Aquaculture 247 (2005) 233 242

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A genetic evaluation of stock enhancement of blue abalone

Haliotis fulgens in Baja California, Mexico
Jose Luis Gutierrez-Gonzalez, Ricardo Perez-EnriquezT
Laboratorio de Genetica Acucola. Centro de Investigaciones Biologicas del Noroeste (CIBNOR). A.P. 128,
La Paz, Baja California Sur 23000, Mexico
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 to recover depleted populations resulting from over-

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 (Mazon-Suastegui et al., 1996), in an effort
T Corresponding author. Tel.: +52 612 123 8484; fax: +52 612
125 3625.
E-mail address: rperez@cibnor.mx (R. Perez-Enriquez).
0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2005.02.021
exploitation (Guzman-del Proo, 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
234 J.L. Gutierrez-Gonzalez, R. Perez-Enriquez / Aquaculture 247 (2005) 233242
the middle of the 1980s. However, there is no
information describing larval survival, or the recap-
ture of individuals after release that could quantify the
success of these stock enhancement programs.
While the isolation of wild organisms for aqua-
culture 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 recommen-
ded 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 micro-
satellite 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 Baha 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 temper-
ature of UV irradiated seawater. Mass spawning was
undertaken at Baha Tortugas, where 1622 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%
 J.L. Gutierrez-Gonzalez, R. Perez-Enriquez / Aquaculture 247 (2005) 233242 235

N
Punta Eugenia
2750'

W E

S
2746'
2742'

Mexico Bahia Tortugas

Gu
Pa

Los Morros
lf o
cif
ic

fC
Oc

ali
2738'

ea

fo
rn
n

ia

2 0 2 4 Km Clam Bay

1154' 11500' 11456' 11452' 11448'

Fig. 1. Location of Baha 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
(Carreon-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 follow-
ing Sweijd et al. (1998). For each individual,
approximately 0.5 g tissue was placed in a micro-
centrifuge 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
236 J.L. Gutierrez-Gonzalez, R. Perez-Enriquez / Aquaculture 247 (2005) 233242
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 micro-
satellites Hka28 and Hka56 developed for Haliotis
kamtschatkana (Miller et al., 2001). DNA amplifica-
tion 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 succes-
sive incubations in solutions containing streptavidin,
biotinalkaline phosphatase, and the chemilumines-
cent 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 differ-
ences. Genetic diversity was measured as the number
of alleles per locus (n A), the observed heterozygosity
(H o), and expected heterozygosity (H e). The poly-
morphic information content (PIC), parentage exclu-
sion 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 accord-
ing 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 organ-
isms 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 Baha 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 Baha
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.
 J.L. Gutierrez-Gonzalez, R. Perez-Enriquez / Aquaculture 247 (2005) 233242 237

3. Results however, the difference with the broodstock further

increased to 25% (Table 1). The average heterozygos-
3.1. Genetic diversity and allele frequencies ity in the families produced in Punta Eugenia, was not
significant lower compared with Baha Tortugas lots,
Average observed heterozygosity and mean num- and number of alleles was quite similar.
ber of alleles per locus were similar in the offspring of The allele frequencies of November and December
November (H o = 0.862, n A = 14) and December broodstocks at both loci were evenly distributed for
(H o = 0.865, n A = 12) at Baha Tortugas (Table 1). most alleles (Fig. 2). The only exception was the most
These estimates of genetic diversity were approx- common allele 115 in November at Hka56, at a
imately 12% lower than those observed in the frequency of 0.275. The alleles present in the off-
spawners of each lot. When the data for both spring represent most of the alleles of their parents,
spawnings of Baha Tortugas were pooled, the number indicating that most breeders contributed to the
of alleles per locus in the offspring increased; spawnings in the Baha Tortugas hatchery.
A heterogeneity test carried out on the frequency of
Table 1 the most common alleles of the November spawning,
Genetic diversity at two microsatellite loci in broodstock and allele 213 for locus Hka28 and alleles 95 and 125 for
offspring stocks at Baha Tortugas (November and December 2000 locus Hka56 (Fig. 2a,b), showed significant differ-
spawnings) and Punta Eugenia hatcheries
ences ( P b 0.05) between offspring and broodstock.
Spawning Locus Mean Even though alleles 211, 214, 233, and 249 of Hka28
Hka28 Hka56 and 121 of Hka56 were not represented in the
Baha Tortugas offspring, the differences were not significant
November/2000 ( P N 0.05), basically because these alleles frequencies
Broodstock (n = 16) were all low (b0.05).
Ho 1.000 0.938 0.969
He 0.899 0.891 0.895
In the offspring of December, alleles at higher
nA 16 16 16 frequencies that showed significant differences with
Offspring (n = 70) parents ( P b 0.05) were: 209, 217, and 219 for locus
Ho 0.725 1.000 0.862 Hka28. The presence of alleles 210 of Hka28 and 95,
He 0.811 0.870 0.841 113, 121, 127, and 129 of Hka56, which are not
nA 13 15 14.0
December/2000
represented in the progenitors (Fig. 2c,d), may have
Broodstock (n = 16) come from the broodstock that were returned to the
Ho 1.000 0.938 0.969 wild and therefore could not be sampled. Once again
He 0.936 0.879 0.907 alleles not represented in offspring were at low
nA 18 12 15 frequency (b0.06) in the parents.
Offspring (n = 57)
Ho 0.788 0.942 0.865
He 0.810 0.855 0.833 3.2. Parentage analysis: power of genetic markers
nA 12 12 12
Both broodstocks (pooled) High average polymorphic information content
Ho 1.000 0.938 0.969 (PIC = 0.822), for the two microsatellites loci was
He 0.917 0.885 0.901
nA 24 21 22.5
observed. The total exclusionary power (PE), i.e. the
Both offspring stocks (pooled) probability of appropriately excluding an unrelated
Ho 0.756 0.971 0.863 parent, combining both loci was 0.780.
He 0.810 0.862 0.836 More than 90% (64 individuals) of the offspring of
nA 17 17 17 November at Baha Tortugas amplified legible prod-
Punta Eugenia Families (n = 50) (offspring)
Ho 0.720 0.633 0.676
ucts to be used in the parentage analysis carried out on
He 0.888 0.846 0.867 both loci (Hka28 and Hka56). Of these, it was
nA 17 10 13.5 possible to unequivocally assign the parental pair that
H o: observed heterozygosity; H e: expected heterozygosity, n A: produced each of them to 77% (49 individuals) (Fig.
allele number, n: sample size. 3). We estimate that 23% of the offspring (15
238 J.L. Gutierrez-Gonzalez, R. Perez-Enriquez / Aquaculture 247 (2005) 233242

