¹Graduate School of Marine Science and Engineering
Nagasaki University, Nagasaki 852, Japan
²Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan
³College of Ocean Sciences, Cheju National University Cheju 590, Korea
ABSTRACT Genetic characteristics of the sea urchin Pseudocentrotus depressus were compared by allozyme analysis for six wild populations: three artificial seed populations (artificial populations), and three mixed populations from the sites where artificial seeds were stocked. Of 25 enzymes analyzed by horizontal starch gel electrophoresis, 9 genetic loci were detected in 7 enzymes (LDH, MDH, IDHP, PGDH, HK, GPI, and PGM). In the 6 wild populations, mean proportion of polymorphic loci and heterozygosity were 0.612 and 0.164, respectively. The divergence points in the dendrogram of Neis genetic distance (D) of 6 populations did not exceed 0.004. The fixation index (FST) was 0.014. These two facts suggest that wild populations were genetically homogeneous.
In the three artificial populations, one showed a remarkable decrease in the proportion of polymorphic loci and heterozygosity. The divergence points in the dendrogram of D of the 3 populations ranged from 0.006 to 0.021. FST value among them was 0.118. This suggests that artificial populations were genetically different from each other, and that artificial populations and wild populations were genetically heterogeneous.
Two of three mixed populations were genetically similar to wild populations. However, in one of the populations, the proportion of polymorphic loci and heterozygosity decreased, and the divergence point in the dendrogram of D was 0.011, suggesting that this population was genetically affected by released artificial seed.
INTRODUCTION Pseudocentrotus depressus is an endemic species of sea urchin found in subtropical Far-Eastern waters, southern Japn, and the south coast of Korea, inhabiting the upper infralittoral zone on rocky shores. The species has been an important target of commercial divers. In southern Japan, mostly along the northwest coast of Kyushu, large numbers of artificially produced seed of P. depressus were released in the last 10 years in response to demands of fishermen to enhance the wild stocks. However, there is a concern from the point of view of population genetics. The artificial seeds are genetically distinct from the wild population as a result of genetic drift which occurred because seeds were produced from a limited number of parents. Release of those genetically different seeds to the sea could affect the genetic structure or composition of the wild population. Release of seeds might cause a decline in genetic variability of the wild population. The greater the use of enhancement, the greater the concern that the genetic variability of the wild populations is adversely affected. These efforts may be greater for animals that have limited mobility, such as sea urchins.
MATERIALS AND METHODS
Sea Urchin CollectionTwelve samples of Pseudocentrotus depressus were collected for the study: 11 from southern Japan and one from Cheju Island, southern Korea. Six are wild populations (W1-W6; Fig. 1 and Table 1) collected by professional divers from the sites where artificial seeds of the species have never been released, except for W3 described below. Three are artificial seeds (A1-A3; Fig. 1 and Table 1) reared entirely in marine farming institutions until release. The last three are mixed populations of wild ones and non-wild ones originated from the released artificial seeds (M1-M3; Fig. 1 and Table 1) collected by the professional divers from the sites where artificial seeds had been released in recent years.
At the site where the mixed populations M1 and M2 were collected, artificial seeds were frequently released during the last 10 years. At the site where the mixed population M3 was collected, artificial seeds that were spawned in autumn 1990 were released only once in the early summer of 1991. Population M3 contains adults from seeds released in 1991, judging from their size and age. One of the wild population, W3, was collected from the same site where M3 was collected, but it did not contain artificial seeds released in 1991 because they were considerably larger than individuals grown from artificial seeds released in 1991.
Live animals were transported to Nagasaki University in insulated containers containing wet seaweed or newspapers with ice cubes or freeze packs. All the sea urchins were dissected within several days after arrival at the laboratory, while they were still alive. Dissected gonad and oesophagus were stored at -40 or -85oC until further analysis. The shell top of all the specimens, including the genital plates, were dried and preserved for age determintion.
Enzyme nomenclature and enzyme commission numbers assigned followed the recommendations of the International Union of Biochemistry (1984). Abbreviations of enzyme and locus and allele symbols followed Shaklee et al. (1990).
Electrophoretic Procedures
Electrophoresis was carried out using standard horizontal starch gel system (Hoelzel 1992). Thawed drips of the two tissues, gonad and oesophagus, were used as crude extracts of enzymes (Numachi 1971). Filter paper wicks saturated with the extracts were inserted in the cut portion of the gel. Eight buffer systems (pH 6.0-9.2) were used. Two of those 8 buffer systems, C-A pH 7.0 (modified from Clayton and Tretiak 1972) and T-C pH 8.0 (modified from Siciliano and Shaw 1976), were used through the study.
