Aquaculture Research, 2016, 1–15 doi:10.1111/are.

13031

Evaluation of two probiotics used during farm

production of white shrimp Litopenaeus vannamei
(Crustacea: Decapoda)

Ram
on Franco1,†, Leonardo Mart
ın2,†, Amilcar Arenal2,†, Dayam
ı Santiesteban1,

Jorge Sotolongo1, Hector Cabrera3, Jaime Mej
ıas3, George Rodr
ıguez2, Angela G Moreno3,
Eulogio Pimentel1 & Nestor M Castillo4
1
Center for Genetic Engineering and Biotechnology, Camag€
uey, Cuba
2
Biochemistry Laboratory, Department of Morphophysiology, Agropecuary Science Faculty, University of Camag€
uey,
Camag€
uey, Cuba
3
Yaguacam Shrimp Hatchery, Yaguanabo, Cumanayagua, Cienfuegos, Cuba
4
Biology Faculty, Department of Biochemistry, University of Havana, Havana, Cuba

Correspondence: R Franco, Center for Genetic Engineering and Biotechnology, P.O. Box 387, Camaguey 70100, Cuba.
E-mail: ramon.franco@cigb.edu.cu
†
These authors contributed equally to this work.

respect to EPICIN 3W treatment in the tank water

Abstract
This study examined the effects of Bacillus licheni-
formis strain CIGBC-232, isolated from the gut of
shrimp Litopenaeus vannamei and having antago-
nistic activity against Vibrio harveyi, on the immu-
nity and larval quality of L. vannamei at various
ontogenetic stages, in two separate experiments:
(1) PL2 to PL17 exposed to the strain CIGBC-232
under laboratory conditions (2) zoea I to PL8
exposed to the strain CIGBC-232 and EPICIN 3W
probiotics under farming conditions. The first
experiment showed that phenoloxidase, peroxidase
and superoxide dismutase activities were improved
in animals grown with CIGBC-232 compared to
the untreated control. In tests, the resistance to
osmotic stress was also enhanced. During the sec-
ond experiment, animals treated with CIGBC-232
exhibited significant (P < 0.05) increases in phe-
noloxidase activity (30–40% higher in zoea I–II,
mysis II–III and PL 2–7) and in the respiratory
burst (30% higher from PL 2 to 7) as compared to
those animals that received EPICIN 3W. There
was no significant difference in the lectins and
agglutinins (except in PL1, 6 and 7), in peroxidase
and superoxide dismutase activities, in the resis-
tance to osmotic stress, nor in the survival rate
among treatments. CIGBC-232 treatment was able
to reduce, the level of presumptive Vibrio spp. with
as well as in animals. At the end of both experi-
ments, the growth of shrimp, i.e. weight and
length was increased by CIGBC-232 treatment.
This study showed the probiotic effect of CIGBC-
232, which appeared to have a better probiotic
performance than EPICIN 3W treatment.
Keywords: shrimp immunity, probiotic, vibrios,
phenoloxidase, larval quality, Bacillus licheni-
formis
Introduction
During the last 20 years, aquaculture has grown
rapidly. In this sector, crustaceans contribute sig-
nificantly, with annual production exceeding
10 million metric tons, having a first-sale commer-
cial value of $40 B (Stentiford, Neil, Peeler,
Shields, Small, Flegel, Vlak, Jones, Morado & Moss
2012). Calculated by production, white leg shrimp
is the most successful internationally introduced
marine crustacean for aquaculture. In 2010,
L. vannamei reached 71.8% of the world produc-
tion of all species of cultivated marine shrimp,
77.9% of which was produced in Asia, (FAO
2012). Despite the explosive growth in world pro-
duction of cultivated shrimp, there have also been
great mortalities and economic losses due to

