Shokita, 1978; Lawrence et al., 1984; Hedgecock, 1987; Bray et al., 1990; Benzie et al., .
1995; Misamore and Browdy, 1997, etc. , no such information is available concerning sea urchins.
Between the two species of Okinawan Echinometra, Ea is the largest, while Ec is smaller. Due to its smaller size and smaller amount of edible gonads, it does not show a
wide acceptance by the consumers. Therefore, these two species were hybridized to explore the possibility of combining desirable traits in the F hybrids. Detailed studies
1
on their larval and adult development, growth performance and aquaculture potential were investigated and the present paper deals with the viability of hybrids in comparison
with their parents under similar rearing conditions.
2. Materials and methods
2.1. Collection and maintenance Mature specimens of Echinometra sp. A and Echinometra sp. C were collected from
Ž
X X
. the Sunabe coast 26
807 N; 127846 E , Okinawa Island at low tide by snorkeling and walking along the sea shore during their natural breeding season from early May to the
end of October, 1996 and from early May to the end of July, 1997. Immediately after collection, the specimens were transported to the laboratory in the Dept. of Biology,
University of the Ryukyus, Okinawa, Japan and maintained in closed aquarium before use for in vitro crosses.
2.2. Cross-fertilization Cross-fertilizations between the gametes of the two Echinometra species were
Ž .
conducted at room temperature 26 8C–288C using all possible combinations of ova and
sperm. For simplicity, when referring to the heterospecific crosses, the maternal species is named first. For example, a hybrid produced by cross-fertilization between the female
of Ea and male of Ec is denoted as Ea = Ec. For each heterospecific fertilization, a conspecific fertilization of eggs from the same female served as a control. In crosses
between female Ea and male Ec for example, 13 control cultures derived from the eggs from 13 females were maintained in parallel with 13 cross-fertilized cultures.
Fertilization was done by mixing two drops of a diluted sperm into one petri dish containing 15 ml conspecific egg suspensions and another dish with 15 ml of heterospe-
cific egg suspensions. The sperm concentration was generally 10
y4
to 10
y3
dilution of Ž
. ‘‘dry’’ sperm Uehara et al., 1990 . The eggs were then layered on the bottom of petri
dishes and incubated at ambient room temperature for 1 h. Aliquots from each dish were then placed on microscope slides, which were examined in equally spaced transects run
from one end of the cover-slip to the other. The first 100 eggs encountered were
Ž classified as ‘‘fertilized’’ if they had reached the 2–4 cell stage. In all cases including
. heterospecific crosses they produced apparently normal plutei in approximately 32 h.
2.3. LarÕal rearing Early stage embryos from the same female were reared in standing cultures in small
glass beakers. When blastulae were seen swimming at the surface of the water, they Ž
. were transferred to glass bottles containing 400 ml of filtered sea water FSW which
was stirred constantly by 10 rpm rotating motors. Larval densities up to the four-armed pluteus stage were maintained at 2–3 individualsrml. When the larvae attained four-
Ž .
armed pluteus stage, they were cultured in the same system 400 or 800 ml glass bottles with a larval density of 1 individualrml. All cultures were carried out in FSW at
26 8C–288C, approximating ambient water temperature. About 50–75 of the culture
water was removed by reverse filtrationrsiphoning every 3 days and replaced with fresh FSW. Larvae were supplemented with a laboratory cultured phytoplankton, Chaetoceros
gracillis at concentrations of 5000, 10,000 and 15,000 cells per ml of medium at four-, Ž
six- and eight-armed stage, respectively, by adjusting the food level every 3 days Wu et .
al., 1990 . 2.4. Metamorphosis
After 20–24 days of rearing, the mature larvae that were deemed competent were used in settlement induction tests. Competence was indicated by the presence of large
juvenile rudiments and a high rate of metamorphosis. Induction of metamorphosis for all Ž
. crosses was performed on coralline red algal stones CRAS , which were immersed into
FSW in the petri dishes containing 30 ml FSW each. Larval density at this stage was maintained at 1 individualr2 ml FSW. The majority of the larvae tended to metamor-
phose within 1 day.
2.5. JuÕenile rearing Five days after completion of metamorphosis the newly produced juveniles were
Ž .
moved to the aerated small glassrplastic aquaria in static water conditions with settlement substrates of crustose CRAS and coral skeletons with filamentous algae for
food. This was continued for up to 3 months. In all rearing, sea water was partially changed every week, and replenished with new sea water to maintain ambient tempera-
Ž .
Ž .
ture 25 8C–288C and salinity 35 ppt .
2.6. Culture of adults Three months after metamorphosis the juveniles of both conspecific and heteropecific
Ž .
crosses were placed in aerated plastic aquaria 36 = 45 = 18 cm with flow through seawater at the Sesoko Station, Tropical Biosphere Research Center, University of the
Ryukyus. These were provided with crustose coralline red algal encrusted coral skele- Ž
tons for food. The experiment was conducted using four treatments two for conspecific .
controls and two for both hybrids with three replications for each. The stocking density
was maintained at 1 individualr1 l of seawater. The algal stones were changed at an interval of 15 days with new ones to supplement them with adequate algal foods. The
culture was continued up to 1 year by which time both the parental species and their reciprocal hybrids contained mature gametes. Growth performance and health conditions
of the cultured urchins were monitored through monthly samplings.
2.7. Data analysis Statistical analysis in this experiment was performed by one way analysis of variance.
Ž .
Comparison between treatment means was carried out by analysis of variance ANOVA Ž
. followed by Duncan’s Multiple Range Test Duncan, 1955 and the significance of
variation between the means was tested using the computerized Stat View version 4.0 program.
3. Results
