JOURNAL OF THE Vol. 41, No.

3
WORLD AQUACULTURE SOCIETY June, 2010

Evaluation of Natural and Commercial Probiotics for Improving

Growth and Survival of the Pearl Oyster, Pinctada mazatlanica,
during Late Hatchery and Early Field Culturing
Oscar L. Aguilar-Macías, Josafat J. Ojeda-Ramírez, Angel I.
Campa-Córdova, and Pedro E. Saucedo1
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa
Palo Santa Rita, La Paz, Baja California Sur 23096, Mexico

Abstract
Survival and growth of pearl oyster, Pinctada mazatlanica, juveniles fed microalgae supplemented
with natural and commercial probiotics were measured for 21 d at the hatchery. Probiotics tested were
(1) a Lactobacillus sp., (2) a mix of two bacilli, Burkholderia cepacia and Pseudomonas aeruginosa,
(3) a marine yeast, Yarrowia lipolytica, (4) Epicin-hatchery® as commercial probiotic, (5) an antibiotic
oxytetracycline, and (6) the control group fed Isochrysis galbana, Pavlova salina, and Chaetoceros
muelleri only. When the hatchery phase ended, the effects of probiotics were followed during the
early stages of field cultivation (90 d). Different from the control group, natural probiotics significantly
improved performance of juveniles during both phases, particularly in the field. The treatment with
Lactobacillus sp. significantly increased survival by 72%, growth in shell height by 63%, and growth in
wet weight by 83% over the control. The marine yeast and mix of bacilli provided intermediate results,
enhancing survival by 55–65%, shell height by 55–58%, and wet weight by 70–76% compared with
the control. Conversely, growth and survival in the treatments with Epicin and oxytetracycline were
significantly lower than in the control. These results show the potential of natural probiotics for
improving hatchery rearing of this pearl oyster.

In aquaculture, probiotics have been tested
in recent decades with promising results for
enhancing the immune system and promot-
ing growth and survival of many marine
species (see reviews by Gómez-Gil et al. 2000;
Balcázar et al. 2006). This is true for fresh-
water and marine fish (see reviews by Ringo
and Gatesoupe 1998; Gatesoupe 2007), penaeid
and nonpenaeid shrimps (Gullian et al. 2003;
Farzanfar 2006; Ochoa-Solano and Olmos-Soto
2006), and bivalve mollusks (Douillet and
Langdon 1994; Riquelme et al. 1997, 2000;
Gibson et al. 1998; Avendaño et al. 2001,
2002). With marine bivalves, however, stud-
ies have been carried out only with lar-
vae and spat of two species of commercial
value, the Chilean scallop, Argopecten purpu-
ratus, and the Pacific oyster, Crassotrea gigas.
Consequently, the real effects of probiotics on
1
Corresponding author.
© Copyright by the World Aquaculture Society 2010
physiological regulation of larvae, spat, or juve-
niles of other bivalve species of economic inter-
est still remain unknown.
Pearl oysters (Pteriidae) have been raised
in hatcheries since the 1970s and success-
ful protocols for controlled production of the
three main pearl-producing species (Pinctada
fucata, Pinctada maxima, and Pinctada mar-
garitifera) are well standardized (see review
by Southgate 2008). Despite this, larvae of
the Pteriidae are susceptible to higher die-offs
from external factors that larvae of other fam-
ilies (e.g., Ostreidae, Pectinidae) resist better
under confinement (Southgate and Beer 1997).
Among some important factors, growth of Vib-
rio and Pseudomonas pathogenic bacteria in
seawater used for hatchery rearing reduces sur-
vival of larvae and final yields of spat, lead-
ing to high economic losses (Saucedo et al.
2007). This is true for P. mazatlanica (Hanley,
1856) in Mexico, where survival of larvae is
3–5% (Saucedo et al. 2005) and development

447
448 AGUILAR-MACÍAS ET AL.

of adequate protocols for preventing bacterial

0.05 × 106 cell/mLc

Concentration
infections and promoting hatchery production

1 × 106 cell/mL
1 × 106 cell/mL

1 × 106 cell/mL
of spat is very recent (Saucedo et al. 2007).
Additionally for this species, knowledge of

10 mg/Lb
10 mg/La
physiological and nutritional needs of hatchery-
reared larvae and postlarvae is still very limited.
As part of a project for hatchery production

of the bacteria constituting the product were not specified by the supplier (Epicore BioNetworks); dose reported by Guerrero-Avalos (2007).
of pearl oysters in Mexico, this study measured

Table 1. Specifications of the different probiotic treatments given to Pinctada mazatlanica juveniles during the late hatchery-culturing phase for 21 d.