© 2016 John Wiley & Sons Ltd 1

Two probiotics enhancing production of L. vannamei R Franco et al.
diseases (Balc
azar, Rojas-Luna & Cunningham
2007; Flegel, Lightner, Lo & Owens 2008). It is
estimated that around 20% of disease losses in
shrimp aquaculture have been caused by bacterial
pathogens (Flegel 2006, 2012). Bacterial disease
outbreaks occur generally as a result of stress and
environmental deterioration associated with an
increase in shrimp farming (Tseng & Chen 2004).
High mortalities during spawning and larval
rearing are a common problem in shrimp hatch-
eries. Many reports have cited larval and postlarval
stages as the most susceptible to diseases (Jira-
vanichpaisal, Puanglarp, Petkon, Donnuea,
S€oderh€all & S€
oderh€ all 2007; Rungrassamee, Klan-
chui, Chaiyapechara, Maibunkaew, Tangphatsorn-
ruang, Jiravanichpaisal & Karoonuthaisiri 2013;
Smith, Brown & Hauton 2003; Soto-Rodriguez,
Gomez-Gil, Lozano, del Rio-Rodr
ıguez, Di
eguez &
Romalde 2012). Their susceptibility could be related
to their immunocompetency. During these L. van-
namei stages important effectors of the immune sys-
tem undergo variations in their levels and activities,
especially in postlarval stages where most of them
seem to decrease (Mart
ın, Castillo, Arenal,
Rodr
ıguez, Franco, Santiesteban, Sotolongo, Forrel-
lat, Espinosa, Carrillo & Cabrera 2012).
Vibriosis, caused by Vibrio harveyi is the most
lethal disease in the stages mentioned above (Ala-
vandi, Manoranjita, Vijayan, Kalaimani & Santi-
ago 2006; Austin & Zhang 2006). In the past,
antibiotics were used extensively to control this
bacterial disease in hatchery systems. However,
the excessive use (misuse) of antibiotics leads to
the emergence of antibiotic resistant bacteria
(Akinbowale, Peng & Barton 2006; Austin 2010;
Kesarcodi-Watson, Kaspar, Lategan & Gibson
2008). Therefore, alternatives such as probiotics
are now widely used as a means of controlling dis-
ease (Qi, Zhang, Boon & Bossier 2009). Probiotics
are microorganisms that, when administrated in
adequate quantities, are able to improve the host’s
health (Ninawe & Selvin 2009). Probiotics provide
benefits through enhancement of growth perfor-
mance (Decamp, Moriarty & Lavens 2008; Liu,
Chiu, Shiu, Cheng & Liu 2010; Wang 2007),
improved digestion and better water quality
(Kesarcodi-Watson et al. 2008). Various lines of
evidence point to immunomodulation as a domi-
nant mode of probiotic action (Newaj-Fyzul, Al-
Harbi & Austin 2014).
The genus Bacillus represents the group of
probiotic species most frequently used in shrimp
2 © 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15
Aquaculture Research, 2016, 1–15
aquaculture, with no deleterious side effects
(G
omez & Shen 2008; Rengpipat, Phianphak, Piy-
atiratitivorakul & Menasveta 1998). They are able
to secrete enzymes (Liu, Chiu, Ho & Wang 2009;
Moriarty 1998; Ochoa-Solano & Olmos-Soto
2006) to improve feed digestion, and water qual-
ity. Studies have shown that when B. subtilis and
B. licheniformis were administrated to larval L. van-
namei and Penaeus monodon, the survival rate was
improved (Decamp et al. 2008). Although several
reports have demonstrated the efficiency of probi-
otics, most of these studies have been performed in
juvenile and adult stages; thus far, little past
research has been directed towards improving the
immune system in shrimp larvae (Liu et al. 2010;
NavinChandran, Iyapparaj, Moovendhan, Rama-
subburayan, Prakash, Immanuel & Palavesam
2014). Also, it must be taken into consideration
that the development of suitable probiotics is not a
simple task, and requires full-scale trials, as well
as development of appropriate monitoring tools
and controlled production (Decamp et al. 2008;
Luis-Villase~nor, Mac
ıas-Rodr
ıguez, G
omez-Gil,
Ascencio-Valle & Campa-C
ordova 2011). In this
context, a better understanding of probiotic influ-
ence on immunity during ontogeny would help in
designing efficient strategies for hatcheries to man-
age disease.
The aim of this study was to investigate the
effects of putative probiotic Bacillus licheniformis
strain CIGBC-232, isolated from the gut of shrimp,
on growth, postlarval quality, and immunity of
L. vannamei postlarvae. This study also evaluated,
the effectiveness of selected probiotics during onto-
geny of white shrimp, L. vannamei, under farming
conditions.
Materials and methods
Bacterial strains
Probiotic candidates were isolated from the intes-
tine of healthy L. vannamei. The contents of the
intestinal tract were aseptically removed from
adult shrimp. The samples were homogenized and
serially diluted, plated on nutrient agar (NA) con-
