A genetic evaluation of stock enhancement of blue abalone Haliotis fulgens in Baja California, Mexico

Aquaculture 247 (2005) 233 – 242 www.elsevier.com/locate/aqua-online

A genetic evaluation of stock enhancement of blue abalone Haliotis fulgens in Baja California, Mexico Jose Luis Gutierrez-Gonzalez, Ricardo Perez-EnriquezT Laboratorio de Gene´tica Acuı´cola. Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR). A.P. 128, La Paz, Baja California Sur 23000, Me´xico Received 15 November 2003; received in revised form 1 February 2005; accepted 1 February 2005

Abstract Stock enhancement programs for Haliotis fulgens have been carried out for several years in Central Baja California to improve depleted populations, usually by the release of competent 5-day old larvae that are raised in hatcheries. In this study we analyzed the genetic diversity at two hatcheries and devised a method to monitor released abalone in the wild based on two microsatellite loci. The genetic diversity of the hatchery-reared abalone was high (mean H o = 0.865, number of alleles per locus = 14) and comparable to that of the broodstock. The number of spawners that contributed genetically to the progeny was more than 80% of the total, indicating that management practices appear adequate to avoid significant reduction in genetic diversity. The presence of released larvae in the wild, assessed by recapture samplings 6, 12, and 18 months after release, was low, indicating that the stock enhancement strategy should be modified to release older juveniles that will have better survival. Low probability of identity (I = 1.7  10 4), estimated with the combination of the two microsatellites, indicates their potential for identification of individuals in the wild in stock enhancement programs. D 2005 Elsevier B.V. All rights reserved. Keywords: Genetic diversity; Stock enhancement; Genetic markers; Abalone; Microsatellites; Haliotis fulgens

1. Introduction As a consequence of drastic reductions in the fishery production of abalone in Mexico (Shepherd et al., 1991, 1998), stock enhancement programs have been carried out for several years in central Baja California (Mazo´n-Sua´stegui et al., 1996), in an effort T Corresponding author. Tel.: +52 612 123 8484; fax: +52 612 125 3625. E-mail address: [email protected] (R. Perez-Enriquez). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.02.021

to recover depleted populations resulting from overexploitation (Guzma´n-del Pro´o, 1992), environmental fluctuations (Vega et al., 1997), predation (Tegner et al., 1992), and natural mortality (Tegner and Butler, 1985). In this region, stock enhancement is carried out by capturing wild breeding animals during the reproductive season, inducing them to spawn in the hatchery, and releasing the resultant offspring into the natural habitat when the larvae are competent to settle at five days. This method of release began in Mexico in the 1960s, and has been practiced consistently from

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the middle of the 1980s. However, there is no information describing larval survival, or the recapture of individuals after release that could quantify the success of these stock enhancement programs. While the isolation of wild organisms for aquaculture provides a potential for domestication and genetic improvement, there is a potential for genetic deterioration of the broodstock if a small effective number of breeding animals is kept within the hatchery (Hedgecock and Sly, 1990; Smith and Conroy, 1992). This is especially important in stock enhancement programs, because if the genetic diversity of the hatchery-reared organisms is low, their release will put at risk the genetic variability of the wild population through inbreeding (FAO, 1993). In spite of the importance of monitoring the composition and genetic diversity of hatchery-reared organisms, there are only a few studies on abalone (Smith and Conroy, 1992; Mgaya et al., 1995; Selvamani et al., 2001). Microsatellites are genetic markers that have proved useful in distinguishing populations (Balloux and Lugon-Moulin, 2002), and in monitoring pedigree and genetic diversity in hatchery-reared organisms, such as fish and shellfish, the objective being to determine the impact of stock enhancement activities on wild populations (Perez-Enriquez and Taniguchi, 1999; Perez-Enriquez et al., 2001; Boudry et al., 2002). The use of these markers has been recommended to follow-up recapture of released juveniles of fish species, such red sea bream (Perez-Enriquez et al., 1999), and could perhaps be used as genetic tags for abalone larvae. Our goal was to establish a general method of genetic evaluation of the stock enhancement programs of blue abalone Haliotis fulgens. This would be achieved by estimating the genetic composition and diversity of hatchery-reared progeny based on microsatellite DNA markers, and by assessing the efficiency of these genetic tags in certifying the hatchery origin of released organisms.

