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. Twentyone
halfsib groups and 60 to 63 fullsib groups of juveniles were obtained
by artificial fertilization of three to five females by single males
based on a nested design. The body weight (g) and test diameter (cm) of
the young was measured 3 and 5 months 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 halfsib correlation analysis ranged from 0.21670.4565 for
weight and 0.20590.4998 for diameter. The results indicate significant
maternal effects. The strength of the nested design and the paternal
halfsib correlation analysis used in this study make the estimate the
most precise and unbiased reported to date.
KEY WORDS: sea urchin. Strongylocentrotus nudus, growth, heritability 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 (Hobson & Chave 1990, Shimabukuro 1991, Hagen 1996). Because of overfishing, interest in aquaculture of sea urchins has increased greatly (Hagen 1998, Lawrence et al. 2001). One of the most important species for aquaculture is Strongylocentrotus nudus (Hagen 1996, Agatsuma 1998). Current culture of S. nudus has used seeds obtained from wild individuals (Gao & Chang 1999, Liao & Qiu, 1999). Analysis of populations of S. nudus in Japanese waters shows considerable range in size of small individuals, presumably of single cohorts (Agatsuma 1997). This could result from inter action 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 halfsib groups because they are less likely to be affected by environmental influence (Gjedrem 1992). Sib analysis techniques have been used for several important aquaculture species (Mallet et al. 1986, Rawson & Hilbish 1990, Hadley et al. 1991, Crenshaw et al. 1991, Newkirk et al. 1977, Benzie et al. 1997). The purpose of this study is to estimate heritability of growth in terms of body weight and diameter of juvenile Strongylocentrotus 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 Strongylocentrotus nudus was mated to 3 to 5 females, therefore generating 63 fullsib families and 21 halfsib 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 months of age. Genetic Analysis The covariance among full and halfsibs provides the basis for the separation of phenotypic variance into genetic and environmental components of variance. The covariance among full and halfsibs are calculated from the observed components of variance obtained from a threelevel 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 threelevel 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.sub.ij.sup.2]/[dn.sub.i])]/(D  S) Effective mean number of offspring in dams: [K.sub.2] = [[summation of]([n.sub.ij.sup.2]/[dn.sub.i])  [summation of] ([n.sub.ij.sup.2]/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 ith sire and jth dam, [dn.sub.1] = number of offspring of ith sire, 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.E]) 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 halfsib 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 halfsib and maternal halfsib and fullsib 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 halfsib: [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.] Maternal halfsib: [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.] Fullsib: [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.] Experimental Animal Collection and Maintenance Parental Strongylocentrotus 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 1822[degrees]C under 500 x 1 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 minutes before 1 mL 0.5 M KCL was injected into the coelomic cavity via the peristomial membrane. They were placed on the tops of flasks filled with sea water and the eggs and sperm collected from each individual for 3060 minutes. The eggs of each female were fertilized with sperm from a single male. Approximately 15,000 eggs from each female were placed in 100L containers. Sperm were diluted 1000fold (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 two to three times to remove excess sperm. The embryos were layered on the bottom of flasks and transferred into a 50L