taining 2.0% [weight/volume] NaCl. The isolated
strains were separated based on differences in col-
ony morphology and screened for inhibitory effects
against Vibrio harveyi, by the two-layer method
(Dopazo, Lemos, Lodeiros, Bolinches, Barja & Tor-
anzo 1988). The selected strain, was identified
Aquaculture Research, 2016, 1–15
based on the morphological and biochemical
characteristics described in the Bergey’s Manual of
Systematic Bacteriology Second Edition (Vos, Gar-
rity, Jones, Krieg, Ludwig, Rainey, Schleifer &
Whitman 2009). Further characterization was
achieved using the 16s rRNA gene. The DNA
sequence was compared against the sequences
available in the NCBI GenBank database using the
BLAST algorithm (http://blast.ncbi.nlm.nih.gov/
Blast.cgi).
All strains were cultured on a nutrient agar
(NA) and in a nutrient broth (NB) supplemented
with 2.0% [w/v] NaCl on a shaker at 28°C. Pure
cultures of all strains were preserved at
70°C
using NA containing 2.0% [w/v] NaCl and 15%
[volume/volume] glycerol until use.
A commercial probiotic formulation, Epicin3W,
(Epicore Bionetworks Inc, Mount Holly, NJ, USA)
was also tested.
Experimental conditions
Experiment I
The experimental design was completely random-
ized with two groups having four replicates per
treatment. The postlarvae 2 (PL2, 2 days after
metamorphosis) were randomly stocked at 75
PL2 L
1 in 4-L plastic tanks containing seawater
at 30  2°C and 30& salinity. In one group, the
Bacillus licheniformis strain (CIGBC-232) was added
directly to the water at a daily dose of
104 cfu mL
1; the other group did not receive any
probiotic. L. vannamei postlarvae were obtained
from the Yag€ uacam hatching facility in
Cumanayag€ ua, Cienfuegos, Cuba. Constant aera-
tion was supplied and 25% of the tank water was
exchanged daily. Throughout the experiment, the
postlarvae were fed on Artemia franciscana nauplii
(Aquatic Eco-Systems Inc., Apopka, FL, USA) at a
rate of 8–10 nauplii for each shrimp larva three
times per day. The postlarvae were reared in these
conditions up to PL17.
Experiment II
Larvae of L. vannamei were reared in Yag€ uacam
hatchery (Cumanayag€ ua, Cienfuegos, Cuba).
L. vannamei was stocked at 175 nauplii L
1 in
fibreglass tanks with a volume of 20 000 L. A
completely randomized experiment was performed
using two groups with eight replicates per group.
Starting from the zoea I (PZ-I) stage, one group
was treated daily using probiotic strain CIGBC-
© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15
Two probiotics enhancing production of L. vannamei R Franco et al.
232 at 1.2 9 104 cfu mL
1, and another group
was treated with a commercial probiotic EPICIN
3W (Epicore Bionetworks Inc) according to the
manufacturer’s instructions. During the experi-
ment, feeding was with diatoms [Chaetoceros gra-
cilis, N-V: 30 000 cells mL
1, PZ-I to PZ-II:
60 000 cells mL
1, PZ-III to PL-2: 50 000
cells mL
1], flagellates [Tetraselmis suecica, N-V to
PZ-II: 2000 cells mL
1, PZ-III to PL-7: 3000 cells
mL
1] and freshly hatched Artemia nauplii
(Aquatic Eco-Systems Inc., Apopka, FL, USA) [PZ-
III: 0.5 nauplii mL
1, M-I to M-II: 1 nauplii
mL
1, M-III to PL-2: 1.5 nauplii mL
1, PL-3 to
PL-4: 3 nauplii mL
1, PL-5 to PL-8: 4 nauplii
mL
1 (four times per day)]. The temperature was
maintained at 30  2°C, salinity at
30  1 g L
1, pH 8.5  0.5, and constant aera-
tion was supplied. From the mysis I (M-I) stage
forward, 50% of each tank volume was
exchanged to eliminate organic detritus. Animals
were maintained in these conditions up to PL8
(8 days after metamorphosis to postlarvae). At
this scale, no negative control was used since
according to the good aquaculture practices, in
the Yag€uacam hatchery, the use of probiotics is
mandatory during the rearing period.
Sampling and conservation
At the end of experiment I, 50 postlarvae were
sampled randomly from each tank for enzymatic
assays. In experiment II, approximately 120 ani-
mals per tank were daily pooled at different larval
development stages from PZ-I to PL7.
All samples were randomly collected at
10:00 h, 2 h after feeding. Samples from experi-
ments I and II were washed three times with cold
sterile H2Odd and immediately frozen at
70°C in
1.5 mL centrifuge tubes (Eppendorf, USA). Frozen
samples were homogenized with a Polytron
homogenizer (Bioblock Scientific, France) in
500 lL H2Odd volumes. Later, the homogenates
were centrifuged at 8050 g for 10 min at 4°C,
and the pellet was resuspended in 100 lL sterile
ice-cold H2Odd. Supernatant and pellet were sepa-
rately preserved at
20°C for measurements and
enzymatic assays.
Soluble protein content