2. Material and methods 2.1. Hatchery spawnings Genetic diversity analyses were carried out at two hatcheries, one located at Bahı´a Tortugas and the

other at Punta Eugenia, Baja California, Mexico (Fig. 1). Mature wild abalones were collected and induced to spawn by desiccation or by increasing the temperature of UV irradiated seawater. Mass spawning was undertaken at Bahı´a Tortugas, where 16–22 breeding animals were separated by sex, and the gametes were collected and mixed for fertilization in a common container. The spawning at Punta Eugenia was done by single pair mating, in which every pair was placed in an individual container where fertilization occurred. Larvae were washed and separated in lots, and left until they became competent, i.e. they began to fix to the bottom, at four to five days old. At this stage, the larvae were transported in plastic bags and released at the selected sites by diving or by placement on the seashore. For this study, we performed genetic analysis on the spawners and larvae of two mass spawnings carried out during November and December 2000. On November, 16 wild breeding animals were used in a 1 : 1 male to female ratio, and yielded 3,250,000 larvae, which were released at low tide at Clam Bay, a small inlet southeast of Tortolo Cape (278 37’ N, 1148 50’ W; Fig. 1). On December, 10 females and 12 males were used to produce 3,850,000 larvae, which were released by boat on the sea surface at Los Morros area (278 39’ N, 1148 52’ W; Fig. 1). The larvae that were not released from either spawning were maintained in the hatchery to the juvenile stage. In March 2001, a sample from each juvenile lot (70 and 57 individuals; mean size 5.5 and 3.06 mm, respectively) was preserved in 95% ethanol for genetic analysis. Muscle tissue from the spawners (without sacrificing them) was also kept in 95% ethanol for genetic analysis. All 16 November spawners were sampled, but only 16 of the 22 December spawners could be obtained, as the rest had been returned to the wild. At the Punta Eugenia hatchery (Fig. 1), spawning was carried out in December 2000. Pairs were set up in individual containers using 11 wild breeding animals, producing four full-sib families and two half-sib families (one of the containers had two females and one male). An unknown quantity of larvae was released in a tide pool near the hatchery, and some individuals were kept in separate tanks until the juvenile stage. At six months, a sample of 10 individuals per tank was taken and preserved in 95%

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N 27°50'

Punta Eugenia W

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Mexico Gu

Los Morros

cif

lf o

Pa n

ia

rn

fo

ea

ali

Oc

fC

ic

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Bahia Tortugas

2

115°4'

0

115°00'

2

4 Km

114°56'

Clam Bay

114°52'

114°48'

Fig. 1. Location of Bahı´a Tortugas and Punta Eugenia hatcheries, and release and recapture sites (Clam Bay, Los Morros, and Punta Eugenia).

ethanol for genetic analysis. Given that spawners were returned to the wild, sampling of adults was not possible. The intertidal areas used for the release of larvae from both hatcheries were composed of platforms of sedimentary rocks that formed plateaus channeled by erosion. Boulders and rocks of minor size tend to settle in these channels. The flora associated with these areas are Macrocystis pyrifera, Phyllospadix torreyi, and Eisenia arborea, while some of the most characteristic fauna consists of the snail Tegula eiseni, sponges, bryozoans, starfish, ophiurae, and chitons (Carreo´n-Palau et al., 2003). 2.2. Monitoring of released abalone juveniles In June and November 2001 (6 and 12 months after release), recapture samplings were carried out. At Clam Bay, five 10  3 m transects perpendicular to the

coast were used, and at Punta Eugenia, the entire tide pool was sampled. Sampling was performed during low tide by checking each of the stones distributed along the transects with surface area from 0.03 up to 0.2 m2, and collecting all juvenile abalones. Samples were maintained in 95% ethanol for genetic analysis. Sampling at Los Morros was carried out by diving (4– 6 m), but was possible only in June because of poor weather conditions in November. 2.3. DNA analysis Genomic DNA was extracted and digested following Sweijd et al. (1998). For each individual, approximately 0.5 g tissue was placed in a microcentrifuge tube, and was digested with TNES buffer (Tris 100 mM, NaCl 400 mM, EDTA 50 mM, SDS 1%, pH 8) and proteinase K (0.5 mg/ml), and incubated at 55 8C for 16 h. The mucopolysaccharide

