- Email: [email protected]
Can ordinary single-day egg collection increase the effective population size in broodstock management programs? Breeder-offspring assignment in black sea bream (Acanthopagrus schlegelii) through two-hourly intervals
Aquaculture 308 (2010) S12–S19
Contents lists available at ScienceDirect
Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Can ordinary single-day egg collection increase the effective population size in broodstock management programs? Breeder-offspring assignment in black sea bream (Acanthopagrus schlegelii) through two-hourly intervals Enrique Blanco Gonzalez a,b, Nobuhiko Taniguchi b, Tetsuya Umino a,⁎ a b
Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan The Research Institute of Marine Bioresources, Fukuyama University, 452-10 Innoshima-Ohama, Onomichi 722-2101, Japan
a r t i c l e
i n f o
Article history: Received 31 July 2009 Received in revised form 14 January 2010 Accepted 25 June 2010 Keywords: Broodstock management Black sea bream Acanthopagrus schlegelii Effective population size Parentage assignment Egg
a b s t r a c t The management of the broodstock used for stock enhancement purposes is essential to preserve the genetic resources of the natural populations. Black sea bream (Acanthopagrus schlegelii) has been extensively released in Japan; nevertheless, the little emphasis paid on the genetic conservation, resulted in important drifts due to the small number of parental fish reared in the hatcheries. The present study shows how simple changes in the broodstock management procedure may greatly contribute to increase the effective number of breeders (Nb) and reduce the rate of inbreeding. In this regard, the collection of eggs at several timings over a single night allowed the identification of those contributors spawning for a short period and with little overall contribution. It enabled identifying those alleles presented in lower frequencies, an essential issue on conservation genetics. The second improvement involved the use of a reasonable large number of individuals as breeders. In this study, the broodstock (BR) comprised 143 specimens, a feasible number to be reared at any hatchery that produces juveniles for stocking in Japan. The BR-offspring assignment confirmed the contribution of an extremely high number of breeders (140), corrected to 136 after considering the differences in sex proportions. The number of offspring comprising each family was similar (up to 3). Nevertheless, large disparities in the contribution were observed among breeders. As a result of the large number of offspring produced by few parental fish, Nb was reduced to 37. Although lower than desirable, the fact that most of breeders took part in the mating process, confirmed the usefulness of this procedure. An alternative method to reduce and simplify the number of egg batches required was investigated. The three groups of eggs sampled in the middle of the sampling period and representing the period the releases were most intensive, were combined (nocturnal group), showing similar results. In this case 136 out of the initial 143 breeders took part in the mating process, and the final Nb was 88. These results confirm that by either of these simple means, most of the genetic diversity would be inherited by the offspring and help to preserving the genetic resources of the natural populations subject to stocking. This experience, although promising, should also be applied to other species to confirm its effectiveness. In addition, it is necessary to confirm the viability and performance of the offspring produced in this way in relation to those collected over the whole spawning season. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The success of aquaculture practices highly depends on the broodstock reared in a hatchery. Their management is essential, especially in stock enhancement programs that aim at preserving natural resources through artificially produced seeds propagation (Allendorf and Phelps, 1980; Taniguchi, 2003, 2004). Stock enhancement programs comprise two main phases. The first one includes the collection of the broodstock, mass production and rearing of the
⁎ Corresponding author. Tel./fax: + 81 824 24 7944. E-mail address: [email protected] (T. Umino). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.06.031
offspring in hatcheries. Secondly, the fish are released into the natural environment once they reach the appropriate size, and are expected to contribute to the breeding population of the natural stock. Initially, most of the efforts had been directed towards increasing the survival of the offspring released into the wild and the fishing yields. However, the importance of preserving all the natural resources, including the genetic diversity of the wild stocks, mean it is necessary to develop a more exhaustive and carefully designed strategy, taking a broader comprehensive approach which integrates enhancements in a more conservative manner (Blankenship and Leber, 1995; Bell et al., 2008; Tringali et al., 2008). Several key strategies have been proposed as a means to maintain the gene pool inherited by the offspring such as the conservation of rare alleles, the use of a large number of native
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
specimens as breeders and the collection of eggs in several days over the spawning season (Allendorf and Ryman, 1987; Doyle et al., 2001; Nugroho and Taniguchi, 2004; Taniguchi, 2004; Fraser, 2008). In this regard, the high cost and space requirements of rearing a large native wild broodstock population have been the main constraint in stock enhancement programs. In Japan, millions of seed of high-valued commercial fish species are released annually throughout the country (Kitada and Kishino, 2006). Nevertheless, in most cases, the small number of breeders maintained in the hatchery has resulted in high levels of inbreeding, compromising the gene pool in the receptive population. In addition, the juveniles for release have usually originated in one night during the spawning peak. Consequently, the contribution of some specimens is missing. As a means to increase the effective number of breeders (Nb) and mitigate the adverse effects in the offspring, it has been suggested to extend the egg collection (Nugroho and Taniguchi, 2004) over an extended period. Black sea bream (Acanthopagrus schlegelii) has a long history of stock enhancement in Japan with releases carried out in almost half of the national prefectures (23 out of 47) since the late 1970s. The seed production and rearing method for this species were first described in the 1960s (Kasahara et al., 1960; Kasahara and Oshimna, 1960; Kasahara and Hibiya, 1967). Several studies have been conducted in order to understand the acclimation, biochemical composition and behavior of hatchery reared juveniles (for a review see Blanco Gonzalez et al., 2008b). Nevertheless, it was not until the development of genetic techniques when the contribution of the broodstock was assessed. The first evaluation of the genetic effects in the first and second generations of hatchery stocks of black sea bream was carried out by Taniguchi et al. (1983) using 42 isozymes. They reported that only between 16 and 26 out of the 102 breeders reared in the hatchery contributed to the first two generations of offspring. Recently, the contribution of the broodstock used for the intensive stock enhancement program conducted in Hiroshima Bay was investigated by DNA fingerprinting (Jeong et al., 2002) and genotyping a few microsatellite markers (Jeong et al., 2007; Blanco Gonzalez et al., 2008a). It was observed that the large number of juveniles released over three decades originated from less than 100 breeders, modified the genetic diversity of the natural stock of black sea bream inhabiting Hiroshima Bay (Blanco Gonzalez and Umino, 2009). Before the release (broodstock = 51), Nb was 20 and 9 in 2000 and 2001, respectively (Jeong et al., 2007). Four years after the release, hatchery reared fish represented 13% of the samples and Nb for the same broodstock was estimated to be 16 (Blanco Gonzalez et al., 2008a). The good acclimation and high survival of the juveniles (Umino et al., 1999; Nakagawa et al., 2000; Jeong et al., 2007) and their presence in the group of spawners (Blanco Gonzalez et al., 2008a), suggested a potential hybridization with their wild counterparts. Therefore, it is essential to develop a proper management plan during the captive phase to minimize the genetic loss in the juveniles to be released. This study aims at improving the broodstock management practices and increasing Nb in black sea bream hatchery released populations. It also aims at identifying those alleles present in lower frequencies and that may require special conservation practices. The
S13
results obtained here should be considered to preserve the gene pool of the natural stocks before conducting large scale releases. 2. Materials and methods 2.1. Broodstock and egg collection The broodstock (BR) used in the present study were reared at the Yamaguchi Prefectural Fisheries Experimental Station (YPFES) and comprised 85 dams and 58 sires. Black sea bream eggs were collected by tank overflowing on May 17, 2006. The first sample was collected at 13:14 (GMT + 9) and 11 additional batches were collected successively every ~2 h, with the last one taken at 11:05 (GMT + 9) on May 18, 2006 (Table 1). Once collected, the wet weight of each batch was measured and ~ 5 g of eggs from each were transferred into 500 ml bin saturated with oxygen and incubated for ~ 2 h to confirm their fertilization. Afterwards, the eggs were preserved in 95% ethanol and stored at 4 °C for later DNA extraction. Egg batches representing the peak of the spawning (19:15–23:15 GMT + 9) were analyzed as independent groups. Meanwhile, several batches of low wet weight collected before and after the peak of the spawning were pooled together to simplify the analysis. At the end, the eggs were divided into six groups based on collection time (see Table 1). On May 27, some scales were removed from the right-ventral side of each breeder during their transfer from indoor tanks to the outdoor conditions, placed into 95% ethanol and stored at 4 °C for later DNA extraction. For the posterior analysis, besides the BR and each of the six groups of eggs, two additional group combinations were considered. The first one represents the pool of all groups of eggs together over one day (“Pooled”) as is the normal practice. The other group (“Nocturnal”) is a combination of the three groups taken in the middle of the collection period (GII, GIII and GIV) and representing the heaviest wet weight. The genetic composition of the nocturnal group was investigated as an alternative way to reduce the number of lots collected during one night without genetic losses. 2.2. DNA extraction and genotyping The DNA extraction was done by two different methods. For the BR, the DNA was obtained following the standard SDS-phenol/ chloroform procedure described by Taggart et al. (1992) as done in our previous study (see details in Blanco Gonzalez et al., 2008a). An alternative Chelex/proteinase K method modified from the one described by Launey and Hedgecock (2001) was followed due the low amount of DNA contained in the eggs. A drop of alcohol was evaporated from the eggs at room temperature. Then, each egg was added to 20 μl of 5% chelex, 3 μl of TE (0.01 M Tris, 1 mM EDTA) and 1 μl of proteinase K (10 mg/ml). The mixture was heated at 55 °C for 2 h and boiled at 100 °C for 10 min. The samples were centrifuged at 4000 rpm for 5 min and the supernatant transferred to clean test tubes and stored at −20 °C for posterior genotyping. The amplification of the DNA was performed by the PCR using the same six loci (ACS3, ACS4, ACS6, ACS9, ACS16 and ACS17) and a
Table 1 Broodstock (BR) and eggs of black sea bream analyzed in this study with detailed information of the collection time and group composition of the eggs.
Date
BR
Eggs
May 27
May 17
May 18
Group
Collection time (GMT + 9)
Nocturnal GI
GII
GIII
GIV
GV
GVI
n = 143
n = 75
n = 75
n = 75
n = 75
n = 75
n = 75
12:45
13:14
19:15
21:10
23:15
1:20
15:14
17:14
3:10
5:08
7:16
9:10
11:05
S14
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
protocol described in our previous study (see details in Blanco Gonzalez et al., 2008a).
2.3. Genetic diversity and pedigree analysis The genetic diversity at each locus was calculated as number of alleles per locus (A) and expected (He) and observed (Ho) heterozygosities for the BR and the eggs using the Excel Microsatellite Toolkit. The allelic richness (Ar) was also calculated between the BR and each group of eggs and between the BR and the pool of eggs in order to correct the differences in sample sizes by FSTAT (Goudet, 2001). Departures from Hardy–Weinberg equilibrium (HWE) at each locus and averaged over all loci were evaluated using a test analogous to Fisher's exact test, with a modified version of the Markov chain method (105 steps and 104 dememorisations) implemented with GENEPOP v4.0 (Raymond and Rousset, 1995). This software was also used to estimate the frequency of null alleles (fn) as well as the inbreeding coefficient, FIS (Weir and Cockerham, 1984). The overall significant value was adjusted following the sequential Bonferroni procedure (Rice, 1989). The overall genetic differentiation among the BR and eggs was evaluated with the AMOVA test with ARLEQUIN v3.0 (Excoffier et al., 2005). The pairwise FST values between the BR and the groups of eggs were also determined using this software. The polymorphic information content (PIC) at each locus implemented with Excel Microsatellite Toolkit was estimated for the BR. The exclusion probability Q (Villanueva et al., 2002) was also calculated. In addition, the probability of identity index (I) described by Paetkau and Strobeck (1994) was tested in the pool of eggs.
