www.elsevier.nlrlocateraqua-online
Comparative viability and growth of hybrids
between two sympatric species of sea urchins
ž
Genus Echinometra in Okinawa
/
M. Aminur Rahman
a, Tsuyoshi Uehara
a,), Laode M. Aslan
ba
Department of Marine and EnÕironmental Sciences, Graduate School of Engineering and Science, UniÕersity of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan
b
Biological Institute, Graduate School of Science, Tohoku UniÕersity, Sendai 980-77, Japan
Received 27 December 1998; accepted 2 August 1999
Abstract
Among the four sympatric but genetically divergent forms of the sea urchins belonging to
Ž . Ž .
Echinometra mathaei sensu lato, Echinometra sp. A Ea and Echinometra sp. C Ec were
studied through a series of cross fertilization experiments and rearing of the resulting hybrids. Detailed investigations on their larval and adult growth, development and viability were carried
Ž .
out. Heterosis was judged at different stages from larval to 1-year-old F hybrids of Ec ova1 =
Ž . Ž . Ž .
Ea sperm and Ea ova =Ec sperm and at the same time they were compared to their parental species controls. For simplicity the maternal species is named first in case of heterogametic crosses. The hybrids mostly appeared to be intermediate between that of the parental species. The
Ž
average performance traits from fertilization to 3-month-old juvenile stages viz. fertilization rate,
. Ž . Ž .
larval survivality, metamorphosis rate, recovery rate of juveniles of Ec ova=Ea sperm hybrids were significantly better than their reciprocal Ea=Ec hybrid but not significantly different from their conpecifics, Ea=Ea and Ec=Ec. After 1 year of rearing, the final mean weight attained by Ea=Ea, Ec=Ea, Ea=Ec and Ec=Ec was 14.7, 13.9, 13.6 and 9.2 g, respectively. Among the crosses, the growth of both the reciprocal hybrids were much faster than the conspecific Ec=Ec and slightly less than Ea=Ea. The weight increase by Ec=Ea and Ea=Ec hybrids were 51.7% and 48.4% higher over the conspecific Ec=Ec, respectively. The fast-growing Ea species thus partially transmitted this trait to the hybrids. Improvement of quality traits in the majority of the
Ž .
performance traits viz. final weight, weight gain, gonad weight in the F hybrids compared to the1
) C orresponding author. T el.: q81-098-895-8897; fax: q81-098-895-8897; e-m ail:
ueharago@sci.u-ryukyu.ac.jp
0044-8486r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
parental species might have important implications to the development of a sea urchin fishery.
q2000 Elsevier Science B.V. All rights reserved.
Keywords: Sea urchin; Echinometra sp.; Hybridization; Viability; Performance traits; Heterosis
1. Introduction
Along the coast of Okinawa Islands, one of the most conspicuous organisms is the echinoid, Echinometra mathaei sensu lato. This species occurs commonly in and around reefs. It is found from central Japan in the north, to southeast Australia in the south, and from Clarion Island off Mexico in the east, and to the gulf of Suez in the west
ŽMortensen, 1943; Kelso, 1970; Clark and Rowe, 1971; Russo, 1977 . Based on.
extensive studies on physiological adaptation and reproduction, external features of eggs and sperms, larval and adult morphologies, karyotypes, spawning seasons and distribu-tion patterns, it has been concluded that sympatric E. mathaei sensu lato in Okinawa must be recognized as four different species, distinguished tentatively as Echinometra
Ž .
species A, B, C and D. Uehara et al., 1990, 1991; Arakaki and Uehara, 1991 . Recent
Ž .
biochemical studies on isozymes, proteins Matsuoka and Suzuki, 1989 , molecular
Ž .
evidence Matsuoka and Hatanaka, 1991; Palumbi and Metz, 1991 , microhabitats
ŽNishihira et al., 1991 , DNA analysis Metz et al., 1991 , and gamete recognition. Ž .
Ž .
protein binding Metz and Palumbi, 1996 also suggested these are separate but closely related species which have recently speciated.
Ž .
Of the four species of Okinawan Echinometra, Echinometra sp. A Ea and
Ž .
Echinometra sp. C Ec can be distinguished from each other by differences in adult
morphology and habitat preferences. Ea is abundant in more or less protected, constantly submerged habitat which is very calm and situated below the level of MLWS, such as tidepools and shallow reef slops or areas protected from strong wave action, whereas Ec inhabits the reef margin or higher places in the intertidal zone or can occupy deep
Ž
burrows, situated above the level of MLWS Tsuchiya and Nishihira, 1984, 1985;
.