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excess was extracted using NaCl (0.75 M) and 1% CTAB (Sigma), incubated at 65 8C for 1 h. DNA was extracted by washing the sample three times with chloroform : isoamyl alcohol (24 : 1) and centrifuging at 4000 rpm for 10 min at each washing step. Cold absolute ethanol was added to the supernatant, and the tube placed in the freezer at 20 8C for 30 min, and centrifuged at 10,000 rpm for 10 min to precipitate the DNA. DNA was washed with 70% ethanol, air dried, resuspended in 200 Al TE Buffer, and kept at 4 8C until used. Genetic analysis was carried out using the microsatellites Hka28 and Hka56 developed for Haliotis kamtschatkana (Miller et al., 2001). DNA amplification was done by PCR in a Progene (Techne) thermal cycler in 25 Al reactions containing 0.5 Al abalone DNA (approximately 50 ng/Al), 0.48 AM of each forward and reverse primers (reverse primers were end-labeled at 5’ with biotin), 80 AM of each dNTP, 0.625 units Roche Taq polymerase, 3 Al PCR buffer 10X and H2O (milliQ). Amplification conditions for PCR at both loci were the following: denaturing at 95 8C for 2 min, 35 cycles of denaturing at 94 8C for 30 s, annealing at 52 8C for 30 s, and extension at 72 8C for 45 s, with a final extension at 72 8C for 2 min. PCR products were separated by 6% polyacrylamide gel electrophoresis. DNA was transferred to a nylon membrane (Nytran-N) by capillarity, and fixed by heating in a hybridization oven at 75 8C for 50 min. DNA was cross-linked to the membrane with UV light for 1 min. The fragments were visualized using the Phototope – Star Detection Kit chemiluminescent protocol (New England Biolabs) (Perez-Enriquez et al., 2001). DNA detection was performed by successive incubations in solutions containing streptavidin, biotin–alkaline phosphatase, and the chemiluminescent substrate (CDP-Star). The membrane was put in plastic foil and the emitted light was detected by exposing it to X-ray films (Kodak Biomax Light Film) for 20 min. Allele sizes were assigned using the sequence ladder of the plasmid pUC19 vector with a biotinylated M13 primer, using the Sequitherm Cycle Sequencing Kit (Epicentre). 2.4. Data analysis Allelic frequencies of broodstocks and offspring were calculated, and a v 2 heterogeneity test was

applied to determine the significance of the differences. Genetic diversity was measured as the number of alleles per locus (n A), the observed heterozygosity (H o), and expected heterozygosity (H e). The polymorphic information content (PIC), parentage exclusion probabilities for the first and second parent for each locus, and the total exclusion probability (PE) for the two loci were calculated using the software CERVUS version 2.0 (Marshall et al., 1998). A parentage analysis among broodstock and offspring that compared the genotypes for each of the offspring at each locus with the genotypes of the breeding animals was done in Microsoft Excel software. The pair(s) that gave a positive match was/were recorded and used to quantify the actual number of parents (N a), i.e. the number of individuals that actually reproduced and contributed genetic material to the next generation (see Fig. 1 in Perez-Enriquez and Taniguchi, 1999 for an example). Afterwards, the effective number of parents N e was estimated according to Gall (1987) with N e = 4N fN m / (N f + N m), where N f and N m are the number of females and males. A correction of N e due to a nonrandom distribution of family size by parents was applied by using N e = 8N e / (4 + V kf + V km), where V kf and V km are the variances in family size for females and males, respectively. The power of the parentage test was calculated by the probability of identity I (Paetkau and Strobeck, 1994). This is the probability of finding two organisms in a population taken at random with the same genotypes. For every locus this probability is given by the following expression I = R i p4i + R i R jNi (2p i p j )2, where p i and p j are the respective frequencies of the ith and jth alleles found in the population. In this case, the population conforms to the pooled set of the wild breeding animals (n = 32) used in the spawnings of November and December 2000 in Bahı´a Tortugas. The total probability of identity is the product of the value obtained at every locus. To determine if the juveniles collected during the recapture samplings in Clam Bay were produced in the hatchery or were of wild origin, a parentage analysis with the November broodstock of Bahı´a Tortugas was carried out. In the case of Punta Eugenia, because the broodstock genotypes were not known, the origin of the juveniles found in the wild was confirmed by comparing their genotypes with those of the families produced in the hatchery.