November
0.3 (a) 0.3 (b)
0.25 Hka28 0.25 Hka56

0.2 0.2

0.15 0.15

0.1 0.1

0.05 0.05

0 0

95
99
209
211
213
214
215
217
218
219
220
221
223
225
233
241
245
249

105
107
111
113
115
117
119
121
123
125
127
129
135
137
Frequency

December
0.3 (c) 0.300 (d)
Hka28 Hka56
0.25 0.250

0.2 0.200

0.15 0.150

0.1 0.100

0.05 0.050

0 0.000
201
203
206
207
208
209
210
211
213
215
217
218
219
220
221
223
231
237
243

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

individuals) might have come from two or more analyzed (49) it appears that the family formed by
possible parental pairs, for which it would be female #12 and male #2 contributed the highest
necessary to use at least an additional microsatellite percentage of offspring (12.5%), and that females
to unequivocally determine their progenitors. Because #12 and #10 formed the highest number of pairs, each
some individuals of the parental broodstock of mating 6 males (Fig. 3). This variance in the number
December of Baha Tortugas were returned to the of offspring per parent reduced N e by 50% (N e = 6.85).
wild immediately after spawning, the parentage
analysis could not be accurate, and, therefore, the 3.4. Monitoring of released juveniles
results are not reported.
In the recapture sampling carried out 12 months
3.3. Effective number of parents after the release of the hatchery-reared offspring, five
juveniles were found in Clam Bay (average length 27
From the parentage analysis, we observed that of mm) in a total sampling area of 150 m2, correspond-
16 breeders used in the spawning of November, 14 ing to a density of 0.03 ind./m2. At Punta Eugenia,
(eight males and six females) are the actual breeders three juveniles were collected (average length 29 mm)
(N a) that genetically contributed to the offspring (Fig. 12 months after release in a total sampling area of 30
3). From the difference in the number of males and m2 (0.1 ind./m2). The lengths of these juveniles,
females, the effective number of parents (N e) was which were similar to the lengths expected for one
13.7, or 87%. From the number of individuals year of age (Shepherd et al., 1995), and also similar to
 J.L. Gutierrez-Gonzalez, R. Perez-Enriquez / Aquaculture 247 (2005) 233242
Fig. 3. Haliotis fulgens parental couples estimated through parentage testing in the November spawning at Baha 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 Baha
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, respec-
tively. The combined value of both loci of
I = 1.7  10 4, indicated that only one in 6000
239
individuals might have had exactly the same com-
pound 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 inbreed-
ing have been suggested as reasons for changes in the
genetic variability of populations produced in captiv-
ity 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).
240 J.L. Gutierrez-Gonzalez, R. Perez-Enriquez / Aquaculture 247 (2005) 233242
In our study, the observed heterozygosity and mean
number of alleles were slightly reduced from parental
stocks to offspring but only significantly for hetero-
zygosity. This is similar to reports on other hatchery-
reared 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 off-
spring (87%), together with the fact there were two
parental stocks spawning at two dates of the repro-
duction 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) demonstra-
ted the power of the two microsatellites as genetic
markers for parentage assignment, and for distinguish-
ing 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 micro-
satellites. 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 Proo 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 (Carreon-
Palau, 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
 J.L. Gutierrez-Gonzalez, R. Perez-Enriquez / Aquaculture 247 (2005) 233242
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 Baha
Tortugas and La Pursima 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 continu-
ous advice and support. Thanks to three anonymous
reviewers that helped improving the manuscript. RPE
received grants SIMAC 990107011, and IFS A/2971-
1. Partial support was also obtained from CONACyT
grant 33018-D to Dr. M.A. del Ro. 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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