Study on heritability of growth in the juvenile sea urchin, strongylocentrotus nudus.

ABSTRACT The heritability of growth of juvenile Strongylocentrotus nudus was analyzed using quantitative genetic methods. Twenty-one half-sib groups and 60-63 full-sib groups of juveniles were obtained by artificial fertilization of 3-5 females by single males based on a nested design. The body weight (g) and test diameter (cm) of the young were measured 3 and 5 mo after metamorphosis. Maternal component estimates are significantly greater than paternal component estimates for both weight and diameter at both ages. Greater maternal components suggest large nonadditive genetic effects that could not be differentiated with the available data. Estimates of heritability in the narrow sense calculated from the additive genetic component using a paternal half-sib correlation analysis ranged from 0.2167 to 0.4565 for weight and 0.2059 to 0.4998 for diameter. The results indicate significant maternal effects. The strength of the nested design and the paternal haft-sib correlation analysis used in this study make the estimate the most precise and unbiased reported to date.

KEY WORDS: growth, heritability, sea urchin, Strongylocentrotus nudus

INTRODUCTION

Sea urchins are one of the most important aquaculture species in the world. The gonads have long been used as a luxury food and as a food source by common people in many countries (Hagen 1996, Hobson et al. 1990, Shimabukuro 1991). Because of over fishing, interest in aquaculture of sea urchins has greatly increased (Hagen 1998, Lawrence 2001). One of the most important species for aquaculture is Strongylocentrotus nudus (Agatsuma 1998, Hagen 1996). Current culture of S. nudus has used seeds obtained from wild individuals (Gao and Chang 1999, Liao and Qiu 1999). Analysis of populations of S. nudus in Japanese waters show considerable range in size of small individuals, presumably of single cohorts (Agatsuma 1997). This could result from interaction of genetic characteristics and the environment. Vadas et al. (2002) found evidence for intrinsic variability in field populations of Strongylocentrotus droebachiensis. It is important to document the degree of heritability of growth in sea urchins because of its implications for both fisheries and aquaculture.

Demonstration of heritability is best done with comparisons of half-sib groups because they are less likely to be affected by environmental influence (Gjedrem 1992). Sib analysis techniques have been used for a variety of important aquaculture species (Benzie et al. 1997, Crenshaw et al. 1991, Hadley et al. 1991, Newkirk et al. 1977, Mallet et al. 1986, Rawson and Hilbish 1990). The purpose of the current study was to estimate heritability of growth in terms of body weight and diameter of juvenile S. nudus.

MATERIALS AND METHODS

Experimental Design

This study used a classic nested mating design developed by Comstock and Robinson (1952) to partition the phenotypic variation in juvenile growth into its genetic and nongenetic causes. In this experiment, each of 21 male S. nudus was mated to 3-5 females, therefore generating 63 full-sib families and 21 half sib families. The effects of males and females nested within males on growth were separated using nested analysis of variance (ANOVA). Juveniles were weighed and their diameters measured at 3 and 5 mo of age.

Genetic Analysis

The covariance among full- and half-sibs provides the basis for the separation of phenotypic variance into genetic and environmental components of variance. The covariance among full- and half sibs are calculated from the observed components of variance obtained from a three-level nested, unbalanced ANOVA (Table 1) and the General Linear Models procedure of the Statistical Analysis System (SAS) (Freund et al. 1986).

The experiment was a three-level classic nested, unbalanced design. Therefore, the number of offspring in dams and in sires and in dams within sires should revise ("revise" means adjust). The Effective Means were computed using the equations:

Effective mean number of offspring in dams within sires:

[K.sub.1] = [N - [summation of]([n.sup.2.sub.ij]/[dn.sub.i]]/(D - S)

Effective mean number of offspring in dams:

[K.sub.2] = [[summation of]([n.sup.2.sub.ij]/[dn.sub.i]] - [summation of]([n.sup.2.sub.ij]/N)]/(S - 1)

Effective mean number of offspring in sires:

[K.sub.3] = (N - [summation of][dn.sup.2.sub.i]/N)/(S - 1)

in which S = number of sires, D = number of dams, [n.sub.ij] = number of offspring of the i-th sire and j-th dam, [dn.sub.1] = number of offspring of i-th sire, and N = sum of number of offspring of all sires or all dams.

The phenotypic variance ([V.sub.P]) was separated into the additive genetic variance ([V.sub.A]), nonadditive genetic variance ([V.sub.N]), and environmental variance ([V.sub.E]), and the environmental variance ([V.sub.E]) was separated into the common environmental variance ([V.sub.EC]) and the specific environmental variance ([V.sub.ES]) using the standard separation of variance components (Falconer 1989). The causal components of variance were estimated from the full- and half-sib covariance using the relationships in Table 2.