Soluble protein concentration was measured using
Bradford0 s method (Bradford 1976). Briefly, 10 lL
of supernatant from each sample were placed into
3
Two probiotics enhancing production of L. vannamei R Franco et al.
a flat-bottom, 96-well, polyethylene plate (Costarâ,
USA). The plate was kept in the dark for 10 min
and then the absorbance was measured at 620 nm
using a multiscan monitor (Titertek MCC/340).
The protein concentration was calculated using a
standard curve of bovine serum albumin (BSA)
(Sigma–Aldrich USA); with three replications. All
the results were expressed as lg of protein per
animal.
Immunological assays
Phenoloxidase assay
PO activity was determined based on chromophore
formation from L-dihydroxyphenylalanine (L-
DOPA) (Sigma–Aldrich, USA) (Hern
andez-L
opez,
Gollas-Galv
an & Vargas-Albores 1996). A negative
control for spontaneous oxidation of the L-DOPA
was used. One unit of enzyme activity was defined
as an increase in absorbance (at 490 nm) of
0.001 min
1 mg protein
1, 20 lL of supernatant
from each sample were used.
Superoxide dismutase assay
SOD activity was measured using the method pro-
posed by Beauchamp and Fridovich (Beauchamp &
Fridovich 1971), using nitroblue tetrazolium
(NBT) in the presence of riboflavin. 20 lL of
supernatant from each sample were used. The
specific activity was expressed as U mg
1 of pro-
tein, where U was defined as a 0.001 increase in
absorbance at 560 nm min
1.
Respiratory burst activity
The respiratory burst was evaluated using Song
and Hsieh0 s method (Song & Hsieh 1994) based
on spectrophotometric detection of formazan from
the NBT-O2
redox reaction. 10 lL of resuspended
pellet from each sample of experiment II were
used. The results were expressed in U lg
1 of pro-
tein, where U was defined as a 0.001 increase in
absorbance at 630 nm min
1.
Peroxidase assay
Peroxidase activity was quantified and calibrated
against a peroxidase activity standard curve
made with radish peroxidase (Heber Biotec sa,
Cuba). Peroxidase assays were performed in flat-
bottom, 96-well, polyethylene plates (Costarâ,
USA) by adding 20 lL of supernatant from each
sample and 50 lL of reaction buffer (Na2HPO4
0.05 M; citric acid 0.02 M; pH 5) containing
4 © 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15
Aquaculture Research, 2016, 1–15
H2O2 (0.15% [V/V]) and o-phenylenediamine
(0.10% [p/V]). The plates were placed 30 min at
room temperature and then the reaction was
stopped with 50 lL sulphuric acid (2 M). The
absorbance was read at 492 nm, using a screen
plate (Titertek Multiskan MCC/340). The results
were expressed in U lg
1 of protein, where U
was defined as the formation of 1 lmol of 2.3-
diaminophenazine, by the oxidation of o-phenyle-
nediamine, per min of reaction per lg of pro-
tein.
Presence of lectins and agglutinin evaluation
To determine the presence of lectins and agglu-
tinin, rabbit blood was collected in 15 mL cen-
trifuge tubes (Corning, USA) in 50 mM EDTA. The
blood was centrifuged at 352 g for 10 min and
the serum was discarded. The erythrocyte pellet
was washed twice with phosphate saline buffer
(PBS) (NaCl 136 mM; KCl 26 mM; Na2HPO4
8 mM; KH2PO4 15 mM; pH 7.2). Subsequently,
an erythrocyte suspension was made (0.2% [V/V])
in PBS. Then, 25 lL from each sample of experi-
ment II were mixed with 25 lL of PBS, and imme-
diately, 25 lL of the erythrocyte suspension were
added to the sample-buffer mixture in a flat-bot-
tom, 96-well plate (Costarâ, USA). The plates were
kept at rest for 20 min at room temperature. The
presence of lectins/agglutinin was detected by
hemagglutination. The assay was performed in
triplicate.
Shrimp postlarval quality
At the end of experiment I, 50 PLs17 from each
replicate were transferred to 2 L of freshwater to
determine the PL resistance to osmotic stress. After
1 h, postlarvae were transferred back to the tank
water and live animals were counted after
30 min. Another 50 postlarvae were taken to
measure their length and weight. Postlarvae were
washed with distilled water, dried with filter paper
and weighed on an analytical balance (Sartorius A
1205) with a precision of 0.1 mg. To determine
the length, postlarvae were measured from the
rostral base to the telson extremity with a preci-
sion of 0.1 mm. At the end of experiment II,
100 PLs 8 samples from each replicate were used
to assess the length, weight and resistance to the
osmotic stress test following the same procedure
described. Gill branch number of 50 PL7 per tank
was determined under a microscope (Olympus
Aquaculture Research, 2016, 1–15 Two probiotics enhancing production of L. vannamei R Franco et al.