The pedigree analysis was performed comparing the genotypes of the BR and each of the 450 eggs at six loci using PAPA software (Duchesne et al., 2002). True parental couples were considered whenever the genotype of both parents coincided with that of the offspring at all loci. Two corrections should be applied to the total number of contributors assigned to the offspring (Lande and Barowwclough, 1987). The first one is due to unequal number of males and females contributing to the offspring. As a result, Nb will be determined by the number of contributors of the less numerous sex. The second correction is because of the variance in the number of offspring per family. Ryman and Laikre (1991) proposed a model to estimate the effective population size (Ne) in stock enhancement programs as the sum of the relative contribution of both wild and hatchery origin individuals. Since the broodstock used in this study comprised only wild specimens Ne = Nb and the inbreeding coefficient (F) was estimated as F = 1/2 Nb. 3. Results 3.1. Genetic diversity In this study, black sea bream showed a high genetic diversity expressed either as allelic (A, Ar) or heterozygosity (Ho) variability at all loci (Table 2). In the BR, allele number ranged from 10 to 27 (mean = 18.33), while each group of eggs inherited 7 to 22 alleles per locus (mean per group ranged 14.3–15.7). After the corresponding adjustment due to differences in sample sizes, the mean Ar for the BR was 16.5. The high conservation of the genetic resources inherited by the eggs was confirmed when all six groups of eggs were combined. In
Table 2 Genetic variability of the BR and eggs of black sea bream at 6 microsatellite loci. * and ** denote P b 0.05 and P b 0.01, respectively. Locus
ACS3
ACS4
ACS6
ACS9
ACS16
ACS17
Mean
Eggs
A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS
BR
GI
GII
GIII
GIV
GV
GVI
Nocturnal
Pooled
n = 143
n = 75
n = 75
n = 75
n = 75
n = 75
n = 75
n = 225
n = 450
27 22.9 0.863 0.909 −0.054 10 9.2 0.813 0.860 − 0.059 13 11.9 0.741 0.783 − 0.057 26 22.4 0.927 0.895 0.035** 18 17.3 0.912 0.958 − 0.051 16 15.2 0.898 0.867 0.035 18.3 ± 6.9 16.5 ± 5.5 0.859 ± 0.06 0.879 ± 0.05 − 0.023
20 20 0.834 0.813 0.025 9 9 0.824 0.813 0.014 11 11 0.715 0.667 0.067 18 18 0.885 0.893 − 0.009 17 17 0.910 0.867 0.048 15 15 0.886 0.893 − 0.008 15.0 ± 4.2 15.0 ± 4.4 0.843 ± 0.06 0.824 ± 0.08 0.022
16 16 0.830 0.747 0.101 8 8 0.793 0.787 0.008 10 10 0.730 0.707 0.032 21 21 0.924 0.907 0.019 16 16 0.903 0.880 0.025 15 15 0.870 0.840 0.035 14.3 ± 4.7 14.3 ± 4.7 0.841 ± 0.07 0.811 ± 0.07 0.036
16 16 0.767 0.787 − 0.026 7 7 0.790 0.813 − 0.029 10 10 0.658 0.707 − 0.075 22 22 0.921 0.947 − 0.028 17 17 0.897 0.893 0.004 15 15 0.875 0.800 0.086* 14.5 ± 5.3 14.5 ± 5.3 0.818 ± 0.09 0.824 ± 0.08 − 0.008
18 18 0.830 0.760 0.084 9 9 0.763 0.787 − 0.031 11 11 0.700 0.733 − 0.048 19 19 0.908 0.920 − 0.013 17 17 0.901 0.813 0.098** 15 15 0.902 0.853 0.054 14.8 ± 4.0 14.8 ± 4.0 0.834 ± 0.8 0.811 ± 0.06 − 0.028
19 19 0.794 0.760 0.044 9 9 0.805 0.920 − 0.143 10 10 0.721 0.667 0.076 20 20 0.926 0.827 0.108* 15 15 0.885 0.893 − 0.009* 16 16 0.860 0.853 0.008 14.8 ± 4.5 14.8 ± 4.5 0.832 ± 0.07 0.820 ± 0.08 0.015
20 20 0.799 0.827 − 0.035 10 10 0.813 0.813 − 0.001 11 11 0.704 0.747 − 0.061 21 21 0.925 0.947 − 0.023 17 17 0.904 0.920 − 0.018 15 15 0.877 0.840 0.043 15.7 ± 4.5 15.7 ± 4.5 0.837 ± 0.07 0.849 ± 0.07 − 0.014
22 20.1 0.812 0.764 0.057 10 9.5 0.783 0.796 − 0.018 11 10.6 0.700 0.716 − 0.026 23 22.3 0.920 0.924 − 0.005 17 16.9 0.904 0.862 0.046 15 14.9 0.885 0.831 0.061 16.3 ± 5.4 15.7 ± 5.1 0.833 ± 0.09 0.816 ± 0.07 0.021
25 21.3 0.809 0.782 0.033 10 9.5 0.800 0.822 − 0.028 12 11.2 0.706 0.704 0.002 25 22.7 0.917 0.907 0.011* 17 16.9 0.902 0.878 0.027 16 15.8 0.881 0.847 0.039 17.5 ± 6.35 16.2 ± 5.3 0.836 ± 0.08 0.823 ± 0.07 0.015*
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
this case, A ranged 10–25 (mean = 17.5). Therefore, over 95% of the alleles observed in the BR were present in the pooled offspring. Similarly, the high level of heterozygosity observed in the BR (Ho = 0.879) was also detected in the offspring (Ho = 0.823), where the loss of heterozygosity accounted for b 7%, confirming a high conservation on the genetic variability in black sea bream. Nevertheless, the pool of eggs produced departures from HWE overall loci that were not observed when each group of eggs was analyzed independently. The deviation from HWE was towards heterozygosity deficit (P = 0.002). The presence of null alleles was not detected in the pool of eggs (Table 5) as well as in any individual group of eggs. The nocturnal group showed slightly lower levels of diversity (A = 16.3; Ar = 15.7; Ho = 0.816) compared to the pooled group (Table 2; Fig. 1); nevertheless, these values were not statistically significant at P b 0.05. Also, the group was in agreement with the HWE overall loci. Significant differences (χ2) on allele frequencies at some major alleles were observed between the BR and each group of eggs except GII (Table 3). These differences were not detected at the locus ACS16. The allele 87 at the locus ACS3 appeared in much higher frequencies in three groups (GIII, GV and GVI) than in the BR. Consequently, the frequency of this allele in the pool of eggs was also significantly higher than that observed in the BR (χ2 = 6.03, d.f. = 1). Besides this excess (χ2 = 4.17, d.f. = 1), the nocturnal group also evidenced the low presence of the allele 100 at ACS6 (χ2 = 5.01, d.f. = 1). The collection of eggs at different timing revealed significant differences in the pairwise FST values among some groups (Table 4) as was the case when GIV was compared to GI and GV. Significant differences were also detected between the BR and GIII. Nevertheless, in all cases, the FST values were very low (b0.008). Once the eggs were pooled, the differences between the egg groups and the BR disappeared. The overall AMOVA averaged over loci also confirmed the homogeneity (P = 0.18). 3.2. Parentage assignment The high PIC scored across all loci (0.8426 ± 0.08) reflects the high polymorphism of the six loci genotyped in the present study (Table 5). The use of six loci revealed very useful to assign the offspring to their true parental fish (Q = 1). In addition, the low probability of finding two individuals sharing the same genotype (I = 2.238E−9), confirmed the strength of the parentage analysis. True parental assignment was possible to all offspring with two exceptions (448 out of 450). The number of contributors per group of eggs ranged from 81 to 87 (41–49 and 36–41 for females and males, respectively) to create between 70 and 74 families per group (Table 6). The fact that most families per group were composed of a single individual increased Nb over 100 except in GI. As a result, the inbreeding rate per group remained very low, between 0.4 and 0.6%. This promising scenario changed drastically when all eggs were pooled together. The
S15
Table 3 Differences in the frequency of major alleles (bp) between the BR and eggs of black sea bream at six microsatellite loci (χ2, d.f. = 1). * and ** denote P b 0.05 and P b 0.01, respectively. Locus
BR
Eggs GI
GII
GIII
GIV
GV
GVI
Nocturnal
Pooled
0.11 0.30 0.12
0.13 0.35 0.10
0.13 0.34 0.11
0.13 0.43** 0.13
0.09 0.36 0.13
0.11 0.41* 0.10
0.05 0.41* 0.11
0.12 0.38* 0.12
0.11 0.38* 0.11
ACS4 69 71 73
0.21 0.28 0.21
0.19 0.23 0.22
0.19 0.28 0.29
0.20 0.31 0.25
0.18 0.37* 0.24
0.25 0.29 0.16
0.22 0.28 0.20
0.19 0.32 0.26
0.21 0.29 0.23
ACS6 98 100 102
0.46 0.12 0.14
0.48 0.14 0.17
0.48 0.10 0.13
0.56 0.09 0.12
0.49 0.03** 0.21
0.48 0.15 0.12
0.51 0.09 0.15
0.51 0.07* 0.15
0.50 0.10 0.15
ACS9 79 83 87
0.08 0.17 0.10
0.09 0.25* 0.15
0.07 0.15 0.13
0.13 0.17 0.04*
0.11 0.22 0.08
0.09 0.15 0.06
0.06 0.17 0.11
0.10 0.18 0.08
0.09 0.18 0.09
ACS16 93 0.12 95 0.16 107 0.11
0.09 0.19 0.12
0.09 0.17 0.12
0.08 0.15 0.13
0.13 0.21 0.13
0.13 0.19 0.14
0.10 0.17 0.15
0.10 0.18 0.12
0.10 0.18 0.13
ACS17 142 0.16 144 0.16 146 0.14
0.11 0.15 0.23*
0.11 0.23 0.20
0.18 0.16 0.21
0.16 0.17 0.10
0.13 0.28** 0.17
0.15 0.21 0.18
0.15 0.19 0.17
0.14 0.20 0.18
ACS3 85 87 89
contribution of most breeders was confirmed (140/143), with only one male (M61) and two females (F26, F72) not contributing to the offspring. A total of 406 small size families were identified. However, a few breeders produced large number of offspring during the night. Consequently, the final Nb dropped to 37, much lower than 136, the value obtained before making the adjustments due to the differential contribution among breeders. In the nocturnal group, the number of breeders identified was 136, and in this case Nb = 88. Special reference to the drastic reduction when all six groups were pooled should be given to females F134 and F137 who contributed to 17 eggs (Fig. 2a), and males M73 and M98 contributing to 18 and 17 eggs, respectively (Fig. 2b). The main contributors within a single group were F129 with 8 offspring at GIV and M73 assigned to 9 eggs at GI. Sires played a more active role than dams during the mating process. On average, each male contributed to 4 groups of eggs (SD = 1.24) while females contributed to 3 groups (SD = 1.42). Therefore, it is deduced that both females and males release their eggs and sperm over several hours within a single day. Nevertheless, we could not identify a clear pattern on their duration or frequency. 4. Discussion
Fig. 1. Average number of A and Ho in the BR and eggs of black sea bream.