Culture media

Silicate sol.
f2 medium
f2 medium
MRS agar

YPD agar
YPD agar
growth and survival of P. mazatlanica juveniles
fed basic diets of microalgae supplemented
with natural and commercial probiotics during

–
–
the late hatchery phase, with a follow-up
period of growth trends during the early field
cultivation phases. The goal is to find improved

Produced at the hatchery

Crassostrea corteziensis
Nodipecten subnodosus

Litopeneaus vannamei
hatchery production protocols for supporting

Bacteria formulationa
CIBNOR collection
Host species
pearl culture activities in northwestern Mexico.

Materials and Methods

Origin of Specimens and Experimental Design

–
Larvae were hatchery-reared in spring fol-
lowing the protocol of Saucedo et al. (2007).
Experiment code

After postlarvae settled (Days 21–23) and

remained under nursery care for 3 wk (Days
OXY
CON
BAC

YAR
LAC

EPI

44–45), a group of 540 healthy juveniles mea-

suring 2–2.5 mm shell height were randomly
selected and divided into six groups of 90 juve-
niles each to test the following treatments of
Strain code

microalgae supplemented with probiotics for 21

NS6.1

YC58
Y021

d: (1) a Lactobacillus sp., (2) a 1:1 mix of two

020
–

–
–

bacilli, Burkholderia cepacia and Pseudomonas

aeruginosa, (3) a marine yeast, Yarrowia lipoly-
tica, (4) a commercial formulation of dried
probiotic bacteria (Epicin-hatchery®; Epicore
Scientific name/commercial name

Pseudomonas aeruginosa

Dose reported by Luna-González et al. (2002).

BioNetworks, Eastampton, NJ, USA), (5) the

Chaetoceros muellerii
Burkholderia cepacia

Yarrowia lipolytica

Isochrysis galbana

antibiotic oxytetracycline (Sigma, St. Louis,

Lactobacillus sp.

Dose reported by Saucedo et al. (2007).

Epicin-hatchery
Oxytetracycline

Pavlova salina

MO, USA; no. O4636), and (6) a control group

consisting of the microalgae Isochrysis gal-
bana, Pavlova salina, and Chaetoceros muel-
lerii given at a 1:1:1 ratio (estimated by cell
count). For all treatments, juveniles received
first the basic diet of microalgae and 15 min
later the corresponding treatment of probi-
otics. Data of each probiotic treatment (Centro
Probiotic type

de Investigaciones Biológicas del Noroeste’s

Marine yeast
Commercial
Lactobacilli

Microalgae

a Details
Antibiotic

(CIBNOR) collection code, experiment code,

Bacilli

culture medium, host species, and concentration

b
c

used) are presented in Table 1.