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3. Results 3.1. Genetic diversity and allele frequencies Average observed heterozygosity and mean number of alleles per locus were similar in the offspring of November (H o = 0.862, n A = 14) and December (H o = 0.865, n A = 12) at Bahı´a Tortugas (Table 1). These estimates of genetic diversity were approximately 12% lower than those observed in the spawners of each lot. When the data for both spawnings of Bahı´a Tortugas were pooled, the number of alleles per locus in the offspring increased; Table 1 Genetic diversity at two microsatellite loci in broodstock and offspring stocks at Bahı´a Tortugas (November and December 2000 spawnings) and Punta Eugenia hatcheries Spawning

Locus Hka28

Mean Hka56

Bahı´a Tortugas November/2000 Broodstock (n = 16) Ho 1.000 0.938 He 0.899 0.891 nA 16 16 Offspring (n = 70) Ho 0.725 1.000 He 0.811 0.870 nA 13 15 December/2000 Broodstock (n = 16) Ho 1.000 0.938 He 0.936 0.879 18 12 nA Offspring (n = 57) Ho 0.788 0.942 He 0.810 0.855 12 12 nA Both broodstocks (pooled) Ho 1.000 0.938 He 0.917 0.885 24 21 nA Both offspring stocks (pooled) Ho 0.756 0.971 0.810 0.862 He nA 17 17 Punta Eugenia Families (n = 50) (offspring) Ho 0.720 0.633 0.888 0.846 He nA 17 10

0.969 0.895 16 0.862 0.841 14.0

0.969 0.907 15 0.865 0.833 12 0.969 0.901 22.5 0.863 0.836 17 0.676 0.867 13.5

H o: observed heterozygosity; H e: expected heterozygosity, n A: allele number, n: sample size.

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however, the difference with the broodstock further increased to 25% (Table 1). The average heterozygosity in the families produced in Punta Eugenia, was not significant lower compared with Bahı´a Tortugas’ lots, and number of alleles was quite similar. The allele frequencies of November and December broodstocks at both loci were evenly distributed for most alleles (Fig. 2). The only exception was the most common allele 115 in November at Hka56, at a frequency of 0.275. The alleles present in the offspring represent most of the alleles of their parents, indicating that most breeders contributed to the spawnings in the Bahı´a Tortugas hatchery. A heterogeneity test carried out on the frequency of the most common alleles of the November spawning, allele 213 for locus Hka28 and alleles 95 and 125 for locus Hka56 (Fig. 2a,b), showed significant differences ( P b 0.05) between offspring and broodstock. Even though alleles 211, 214, 233, and 249 of Hka28 and 121 of Hka56 were not represented in the offspring, the differences were not significant ( P N 0.05), basically because these alleles’ frequencies were all low (b0.05). In the offspring of December, alleles at higher frequencies that showed significant differences with parents ( P b 0.05) were: 209, 217, and 219 for locus Hka28. The presence of alleles 210 of Hka28 and 95, 113, 121, 127, and 129 of Hka56, which are not represented in the progenitors (Fig. 2c,d), may have come from the broodstock that were returned to the wild and therefore could not be sampled. Once again alleles not represented in offspring were at low frequency (b0.06) in the parents. 3.2. Parentage analysis: power of genetic markers High average polymorphic information content (PIC = 0.822), for the two microsatellites loci was observed. The total exclusionary power (PE), i.e. the probability of appropriately excluding an unrelated parent, combining both loci was 0.780. More than 90% (64 individuals) of the offspring of November at Bahı´a Tortugas amplified legible products to be used in the parentage analysis carried out on both loci (Hka28 and Hka56). Of these, it was possible to unequivocally assign the parental pair that produced each of them to 77% (49 individuals) (Fig. 3). We estimate that 23% of the offspring (15

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November

137

127

125

123

121

119

117

115

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99

95

249

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Hka56

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Hka28

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Hka56

95 99 111 113 114 115 116 117 119 120 121 123 124 125 126 127 129

201 203 206 207 208 209 210 211 213 215 217 218 219 220 221 223 231 237 243

Frequency

0.25

(b)

135

0.3

Hka28

129

(a)