Detailed information on the parental contribution is required during the broodstock management in order to minimize the deleterious genetic effects of inbreeding, small population sizes in the offspring to be released. Allendorf and Phelps (1980) recognized three indicators to measure the loss of genetic variation in a hatchery strain: a reduction in the proportion of polymorphic loci, in the number of alleles per locus and in the heterozygosity. In the present study, the identification of most parental fish was possible due to the hyper polymorphism showed by the six microsatellite DNA loci genotyped. Most of the genetic variability observed in the BR (A = 18.3 and Ho = 0.879) was transferred to the offspring, except 5% of the A and 7% of the Ho, that were lost. This reduction is 2–3-fold
S16
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
Table 4 Pairwise FST values (below diagonal) and associated P-value (above diagonal) between BR and eggs of black sea bream. Eggs BR
GI
n = 143 BR GI GII GIII GIV GV GVI Nocturnal Pooled
n = 75
GII n = 75
0.144 0.008 − 0.000 0.002* 0.002 0.000 − 0.001 0.000 − 0.000
0.001 0.004 0.005* 0.005 0.001
0.730 0.207 0.002 0.004 0.001 − 0.001
GIII
GIV
GV
GVI
Nocturnal
Pooled
n = 75
n = 75
n = 75
n = 75
n = 225
n = 450
0.000 0.009 0.117
0.027 0.000 0.018 0.027
0.757 0.234 0.703 0.297 0.243 0.793
0.405
0.559
0.005 0.002 0.000
0.006* 0.001
0.234 0.027 0.378 0.045 0.000 − 0.001
*P b 0.002 after Bonferroni correction, k = 21.
Table 5 Polymorphic information content (PIC) of microsatellite loci base on the data of BR, exclusion probability (Q), probability of identity index (I) and frequency of null alleles (fn) at each locus determined for the pooled eggs. Locus ACS3
ACS4
ACS6
ACS9
ACS16
ACS17
Mean
PIC
0.8493
0.7838
0.7158
0.9191
0.9014
0.8859
0.8426 ± 0.08
Q I fn
0.9049 0.051 0.0105
0.8076 0.0687 0.0045
0.7623 0.1109 0.0049
0.9621 0.0126 0.0103
0.9450 0.0178 0.0493
0.9304 0.0256 0.0231
Total
~1 2.238E−9
lower than that reported in black sea bream juveniles produced as part of the stock enhancement program carried out in Hiroshima Bay, either before (Jeong et al., 2007) or after their release (Blanco Gonzalez et al, 2008a). Stock enhancement programs using hatchery reared juveniles requires special attention in order to preserve the genetic diversity of the wild population (Allendorf and Ryman, 1987; Ryman, 1997; Taniguchi, 2003). They proposed the use of large number of breeders from the local population as an essential prerequisite to maintain the genetic resources of the natural stocks. Here, it was confirmed that rearing 143 breeders resulted in a larger gene pool than rearing only 51 specimens (Blanco Gonzalez et al., 2008a). In the present study, the average number of alleles was also larger than those reported from several natural populations (e.g. Jeong et al., 2003). The lower variability observed by Jeong et al. (2003) may be attributed to the small sample size per location (n = 50). Nevertheless, this argument is not applicable when the results obtained here are compared to those reported in Hiroshima Bay analyzing larger samples (Blanco Gonzalez et al., 2008a; Blanco Gonzalez and Umino, 2009). In this bay, the long and intensive releases of juveniles produced from a limited number of breeders has not only seriously eroded the genetic resources after a bottleneck
episode, but also reduced the size-at-age of the natural stock (Blanco Gonzalez et al., 2009). Therefore, the natural population inhabiting Hiroshima Bay is likely to have suffered remarkable inbreeding and genetic drift problems due to inappropriate broodstock management practices, which would explain their lower diversity when compared to that observed in the present study. Beside the conservation improvement achievements using larger number of breeders, the multiple-egg-collection strategy confirmed its usefulness of identifying those alleles inherited in lower frequency, crucial information to preserve the founders’ gene pool. As a result of the large number of alleles scored per group of egg (Table 2), slight differences in the pairwise FST values among some groups of eggs and the BR were detected (Table 4), disappearing when the groups of eggs were pooled. However, the pool of all groups of eggs produced departures from the HWE due to heterozygosity deficit. The presence of null alleles was not detected (Table 2), suggesting that deviations from HWE may be a consequence of an increase in genetic drift. A study conducted on Atlantic cod (Gadus morhua) larvae showed how the combination of genetically heterogeneous cohorts resulted in a homogeneous group that presented departures from HWE (Ruzzante et al., 1996). Difference in reproductive success associated to oceanographic and environmental conditions were suggested to explain those results. Nugroho and Taniguchi (2004) reported some inbreeding and differences in the genetic composition of red sea bream (Pagrus major) offspring collected at different dates during the spawning peak. In that study, they referred to the low contribution of the breeders per collection day and some bottleneck effects and genetic drift to explain their result. They suggested pooling the eggs from several days as a mean to enhance the genetic variability and reduce the harmful consequences. In the present study, coancestry among breeders was not estimated, although considering the wild origin and differences in age classes of the BR, it seems not likely to be the cause of the inbreeding. Errors during egg collection may have favored some families, though the random sampling of the eggs
Table 6 Results of the pedigree analysis of black sea bream. Eggs n
Number of contributors Female (Nf) Male (Nm) Total number of families Nb (unbalanced family size) Nb F
GI
GII
GIII
GIV
GV
GVI
Nocturnal
Pooled
75
75
75
75
75
75
225
450
83 (58.0%) 42 (49.4%) 41 (70.7%) 74 83.0 84.8 0.006
87 (60.8%) 46 (54.15) 41 (70.7%) 74 86.7 113.2 0.004
85 (59.4%) 49 (57.6%) 36 (62.1%) 74 83.0 109.1 0.005
87 (60.8%) 46 (54.1%) 41 (70.7%) 72 86.7 103.2 0.005
85 (59.4%) 43 (50.6%) 42 (72.4%) 72 85.0 108.0 0.005
81 (56.6%) 41 (48.2%) 40 (69.0%) 70 81.0 104.4 0.005
136 (95.1%) 80 (94.1%) 56 (96.5%) 215 131.8 87.7 0.006
140 (97.9%) 83 (97.6%) 57 (98.3%) 406 135.2 36.6 0.014
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
S17
Fig. 2. Contribution of each parental female (a) and male (b) to the groups of eggs.
makes us to reject that hypothesis too. The answer seems to come out when looking at the family contribution of parental females and males (Fig. 2a, b) and the correction made due to the differential contribution among breeders that established the final Nb in scarcely 37, lower than the threshold number of 50 recommended by FAO/ UNEP (1981) for short-term enhancement programs. Large variation in individual reproductive success has been postulated to explain the uneven parental contribution in several marine organisms (Hedgecock, 1994; Ruzzante et al., 1996; Li and Hedgecock, 1998; Boudry et al., 2002). Such variation was suggested to be caused by differences in the gamete quality, sperm-egg interaction and viability between families (Boudry et al., 2002). In the present study the large contribution of a few breeders seems to be responsible of the final F (0.014), although it is significantly more promising than the 2.5% and 5.6% reported for the offspring prior to their release in Hiroshima Bay in 2000 and 2001, respectively (Jeong et al., 2007). In that study, Jeong et al. (2007) could only detect the contribution of 63% and 59% of the original BR, a proportion similar to that observed in the present study when each group of eggs was analyzed independently (57–61%), resulting in Nb = 20 and 9 in 2000 and 2001, respectively. The value of the inbreeding coefficient was also much lower than the 3% obtained in hatchery fish four years after their release (Blanco Gonzalez et al., 2008a). In addition, the parentage analysis conducted in black sea
bream reared at the YPFES confirmed a much larger proportion of breeders contributing to the offspring in this specie (140/143) when compared to that reported in other marine finfishes (Pérez-Enriquez et al., 1999; Hara and Sekino, 2003; Jackson et al., 2003; OrtegaVillaizán Romo et al., 2005, 2006; Kim et al., 2007). Consequently, the Nb scored for black sea bream here was also larger than all those studies, except for red sea bream (Pérez-Enriquez et al., 1999). In that species, the contribution of 91 out of 250 breeders produced a final Nb = 63.7, with only 0.8% of inbreeding. Despite this promising low rate of inbreeding, red sea bream offspring showed a reduction of 25% of the alleles scored in their founders. Therefore, although higher rates of inbreeding were observed in this study in black sea bream, the fact that almost all breeders contributed to the mating and that most of alleles were inherited gives room for optimism. In addition, the collection of three groups of eggs during the most intense period of spawning (nocturnal group) would not only simplify the offspring collection practices, but also increased Nb to 88, reducing the rate of inbreeding to b0.6%. Although it seems to be useful, the risk of losing some important genetic information makes us to be very cautious to suggest the adoption of this strategy before confirming its usefulness in other species. In addition, it is necessary to confirm the viability and performance of the offspring produced in this way in relation to those collected over the whole spawning season.