 EVALUATION OF PROBIOTICS FOR PINCTADA MAZATLANICA JUVENILES
Probiotic treatments consisted of 4-L plastic
containers holding 30 specimens each. For
each treatment, containers were managed in
triplicate. Containers received fresh, gently
aerated seawater (27 ± 1 C; salinity of 37),
further filtered at 1 μm and sterilized by
UV radiation. Every third day, containers
were drained, washed, and refilled with fresh
seawater at the same temperature and salinity.
Culture of Bacterial Strains
Bacteria were isolated from different host
species (Table 1). Each strain was first selected
from in vitro antagonism tests against the
pathogenic bacteria Vibrio alginolyticus and
Vibrio harveyi and then from hemolytic activ-
ity tests with bovine erythrocytes to confirm
that none were toxic. Strains were stored in
Man, Rogosa and Sharpe (MRS) agar medium
(Difco Laboratories/BD Microbiology Systems,
Detroit, MI, USA; no. 288130) or yeast pep-
tone dextrose (YPD) agar (Sigma, no. Y-1500)
supplemented with 15% glycerol at −80 C until
used. Each strain was identified by the BIOLOG
system (Biolog, Hayward, CA, USA).
Preparation of Probiotic Treatments
Probiotics were thawed and incubated in
agar media (Table 1) at 30 C for 24–48 h.
The cells were removed from the culture
medium by centrifugation (14,000 g, 5 min
at 4 C) and suspended in 3% sterile saline
solution. Proper optical density at 540 nm
allowed having a stock density to 1 × 109
CFU/mL (Ausubel 1999), further adjusted to
1 × 106 cell/mL for addition to culturing
tanks. Additionally, a stock solution of 10 mg/L
was used for the Epicin and oxytetracycline
treatments, as suggested for whiteleg shrimp
postlarvae Litopenaeus vannamei (Guerrero-
Avalos 2007) and veliger larvae of four bivalve
species (Luna-González et al. 2002).
Monitoring of Juveniles at the Hatchery
From the stock sample of 540 juveniles to
be fed with different probiotic treatments, a
subsample of 150 randomly selected specimens
449
were collected when starting the trials (Day 1
or t0 ) for obtaining data of shell height (to the
nearest 0.1 mm) and wet weight (to the near-
est 0.01 g). Subsequent measurements of these
variables were taken at Days 7 (t1 ), 14 (t2 ), and
21 (t3 ) on groups of 30 randomly selected spec-
imens from each treatment (10 per replicate).
Absolute growth and survival of specimens
under each treatment were estimated with these
data. Growth rates were estimated by the dif-
ference between final and initial values, divided
by the experimental time at the hatchery (21 d).
Transferring Juveniles to the Field
At the end of the hatchery phase, juveniles
were transferred to the field for a follow-
up cultivation period lasting 60 d. Juveniles
were placed on plastic trays (Nestier, Buckhorn,
Milford, OH, USA) and kept on a submarine
trestle at 10-m depth in Bahía de La Paz, Gulf of
California, Mexico (24◦ 16 N, 110◦ 19 W). Trays
were managed in triplicate at each treatment.
Field samples were taken every 15 d (for t4 and
t5 ) and then every 30 d (for t6 and t7 ). Juveniles
under each treatment were counted to estimate
survival rates and then measured (0.1 mm) and
weighed (0.01 g) in the same way described for
the hatchery phase for estimation of absolute
growth and growth rates in the field.
Statistical Analysis
Group normality (shell height and wet
weight) was initially analyzed with the Kol-
mogorov–Smirnov test and then confirmed
with the Levene test for homogeneity of vari-
ances (Sokal and Rohlf 1981). Thereafter, one-
way ANOVA was applied to assess significant
differences in survival and growth of speci-
mens as a function of the probiotic treatment.
The analysis was performed separately for the
hatchery and field culturing phases. When nec-
essary, post hoc, multiple-range comparisons
using the Tukey’s test were included. The level
of significance was set at P < 0.05.
Results
Figure 1 shows survival rate of specimens
from all treatments during nursery and field
450 AGUILAR-MACÍAS ET AL.
Figure 1. Survival rate of Pinctada mazatlanica juveniles
fed microalgae supplemented with different probiotic
treatments during late hatchery and early field culturing
phases. LAC = Lactobacillus sp.; YAR = Yarrowia
lipolytica; BAC = mix of Burkholderia cepacia and
Pseudomonas aeruginosa; EPI = Epicin-hatchery; OXY
= oxytetracycline; CON = control group. Bars denote
standard deviation. Survival means sharing superscripts
are not significantly different (P > 0 .05 ).
phases. At the end of the study, juveniles fed
the LAC treatment had the highest survival
(68.8%), whereas the lowest survival (32%)
occurred in the OXY treatment. Survival was
significantly affected by the probiotic treat-
ment used at the hatchery (F = 4.86) and field