Arakaki and Uehara, 1991; Nishihira et al., 1991 . The reproductive season of these two
Ž .
species is the same Arakaki and Uehara, 1991 . Ea differs conspicuously from Ec on the basis of color patterns, spicule characteristics of gonad and tube feet, gamete sizes,
Ž .
test dimensions and growth performances Uehara and Shingaki, 1985; Rahman, 1997 . The gonads of sea urchins either fresh or in the form of food paste have long been
Ž .
used as luxury foods in Japan Shimabukuro, 1991 . The average wholesale price of
Ž .
fresh Japanese sea urchin gonads regardless of sex is approximately ¥14,000rkg,
Ž .
making it one of the most valuable sea foods in the world Hagen, 1996 . Echinometra
Ž
spp. have also been used as a food source by the common people in Hawaii Hobson and
. Ž .
Chave, 1990 and in Indonesia Arakaki, pers. commun. . During our experimental samplings in the sea, we also observed that some of the local Okinawan people dissected out gonads directly from the live specimens of Echinometra spp. at low tide from the shallow intertidal zone. Although, a large number of studies have been conducted on
Ž
hybridization in fishes for review see Mukhopadhyay and Dehadrai, 1987; Hecht et al.,
. Ž
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 studies1 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 26807 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 268C–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 10y4 to 10y3 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
.
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 268C–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
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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 258C–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
.
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
Cross-fertilization between the two sympatric Echinometra sp. A and Echinometra sp. C was conducted using all possible combinations. During each cross-fertilization experiment, eggs from the same female and sperm from the same male were used and gametes from different individuals of the same species were not mixed together. In crosses with the eggs of 13 female Echinometra sp. A, the percent fertilization of the
Ž .
conspecific controls Ea=Ea averaged 99.78% and was higher than that obtained from
Ž . Ž .
the corresponding Ec=Ea crosses 92.82% , but was not significantly so P)0.05 .
Ž . Ž
The mean percentage of fertilization of Ea=Ea 99.78% differed significantly P
-. Ž . Ž .
0.05 from that of the Ea=Ec crosses 42.38% Table 1 . In crosses with the eggs of 13 females of Echinometra sp. C, the percent fertilization of the conspecific controls
Table 1
Performance of hybrid groups and their conspecific controls from fertilization to 3-month-old juvenile urchins All values represent mean"S.D. with ranges in parentheses. Mean values for groups within each experiment
Ž .
with common superscripts in the same column are not significantly different P)0.05 .
1 2
Ž . Ž . Ž . Ž .
Treatment groups Fertilization % Survival % Metamorphosis % Recovery %
a a a a
Matured larvae that were deemed competent for metamorphosis after a 20- to 24-day culture period in laboratory condition.
2
ŽEc=Ec averaged 99.31% and was significantly different. ŽP-0.05 from that ob-.
tained from the corresponding Ea=Ec crosses, but it did not differ significantly
ŽP)0.05 from that of Ec. =Ea crosses Table 1 . Therefore, cross fertilization wasŽ .
highly asymmetrical. Ec ova are easily fertilized by either Ec or Ea sperm, but even a
Ž y1 .
very high concentration of Ec sperm above 10 dilution of ‘dry’ sperm produced a
Ž .
very low percent of fertilization 42.38% of Ea ova. This reduction, at least, may be due to the presence of gamete recognition protein binding system as reported by Metz and
Ž . Ž .
Palumbi 1996 and Metz et al. 1994 . These results followed the same trends as
Ž .
reported by Uehara et al. 1990 .
Survival of mature larvae for the hybrid produced using the eggs of Ec was not
Ž .
significantly different P)0.05 from the pure parental matings. On the other hand, the
Ž . Ž .
survival rate for the reciprocal hybrid Ea=Ec was significantly P-0.05 lower
ŽTable 1 . In this case mortality usually occurred during various stages of larval.
development.
The laboratory-raised larvae of the parental species and the hybrids reached a state of metamorphosis at about 20–24 days of age as evidenced by having large rudiment. Complete metamorphosis from feeding larvae to feeding adult took place in about 1 day after settlement on red algal substratum. This included the complete development of adult internal organs as well as the formation of the adult mouth, anus, tubefeet, and spines. Completion of metamorphosis was the same and the rate of development was
Ž .
equivalent among all treatments Table 1 . Moreover, the time required to complete development from the eight armed stage to metamorphosed juvenile was similar among all treatments. The majority of the larvae were metamorphosed to young juveniles on
Ž .