Heritabilities were computed using the relationships: [h.sup.2] = [V.sub.A]/ [[V.sub.A] + [V.sub.NA] + [V.sub.E]]

Thus, heritabilites in the narrow sense of paternal half-sib and maternal half-sib and full-sib were computed using the respective relationships:

[h.sup.2.sub.HS(S)] = 4 x [[sigma].sup.2.sub.S]/([[sigma].sup.2.sub.S] + [[sigma].sup.2.sub.D] + [[sigma].sup.2])

[h.sup.2.sub.HS(D)] = 4 x [[sigma].sup.2.sub.D]/ ([[sigma].sup.2.sub.S] + [[sigma].sup.2.sub.D] + [[sigma].sup.2])

[h.sup.2.sub.FS(D/S)] = 2 x ([[sigma].sup.2.sub.S] + [[sigma].sup.2.sub.D])/([[sigma].sup.2.sub.S] + [[sigma].sup.2.sub.D] + [[sigma].sup.2])

Test of significant of heritability: t = [h.sup.2]/[[sigma].sub.h.sup.2]

Paternal half-sib:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]

Maternal half-sib:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]

Full-sib:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]

Experimental Animal

Collection and Maintenance

Parental S. nudus were taken from a cultured population in Dalian Bay on the northern coast of the Yellow Sea on 10 September 2001. These individuals were held at 18-22[degrees]C under 500 lx illumination and fed Laminaria japonica ad libitum for 32 days before spawning on 12 October 2001.

Fertilization

Individuals were removed from the aquaria and allowed to drain for 30 min before 1 mL 0.5 M KCI was injected into the coelomic cavity via the peristomial membrane. They were placed on the tops of flasks filled with seawater, and the eggs and sperm were collected from each individual for 30-60 min.

The eggs of each female were fertilized with sperm from a single male. Approximately 150,000 eggs from each female were placed in 100-L containers. Sperm were diluted 1000-fold (Uehara et al. 1990), and a small amount of the diluted sperm was added to the eggs. Fertilization success was examined microscopically. The fertilized eggs were washed 2-3 times to remove excess sperm. The embryos were layered on the bottom of flasks and transferred into a 50-L container to develop at 17-21[degrees]C at a density of 4-5 individuals/mL. Normal plutei developed in 30-35 h (Rahman et al. 2000, Rahman et al. 2001).

Rearing

The larvae were transferred to 100-L containers of filtered seawater at 16[degrees]C. Densities of larvae from the 2-arm to the 8-arm stage were maintained at 1-2 individuals/mL. The water was changed twice daily. The larvae were fed Chaetoceros gracilis. Light was maintained at <300 lx. A small amount of air was bubbled into the water. After three months the juveniles from each fertilization group were placed separately into plastic cages suspended in a large pool. The ,juveniles were fed fresh Laminara japonica. The cages were changed every 2 mo. The juveniles were weighed and their diameter measured at ages 3 and 5 mo.

RESULTS

Growth: Increase in Body Weight and Test Diameter

Mean and standard deviation of the increase in body weight and test diameter of offspring at 3 and 5 mo of age are given in Table 3.

Analysis of Variance of Body Weight and Test Diameter of Offspring

Analysis of variance demonstrated great differences in body weight and diameter of juveniles from different females mated with the same male and between males at both 3 and 5 mo of age (Table 4).

Effective mean number of offspring for sires and dams after 3 mo was computed as follows: effective mean number of offspring in dams within sire is [K.sub.1] = 34.689, in dams is [K.sub.2] = 38.821, and in sires is [K.sub.3] = 107.719. Effective mean number of offspring for sires and dams after 5 mo was computed as follows: [K.sub.1] = 33.505, [K.sub.2] = 36.831, and [K.sub.1] = 109.268.

The causal components of variance were estimated from the full- and half-sib covariance using the relationships in Table 5.

Estimations of Heritability of Body Weight and Test Diameter of Offspring

Heritabilities in the narrow sense of paternal half-sib and maternal half-sib and full-sib of body weight and diameter of 3-mo-old S. nudus were calculated on the result of component of variance and test (t = [h.sup.2]/[[sigma].sub.h.sup.2]) of significance of heritability, respectively, as shown in Table 6.

All of the heritability in the narrow sense of sire half-sib and the heritability in the broad sense of dam full-sib of body weight and test diameter of S. intermedius were significantly different from zero (t-test, P < 0.01).