S18
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
Several means have been proposed to maintain the genetic resources and increase the Nb of hatchery strains for release: increase the number of breeders, collect the eggs in several days over the spawning season, apply the minimal kinship criteria among breeders, or maintain 1:1 sex ratio in the breeders (Allendorf and Ryman, 1987; Doyle et al., 2001; Nugroho and Taniguchi, 2004; Taniguchi, 2004; Fraser, 2008). In addition to these recommendations, based on our results, we suggest the collection of eggs at different times over a single night as part of the broodstock management design in case the collection of the eggs cannot be extended over the whole spawning season. Besides identifying the main contributors during the night the eggs were collected, this simple strategy may also identify those breeders only contributing in small proportion. This information may be crucial in case of alleles present at low frequencies in the wild, and hence highly relevant to preserve the gene pool of the natural population subject to enhancement. The strategy proposed would be very useful to recognize those families over-represented and equalize the parental contribution which would increase Nb and reduce F; however, posterior selection processes cannot be neglected. Considering that the eggs are produced in a single night and selecting the lots that conserve the larger genetic variability, several lots could be combined and reared under the same conditions, reducing cost and space requirements in hatcheries with limited capacity. 5. Conclusions The genetic benefits of rearing a larger number of breeders have been confirmed in this study. The broodstock maintained at the YPFES (n = 143) showed larger allele number than that reported in Hiroshima Bay (BR = 51) at six polymorphic microsatellites (Blanco Gonzalez et al., 2008a). A general concern exists regarding the inheritance of those alleles present in lower frequencies as well as the reduction of heterozygote in the offspring due to the hatchery conditions (Allendorf and Phelps, 1980; Allendorf and Ryman, 1987). The collection of eggs in several lots over the night revealed an extremely high contribution of the breeders (140/143, 98%). Consequently, most of the genetic variability was transferred to the offspring. The high rate of inheritance, in addition to the use of a large BR, has considerably helped to minimize the erosion of the genetic resources. The adoption of these simple means in the BR management plan of black sea bream has confirmed their usefulness to increase Nb (37) and reduced the rate of inbreeding (1.4%) when compared to the levels reported for this species in Hiroshima Bay (Jeong et al., 2007; Blanco Gonzalez et al., 2008a). Moreover, it was observed that the effective number of parents exceeded 100 (Nb = 136). Therefore, if we are able to balance the contribution of the over-represented breeders (i.e., by tracing the pedigree and selecting the lots of eggs that maximize Nb), the BR used in the present study confirmed its potential to produce offspring with similar genetic composition to enhance the wild stocks. The results, although promising, require a lot of caution and need to be tested in every species before their implementation. Further research should also be routinely done with larger samples of offspring in order to get a more accurate estimation of their genetic inheritance. In addition, it is necessary to confirm the viability and performance of the offspring produced in this way in relation to those collected over the whole spawning season. The collection of several batches of eggs over a single night should not exclude other strategies previously suggested for broodstock management (Allendorf and Ryman, 1987; Doyle et al., 2001; Nugroho and Taniguchi, 2004; Taniguchi, 2004; Fraser, 2008). Acknowledgement We wish to thank Mr. Hiroshi Shiozaki and the staff at the YPFES for their assistant during the sample collection. This study was partly supported by a Grant-in-Aid for Scientific Research (C) from the Japan
Society for the Promotion of Science (JSPS) to T.U. (Nos. 145600152 and 19580205). References Allendorf, F.W., Phelps, S.R., 1980. Loss of genetic variation in a hatchery stock of cutthroat trout. Trans. Am. Fish. Soc. 109, 537–543. Allendorf, F.W., Ryman, N., 1987. Genetic Management of Hatchery Stocks. In: Ryman, N., Utter, F.M. (Eds.), Population Genetics and Fishery Management. University of Washington Press, Seattle, pp. 141–159. Bell, J.D., Leber, K.M., Blankenship, H.L., Loneragan, N.R., Masuda, R., 2008. A new era for restocking, stock enhancement and sea ranching of coastal fisheries resources. Rev. Fish. Sci. 16, 1–8. Blanco Gonzalez, E., Murakami, T., Yoneji, T., Nagasawa, K., Umino, T., 2009. Reduction in size-at-age of black sea bream (Acanthopagrus schlegelii) following intensive releases of cultured juveniles in Hiroshima Bay, Japan. Fish. Res. 99, 130–133. Blanco Gonzalez, E., Nagasawa, K., Umino, T., 2008a. Stock enhancement program for black sea bream (Acanthopagrus schlegelii) in Hiroshima Bay: monitoring the genetic effects. Aquaculture 276, 36–43. Blanco Gonzalez, E., Umino, T., 2009. Fine-scale genetic structure derived from stocking black sea bream, Acanthopagrus schlegelii (Bleeker, 1854), in Hiroshima Bay, Japan. J. App. Icht. 25, 407–410. Blanco Gonzalez, E., Umino, T., Nagasawa, K., 2008b. Stock enhancement program for black sea bream (Acanthopagrus schlegelii) in Hiroshima Bay, Japan: a review. Aquac. Res. 39, 1307–1315. Blankenship, H.L., Leber, K.M., 1995. A responsible approach to marine stock enhancement. Am. Fish. Soc. Symp. 15, 167–175. Boudry, P., Collet, B., Cornette, F., Hervouet, V., Bonhomme, F., 2002. High variance in reproductive success of Pacific oyster (Crassostrea gigas, Thunberg) revealed by microsatellite-based parentage analysis of multifactorial crosses. Aquaculture 204, 283–296. Doyle, R.W., Pérez-Enriquez, R., Takagi, M., Taniguchi, N., 2001. Selective recovery of founder genetic diversity in aquacultural broodstocks and captive, endangered fish populations. Genetics 111, 291–304. Duchesne, P., Godbout, M.H., Bernatchez, L., 2002. PAPA (package for the analysis of parental allocation): a computer program for simulated and real parental allocation. Mol. Ecol. Notes 2, 191–193. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol. Bioinform. Online 1, 47–50. FAO/UNEP, 1981. Conservation of the genetic resources of fish: problems and recommendations. Report of the Expert Consultation on the genetic resources of fish. Rome, 9-13 June 1980. FAO Fish. Tech. Pap. 217, 1–43. Fraser, D.J., 2008. How well can captive breeding programs conserve biodiversity? A review of salmonids. Evol. App. 1, 535–585. Goudet, J., 2001. FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3). Available: http://www2.unil.ch/popgen/softwares/fstat. htm. Hara, M., Sekino, M., 2003. Efficient detection of parentage in a cultured Japanese flounder Paralichthys olivaceus using microsatellite DNA marker. Aquaculture 217, 107–114. Hedgecock, D., 1994. Does Variance in Reproductive Success Limit Effective Population Sizes of Marine Organisms? In: Beaumont, A.R. (Ed.), Genetics and Evolution of Aquatic Organisms. Chapman and Hall, London, pp. 122–134. Jackson, T.R., Martin-Robichaud, D.J., Reith, M.E., 2003. Application of DNA markers to the management of Atlantic halibut (Hippoglossus hippoglossus) broodstock. Aquaculture 220, 245–259. Jeong, D.-S., Blanco Gonzalez, E., Morishima, K., Arai, K., Umino, T., 2007. Parentage assignment of stocked black sea bream, Acanthopagrus schlegelii in Hiroshima Bay using microsatellite DNA markers. Fish. Sci. 73, 823–830. Jeong, D.-S., Hayashi, M., Umino, T., Nakagawa, H., Morishima, K., Arai, K., 2002. Comparison of genetic variability between wild and hatchery-stocked black sea bream Acanthopagrus schlegeli using DNA fingerprinting. Fish Genet. Breed. Sci. 32, 135–139 (in Japanese with English abstract). Jeong, D.-S., Umino, T., Kuroda, K., Hayashi, M., Nakagawa, H., Kang, J.-C., Morishima, K., Arai, K., 2003. Genetic divergence and population structure of black sea bream Acanthopagrus schlegeli inferred from microsatellite analysis. Fish. Sci. 69, 896–902. Kasahara, S., Hibiya, T., 1967. Fundamental studies on techniques of mass-production of fry of the black porgy, Mylio macrocephalus — I. On the spawning induced by injection of a preparation of mammalian gonadotropic hormones. J. Fac. Fish. Anim. Husb. Hiroshima Univ. 7, 105–111 (in Japanese). Kasahara, S., Oshimna, Y., 1960. Black sea bream culture: mass production and other problems related to seed production. Suisanzoshoku 7, 41–47 (in Japanese). Kasahara, S., Hirano, R., Ooshima, Y., 1960. A study on the growth and rearing methods of the fry of black porgy, Mylio macrocephalus (Basilewsky). Bull. Jap. Soc. Sci. Fish. 26, 239–244 (in Japanese with English abstract). Kim, S.G., Morishima, K., Satoh, N., Fujioka, T., Saito, S., Arai, K., 2007. Parentage assignment in hatchery population of brown sole Pleuronectes herzensteini by microsatellite DNA markers. Fish. Sci. 73, 1087–1093. Kitada, S., Kishino, H., 2006. Lessons learned from Japanese marine finfish stock enhancement programmes. Fish. Res. 80, 101–112. Lande, R., Barowwclough, G.F., 1987. Effective population size, genetic variation, and their use in population management. In: Soulé, M. (Ed.), Viable Populations for Conservation. Cambridge University Press, Cambridge, pp. 87–124. Launey, S., Hedgecock, D., 2001. High genetic load in the Pacific oyster Crassostrea gigas. Genetics 159, 255–265.
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19 Li, G., Hedgecock, D., 1998. Genetic heterogeneity, detected by PCR SSCP, among samples of larval Pacific oyster (Crassostrea gigas) supports the hypothesis of large variance in reproductive success. Can. J. Fish. Aquat. Sci. 55, 1025–1033. Nakagawa, H., Umino, T., Hayashi, M., Sasaki, T., Okada, K., 2000. Changes in biochemical composition of black sea bream released at 20 mm size in Daio Bay, Hiroshima. Suisanzoshoku 48, 643–648. Nugroho, E., Taniguchi, N., 2004. Daily change of genetic variability in hatchery offspring of red sea bream during spawning season. Fish. Sci. 70, 638–644. Paetkau, D., Strobeck, C., 1994. Microsatellite analysis of genetic variation in black bear populations. Mol. Ecol. 489–495. Pérez-Enriquez, R., Takagi, M., Taniguchi, N., 1999. Genetic variability and pedigree tracing of a hatchery-reared stock of red sea bream (Pagrus major) used for stock enhancement, based on microsatellite DNA markers. Aquaculture 173, 413–423. Ortega-Villaizán Romo, M.M., Aritaki, M., Taniguchi, N., 2006. Pedigree analysis of recaptured fish in the stock enhancement program of spotted halibut Verasper variegatus. Fish. Sci. 72, 48–52. Ortega-Villaizán Romo, M.M., Suzuki, S., Ikeda, M., Nakajima, M., Taniguchi, N., 2005. Monitoring of the genetic variability of the hatchery and recaptured fish in the stock enhancement program of the rare species barfin flounder Verasper moseri. Fish. Sci. 71, 1120–1130. Raymond, M., Rousset, F., 1995. GENEPOP (version 3.4): population genetics software for exact tests and ecumenicism. J. Hered. 86, 248–249. Rice, W.R., 1989. Analyzing tables of statistical tests. Evolution 43, 223–225. Ruzzante, D.E., Taggart, C.T., Cook, D., 1996. Spatial and temporal variation in the genetic composition of a larval cod (Gadus morhua) aggregation: cohort contribution and genetic stability. Can. J. Fish. Aquat. Sci. 53, 2695–2705.
S19
Ryman, N., 1997. Minimizing adverse effects of fish culture: understanding the genetics of populations with overlapping generations. ICES J. Mar. Sci. 54, 1149–1159. Ryman, N., Laikre, L., 1991. Effects of supportive breeding on the genetically effective population size. Conserv. Biol. 5, 325–329. Taggart, J.B., Hynes, R.A., Prodohl, P.A., Ferguson, A., 1992. A simplified protocol for routine total DNA isolation from salmonid fishes. J. Fish Biol. 40, 963–965. Taniguchi, N., 2003. Genetic factors in broodstock management for seed production. Rev. Fish Biol. Fish. 13, 177–185. Taniguchi, N., 2004. Broodstock management for stock enhancement programs of marine fish with assistance of DNA marker (a review). In: Leber, K.M., Kitada, S., Blankenship, H.L., Svasand, T. (Eds.), Stock Enhancement and Sea Ranching. Developments, Pitfalls and Opportunities. Blackwell Publishing, Oxford, pp. 329–338. Taniguchi, N., Sumantadinata, K., Iyama, S., 1983. Genetic change in the first and second generations of hatchery stock of black seabream. Aquaculture 35, 309–320. Tringali, M., Leber, K.M., Halstead, W.G., McMichael, R., O'Hop, J., Winner, B., Cody, R., Young, C., Neidig, C.N., Wolfe, H., Forstchen, A., Barbieri, L., 2008. Marine stock enhancement in Florida: A multi-disciplinary, stakeholder-supported, accountabilitybased approach. Rev. Fish. Sci. 16, 51–57. Umino, T., Hayashi, M., Miyatake, J., Nakayama, K., Sasaki, T., Okada, K., Nakagawa, H., 1999. Significance of release of black sea bream at 20-mm size on stock enhancement in Daiô Bay, Hiroshima. Suisanzoshoku 47, 337–342. Villanueva, B., Verspoor, E., Visscher, P.M., 2002. Parental assignment in fish using microsatellite genetic markers with finite numbers of parents and offspring. Anim. Gen. 33, 33–41. Weir, B.S., Cockerham, C.C., 1984. Estimating F-statistics for the analysis of population structure. Evolution 38, 1358–1370.