CRAS collected from the shallow sea within 1 day and there were no particular deformitiesrdefects observed in the metamorphosed juvenile hybrids, Ec=Ea and
Ž .
Ea=Ec. The Ea=Ec hybrids showed significantly P-0.05 lower metamorphosis rate compared with other groups, but Ec=Ea hybrids did not differ significantly
ŽP)0.05 from their pure parental species..
After 3 months of rearing, the juvenile urchins were transferred to a grow out system with flow-through sea water. It was found that the recovery of Ea=Ec hybrids was
Ž .
significantly P-0.05 lower compared to parental species controls, but the hybrids
ŽEc=Ea did not differ significantly P. )0.05 from these..
The detailed growth and survival of the hybrids and their parental species at the end of the experimental period are summarized in Table 2 while the growth trend is depicted in Fig. 1. It was observed that though the hybrids were intermediate to their parental species, the growth of hybrids was much faster than the slow-growing conspecific Ec=Ec. and slightly less than the fast-growing Ea=Ea cross under identical rearing
Ž .
conditions Table 2 and Fig. 1 . The mean total weight gained by Ea=Ea, Ec=Ec, Ec=Ea, and Ea=Ec was 14.5"0.12, 9.1"0.01, 13.8"0.09, and 13.5"0.03 g respectively during the culture period of 1 yr. Between the two hybrids, Ec=Ea values were consistently higher than Ec=Ec and reciprocal cross Ea=Ec although, slightly
Ž . Ž .
but statistically significantly P-0.05 less than Ea=Ea Table 2 . The mean weight
Ž .
gained by the hybrids and parental species differed significantly P-0.05 . Among the four groups, the weight gain between Ec=Ea and Ea=Ec was not significantly
Ž . Ž .
Table 2
Ž . Ž .
Echinometra sp. A Ea and Echinometra sp. C Ec
Details of growth and survival of the conspecific controls and hybrids produced experimentally at the end of
Ž .
the study period. Standard error S.E. and ranges are in parentheses. A total of 30 specimens were measured for weight and volume with 10 per replicate for each treatment.
Parametersrtreatments Ea=Ea Ec=Ea Ea=Ec Ec=Ec
U
a ab bc c
Ž . Ž . Ž . Ž . Ž .
Mean initial weight g 0.16 0.003 0.15 0.002 0.14 0.003 0.13 0.002
Ž0.15–0.16. Ž0.14–0.15. Ž0.13–0.14. Ž0.12–0.13.
a b b c
Ž . Ž . Ž . Ž . Ž .
Mean final weight g 14.7 0.12 13.9 0.09 13.6 0.03 9.2 0.10
Ž14.6–14.8. Ž13.8–14.0. Ž13.6–13.7. Ž9.1–9.3.
a b b c
Ž . Ž . Ž . Ž . Ž .
Weight gain g 14.5 0.12 13.8 0.09 13.5 0.03 9.1 0.01
Ž14.4–14.6. Ž13.7–13.90 Ž13.4–13.5. Ž9.0–9.2.
a b b c
Ž . Ž . Ž . Ž . Ž .
Wet gonad weight g 2.39 0.030 2.27 0.027 2.22 0.025 1.30 0.017
Ž2.25–2.53. Ž2.18–2.40. Ž2.10–2.35. Ž1.21–1.40
3 a b b c
Ž . Ž . Ž . Ž . Ž .
Test volume cm 11.4 0.17 10.4 0.12 10.1 0.01 6.4 0.04
Ž11.2–11.6. Ž10.2–10.5. Ž10.0–10.2. Ž6.4–6.5.
a a b a
Ž . Ž . Ž . Ž . Ž .
Survival % 84.44 1.92 77.78 1.11 70.00 1.92 82.22 2.94
Ž83.33–86.67. Ž76.67–80.0. Ž66.67–73.33. Ž76.67–86.67.
U
Ž .
Mean values in each row having same superscript were not significantly different P)0.05 .
the values were comparatively much closer to their fast growing parental Ea species.
Ž .
Test volumes length=width=height also followed the same trends as those of weight
Ž . Ž .
gain Table 2 . The fresh weight of edible gonad was significantly P-0.05 lowest in Ec=Ec. The hybrids contained a large amount of gonad, but slightly less than Ea=Ea,
Ž .