Estimated heritability was somewhat different among the sire heritability, dam heritability, and the pooled (combined) heritability for all three traits. The estimates of dam heritability were higher than those of sire heritability, and the pooled (combined) heritability was moderate.

DISCUSSION

Predicted heritabilities of larval growth have been reported for full-sib correlation analysis for Crassostrea virginica (Hadley et al. 1975, Lannan 1972, Newkirk et al. 1977) and Penaeus vannamei (Carr et al. 1997) and half-sib correlation analysis for Mercenaria mercenaria (Rawson and Hilbish 1990), Macrobrachium rosenbergii (Malecha et al. 1984), Penaeus vannamei (Benzie et al. 1996, Carr et al. 1997), and Penaeus stylirostri (Benzie et al. 1996). Predicted heritabilities of shell traits in wild Littorina saxatilis populations have been reported for full-sib correlation analysis and offspring-mother regression (Carballo et al. 2001). Estimates of heritabilities in the narrow sense generally ranged from 0.2 to 0.7. Realized heritability for increase in rate of growth in northern quahog and Argopecten irradians concetricus (Crenshaw et al. 1991, Crenshaw et al. 1996) and realized heritability estimates for growth in the Chilean oyster Ostrea chilensis (Torn et al. 1995). In our study, the estimates of heritabilities in the narrow sense for body weight at 3-5 mo of age ranged from 0.217 to 0.457, consistent with those reported for other species. However, estimates based on full-sib families bias heritabilities upwards when dominance and maternal effects are present (Lester 1988). Because of the nested design and a paternal half-sib correlation analysis used, the estimate reported here is more precise and unbiased. This is the first report of heritability in the narrow sense reported for sea urchins.

An animal model includes a random effect for the additive genetic effect of each individual; incorporates a complete set of additive genetic relationships among all individuals; and allows an unbiased estimation of variance components, even for the data involving selection and nonrandom mating (Gall and Bakar 2002, Sorensen and Kennedy 1986, Su et al. 1997). In the current investigation, full-sib family was taken as a random effect in a simple random model to account for the covariance among full-sibs caused by common environmental, dam, and nonadditive genetic effects, and half-sib family was used to account for covariance among half-sibs caused by common environmental, dam, sire, and nonadditive genetic effects. The results from the analyses based on this model were expected to be unbiased estimates of genetic parameters for the base population.

The much larger heritabilities computed from the female additive genetic component indicate the female genetic component still contains common environmental effects, maternal effects, or non-additive genetic variance in the body weight or diameter. Dam effects are omnipresent in the study. The fact that the juvenile phase may indicate that the quality of yolk reserves plays a role in early development. Dam effects may persist after the onset of exogenous feeding. Crandell and Gall (1993) reported that dam effects persist up to 2 y in rainbow trout and up to 18 mo in Arctic char (Nilsson, 1994).

The estimated heritability indicates significant additive genetic variation for body weight and test size over all the sampling periods. Sire heritability for the different variables were lower than dam heritability in many cases. However, all heritabilities in the narrow sense of body weight and test diameter of & intermedius were significantly different from zero (P < 0.01). The heritabilities in the narrow sense estimate obtained from this experiment indicate there is sufficient variation in the base population of sea urchins to respond to natural or artificial selection on juvenile growth weight. This justifies selection of juveniles based on growth characteristics for cultured brood stock.

TABLE 1.
Analysis of variance for components of phenotypic variation.

    Source of         Degrees of         Sum of        Mean Square
    Variance         Freedom (df)     Squares (SS)        (MS)

Dams                F x M - 1         S[S.sub.M]
Sires               F - 1             S[S.sub.F]      M[S.sub.F]
Dams/sires          F x (M - 1)       S[S.sub.M(F)]   M[S.sub.M(F)]
Offspring (error)   F x M x (n - 1)   SS              MS
Total               N - 1             S[S.sub.T]

    Source of                       Expected Mean
    Variance                        Square E(MS)

Dams
Sires               [[sigma].sup.2] + [k.sub.2] [[sigma].sup.2.sub.M]
                      + [k.sub.3] [[sigma].sup.2.sub.F]
Dams/sires          [[sigma].sup.2] + [k.sub.1] [[sigma].sup.2.sub.M]
Offspring (error)   [[sigma].sup.2]
Total

TABLE 2.
Relationships between the covariance of full- and half-sibs
and causal components of phenotypic variance.