container to develop at 1721[degrees]C at a density of four to five individuals/mL. Normal plutei developed in 3035 h. (Rahman et al. 2000, Rahman et al. 2001). Rearing The larvae were transferred to 100L containers of filtered seawater at 16[degrees]C. Densities of larvae from the 2arm to the 8arm stage were maintained at one to two 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 babbled into the water, After 3 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 months. The juveniles were weighed and their diameter measured at ages of 3 and 5 months. RESULTS GrowthIncrease in Bodyweight and Test Diameter Mean and standard deviation of the increase in body weight and test diameter of offspring at 3 and 5 months 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 months of age (Table 4). Effective mean number of offspring for sires and clams after 3 months was computed as follows: effective mean number of offspring in darns 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 months was computed as follows: [K.sub.1] = 33.505: [K.sub.2] = 36.831; [K.sub.3] = 109.268. The causal components of variance were estimated from the full and halfsib 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 halfsib and maternal halfsib and fullsib of body weight and diameter of 3monthold Strongylocentrotus nudus were calculated on the result of component of variance and test (t = [h.sub.2]/[[sigma].sub.h.sup.2) of significant of heritability respectively as Table 6. All of the heritability in the narrow sense of sire halfsib and the heritability in the broad sense of dam fullsib of body weight, test diameter of S. intermedius were significantly different from zero (ttest, 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 that of sire heritability, and the pooled (combined) heritability was moderate. DISCUSSION Predicted heritabilities of larval growth have been reported for fullsib correlation analysis for Crassostrea virginiica (Lannan 1972, Haley et al, 1975, Newkirdk et al. 1977) and Penaeus vannamei (Carr et al. 1997) and halfsib correlation analysis for Mercenaria mercenaria (Rawson & Hilbish 1990), Macrobrachium rosenbergii (Malecha et al. 1984), Penaeus vannamei (Carr et al. 1997, Benzie et al. 1996), and Penaeus stylirostri (Benzie et al. 1996). Predicted heritabilities of shell traits in wild littorina saxatilis populations have been reported for fullsib correlation analysis and offspring mother regression (Carballo et al. 2001). Estimates of heritabilities in the narrow sense generally ranged from 0.20.7. Realized heritability for increase in rate of growth in northern quahog and Argopecten irradians concetricus (Crenshaw et al. 1996, Crenshaw et al. 1991) and realized heritability estimates for growth in the Chilean oyster Ostrea chilensis (Toro et al. 1995). In our study, the estimates of heritabilities in the narrow sense for body weight at 35 months of age ranged from 0.2170.457, consistent with those reported for other species. However, estimates are based on fullsib families bias heritabilities upwards when dominance and maternal effects are present (Lester, 1988). Because of the nested design and a paternal halfsib 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; and 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 & Bakar 2002, Sorensen & Kennedy 1986, Su et al. 1997). In this investigation fullsib family was taken as a random effect in a simple random model to account for the covariance among fullsibs caused by common environmental, dam and nonadditive genetic effects, and halfsib 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 contain common environmental effects, maternal effects or nonadditive 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 years in rainbow trout and up to 18monthold Arctic char (Nilsson 1994). The estimated heritability indicates significant additive genetic variation for body weight and test size all over 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, test diameter of S. 