Ž .
Ž . Ž
. Ž
. Ž
. Ž
. Group C
0 0 29 16
44 24 28 15
23 13 Ž .
Ž . Ž .
Ž .
Ž .
Ž .
Group D 0 0
4 2 24 13
66 35 77 43
classified into the following four groups: Group A sperms showed no apparent change in Ž
. Ž
. morphology Fig. 4a ; Group B sperms had a missing flagellum Fig. 4b ; Group C
Ž .
sperms had an abnormal acrosome Fig. 4c ; and Group D sperms had both a missing Ž
. flagellum and an abnormal acrosome Fig. 4d . For each irradiation duration, more than
50 randomly selected mature sperms were used to classify the sperms into groups A–D. Table 1 shows the changes in percentage of the four groups of sperms in different UV
irradiation groups. The group A type accounted for 98 of the control group sperms, but this percentage decreased significantly with increasing exposure time. The percent-
age of group B sperms was consistently low and changed little in all groups. Group C sperms were not observed in the control group but occurred in all irradiation groups,
accounting for 29, 44, 28 and 23 of sperms in the 30, 60, 90 and 120 s irradiation groups, respectively. Group D sperms markedly increased with increasing
irradiation duration, reaching a maximum proportion of 77 in the 120-s irradiation group.
4. Discussion
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