      Component of
        Variance               Covariance Components

[[sigma].sup.2.sub.F]       CO[V.sub.HS]
[[sigma].sup.2.sub.M]       CO[V.sub.FS] - CO[V.sub.HS]
[[sigma].sup.2.sub.E]       [V.sub.P] - CO[V.sub.FS]
[[sigma].sup.2.sub.T] =     [V.sub.P]
  [[sigma].sup.2.sub.F] +
  [[sigma].sup.2.sub.M] +
  [[sigma].sup.2]
[[sigma].sup.2.sub.F] +     CO[V.sub.FS]
  [[sigma].sup.2.sub.M]

      Component of                            Causal
        Variance                            Components

[[sigma].sup.2.sub.F]       1/4 [V.sub.A]
[[sigma].sup.2.sub.M]       1/4[V.sub.A] + 1/4[V.sub.NA] + [V.sub.EC]
[[sigma].sup.2.sub.E]       1/2[V.sub.A] + 3/4[V.sub.D] + [V.sub.ES]
[[sigma].sup.2.sub.T] =     [V.sub.A] + [V.sub.NA] + [V.sub.EC] +
  [[sigma].sup.2.sub.F] +     [V.sub.ES]
  [[sigma].sup.2.sub.M] +
  [[sigma].sup.2]
[[sigma].sup.2.sub.F] +     1/2[V.sub.A] + 1/4[V.sub.D] + [V.sub.EC]
  [[sigma].sup.2.sub.M]

      Component of                      Calculation of
        Variance                     Component of Variance

[[sigma].sup.2.sub.F]       {M[S.sub.F] - [(M[S.sub.M(F)] -
                              M[S.sub.E])/[k.sub.1] x [k.sub.2] -
                              M[S.sub.E]}/[k.sub.3]
[[sigma].sup.2.sub.M]       (M[S.sub.M(F)] - M[S.sub.E])/[k.sub.1]
[[sigma].sup.2.sub.E]       M[S.sub.E]
[[sigma].sup.2.sub.T] =
  [[sigma].sup.2.sub.F] +
  [[sigma].sup.2.sub.M] +
  [[sigma].sup.2]
[[sigma].sup.2.sub.F] +
  [[sigma].sup.2.sub.M]

TABLE 3.
Body weight and test diameter of offspring at 3 and 5 mo of age.

                  Body Weight (g)     Test Diameter (men)

                          Standard              Standard
Growth Phase   Average    Deviation   Average   Deviation

3 mo           0.014484    0.0103      2.916      0.945
5 mo           1.366       0.377       8.492      2.841

TABLE 4.
Analysis of variance for components of phenotypic variation of
Strongylocentrotus nudus at 3 and 5 mo of age.

                                    Body Weight

                           Degrees of         Mean Square
  Source of Variance      Freedom (df)           (MS)

3 mo
  Dam                           62       4.08340 x [10.sup.-3]
  Sire                          20       7.26634 x [10.sup.-3]
  Dams within sires             42       2.56772 x [10.sup.-3]
  Full-sibs within dams       2210       4.12753 x [10.sup.-4]
  Total                       2272
5 mo
  Dams                          59       0.79976
  Sire                          20       1.31728
  Dams within sires             39       0.53436
  Full-sibs within dams       2045       0.10551
  Total                       2104

                                    Test Diameter

                                        Mean
  Source of Variance      F-Value    Square (MS)   F-Value

3 mo
  Dam                      9.893 *      6.01406     6.737 *
  Sire                    17.605 *     10.71717    12.006 *
  Dams within sires        6.221 *      3.77449     4.228 *
  Full-sibs within dams                 0.89266
  Total
5 mo
  Dams                     7.580 *     69.16935     8.570 *
  Sire                    12.485 *    108.2978     13.418 *
  Dams within sires        5.065 *     49.10348     6.084 *
  Full-sibs within dams                 8.07108
  Total

                                     Test Diameter

                                  Expected Mean Square
  Source of Variance                     E (MS)

3 mo
  Dam
  Sire                    [[sigma].sup.2] + [k.sub.2]
                            [[sigma].sup.2.sub.M] + [k.sub.3]
                            [[sigma].sup.2.sub.M]
  Dams within sires       [[sigma].sup.2] + [k.sub.2]
                            [[sigma].sup.2.sub.M]
  Full-sibs within dams   [[sigma].sup.2]
  Total
5 mo
  Dams
  Sire                    [[sigma].sup.2] + [k.sub.2]
                            [[sigma].sup.2.sub.M] +
                            [k.sub.3][[sigma].sup.2.sub.F]
  Dams within sires       [[sigma].sup.2] + [k.sub.1]
                            [[sigma].sup.2.sub.M]
  Full-sibs within dams   [[sigma].sup.2]
  Total

* P < 0.01: S = sires. D = dams; [k.sub.1] is the weighed mean
offspring number of females, [k.sub.2] is the weighed offspring
number of females within sire, [k.sub.3] is the weighed mean
offspring number of sires.