phases (F = 6.88).
Absolute growth and growth rates, deter-
mined by shell height, are presented in Figure 2.
Specimens fed with the LAC treatment grew
larger (18.5 mm) and faster (0.39 mm/d) than
those provided with EPI treatment, which
grew less (11.3 mm) and slower (0.24 mm/d).
These size differences were significant during
the hatchery (F = 8.89) and field (F = 4.18)
phases.
Results of growth in wet live weight are
presented in Figure 3. During the hatchery
phase, greater gain in wet biomass occurred in
the YAR (23.5 mg and 0.96 mg/d) and LAC
(19.1 mg and 0.75 mg/d) treatments and less
biomass in the EPI treatment (10.4 mg and
0.33 mg/d). During the field phase, both treat-
ments yielded similar body biomass increase
(ca. 88 mg and 0.77 mg/d); less biomass
gain occurred in the control group (55.9 mg;
0.66 mg/d) during both phases. These weight
differences were significant at the hatchery
Figure 2. Absolute growth and growth rate for shell
height of Pinctada mazatlanica juveniles fed microalgae
supplemented with different probiotic treatments during
late hatchery and early field culturing phases. LAC =
Lactobacillus sp.; YAR = Yarrowia lipolytica; BAC = mix
of Burkholderia cepacia and Pseudomonas aeruginosa;
EPI = Epicin-hatchery; OXY = oxytetracycline; CON
= control group. Bars denote standard deviation. Means
within columns sharing superscripts are not significantly
different (P > 0 .05 ).
(F = 5.89) and field (F = 3.22) phases, but
significance at the field was marginal.
Discussion
While some Vibrio and Pseudomonas bac-
teria are commensals with no negative effects
on the host, some others are pathogenic bac-
teria causing large economic losses in bivalve
hatcheries (Riquelme et al. 1997; Gibson et al.
1998; Luna-González et al. 2002) and mari-
culture farms (Beleneva et al. 2007). At CIB-
NOR’s experimental hatchery in Bahía de La
Paz (Mexico), pathogen Vibrio occurs in sur-
face seawater throughout the year at counts of
approximately 35 × 103 CFU/mL and peaks
at 80 × 103 CFU/mL during the summer
 EVALUATION OF PROBIOTICS FOR PINCTADA MAZATLANICA JUVENILES
Figure 3. Absolute growth and wet live growth rate of
Pinctada mazatlanica juveniles fed microalgae supple-
mented with different probiotic treatments during late
hatchery and early field culturing phases. LAC = Lac-
tobacillus sp.; YAR = Yarrowia lipolytica; BAC = mix
of Burkholderia cepacia and Pseudomonas aeruginosa;
EPI = Epicin-hatchery; OXY = oxytetracycline; CON =
control group. Bars denote standard deviation. Means
within columns sharing superscripts are not significantly
different (P > 0 .05 ).
(Sainz-Hernández and Maeda-Martínez 2005).
These concentrations usually cause high die-
offs of P. mazatlanica larvae during the sum-
mer reproductive season and affect hatchery
propagation of the species (Saucedo et al. 2007;
Abasolo-Pacheco and Saucedo 2009). In the
Chesapeake Bay, Wright et al. (1996) also
found higher concentrations of Vibrio vulnifi-
cus during the warmer months of May, July,
and September, which are much lower than at
CIBNOR’s hatchery (3.0 × 101 to 2.1 × 102
CFU/mL). Because of these findings, we tested
whether oysters fed a basic diet of microalgae
supplemented with probiotics in months where
the activity of pathogenic bacteria is lower
(spring) would yield better results during the
hatchery and field stages, as occur with other
451
bivalves such as the Chilean scallop, A. purpu-
ratus (Riquelme et al. 1997, 2000; Avendaño
et al. 2002) and the Pacific oyster, C. gigas
(Douillet and Langdon 1994).
At the start of the trials, juvenile P. maza-
tlanica were approximately 45 d old and
measured 2–2.5 mm shell height. This size
range is consistent with sizes reported for
other Pteriidae of the same age produced in
hatcheries (see review by Southgate 2008).
At the end of the hatchery phase (66 d
old), juveniles reached 8.9 mm shell height
and weighed 13.4 mg wet weight, yielding
growth rates of 0.3 mm/d and 0.48 mg/d,
regardless of the treatment. Alagarswami et al.
(1983) and Southgate and Beer (1997) reported
growth rates of approximately 0.4 mm/d for
hatchery-reared P. fucata and P. margaritifera
on transfer to the sea. In contrast, shell growth
rates declined by 27% and weight growth rates
increased by 14% at the end of the field phase
(again, regardless of the treatment). This result
suggests that the change from the controlled
hatchery environment to a highly variable field
environment differently affected gain in hard
(shell) and soft (body tissue) biomass. As the
trend was the same for all probiotic treatments
and was not related to the nutritional status
of the specimens, the explanation probably
comes from the difference of 4 C between the
hatchery (26 C) and the field (22 C) at the