Twenty-five enzymes were examined in extracts of gonad and intestine. Enzyme staining procedures followed those of Siciliano and Shaw (1976) for ADH (EC: 1.1.1.1), G3PDH (1.1.1.8), LDH (1.1.1.27), GLYDH (1.1.1.29), MDH (1.1.1.37), MEP (1.1.1.40), IDHP (1.1.1.42), PGDH (1.1.1.44), G6PD (1.1.1.49), SOD (1.15.1.1), AK (2.7.4.3), ACP (3.1.3.2), TPI (5.3.1.1), MPI (5.3.1.8), GPI (5.3.1.9), PGM (5.1.2.2); those of Shaw and Prasad (1970) for IDDH (1.1.1.14), XDH (1.2.1.37), CAT (1.11.1.6), HK (2.7.1.1), EST (3.1.1.*), ALP (3.1.3.1); those of Ayala et al. (1972) for ODH (1.1.1.73); those of Christfferson et al. (1978) for AAT (2.6.1.1); and those of Kayano (1978) for LAP (3.4.11.1).
Statistical Analysis
Significance of departure from Hardy-Weinberg proportions was tested using the AIC (Akaike Information Criterion, Akaike 1973) value of observed and expected allelic frequency at each locus in each population sample.
Proportions of polymorphic loci (P0.95; frequency of the most common allele <0.95), number of alleles per locus (A), and mean heterozygosity; both the observed (HO) and the expected (HE) were calculated for each population.
The amount of genetic divergence among populations was calculated by the genetic variance statistics FST (Wright 1965) at each locus for the three groups: wild, artificial, and mixed populations. In addition, a mean FST for all loci was obtained for each group.
Neis genetic distance (D) was calculated between all pairs of 12 populations (Nei 1972). A dendrogram based on the genetic distance was constructed using the unweighted pair-group arithmetic average (UPGMA) clustering (Sneath and Sokal 1973).
RESULTS
Enzyme Activity and Zymogram InterpretationTen enzymes (G3PDH, IDDH, GLYDH, MEP, XDH, AAT, AK, ALP, ACP, and TPI) did not show any activity in any of the eight buffer systems. Eight enzymes (ADH, G6PD, ODH, CAT, SOD, EST, LAP, and MPI) showed some activity, but resolution of zymograms was not improved by any buffer systems or activities were not stable and could not be resolved for all individuals. Seven enzymes (LDH, MDH, IDHP, PGDH, HK, GPI, and PGM) showed both stable activity and good resolution of zymograms.
Polymerization structure, detected locus, and number of alleles at each locus were interpreted as follows: LDH: monomorphic, one locus LDH*; MDH: polymorphic, dimer, two loci MDH-1* and MDH-2*, each two alleles; IDHP: polymorphic, dimer, one locus IDHP*, three alleles; PGDH: polymorphic, dimer, one locus PGDH*, three alleles; HK: polymorphic, monomer, one locus HK*, three alleles; GPI: polymorphic, dimer, one locus GPI*, five alleles; PGM: polymorphic, monomer, two loci PGM-1* and PGM-2*, each three alleles.
Genetic Variability
Allelic frequencies and genetic variability (P0.95, A, HO , and HE) for 12 populations of Pseudocentrotus depressus are shown in Table 2.
Artificial population 1 (A1) and mixed population 3 (M3) had discernibly less genetic variation; and P0.95 was 0.222 and 0.333, respectively. In contrast, values of the other 10 populations were 0.5-0.7. In addition, the number of alleles per locus of these two populations was less than 2 for both populations.
In all populations except W2 and A1, observed heterozygosities (HO) were similar and were from 0.135 to 0.169. In the remaining two, W2 and A1, HO was 0.047 and 0.206, respectively. Mean HO of the 12 populations was 0.1480 (SD, 0.0340). The mean value of HO/HE was 1.0105 (SD, 0.0815).
FST for the three groups, wild (six populations, W1-W6), artificial (three populations, A1-A3), and mixed (three, M1-M3) are shown in Table 3. In 6 wild populations, the FST of each locus ranged from 0.0056 to 0.0475 and the mean was 0.0139. Those in artificial group were 0.1178 (mean, 0.0107 (min.), and 0.2450 (max.). In the mixed group, they were 0.0339 (mean), 0.0049 (min.) and 0.0744 (max.). The means were largest in the artificial and smallest in the wild populations.