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 Degree of Sum of Variance Freedom (df) Squares (SS) Dams F x M  1 S[S.sub.M] Sires F  1 S[S.sub.F] Dams/sires F x (M  1) S[S.sub.M(F)] Offspring (error) F x M x (n  1) SS Total N  1 S[S.sub.T] Source of Mean Square Expected Mean Variance (MS) Square E (MS) Dams Sires M[S.sub.F] [[sigma].sup.2] + [k.sub.2] [[sigma]. sup.2.sub.M] + [k. sub.3] + [[sigma]. sup.2.sub.F] Dams/sires M[S.sub.M(F)] [[sigma].sup.2] + [k.sub.1] [[sigma]. sup.2.sub.F] Offspring (error) MS [[sigma].sup.2] Total TABLE 2. Relationships between the covariance of full and halfsibs and causal components of phenotypic variance. Component of Covariance Variance 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 months of age. Body Weight (g) Test Diameter (mm) Growth Standard Standard Phase Average Deviation Average Deviation 3 months 0.014484 0.0103 2.916 0.945 5 months 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 months of age. Body Weight Source of Degrees of Mean Variance Freedom (df) Square (MS) FValue 3 months Dam 62 4.08340 x 9.893 ** [10.sup.3] Sire 20 7.26634 x 17.605 ** [10.sup.3] Dams within 42 2.56772 x 6.221 ** sires [10.sup.3] Fullsibs 2210 4.12753 x within dams [10.sup.4] Total 2272 5 months Dams 59 0.79976 7.580 ** Sire 20 1.31728 12.485 ** Dams within 39 0.53436 5.065 ** sires Fullsibs 2045 0.10551 within dams Total 2104 Body Weight Test Diameter Source of Mean Expected Mean Variance Square (MS) FValue Square E (MS) 3 months Dam 6.01406 6.737 ** Sire 10.71717 12.006 ** [[sigma].sup.2] + [k.sub.2] [[sigma]. sup.2.sub.M] + [k.sub.3][[sigma]. sup.2.sub.M] Dams within 3.77449 4.228 ** [[sigma].sup.2] + sires [k.sub.1] [[sigma. sup.2.sub.M] Fullsibs 0.89266 [[sigma].sup.2] within dams Total 5 months Dams 69.16935 8.570 ** Sire 108.2978 13.418 ** [[sigma].sup.2] + [k.sub.2] [[sigma]. sup.2.sub.M] + [k.sub.3][[sigma]. sup.2.sub.F] Dams within 49.10348 6.084 ** [[sigma].sup.2] + sires [k.sub.1] [[sigma. sup.2.sub.M] Fullsibs 8.07108 [[sigma].sup.2] within dams 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 halfsibs and causal components of phenotypic variance. Component of Causal Covariance Variance Components Components [[sigma].sup.2.sub.F] 1/4 [V.sub.A] [Cov.sub.HS] [[sigma].sup.2.sub.M] l/4[V.sub.A] + [Cov.sub.FS] 1/4 [V.sub.NA]  [Cov.sub + [V.sup.EC] .HS] [[sigma].sup.2] 1/2[V.sub.A] [V.sub.P]  + 3/4[V.sub.D] [Cov.sub. + [V.sub.EW] FS] [[sigma].sup.2.sub.P] = [V.sub.A] + [V.sub.P] [[sigma].sup.2.sub.F] [V.sub.NA] + [[sigma].sup.2.sub.M] + [V.sub.EC] [[sigma].sup.2] + [V.sub.EW] [[sigma].sup.2.sub.F] + 1/2[V.sub.A] CO[V.sub.FS] [[sigma].sup.2.sub.M] + 1/4[V.sub.D] + [V.sub.EC] Result of Component of Variance 3 Month 5 Month Component of Test Body Test Variance Body Weight Diameter Weight Diameter [[sigma].sup.2.sub.F] 4.13895 x 0.06027 0.00678 0.50447 [10.sup.5] [[sigma].sup.2.sub.M] 6.16974 x 0.08308 0.01280 1.22465 [10.sup.5] [[sigma].sup.2] 4.275 x 0.89264 0.10551 8.07108 [10.sup.4] [[sigma].sup.2.sub.P] = 5.40587 x 1.03598 0.12509 9.80020 [[sigma].sup.2.sub.F] [10.sup.4] [[sigma].sup.2.sub.M] [[sigma].sup.2] [[sigma].sup.2.sub.F] + 1.03087 x 0.14335 0.01958 1.72912 [[sigma].sup.2.sub.M] [10.sup.4] TABLE 6. Heritabilitics in narrow sense ([h.sup.2]) and standard error ([[sigma].sub.h.sup.2]). 3 Months 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 Months of Age Body Weight Diameter [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 ** Denotes very significance between [h.sub.2] with zero (P < 0.01); [t.sub.0.01] [infinity] = 2.576. ACKNOWLEDGMENTS The authors 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. They also thank J. M. Lawrence for editing the paper. LITERATURE CITED Agatsuma. Y. 1998. 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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 SciTech University of Agriculture and Forestry, Yangling, Shaanxi 712100, China This is contribution number G1999012009 of 973 from the Chinese National Fundamental research project and Chinese High Technology Plan (2002AA628170). * Corresponding author. Email: jhxiang@ms.qdiao.ac.cn 

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