Contents lists available at ScienceDirect
Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Can ordinary single-day egg collection increase the effective population size in broodstock management programs? Breeder-offspring assignment in black sea bream (Acanthopagrus schlegelii) through two-hourly intervals Enrique Blanco Gonzalez a,b, Nobuhiko Taniguchi b, Tetsuya Umino a,⁎ a b
Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan The Research Institute of Marine Bioresources, Fukuyama University, 452-10 Innoshima-Ohama, Onomichi 722-2101, Japan
a r t i c l e
i n f o
Article history: Received 31 July 2009 Received in revised form 14 January 2010 Accepted 25 June 2010 Keywords: Broodstock management Black sea bream Acanthopagrus schlegelii Effective population size Parentage assignment Egg
a b s t r a c t The management of the broodstock used for stock enhancement purposes is essential to preserve the genetic resources of the natural populations. Black sea bream (Acanthopagrus schlegelii) has been extensively released in Japan; nevertheless, the little emphasis paid on the genetic conservation, resulted in important drifts due to the small number of parental fish reared in the hatcheries. The present study shows how simple changes in the broodstock management procedure may greatly contribute to increase the effective number of breeders (Nb) and reduce the rate of inbreeding. In this regard, the collection of eggs at several timings over a single night allowed the identification of those contributors spawning for a short period and with little overall contribution. It enabled identifying those alleles presented in lower frequencies, an essential issue on conservation genetics. The second improvement involved the use of a reasonable large number of individuals as breeders. In this study, the broodstock (BR) comprised 143 specimens, a feasible number to be reared at any hatchery that produces juveniles for stocking in Japan. The BR-offspring assignment confirmed the contribution of an extremely high number of breeders (140), corrected to 136 after considering the differences in sex proportions. The number of offspring comprising each family was similar (up to 3). Nevertheless, large disparities in the contribution were observed among breeders. As a result of the large number of offspring produced by few parental fish, Nb was reduced to 37. Although lower than desirable, the fact that most of breeders took part in the mating process, confirmed the usefulness of this procedure. An alternative method to reduce and simplify the number of egg batches required was investigated. The three groups of eggs sampled in the middle of the sampling period and representing the period the releases were most intensive, were combined (nocturnal group), showing similar results. In this case 136 out of the initial 143 breeders took part in the mating process, and the final Nb was 88. These results confirm that by either of these simple means, most of the genetic diversity would be inherited by the offspring and help to preserving the genetic resources of the natural populations subject to stocking. This experience, although promising, should also be applied to other species to confirm its effectiveness. In addition, it is necessary to confirm the viability and performance of the offspring produced in this way in relation to those collected over the whole spawning season. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The success of aquaculture practices highly depends on the broodstock reared in a hatchery. Their management is essential, especially in stock enhancement programs that aim at preserving natural resources through artificially produced seeds propagation (Allendorf and Phelps, 1980; Taniguchi, 2003, 2004). Stock enhancement programs comprise two main phases. The first one includes the collection of the broodstock, mass production and rearing of the
⁎ Corresponding author. Tel./fax: + 81 824 24 7944. E-mail address: [email protected] (T. Umino). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.06.031
offspring in hatcheries. Secondly, the fish are released into the natural environment once they reach the appropriate size, and are expected to contribute to the breeding population of the natural stock. Initially, most of the efforts had been directed towards increasing the survival of the offspring released into the wild and the fishing yields. However, the importance of preserving all the natural resources, including the genetic diversity of the wild stocks, mean it is necessary to develop a more exhaustive and carefully designed strategy, taking a broader comprehensive approach which integrates enhancements in a more conservative manner (Blankenship and Leber, 1995; Bell et al., 2008; Tringali et al., 2008). Several key strategies have been proposed as a means to maintain the gene pool inherited by the offspring such as the conservation of rare alleles, the use of a large number of native
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
specimens as breeders and the collection of eggs in several days over the spawning season (Allendorf and Ryman, 1987; Doyle et al., 2001; Nugroho and Taniguchi, 2004; Taniguchi, 2004; Fraser, 2008). In this regard, the high cost and space requirements of rearing a large native wild broodstock population have been the main constraint in stock enhancement programs. In Japan, millions of seed of high-valued commercial fish species are released annually throughout the country (Kitada and Kishino, 2006). Nevertheless, in most cases, the small number of breeders maintained in the hatchery has resulted in high levels of inbreeding, compromising the gene pool in the receptive population. In addition, the juveniles for release have usually originated in one night during the spawning peak. Consequently, the contribution of some specimens is missing. As a means to increase the effective number of breeders (Nb) and mitigate the adverse effects in the offspring, it has been suggested to extend the egg collection (Nugroho and Taniguchi, 2004) over an extended period. Black sea bream (Acanthopagrus schlegelii) has a long history of stock enhancement in Japan with releases carried out in almost half of the national prefectures (23 out of 47) since the late 1970s. The seed production and rearing method for this species were first described in the 1960s (Kasahara et al., 1960; Kasahara and Oshimna, 1960; Kasahara and Hibiya, 1967). Several studies have been conducted in order to understand the acclimation, biochemical composition and behavior of hatchery reared juveniles (for a review see Blanco Gonzalez et al., 2008b). Nevertheless, it was not until the development of genetic techniques when the contribution of the broodstock was assessed. The first evaluation of the genetic effects in the first and second generations of hatchery stocks of black sea bream was carried out by Taniguchi et al. (1983) using 42 isozymes. They reported that only between 16 and 26 out of the 102 breeders reared in the hatchery contributed to the first two generations of offspring. Recently, the contribution of the broodstock used for the intensive stock enhancement program conducted in Hiroshima Bay was investigated by DNA fingerprinting (Jeong et al., 2002) and genotyping a few microsatellite markers (Jeong et al., 2007; Blanco Gonzalez et al., 2008a). It was observed that the large number of juveniles released over three decades originated from less than 100 breeders, modified the genetic diversity of the natural stock of black sea bream inhabiting Hiroshima Bay (Blanco Gonzalez and Umino, 2009). Before the release (broodstock = 51), Nb was 20 and 9 in 2000 and 2001, respectively (Jeong et al., 2007). Four years after the release, hatchery reared fish represented 13% of the samples and Nb for the same broodstock was estimated to be 16 (Blanco Gonzalez et al., 2008a). The good acclimation and high survival of the juveniles (Umino et al., 1999; Nakagawa et al., 2000; Jeong et al., 2007) and their presence in the group of spawners (Blanco Gonzalez et al., 2008a), suggested a potential hybridization with their wild counterparts. Therefore, it is essential to develop a proper management plan during the captive phase to minimize the genetic loss in the juveniles to be released. This study aims at improving the broodstock management practices and increasing Nb in black sea bream hatchery released populations. It also aims at identifying those alleles present in lower frequencies and that may require special conservation practices. The
S13
results obtained here should be considered to preserve the gene pool of the natural stocks before conducting large scale releases. 2. Materials and methods 2.1. Broodstock and egg collection The broodstock (BR) used in the present study were reared at the Yamaguchi Prefectural Fisheries Experimental Station (YPFES) and comprised 85 dams and 58 sires. Black sea bream eggs were collected by tank overflowing on May 17, 2006. The first sample was collected at 13:14 (GMT + 9) and 11 additional batches were collected successively every ~2 h, with the last one taken at 11:05 (GMT + 9) on May 18, 2006 (Table 1). Once collected, the wet weight of each batch was measured and ~ 5 g of eggs from each were transferred into 500 ml bin saturated with oxygen and incubated for ~ 2 h to confirm their fertilization. Afterwards, the eggs were preserved in 95% ethanol and stored at 4 °C for later DNA extraction. Egg batches representing the peak of the spawning (19:15–23:15 GMT + 9) were analyzed as independent groups. Meanwhile, several batches of low wet weight collected before and after the peak of the spawning were pooled together to simplify the analysis. At the end, the eggs were divided into six groups based on collection time (see Table 1). On May 27, some scales were removed from the right-ventral side of each breeder during their transfer from indoor tanks to the outdoor conditions, placed into 95% ethanol and stored at 4 °C for later DNA extraction. For the posterior analysis, besides the BR and each of the six groups of eggs, two additional group combinations were considered. The first one represents the pool of all groups of eggs together over one day (“Pooled”) as is the normal practice. The other group (“Nocturnal”) is a combination of the three groups taken in the middle of the collection period (GII, GIII and GIV) and representing the heaviest wet weight. The genetic composition of the nocturnal group was investigated as an alternative way to reduce the number of lots collected during one night without genetic losses. 2.2. DNA extraction and genotyping The DNA extraction was done by two different methods. For the BR, the DNA was obtained following the standard SDS-phenol/ chloroform procedure described by Taggart et al. (1992) as done in our previous study (see details in Blanco Gonzalez et al., 2008a). An alternative Chelex/proteinase K method modified from the one described by Launey and Hedgecock (2001) was followed due the low amount of DNA contained in the eggs. A drop of alcohol was evaporated from the eggs at room temperature. Then, each egg was added to 20 μl of 5% chelex, 3 μl of TE (0.01 M Tris, 1 mM EDTA) and 1 μl of proteinase K (10 mg/ml). The mixture was heated at 55 °C for 2 h and boiled at 100 °C for 10 min. The samples were centrifuged at 4000 rpm for 5 min and the supernatant transferred to clean test tubes and stored at −20 °C for posterior genotyping. The amplification of the DNA was performed by the PCR using the same six loci (ACS3, ACS4, ACS6, ACS9, ACS16 and ACS17) and a
Table 1 Broodstock (BR) and eggs of black sea bream analyzed in this study with detailed information of the collection time and group composition of the eggs.
Date
BR
Eggs
May 27
May 17
May 18
Group
Collection time (GMT + 9)
Nocturnal GI
GII
GIII
GIV
GV
GVI
n = 143
n = 75
n = 75
n = 75
n = 75
n = 75
n = 75
12:45
13:14
19:15
21:10
23:15
1:20
15:14
17:14
3:10
5:08
7:16
9:10
11:05
S14
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
protocol described in our previous study (see details in Blanco Gonzalez et al., 2008a).
2.3. Genetic diversity and pedigree analysis The genetic diversity at each locus was calculated as number of alleles per locus (A) and expected (He) and observed (Ho) heterozygosities for the BR and the eggs using the Excel Microsatellite Toolkit. The allelic richness (Ar) was also calculated between the BR and each group of eggs and between the BR and the pool of eggs in order to correct the differences in sample sizes by FSTAT (Goudet, 2001). Departures from Hardy–Weinberg equilibrium (HWE) at each locus and averaged over all loci were evaluated using a test analogous to Fisher's exact test, with a modified version of the Markov chain method (105 steps and 104 dememorisations) implemented with GENEPOP v4.0 (Raymond and Rousset, 1995). This software was also used to estimate the frequency of null alleles (fn) as well as the inbreeding coefficient, FIS (Weir and Cockerham, 1984). The overall significant value was adjusted following the sequential Bonferroni procedure (Rice, 1989). The overall genetic differentiation among the BR and eggs was evaluated with the AMOVA test with ARLEQUIN v3.0 (Excoffier et al., 2005). The pairwise FST values between the BR and the groups of eggs were also determined using this software. The polymorphic information content (PIC) at each locus implemented with Excel Microsatellite Toolkit was estimated for the BR. The exclusion probability Q (Villanueva et al., 2002) was also calculated. In addition, the probability of identity index (I) described by Paetkau and Strobeck (1994) was tested in the pool of eggs.