TABLE 5.
Relationships between the covariance of full- and half-sibs and causal
components of phenotypic variance.

        Component                             Causal
       of Variance                          Components

[[sigma].sup.2.sub.F]       1/4 [V.sub.A]
[[sigma].sup.2.sub.M]       1/4 [V.sub.A] + 1/4[V.sub.NA] + [V.sub.EC]
[[sigma].sup.2]             1/2 [V.sub.A] + 3/4[V.sub.D] + [V.sub.EW]
[[sigma].sup.2.sub.P]       [V.sub.A] + [V.sub.NA] + [V.sub.EC] +
  = [[sigma].sup.2.sub.F]     [V.sub.EW]
  + [[sigma].sup.2.sub.M]
  + [[sigma].sup.2]
[[sigma].sup.2.sub.F]       1/2 [V.sub.A]

                                              Result of Component
                                                  of Variance

                                                     3 Mo

        Component             Covariance
       of Variance            Components          Body Weight

[[sigma].sup.2.sub.F]       [Cov.sub.HS]     4.13895 x [10.sup.-5]
[[sigma].sup.2.sub.M]       [Cov.sub.FS] -   6.16974 x [10.sup.-5]
                              [Cov.sub.HS]
[[sigma].sup.2]             [V.sub.P] -        4.275 x [10.sup.-4]
                              [Cov.sub.FS]
[[sigma].sup.2.sub.P]       [V.sub.P]        5.40587 x [10.sup.-4]
  = [[sigma].sup.2.sub.F]
  + [[sigma].sup.2.sub.M]
  + [[sigma].sup.2]
[[sigma].sup.2.sub.F]       [COV.sub.FS]     1.03087 x [10.sup.-4]

                                  Result of Component of Variance

                                3 Mo                   5 Mo

        Component
       of Variance          Test Diameter   Body Weight   Test Diameter

[[sigma].sup.2.sub.F]          0.06027        0.00678        0.50447
[[sigma].sup.2.sub.M]          0.08308        0.01280        1.22465

[[sigma].sup.2]                0.89264        0.10551        8.07108

[[sigma].sup.2.sub.P]          1.03598        0.12509        9.80020
  = [[sigma].sup.2.sub.F]
  + [[sigma].sup.2.sub.M]
  + [[sigma].sup.2]
[[sigma].sup.2.sub.F]          0.14335        0.01958        1.72912

TABLE 6.
Heritabilities in narrow sense ([h.sup.2]) and standard error
([[sigma].sub.h.sup.2]).

                                     3 Mo of Age

                         Body Weight               Diameter

[h.sup.2.sub.S]    0.306 * [+ or -] 0.055   0.233 * [+ or -] 0.046
[h.sup.2.sub.D]    0.457 * [+ or -] 0.071   0.321 * [+ or -] 0.046
[h.sup.2.sub.FS]   0.381 * [+ or -] 0.063   0.277 * [+ or -] 0.051

                                     5 Mo of Age

[h.sup.2.sub.S]    0.217 * [+ or -] 0.042   0.206 * [+ or -] 0.041
[h.sup.2.sub.D]    0.409 * [+ or -] 0.064   0.500 * [+ or -] 0.072
[h.sup.2.sub.FS]   0.313 * [+ or -] 0.054   0.353 * [+ or -] 0.058

* Very significant between [h.sup.2] with zero (P < 0.01);
[t.sub.0.01], [varies] = 2.576.

ACKNOWLEDGMENTS

This is contribution number G1999012009 of 973 from the Chinese National Fundamental Research project and Chinese High Technology Plan (2002AA628170). We would like to thank J. Song, G. D. Wang, R. L. Xing, and S. G. Yan for their invaluable assistance in the laboratory production of the family lines. The authors thank J. M. Lawrence for editing the paper.

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LIU XIAOLIN, (1,3) CHANG YAQING, (2) XIANG JIANHAI, (1), * DING JUN, (2) AND CAO XUEBIN (2)

(1) Experimental Marine Biology Laboratory, Institute of Oceanology, CAS, Qingdao 266071, China; (2) Key Lab of Mariculture and Biotechnology of the Ministry of Agriculture, Dalian Fisheries University, Dalian 116023, China; (3) College of Animal Science and Technology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi 712100, China

* Corresponding author. E-mail: [e-mail]