Genetic Distance and Dendrogram
Neis genetic distance (D), calculated between all pairs of 12 populations, are shown in Table 4. A dendrogram based on genetic distance using the unweighted pair-group method of analysis (UPGMA, Sneath and Sokal 1973) is shown in Figure 2.
None of the values of diverging points for the six wild populations, W1 to W6, exceeded 0.004. Judging from Figure 2, two of the three mixed population (M1 and M2) were genetically similar to the wild ones. However, the diverging point of M3 population was fairly large. In contrast, those values for the three artificial populations, A1 to A3, were all large. Among the three, A2 is remarkably different from the others. Genetic distance of the diverging point between A2 and the other 11 populations all exceeded 0.02 (Fig. 2).
DISCUSSION
Mean H for the 6 wild populations used in this study were fairly high: 0.165 for HO (range: 0.138-0.206; SD: 0.024) and 0.163 for HE (0.139-0.191; 0.018) in comparison to reported values for other species of the class Echinoidea. A range of mean values of 0-0.05 (mean: 0.02) has been reported for the four species, Toxopneustes pileolus, Tripneustes gratilla, Pseudoboletia maculata, and Pseudocentrotus depressus (Matsuoka 1985); 0-0.055 (mean: 0.033) for the two species, Echinostrephus aciculatus and E. molaris (Matsuoka and Suzuki 1987); 0.031-0.043 (mean: 0.035) for Diadema setosum (Matsuoka 1989); 0.035 for Diadema savignyi (Matsuoka 1989); 0.027-0.056 (mean: 0.0415) for Echinothrix calamaris (Matsuoka 1989); 0.035 for Echinothrix diadema (Matsuoka 1989); 0.028-0.088 (mean: 0.0632) for Anthocidaris crassispina (Matsuoka and Suzuki 1989); 0.072 for Glyptocidaris crenularis (Matsuoka and Nakamura 1990); 0.0126-0.0127 (Watts et al. 1990) and 0.055-0.07 (mean: 0.0653) for Echinometra mathaei (Matsuoka and Hatanaka 1991); 0.032-0.038 (mean: 0.035) for Stomopneustes variolaris (Matsuoka and Nakamura 1991). This difference might be caused by an insufficient number of loci evaluated in the present study. More than 15 loci were used in the reported studies.
As shown by the low FST value (0.0139) for the 6 wild populations and their dendrogram (Fig. 2), the gene pool of wild populations of Pseudocentrotus depressus is geographically homogeneous, in spite of their benthic behavior an poor mobility after settlement. However, this homogeneity may be attributable to the fairly long planktonic larval stages that occur before settlement. Therefore, it was not unexpected that the FST value was highest in the artificial and lowest in the wild population.
The dendrogram resulting from this study (Fig. 2) suggests certain relationships among the three mixture populations. Unexpectedly, two of the three mixed populations, M1 and M2, were genetically similar to the wild ones, while the diverging point of the M3 was fairly large and isolated from populations M1, M2, and W1-W6. The populations W3 and M3 were collected from the same site but differ from each other in size as shown in Table 1. According to the age determination as derived from the annual ring appeared in the genital plates (Chung and Natsukari, unpublished), all individuals of M3 were 2 years old or less, and all of W3 were more than 3 years old. A year prior to the collection of M3, 1-year-old seeds that were fertilized in 1990 and whose origin was the same as artificial population A2 were released at the site. No other seed release was made except this release in 1991. Thus, W3 does not include released artificial seed, and all collected were wild individuals. The population M3 should be a mixture of wild and artifically reared urchins. Considering the above facts, M3 was probably influenced genetically by the released seed that had the same origin as artificial seed A2.
Contrary to our expectations, the two mixed populations M1 and M2 were not genetically different from the six wild populations. Artificial seeds were frequently released extending over a 10-year period at the site where the two were collected. The failure of M1 and M2 populations to be distinct from wild populations is attributable to the cumulataive effects of multiple seed releases.
ACKNOWLEDGMENTS
This study was financially supported by the Nippon Life Insurance Foundation Grant and could not have been attempted without considerable cooperation. We are grateful to the Nippon Life Insurance Foundation and the staffs of the institutions and the fishery cooperative associations who supported us in obtaining specimens used in the study.
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