The pedigree analysis was performed comparing the genotypes of the BR and each of the 450 eggs at six loci using PAPA software (Duchesne et al., 2002). True parental couples were considered whenever the genotype of both parents coincided with that of the offspring at all loci. Two corrections should be applied to the total number of contributors assigned to the offspring (Lande and Barowwclough, 1987). The first one is due to unequal number of males and females contributing to the offspring. As a result, Nb will be determined by the number of contributors of the less numerous sex. The second correction is because of the variance in the number of offspring per family. Ryman and Laikre (1991) proposed a model to estimate the effective population size (Ne) in stock enhancement programs as the sum of the relative contribution of both wild and hatchery origin individuals. Since the broodstock used in this study comprised only wild specimens Ne = Nb and the inbreeding coefficient (F) was estimated as F = 1/2 Nb. 3. Results 3.1. Genetic diversity In this study, black sea bream showed a high genetic diversity expressed either as allelic (A, Ar) or heterozygosity (Ho) variability at all loci (Table 2). In the BR, allele number ranged from 10 to 27 (mean = 18.33), while each group of eggs inherited 7 to 22 alleles per locus (mean per group ranged 14.3–15.7). After the corresponding adjustment due to differences in sample sizes, the mean Ar for the BR was 16.5. The high conservation of the genetic resources inherited by the eggs was confirmed when all six groups of eggs were combined. In
Table 2 Genetic variability of the BR and eggs of black sea bream at 6 microsatellite loci. * and ** denote P b 0.05 and P b 0.01, respectively. Locus
ACS3
ACS4
ACS6
ACS9
ACS16
ACS17
Mean
Eggs
A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS A Ar He Ho FIS
BR
GI
GII
GIII
GIV
GV
GVI
Nocturnal
Pooled
n = 143
n = 75
n = 75
n = 75
n = 75
n = 75
n = 75
n = 225
n = 450
27 22.9 0.863 0.909 −0.054 10 9.2 0.813 0.860 − 0.059 13 11.9 0.741 0.783 − 0.057 26 22.4 0.927 0.895 0.035** 18 17.3 0.912 0.958 − 0.051 16 15.2 0.898 0.867 0.035 18.3 ± 6.9 16.5 ± 5.5 0.859 ± 0.06 0.879 ± 0.05 − 0.023
20 20 0.834 0.813 0.025 9 9 0.824 0.813 0.014 11 11 0.715 0.667 0.067 18 18 0.885 0.893 − 0.009 17 17 0.910 0.867 0.048 15 15 0.886 0.893 − 0.008 15.0 ± 4.2 15.0 ± 4.4 0.843 ± 0.06 0.824 ± 0.08 0.022
16 16 0.830 0.747 0.101 8 8 0.793 0.787 0.008 10 10 0.730 0.707 0.032 21 21 0.924 0.907 0.019 16 16 0.903 0.880 0.025 15 15 0.870 0.840 0.035 14.3 ± 4.7 14.3 ± 4.7 0.841 ± 0.07 0.811 ± 0.07 0.036
16 16 0.767 0.787 − 0.026 7 7 0.790 0.813 − 0.029 10 10 0.658 0.707 − 0.075 22 22 0.921 0.947 − 0.028 17 17 0.897 0.893 0.004 15 15 0.875 0.800 0.086* 14.5 ± 5.3 14.5 ± 5.3 0.818 ± 0.09 0.824 ± 0.08 − 0.008
18 18 0.830 0.760 0.084 9 9 0.763 0.787 − 0.031 11 11 0.700 0.733 − 0.048 19 19 0.908 0.920 − 0.013 17 17 0.901 0.813 0.098** 15 15 0.902 0.853 0.054 14.8 ± 4.0 14.8 ± 4.0 0.834 ± 0.8 0.811 ± 0.06 − 0.028
19 19 0.794 0.760 0.044 9 9 0.805 0.920 − 0.143 10 10 0.721 0.667 0.076 20 20 0.926 0.827 0.108* 15 15 0.885 0.893 − 0.009* 16 16 0.860 0.853 0.008 14.8 ± 4.5 14.8 ± 4.5 0.832 ± 0.07 0.820 ± 0.08 0.015
20 20 0.799 0.827 − 0.035 10 10 0.813 0.813 − 0.001 11 11 0.704 0.747 − 0.061 21 21 0.925 0.947 − 0.023 17 17 0.904 0.920 − 0.018 15 15 0.877 0.840 0.043 15.7 ± 4.5 15.7 ± 4.5 0.837 ± 0.07 0.849 ± 0.07 − 0.014
22 20.1 0.812 0.764 0.057 10 9.5 0.783 0.796 − 0.018 11 10.6 0.700 0.716 − 0.026 23 22.3 0.920 0.924 − 0.005 17 16.9 0.904 0.862 0.046 15 14.9 0.885 0.831 0.061 16.3 ± 5.4 15.7 ± 5.1 0.833 ± 0.09 0.816 ± 0.07 0.021
25 21.3 0.809 0.782 0.033 10 9.5 0.800 0.822 − 0.028 12 11.2 0.706 0.704 0.002 25 22.7 0.917 0.907 0.011* 17 16.9 0.902 0.878 0.027 16 15.8 0.881 0.847 0.039 17.5 ± 6.35 16.2 ± 5.3 0.836 ± 0.08 0.823 ± 0.07 0.015*
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
this case, A ranged 10–25 (mean = 17.5). Therefore, over 95% of the alleles observed in the BR were present in the pooled offspring. Similarly, the high level of heterozygosity observed in the BR (Ho = 0.879) was also detected in the offspring (Ho = 0.823), where the loss of heterozygosity accounted for b 7%, confirming a high conservation on the genetic variability in black sea bream. Nevertheless, the pool of eggs produced departures from HWE overall loci that were not observed when each group of eggs was analyzed independently. The deviation from HWE was towards heterozygosity deficit (P = 0.002). The presence of null alleles was not detected in the pool of eggs (Table 5) as well as in any individual group of eggs. The nocturnal group showed slightly lower levels of diversity (A = 16.3; Ar = 15.7; Ho = 0.816) compared to the pooled group (Table 2; Fig. 1); nevertheless, these values were not statistically significant at P b 0.05. Also, the group was in agreement with the HWE overall loci. Significant differences (χ2) on allele frequencies at some major alleles were observed between the BR and each group of eggs except GII (Table 3). These differences were not detected at the locus ACS16. The allele 87 at the locus ACS3 appeared in much higher frequencies in three groups (GIII, GV and GVI) than in the BR. Consequently, the frequency of this allele in the pool of eggs was also significantly higher than that observed in the BR (χ2 = 6.03, d.f. = 1). Besides this excess (χ2 = 4.17, d.f. = 1), the nocturnal group also evidenced the low presence of the allele 100 at ACS6 (χ2 = 5.01, d.f. = 1). The collection of eggs at different timing revealed significant differences in the pairwise FST values among some groups (Table 4) as was the case when GIV was compared to GI and GV. Significant differences were also detected between the BR and GIII. Nevertheless, in all cases, the FST values were very low (b0.008). Once the eggs were pooled, the differences between the egg groups and the BR disappeared. The overall AMOVA averaged over loci also confirmed the homogeneity (P = 0.18). 3.2. Parentage assignment The high PIC scored across all loci (0.8426 ± 0.08) reflects the high polymorphism of the six loci genotyped in the present study (Table 5). The use of six loci revealed very useful to assign the offspring to their true parental fish (Q = 1). In addition, the low probability of finding two individuals sharing the same genotype (I = 2.238E−9), confirmed the strength of the parentage analysis. True parental assignment was possible to all offspring with two exceptions (448 out of 450). The number of contributors per group of eggs ranged from 81 to 87 (41–49 and 36–41 for females and males, respectively) to create between 70 and 74 families per group (Table 6). The fact that most families per group were composed of a single individual increased Nb over 100 except in GI. As a result, the inbreeding rate per group remained very low, between 0.4 and 0.6%. This promising scenario changed drastically when all eggs were pooled together. The
S15
Table 3 Differences in the frequency of major alleles (bp) between the BR and eggs of black sea bream at six microsatellite loci (χ2, d.f. = 1). * and ** denote P b 0.05 and P b 0.01, respectively. Locus
BR
Eggs GI
GII
GIII
GIV
GV
GVI
Nocturnal
Pooled
0.11 0.30 0.12
0.13 0.35 0.10
0.13 0.34 0.11
0.13 0.43** 0.13
0.09 0.36 0.13
0.11 0.41* 0.10
0.05 0.41* 0.11
0.12 0.38* 0.12
0.11 0.38* 0.11
ACS4 69 71 73
0.21 0.28 0.21
0.19 0.23 0.22
0.19 0.28 0.29
0.20 0.31 0.25
0.18 0.37* 0.24
0.25 0.29 0.16
0.22 0.28 0.20
0.19 0.32 0.26
0.21 0.29 0.23
ACS6 98 100 102
0.46 0.12 0.14
0.48 0.14 0.17
0.48 0.10 0.13
0.56 0.09 0.12
0.49 0.03** 0.21
0.48 0.15 0.12
0.51 0.09 0.15
0.51 0.07* 0.15
0.50 0.10 0.15
ACS9 79 83 87
0.08 0.17 0.10
0.09 0.25* 0.15
0.07 0.15 0.13
0.13 0.17 0.04*
0.11 0.22 0.08
0.09 0.15 0.06
0.06 0.17 0.11
0.10 0.18 0.08
0.09 0.18 0.09
ACS16 93 0.12 95 0.16 107 0.11
0.09 0.19 0.12
0.09 0.17 0.12
0.08 0.15 0.13
0.13 0.21 0.13
0.13 0.19 0.14
0.10 0.17 0.15
0.10 0.18 0.12
0.10 0.18 0.13
ACS17 142 0.16 144 0.16 146 0.14
0.11 0.15 0.23*
0.11 0.23 0.20
0.18 0.16 0.21
0.16 0.17 0.10
0.13 0.28** 0.17
0.15 0.21 0.18
0.15 0.19 0.17
0.14 0.20 0.18
ACS3 85 87 89
contribution of most breeders was confirmed (140/143), with only one male (M61) and two females (F26, F72) not contributing to the offspring. A total of 406 small size families were identified. However, a few breeders produced large number of offspring during the night. Consequently, the final Nb dropped to 37, much lower than 136, the value obtained before making the adjustments due to the differential contribution among breeders. In the nocturnal group, the number of breeders identified was 136, and in this case Nb = 88. Special reference to the drastic reduction when all six groups were pooled should be given to females F134 and F137 who contributed to 17 eggs (Fig. 2a), and males M73 and M98 contributing to 18 and 17 eggs, respectively (Fig. 2b). The main contributors within a single group were F129 with 8 offspring at GIV and M73 assigned to 9 eggs at GI. Sires played a more active role than dams during the mating process. On average, each male contributed to 4 groups of eggs (SD = 1.24) while females contributed to 3 groups (SD = 1.42). Therefore, it is deduced that both females and males release their eggs and sperm over several hours within a single day. Nevertheless, we could not identify a clear pattern on their duration or frequency. 4. Discussion
Fig. 1. Average number of A and Ho in the BR and eggs of black sea bream.