time of transfer of specimens. Christophersen
and Magnesen (2001) observed that survival
of Pecten maximus during the first days of
field cultivation depended not only on the
temperature changes but also on the length
of the acclimation time given to spat at the
hatchery; specimens acclimated for 2–3 wk had
64–75% better survival than those receiving
either none or 1-wk acclimation. We kept
P. mazatlanica under nursery care for 3 wk,
which ensured 46% survival in the field for the
control group and 65% in the LAC group (the
difference is significant). While the acclimation
frame is consistent with the protocol of Pit and
Southgate (2000) for rearing P. margaritifera
spat, our final survival rates are approximately
50% higher than those reported for spat of
452 AGUILAR-MACÍAS ET AL.
this species (26–34%) after transfer to the sea
(Southgate and Beer 1997).
When compared with the control group, the
LAC strain significantly increased shell growth
rates by 63% and wet growth rates by 83% at
the end of the trials. The YAR and BAC treat-
ments also enhanced survival (55–65%), shell
height (55–58%), and wet weight (70–76%)
over the control. Better results with the LAC
treatment were probably obtained because this
strain was isolated from the gut of a bivalve
species (lions-paw scallop, Nodipecten subno-
dosus) and did not include mixtures with strains
extracted from other zoological groups, such
as whiteleg shrimp, L. vannamei (in the case
of the BAC treatment). There are no available
studies reporting efficacy of bacilli and yeasts
with marine bivalves, but some bacteria have
given satisfactory results as probiotics. Douil-
let and Langdon (1994) and Gómez-Gil et al.
(2000) reported that growth of C. gigas larvae
was enhanced by adding the strain CA2 (likely
Alteromonas spp.) as a supplement to the diet
of microalgae. With the same species, Gibson
et al. (1998) observed similar protective effects
of Aeromonas media (strain A199) against Vib-
rio bacteria when rearing larvae at the hatch-
ery. Moreover, a probiotic strain of Vibrio sp.
helped increase survival of A. purpuratus larvae
by 240% over a control fed only with microal-
gae during the settlement stage (Avendaño et al.
2002), and at the same time provided pro-
tection against successive challenges from V.
anguillarum (Riquelme et al. 1997, 2000). In
contrast, probiotic yeasts appear to yield better
results as growth promoters of larvae of some
marine and freshwater fish, including sea bass,
Dicentrarchus labrax (Tovar et al. 2002, 2004)
and Nile tilapia, Oreochromis niloticus (Lara-
Flores et al. 2003).
Significantly poor growth and survival occur-
red with the EPI and OXY treatments, compared
with the control group. Epicin is a formu-
lation of dried bacteria (scientific names are
not specified by the supplier) that is routinely
used at shrimp farms in northwestern Mexico
to enhance growth and survival of L. van-
namei postlarvae. Although the optimal dose
for postlarval shrimp is 10 mg/L, recent stud-
ies with this product report inconsistent results.
Guerrero-Avalos (2007) reported that growth
of shrimp postlarvae could only be achieved
when the dose was increased to 30–50 mg/L. In
marine bivalves, Abasolo-Pacheco and Saucedo
(2009) observed that microalgae diets sup-
plemented with Epicin at 30–50 mg/L were
harmful (not lethal) for A. ventricosus, N. subn-
odosus, and P. mazatlanica larvae rose at the
hatchery. In contrast, Granados-Amores (2008)
reported that growth of hatchery-raised N. subn-
odosus juveniles was significantly improved
at concentrations of Epicin at approximately
3–6 mg/L. Hence, we suspect that the doses
used as adequate for shrimp postlarvae (either
10 or 30–50 mg/L) may be adequate for
bivalve mollusks during their adult stage only
but not during their juvenile stages. We also
believe that the dose of OXY (10 mg/L) was
excessive or toxic to juveniles.
In summary, all natural probiotics (particu-
larly lactobacilli) were beneficial and improved
survival and growth of juvenile P. mazatlanica
when used at the hatchery (further followed
at the field). These results will help improve
the protocols for rearing this pearl oyster, the
species in our experimental hatchery. Addi-
tional tests on the immune response of the
species are recommended, particularly during
the larval stage.
Acknowledgments
We thank Mario Osuna and Pablo Ormart at
CIBNOR for technical support during hatchery
rearing of spat and Ira Fogel for editorial review
of this manuscript. The research was carried out
with a grant from CIBNOR and is part of a
BSc project supported by a scholarship from
CONACYT to the first and second authors.
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