105

0.3

Alleles Fig. 2. Allele frequencies of Hka28 and Hka56 loci of Haliotis fulgens broodstock (white bars), and offspring (dark bars) of November (a, b) and December (c, d) spawnings at Bahı´a Tortugas.

individuals) might have come from two or more possible parental pairs, for which it would be necessary to use at least an additional microsatellite to unequivocally determine their progenitors. Because some individuals of the parental broodstock of December of Bahı´a Tortugas were returned to the wild immediately after spawning, the parentage analysis could not be accurate, and, therefore, the results are not reported. 3.3. Effective number of parents From the parentage analysis, we observed that of 16 breeders used in the spawning of November, 14 (eight males and six females) are the actual breeders (N a) that genetically contributed to the offspring (Fig. 3). From the difference in the number of males and females, the effective number of parents (N e) was 13.7, or 87%. From the number of individuals

analyzed (49) it appears that the family formed by female #12 and male #2 contributed the highest percentage of offspring (12.5%), and that females #12 and #10 formed the highest number of pairs, each mating 6 males (Fig. 3). This variance in the number of offspring per parent reduced N e by 50% (N e = 6.85). 3.4. Monitoring of released juveniles In the recapture sampling carried out 12 months after the release of the hatchery-reared offspring, five juveniles were found in Clam Bay (average length 27 mm) in a total sampling area of 150 m2, corresponding to a density of 0.03 ind./m2. At Punta Eugenia, three juveniles were collected (average length 29 mm) 12 months after release in a total sampling area of 30 m2 (0.1 ind./m2). The lengths of these juveniles, which were similar to the lengths expected for one year of age (Shepherd et al., 1995), and also similar to

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Fig. 3. Haliotis fulgens parental couples estimated through parentage testing in the November spawning at Bahı´a Tortugas. The number of offspring per couple is in parenthesis.

the size of the juveniles kept in both hatcheries, allowed us to suggest that they were born in the same spawning season. None of the five animals collected in Clam Bay gave a positive match in the parentage analysis performed with the November broodstock from Bahı´a Tortugas, indicating that they were very probably not produced in the hatchery, but in the wild. In contrast, two of the three juveniles collected at Punta Eugenia had two-loci compound genotypes same to the compound genotypes of the individuals of one of the families produced in the hatchery, indicating that these two organisms were probably released at this location in December 2000. To discard the idea that the genotypes of these two individuals could have come from the wild, we calculated the probability of identity (I) of genotypes, considering the number of alleles at each locus (Table 1, broodstock pooled) giving probability values of 0.009 and 0.018, respectively. The combined value of both loci of I = 1.7  10 4, indicated that only one in 6000

individuals might have had exactly the same compound genotype.

4. Discussion The use of aquaculture for stock enhancement of abalone, by releasing hatchery-reared abalone larvae to the wild, is now a common practice. However, concerns over the genetic impacts of this practice have been expressed. Genetic drift associated with the effects of bottleneck, reduction in the effective population size, and effects of selection and inbreeding have been suggested as reasons for changes in the genetic variability of populations produced in captivity that are used in stock enhancement programs (Allendorf and Ryman, 1987; Ryman, 1991; Hindar et al., 1991). Some genetic effects caused by reduction in genetic diversity have already been reported in fish (Poteaux et al., 1999) and in abalone (Smith and Conroy, 1992; Mgaya et al., 1995).