Fig. 1. Mean live weights g attained by the parental species controls and hybrids produced experimentally
Ž . Ž .
using Echinometra sp. A Ea and Echinometra sp. C Ec during the culture period of 1 year when they contained mature gametes; maternal species are named first. A total of 30 specimens were measured every month with 10 per replicate for each treatment. ANOVA analysis showed that growth of the hybrids, both of
Ž .
the reciprocal crosses were not statistically significant P)0.05 but that they both differed significantly from
Ž .
Ž .
although the values were significantly different P-0.05 . The reciprocal hybrids did not differ significantly. The gonad production showed an increment of 21.6% in F1
Ž .
hybrids over mid-parents mean of both parental species . While it showed an increase of 74.6% in F hybrid of Ec1 =Ea and 70.8% in F hybrid of Ea1 =Ec over the inferior parents Ec. Survival was highest in Ea=Ea followed by Ec=Ec, and Ec=Ea in that
Ž . Ž .
Fig. 2. Echinometra sp. A Ea and Echinometra sp. C Ec and their reciprocal hybrids, produced
Ž . Ž . Ž .
experimentally, 1 year after metamorphosis; maternal species named first. A Ea=Ea; B Ea=Ec; C
Ž .
Ž .
order, which were not significantly different P)0.05 but the hybrid, Ea=Ec differed
Ž . Ž .
significantly P-0.05 from other groups Table 2 . Therefore, the results of growth
Ž .
and survival indicated that the hybrids Fig. 2 in both directions were viable in lab-reared conditions. The 1-year-old mature hybrids exhibited color patterns similar to those of their maternal colorations, although the reciprocal hybrids were closer to Ea in
Ž .
size Fig. 2 .
4. Discussion
Ž .
The present hybridization experiment indicated that the hybrids, Ec ova =Ea
Žsperm showed better results from fertilization to adult stage in all performance traits.
Ž .
similar to those of their parental species Ea. The reciprocal hybrids, Ea ova =Ec
Žsperm were inferior in many performance traits, especially in larval stages, although.
they showed better performances in advanced stages. Similar trends were observed in
Ž
fishes for review see Mukhopadhyay and Dehadrai, 1987; Laywonyawut et al., 1992;
.
Rahman et al., 1995 .
The growth pattern and survival of all four combinations showed similar trends in triplicate aquaria. It was observed that Ea=Ea registered faster growth than the other groups throughout the experimental period, while the slowest growth was observed in Ec=Ec. The growth of reciprocal hybrids was almost the same throughout the culture period of 1 year. Subsequently, the growth of Ec=Ea was surpassed by Ea=Ec and maintained the same trend until the end of the experiment. Growth of the reciprocal hybrids mostly appeared to be intermediate between that of parental species. The fast-growing parent partially transmitting this trait to hybrids. The growth data, however, confirmed that hybridization between Ea and Ec was successful in laboratory-rearing conditions and the hybrids were viable and showed superior performances over the mid
Ž
parent values. Similar results were also observed in fishes for review see Konda Reddy, 1977; Chevassus, 1983; Varghese et al., 1984; Bhowmick et al., 1987; Maheshwari et
.
al., 1990; Basavaraju et al., 1995, etc. .
Although the gonad weight in 1-year-old cultured hybrids was closer to the Ea
Ž .
parental species, the values differed significantly P-0.05 . Ec contained a less gonadal tissue. The hybrids displayed better performance in edible gonad production compared to those of the parental Ec species. On the contrary, although they produced gonads within 1 year, it may be necessary to cultivate them at least 2 years to get an adequate amount of gonads. For further study, factors controlling gonad weight or volume such as food, age, temperature should be determined.
Among the commercial sea urchins of Japan, Tripneustes gratilla is considered one of the most important species fished in the Okinawa and the Amami Islands. Its gonad is
Ž .
the gonad of these species. To fulfill the demand of the increasing population, people might pay attention to use this species as a source of food in near future. Sea urchins have long been used as a capture fishery in the world. The majority of the common consumers harvest them from the sea. Due to difficulties with rearing urchins, no one shows an interest to culture them in captivity. Our overall culture method presented here is very convenient, which would provide useful guidelines for those who are interested to culture sea urchins. On the other hand, this is the first successful attempt to produce viable hybrids through cross fertilization between two genetically divergent species of sea urchins. Until now no one has reported these types of experiment in sea urchins. The main findings of this experiment is the superior performance of the F1 hybrids, compared to mid parent values. Hence these hybrids may be considered for future use in aquaculture.
Acknowledgements
We wish to express our gratitude to Dr. K. Takano and Staffs of Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, for use of their facilities to culture the urchins. Special thanks are also due to Dr. Nurul Khan and Adriane Fink for kindly reading this manuscript.
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