Detailed information on the parental contribution is required during the broodstock management in order to minimize the deleterious genetic effects of inbreeding, small population sizes in the offspring to be released. Allendorf and Phelps (1980) recognized three indicators to measure the loss of genetic variation in a hatchery strain: a reduction in the proportion of polymorphic loci, in the number of alleles per locus and in the heterozygosity. In the present study, the identification of most parental fish was possible due to the hyper polymorphism showed by the six microsatellite DNA loci genotyped. Most of the genetic variability observed in the BR (A = 18.3 and Ho = 0.879) was transferred to the offspring, except 5% of the A and 7% of the Ho, that were lost. This reduction is 2–3-fold
S16
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
Table 4 Pairwise FST values (below diagonal) and associated P-value (above diagonal) between BR and eggs of black sea bream. Eggs BR
GI
n = 143 BR GI GII GIII GIV GV GVI Nocturnal Pooled
n = 75
GII n = 75
0.144 0.008 − 0.000 0.002* 0.002 0.000 − 0.001 0.000 − 0.000
0.001 0.004 0.005* 0.005 0.001
0.730 0.207 0.002 0.004 0.001 − 0.001
GIII
GIV
GV
GVI
Nocturnal
Pooled
n = 75
n = 75
n = 75
n = 75
n = 225
n = 450
0.000 0.009 0.117
0.027 0.000 0.018 0.027
0.757 0.234 0.703 0.297 0.243 0.793
0.405
0.559
0.005 0.002 0.000
0.006* 0.001
0.234 0.027 0.378 0.045 0.000 − 0.001
*P b 0.002 after Bonferroni correction, k = 21.
Table 5 Polymorphic information content (PIC) of microsatellite loci base on the data of BR, exclusion probability (Q), probability of identity index (I) and frequency of null alleles (fn) at each locus determined for the pooled eggs. Locus ACS3
ACS4
ACS6
ACS9
ACS16
ACS17
Mean
PIC
0.8493
0.7838
0.7158
0.9191
0.9014
0.8859
0.8426 ± 0.08
Q I fn
0.9049 0.051 0.0105
0.8076 0.0687 0.0045
0.7623 0.1109 0.0049
0.9621 0.0126 0.0103
0.9450 0.0178 0.0493
0.9304 0.0256 0.0231
Total
~1 2.238E−9
lower than that reported in black sea bream juveniles produced as part of the stock enhancement program carried out in Hiroshima Bay, either before (Jeong et al., 2007) or after their release (Blanco Gonzalez et al, 2008a). Stock enhancement programs using hatchery reared juveniles requires special attention in order to preserve the genetic diversity of the wild population (Allendorf and Ryman, 1987; Ryman, 1997; Taniguchi, 2003). They proposed the use of large number of breeders from the local population as an essential prerequisite to maintain the genetic resources of the natural stocks. Here, it was confirmed that rearing 143 breeders resulted in a larger gene pool than rearing only 51 specimens (Blanco Gonzalez et al., 2008a). In the present study, the average number of alleles was also larger than those reported from several natural populations (e.g. Jeong et al., 2003). The lower variability observed by Jeong et al. (2003) may be attributed to the small sample size per location (n = 50). Nevertheless, this argument is not applicable when the results obtained here are compared to those reported in Hiroshima Bay analyzing larger samples (Blanco Gonzalez et al., 2008a; Blanco Gonzalez and Umino, 2009). In this bay, the long and intensive releases of juveniles produced from a limited number of breeders has not only seriously eroded the genetic resources after a bottleneck
episode, but also reduced the size-at-age of the natural stock (Blanco Gonzalez et al., 2009). Therefore, the natural population inhabiting Hiroshima Bay is likely to have suffered remarkable inbreeding and genetic drift problems due to inappropriate broodstock management practices, which would explain their lower diversity when compared to that observed in the present study. Beside the conservation improvement achievements using larger number of breeders, the multiple-egg-collection strategy confirmed its usefulness of identifying those alleles inherited in lower frequency, crucial information to preserve the founders’ gene pool. As a result of the large number of alleles scored per group of egg (Table 2), slight differences in the pairwise FST values among some groups of eggs and the BR were detected (Table 4), disappearing when the groups of eggs were pooled. However, the pool of all groups of eggs produced departures from the HWE due to heterozygosity deficit. The presence of null alleles was not detected (Table 2), suggesting that deviations from HWE may be a consequence of an increase in genetic drift. A study conducted on Atlantic cod (Gadus morhua) larvae showed how the combination of genetically heterogeneous cohorts resulted in a homogeneous group that presented departures from HWE (Ruzzante et al., 1996). Difference in reproductive success associated to oceanographic and environmental conditions were suggested to explain those results. Nugroho and Taniguchi (2004) reported some inbreeding and differences in the genetic composition of red sea bream (Pagrus major) offspring collected at different dates during the spawning peak. In that study, they referred to the low contribution of the breeders per collection day and some bottleneck effects and genetic drift to explain their result. They suggested pooling the eggs from several days as a mean to enhance the genetic variability and reduce the harmful consequences. In the present study, coancestry among breeders was not estimated, although considering the wild origin and differences in age classes of the BR, it seems not likely to be the cause of the inbreeding. Errors during egg collection may have favored some families, though the random sampling of the eggs
Table 6 Results of the pedigree analysis of black sea bream. Eggs n
Number of contributors Female (Nf) Male (Nm) Total number of families Nb (unbalanced family size) Nb F
GI
GII
GIII
GIV
GV
GVI
Nocturnal
Pooled
75
75
75
75
75
75
225
450
83 (58.0%) 42 (49.4%) 41 (70.7%) 74 83.0 84.8 0.006
87 (60.8%) 46 (54.15) 41 (70.7%) 74 86.7 113.2 0.004
85 (59.4%) 49 (57.6%) 36 (62.1%) 74 83.0 109.1 0.005
87 (60.8%) 46 (54.1%) 41 (70.7%) 72 86.7 103.2 0.005
85 (59.4%) 43 (50.6%) 42 (72.4%) 72 85.0 108.0 0.005
81 (56.6%) 41 (48.2%) 40 (69.0%) 70 81.0 104.4 0.005
136 (95.1%) 80 (94.1%) 56 (96.5%) 215 131.8 87.7 0.006
140 (97.9%) 83 (97.6%) 57 (98.3%) 406 135.2 36.6 0.014
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
S17
Fig. 2. Contribution of each parental female (a) and male (b) to the groups of eggs.
makes us to reject that hypothesis too. The answer seems to come out when looking at the family contribution of parental females and males (Fig. 2a, b) and the correction made due to the differential contribution among breeders that established the final Nb in scarcely 37, lower than the threshold number of 50 recommended by FAO/ UNEP (1981) for short-term enhancement programs. Large variation in individual reproductive success has been postulated to explain the uneven parental contribution in several marine organisms (Hedgecock, 1994; Ruzzante et al., 1996; Li and Hedgecock, 1998; Boudry et al., 2002). Such variation was suggested to be caused by differences in the gamete quality, sperm-egg interaction and viability between families (Boudry et al., 2002). In the present study the large contribution of a few breeders seems to be responsible of the final F (0.014), although it is significantly more promising than the 2.5% and 5.6% reported for the offspring prior to their release in Hiroshima Bay in 2000 and 2001, respectively (Jeong et al., 2007). In that study, Jeong et al. (2007) could only detect the contribution of 63% and 59% of the original BR, a proportion similar to that observed in the present study when each group of eggs was analyzed independently (57–61%), resulting in Nb = 20 and 9 in 2000 and 2001, respectively. The value of the inbreeding coefficient was also much lower than the 3% obtained in hatchery fish four years after their release (Blanco Gonzalez et al., 2008a). In addition, the parentage analysis conducted in black sea
bream reared at the YPFES confirmed a much larger proportion of breeders contributing to the offspring in this specie (140/143) when compared to that reported in other marine finfishes (Pérez-Enriquez et al., 1999; Hara and Sekino, 2003; Jackson et al., 2003; OrtegaVillaizán Romo et al., 2005, 2006; Kim et al., 2007). Consequently, the Nb scored for black sea bream here was also larger than all those studies, except for red sea bream (Pérez-Enriquez et al., 1999). In that species, the contribution of 91 out of 250 breeders produced a final Nb = 63.7, with only 0.8% of inbreeding. Despite this promising low rate of inbreeding, red sea bream offspring showed a reduction of 25% of the alleles scored in their founders. Therefore, although higher rates of inbreeding were observed in this study in black sea bream, the fact that almost all breeders contributed to the mating and that most of alleles were inherited gives room for optimism. In addition, the collection of three groups of eggs during the most intense period of spawning (nocturnal group) would not only simplify the offspring collection practices, but also increased Nb to 88, reducing the rate of inbreeding to b0.6%. Although it seems to be useful, the risk of losing some important genetic information makes us to be very cautious to suggest the adoption of this strategy before confirming its usefulness in other species. In addition, it is necessary to confirm the viability and performance of the offspring produced in this way in relation to those collected over the whole spawning season.