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In our study, the observed heterozygosity and mean number of alleles were slightly reduced from parental stocks to offspring but only significantly for heterozygosity. This is similar to reports on other hatcheryreared stocks, such as red sea bream Pagrus major (Perez-Enriquez et al., 1999), and pearl oyster Pinctada margaritifera (Durand et al., 1993). The maintenance of levels of genetic diversity was a consequence of the large number of parents that reproduced and genetically contributed to the offspring (87%), together with the fact there were two parental stocks spawning at two dates of the reproduction season, making a total of 38 spawners. This assures a wider spectrum of genetic variability represented in offspring (Taniguchi et al., 2003). However, in the November spawning the effective number of breeders (N e) was reduced by 50%, after correction for unequal number of males and females, and for nonrandom distribution of family size (Gall, 1987). Even though this reduction in N e results in an increase in the inbreeding coefficient ( F = 1/2 N e) from 0.07 to 0.15, the effect in the wild could be diluted when several spawnings are carried out during the reproduction season. This increase in inbreeding might not be very different to what happens in the wild, where the reproductive behavior indicates that it is very probable that mating does not involve a 1 : 1 male to female ratio, but rather occur as mass spawnings. At least this is the case for other abalone species (Sasaki and Shepherd, 1995). Nevertheless, care should be taken by the hatcheries located on the Pacific coast of Baja California to maintain genetic variability using larger numbers of spawners, at least 50 (FAO, 1993), and to increase couple mating to reduce inbreeding rate for subsequent generations. The high estimated probability of exclusion (i.e. the exclusion of unrelated parents to an offspring in a parentage test, leaving only related ones) demonstrated the power of the two microsatellites as genetic markers for parentage assignment, and for distinguishing organisms captured in the wild as being of hatchery origin. Nevertheless, the use of two markers was not sufficient for a 100% parental assignment. Selvamani et al. (2001) reported similar results using two loci, but they were able to assign up to 100% of offspring to the correct parents using three microsatellites. Therefore, if at least one or two more highly polymorphic microsatellites were available for H.

fulgens, we would more than likely be able to assign parentage to the unassigned 23% of offspring of the November spawning that showed more than one possible parental pair. This also would allow a lower probability of identity to be obtained, increasing the precision of individual identification, as reported by Pearse et al. (2001), Perez-Enriquez and Taniguchi (1999) and Paetkau and Strobeck (1994) who used five, five, and four microsatellites respectively with other species. Our group is now working on isolating additional polymorphic microsatellites for H. fulgens. Mortality and larval dispersal are the main reasons that could explain the absence of released juveniles at Clam Bay. In the first case, Gonzalez-Aviles and Shepherd (1996) and Shepherd et al. (1991) reported high natural mortality of released abalone larvae. This mortality rate might be increased if the seeding method or the selection of the release site, important in providing a suitable microhabitat (De Waal et al., 2003), is not adequate. In the second case, larvae are passively transported by coastal currents (McShane, 1992; Guzman del Pro´o et al., 2000), removing them from the release area. A third explanation could be that, because of the cryptic behavior of abalone (McShane, 1995), juveniles actually exist within the release site, but are hidden in caves and small crevices, preventing recovery. Nevertheless, this explanation is an unlikely cause of low recovery of juveniles since the density of individuals collected at Clam Bay (0.03 ind./m2) is similar to the abundance of the natural population recorded in 1997 (Carreo´nPalau, 2000). Therefore, we suggest that the released larvae either died during and after settling, and/or settled in a different site from the release. Even though we do not know how many larvae were released in Punta Eugenia, the recapture of released juveniles in this area was probably facilitated because the release site was a semi-protected area in which larval dispersal could be limited (Prince et al., 1988). Tong et al. (1987) in a study of recapture of released abalone H. iris, emphasized that the survival rate is higher when the organisms are released at larger sizes. Kojima (1995) and De Waal and Cook (2001) also observed a higher survival rate when the size of the release was increased, and emphasized that physical and biological factors must be considered during the stock enhancement activities to get a better understanding of factors that affect survival of abalone

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seed. These observations suggest that hatcheries in Baja California could try releasing juveniles longer than 24 mm (De Waal and Cook, 2001) to increase survival rates. In summary, our results indicate that even though genetic diversity within stock enhancement programs at two Baja California hatcheries is not compromised, hatchery managers are required to exercise care to avoid it decreasing. Also, release strategies should be modified to increase larval survival, and better ensure cost effectiveness of these programs.

Acknowledgments The authors thank hatchery technicians from Bahı´a Tortugas and La Purı´sima Cooperatives, and the Instituto Nacional de la Pesca for their support. Thanks to Dr. R.E Withler of the Pacific Biological Station, Nanaimo, B.C., Canada for testing pinto abalone (H. kamtschatkana) microsatellites in H. fulgens. Thanks to N. Elliott of CSIRO for helpful comments and A.M. Ibarra of CIBNOR for continuous advice and support. Thanks to three anonymous reviewers that helped improving the manuscript. RPE received grants SIMAC 990107011, and IFS A/29711. Partial support was also obtained from CONACyT grant 33018-D to Dr. M.A. del Rı´o. The first author is a CONACyT graduate fellow (114893). S. Avila provided technical support and editing staff at CIBNOR improved the English text.

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