S18
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19
Several means have been proposed to maintain the genetic resources and increase the Nb of hatchery strains for release: increase the number of breeders, collect the eggs in several days over the spawning season, apply the minimal kinship criteria among breeders, or maintain 1:1 sex ratio in the breeders (Allendorf and Ryman, 1987; Doyle et al., 2001; Nugroho and Taniguchi, 2004; Taniguchi, 2004; Fraser, 2008). In addition to these recommendations, based on our results, we suggest the collection of eggs at different times over a single night as part of the broodstock management design in case the collection of the eggs cannot be extended over the whole spawning season. Besides identifying the main contributors during the night the eggs were collected, this simple strategy may also identify those breeders only contributing in small proportion. This information may be crucial in case of alleles present at low frequencies in the wild, and hence highly relevant to preserve the gene pool of the natural population subject to enhancement. The strategy proposed would be very useful to recognize those families over-represented and equalize the parental contribution which would increase Nb and reduce F; however, posterior selection processes cannot be neglected. Considering that the eggs are produced in a single night and selecting the lots that conserve the larger genetic variability, several lots could be combined and reared under the same conditions, reducing cost and space requirements in hatcheries with limited capacity. 5. Conclusions The genetic benefits of rearing a larger number of breeders have been confirmed in this study. The broodstock maintained at the YPFES (n = 143) showed larger allele number than that reported in Hiroshima Bay (BR = 51) at six polymorphic microsatellites (Blanco Gonzalez et al., 2008a). A general concern exists regarding the inheritance of those alleles present in lower frequencies as well as the reduction of heterozygote in the offspring due to the hatchery conditions (Allendorf and Phelps, 1980; Allendorf and Ryman, 1987). The collection of eggs in several lots over the night revealed an extremely high contribution of the breeders (140/143, 98%). Consequently, most of the genetic variability was transferred to the offspring. The high rate of inheritance, in addition to the use of a large BR, has considerably helped to minimize the erosion of the genetic resources. The adoption of these simple means in the BR management plan of black sea bream has confirmed their usefulness to increase Nb (37) and reduced the rate of inbreeding (1.4%) when compared to the levels reported for this species in Hiroshima Bay (Jeong et al., 2007; Blanco Gonzalez et al., 2008a). Moreover, it was observed that the effective number of parents exceeded 100 (Nb = 136). Therefore, if we are able to balance the contribution of the over-represented breeders (i.e., by tracing the pedigree and selecting the lots of eggs that maximize Nb), the BR used in the present study confirmed its potential to produce offspring with similar genetic composition to enhance the wild stocks. The results, although promising, require a lot of caution and need to be tested in every species before their implementation. Further research should also be routinely done with larger samples of offspring in order to get a more accurate estimation of their genetic inheritance. In addition, it is necessary to confirm the viability and performance of the offspring produced in this way in relation to those collected over the whole spawning season. The collection of several batches of eggs over a single night should not exclude other strategies previously suggested for broodstock management (Allendorf and Ryman, 1987; Doyle et al., 2001; Nugroho and Taniguchi, 2004; Taniguchi, 2004; Fraser, 2008). Acknowledgement We wish to thank Mr. Hiroshi Shiozaki and the staff at the YPFES for their assistant during the sample collection. This study was partly supported by a Grant-in-Aid for Scientific Research (C) from the Japan
Society for the Promotion of Science (JSPS) to T.U. (Nos. 145600152 and 19580205). References Allendorf, F.W., Phelps, S.R., 1980. Loss of genetic variation in a hatchery stock of cutthroat trout. Trans. Am. Fish. Soc. 109, 537–543. Allendorf, F.W., Ryman, N., 1987. Genetic Management of Hatchery Stocks. In: Ryman, N., Utter, F.M. (Eds.), Population Genetics and Fishery Management. University of Washington Press, Seattle, pp. 141–159. Bell, J.D., Leber, K.M., Blankenship, H.L., Loneragan, N.R., Masuda, R., 2008. A new era for restocking, stock enhancement and sea ranching of coastal fisheries resources. Rev. Fish. Sci. 16, 1–8. Blanco Gonzalez, E., Murakami, T., Yoneji, T., Nagasawa, K., Umino, T., 2009. Reduction in size-at-age of black sea bream (Acanthopagrus schlegelii) following intensive releases of cultured juveniles in Hiroshima Bay, Japan. Fish. Res. 99, 130–133. Blanco Gonzalez, E., Nagasawa, K., Umino, T., 2008a. Stock enhancement program for black sea bream (Acanthopagrus schlegelii) in Hiroshima Bay: monitoring the genetic effects. Aquaculture 276, 36–43. Blanco Gonzalez, E., Umino, T., 2009. Fine-scale genetic structure derived from stocking black sea bream, Acanthopagrus schlegelii (Bleeker, 1854), in Hiroshima Bay, Japan. J. App. Icht. 25, 407–410. Blanco Gonzalez, E., Umino, T., Nagasawa, K., 2008b. Stock enhancement program for black sea bream (Acanthopagrus schlegelii) in Hiroshima Bay, Japan: a review. Aquac. Res. 39, 1307–1315. Blankenship, H.L., Leber, K.M., 1995. A responsible approach to marine stock enhancement. Am. Fish. Soc. Symp. 15, 167–175. Boudry, P., Collet, B., Cornette, F., Hervouet, V., Bonhomme, F., 2002. High variance in reproductive success of Pacific oyster (Crassostrea gigas, Thunberg) revealed by microsatellite-based parentage analysis of multifactorial crosses. Aquaculture 204, 283–296. Doyle, R.W., Pérez-Enriquez, R., Takagi, M., Taniguchi, N., 2001. Selective recovery of founder genetic diversity in aquacultural broodstocks and captive, endangered fish populations. Genetics 111, 291–304. Duchesne, P., Godbout, M.H., Bernatchez, L., 2002. PAPA (package for the analysis of parental allocation): a computer program for simulated and real parental allocation. Mol. Ecol. Notes 2, 191–193. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol. Bioinform. Online 1, 47–50. FAO/UNEP, 1981. Conservation of the genetic resources of fish: problems and recommendations. Report of the Expert Consultation on the genetic resources of fish. Rome, 9-13 June 1980. FAO Fish. Tech. Pap. 217, 1–43. Fraser, D.J., 2008. How well can captive breeding programs conserve biodiversity? A review of salmonids. Evol. App. 1, 535–585. Goudet, J., 2001. FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3). Available: http://www2.unil.ch/popgen/softwares/fstat. htm. Hara, M., Sekino, M., 2003. Efficient detection of parentage in a cultured Japanese flounder Paralichthys olivaceus using microsatellite DNA marker. Aquaculture 217, 107–114. Hedgecock, D., 1994. Does Variance in Reproductive Success Limit Effective Population Sizes of Marine Organisms? In: Beaumont, A.R. (Ed.), Genetics and Evolution of Aquatic Organisms. Chapman and Hall, London, pp. 122–134. Jackson, T.R., Martin-Robichaud, D.J., Reith, M.E., 2003. Application of DNA markers to the management of Atlantic halibut (Hippoglossus hippoglossus) broodstock. Aquaculture 220, 245–259. Jeong, D.-S., Blanco Gonzalez, E., Morishima, K., Arai, K., Umino, T., 2007. Parentage assignment of stocked black sea bream, Acanthopagrus schlegelii in Hiroshima Bay using microsatellite DNA markers. Fish. Sci. 73, 823–830. Jeong, D.-S., Hayashi, M., Umino, T., Nakagawa, H., Morishima, K., Arai, K., 2002. Comparison of genetic variability between wild and hatchery-stocked black sea bream Acanthopagrus schlegeli using DNA fingerprinting. Fish Genet. Breed. Sci. 32, 135–139 (in Japanese with English abstract). Jeong, D.-S., Umino, T., Kuroda, K., Hayashi, M., Nakagawa, H., Kang, J.-C., Morishima, K., Arai, K., 2003. Genetic divergence and population structure of black sea bream Acanthopagrus schlegeli inferred from microsatellite analysis. Fish. Sci. 69, 896–902. Kasahara, S., Hibiya, T., 1967. Fundamental studies on techniques of mass-production of fry of the black porgy, Mylio macrocephalus — I. On the spawning induced by injection of a preparation of mammalian gonadotropic hormones. J. Fac. Fish. Anim. Husb. Hiroshima Univ. 7, 105–111 (in Japanese). Kasahara, S., Oshimna, Y., 1960. Black sea bream culture: mass production and other problems related to seed production. Suisanzoshoku 7, 41–47 (in Japanese). Kasahara, S., Hirano, R., Ooshima, Y., 1960. A study on the growth and rearing methods of the fry of black porgy, Mylio macrocephalus (Basilewsky). Bull. Jap. Soc. Sci. Fish. 26, 239–244 (in Japanese with English abstract). Kim, S.G., Morishima, K., Satoh, N., Fujioka, T., Saito, S., Arai, K., 2007. Parentage assignment in hatchery population of brown sole Pleuronectes herzensteini by microsatellite DNA markers. Fish. Sci. 73, 1087–1093. Kitada, S., Kishino, H., 2006. Lessons learned from Japanese marine finfish stock enhancement programmes. Fish. Res. 80, 101–112. Lande, R., Barowwclough, G.F., 1987. Effective population size, genetic variation, and their use in population management. In: Soulé, M. (Ed.), Viable Populations for Conservation. Cambridge University Press, Cambridge, pp. 87–124. Launey, S., Hedgecock, D., 2001. High genetic load in the Pacific oyster Crassostrea gigas. Genetics 159, 255–265.
E. Blanco Gonzalez et al. / Aquaculture 308 (2010) S12–S19 Li, G., Hedgecock, D., 1998. Genetic heterogeneity, detected by PCR SSCP, among samples of larval Pacific oyster (Crassostrea gigas) supports the hypothesis of large variance in reproductive success. Can. J. Fish. Aquat. Sci. 55, 1025–1033. Nakagawa, H., Umino, T., Hayashi, M., Sasaki, T., Okada, K., 2000. Changes in biochemical composition of black sea bream released at 20 mm size in Daio Bay, Hiroshima. Suisanzoshoku 48, 643–648. Nugroho, E., Taniguchi, N., 2004. Daily change of genetic variability in hatchery offspring of red sea bream during spawning season. Fish. Sci. 70, 638–644. Paetkau, D., Strobeck, C., 1994. Microsatellite analysis of genetic variation in black bear populations. Mol. Ecol. 489–495. Pérez-Enriquez, R., Takagi, M., Taniguchi, N., 1999. Genetic variability and pedigree tracing of a hatchery-reared stock of red sea bream (Pagrus major) used for stock enhancement, based on microsatellite DNA markers. Aquaculture 173, 413–423. Ortega-Villaizán Romo, M.M., Aritaki, M., Taniguchi, N., 2006. Pedigree analysis of recaptured fish in the stock enhancement program of spotted halibut Verasper variegatus. Fish. Sci. 72, 48–52. Ortega-Villaizán Romo, M.M., Suzuki, S., Ikeda, M., Nakajima, M., Taniguchi, N., 2005. Monitoring of the genetic variability of the hatchery and recaptured fish in the stock enhancement program of the rare species barfin flounder Verasper moseri. Fish. Sci. 71, 1120–1130. Raymond, M., Rousset, F., 1995. GENEPOP (version 3.4): population genetics software for exact tests and ecumenicism. J. Hered. 86, 248–249. Rice, W.R., 1989. Analyzing tables of statistical tests. Evolution 43, 223–225. Ruzzante, D.E., Taggart, C.T., Cook, D., 1996. Spatial and temporal variation in the genetic composition of a larval cod (Gadus morhua) aggregation: cohort contribution and genetic stability. Can. J. Fish. Aquat. Sci. 53, 2695–2705.
S19
Ryman, N., 1997. Minimizing adverse effects of fish culture: understanding the genetics of populations with overlapping generations. ICES J. Mar. Sci. 54, 1149–1159. Ryman, N., Laikre, L., 1991. Effects of supportive breeding on the genetically effective population size. Conserv. Biol. 5, 325–329. Taggart, J.B., Hynes, R.A., Prodohl, P.A., Ferguson, A., 1992. A simplified protocol for routine total DNA isolation from salmonid fishes. J. Fish Biol. 40, 963–965. Taniguchi, N., 2003. Genetic factors in broodstock management for seed production. Rev. Fish Biol. Fish. 13, 177–185. Taniguchi, N., 2004. Broodstock management for stock enhancement programs of marine fish with assistance of DNA marker (a review). In: Leber, K.M., Kitada, S., Blankenship, H.L., Svasand, T. (Eds.), Stock Enhancement and Sea Ranching. Developments, Pitfalls and Opportunities. Blackwell Publishing, Oxford, pp. 329–338. Taniguchi, N., Sumantadinata, K., Iyama, S., 1983. Genetic change in the first and second generations of hatchery stock of black seabream. Aquaculture 35, 309–320. Tringali, M., Leber, K.M., Halstead, W.G., McMichael, R., O'Hop, J., Winner, B., Cody, R., Young, C., Neidig, C.N., Wolfe, H., Forstchen, A., Barbieri, L., 2008. Marine stock enhancement in Florida: A multi-disciplinary, stakeholder-supported, accountabilitybased approach. Rev. Fish. Sci. 16, 51–57. Umino, T., Hayashi, M., Miyatake, J., Nakayama, K., Sasaki, T., Okada, K., Nakagawa, H., 1999. Significance of release of black sea bream at 20-mm size on stock enhancement in Daiô Bay, Hiroshima. Suisanzoshoku 47, 337–342. Villanueva, B., Verspoor, E., Visscher, P.M., 2002. Parental assignment in fish using microsatellite genetic markers with finite numbers of parents and offspring. Anim. Gen. 33, 33–41. Weir, B.S., Cockerham, C.C., 1984. Estimating F-statistics for the analysis of population structure. Evolution 38, 1358–1370.