BH-2, Japan). The survival per cent was calculated

in each pond, from the final number of animals
compared to the initial number of stocked animals.

Vibrio spp. levels in postlarvae and culture water

In experiment II, samples of water from each tank
and 50 individuals were taken daily. Serial dilu-
tion of water samples were made in sterile PBS
and plates containing thiosulfate-citrate-bile salts-
sucrose (TCBS) media were inoculated with 10 lL
of each sample dilution. For the evaluation of bac-
teria in the animals of experiment II, the samples
were homogenized (Wiggenhauser, D-130, Malay-
sia) in 500 lL of PBS. Serial dilution (1:10) were
made in sterile PBS and plates containing TCBS
media were inoculated with 10 lL of each sample
dilution. Plates were incubated at 28°C for 24 h
and results were expressed as colony-forming units
(CFU) mL
1. Figure 1 Inhibition zone of probiotic CIGBC-232
against V. harveyi, with a 13  1 mm zone of inhibi-
tion. Colony at the center corresponds to Vibrio harveyi
Statistical analysis (used as negative control), while colonies near the edge
are CIGBC-232.
Variance in enzymatic activities and numerical
parameters (length, weight and bacterial count
in water and animals) were compared statisti- Table 1 Physiological characteristics of the CIGBC-232
cally using the F test, and means were com- strain
pared by Student0 s t-Test. The correlation
between postlarval soluble protein and age in Test Results Test Results
days was evaluated by a simple linear regression
Catalase + Growth in NaCl:
analysis. The Newman–Keuls multiple range test Voges-proskauer + 2% +
was used to compare the regression slopes. Other Nitrate reduction + 5% +
parameters of quality, such as % survival, were Acid from: 7% +
evaluated by a 2 9 4 v2 contingency table. The Glucose + 7.5%

Xylose + Growth at:
number of gill branches was evaluated by Kol-
Arabinose + 10°C +
mogorov-Smirnov0 s frequency test. The same test Mannitol + 30°C +
was used to evaluate hemagglutination. All sta- Hydrolysis of Gelatin + 55°C +
tistical analyses were made by statistics software Hydrolysis of starch + 60°C

STATGRAPHICS PLUS 5.1. Hydrolysis of casein + pH 5.7 +
pH 6.8 +

Results

biochemical characteristics determined by con-

Isolation and identification of strains
Only strain CIGBC-232 was able to inhibit the
growth of V. harveyi, with a 13  1 mm zone of
inhibition (Fig. 1). Strain CIGBC-232 consisted of
facultative anaerobic, Gram-positive, rod-shaped
cells, forming spores that lie centrally in unswol-
len sporangia. Colonies on trypticase soy agar
(TSA) are opaque, beige, irregular in shape with
undulate edges. Table 1 shows the results of the
ventional methods. Strain CIGBC-232 shared the
highest level of 16S rRNA gene sequence simi-
larity (99.9%) with Bacillus licheniformis strains
DSM 13T and BCRC 11702 (NCBI references
sequences: NR_118996.1 and NR_116023.1
respectively). The partial 16S rRNA and
sequences derived in this study have been depos-
ited into the GenBank database under the acces-
sion number KT924442.

© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15 5

Two probiotics enhancing production of L. vannamei R Franco et al. Aquaculture Research, 2016, 1–15

without significant differences between the treated

Soluble protein content
groups. Nevertheless, from PL2 to PL7, the respira-
Administration of the probiotic strain CIGBC-232 tory burst was significantly increased in the ani-
significantly increased soluble protein content (1.4- mals treated with strain CIGBC-232; 78% in PL2
fold) over the untreated group (experiment I). In to 91% in PL7 (Fig. 4).
experiment II, the soluble protein content increased
SOD specific activity
with a linear tendency from the PZ-I to the PL7
stage in both treated groups. There were no signifi- The SOD and peroxidase activities in postlarvae
cant differences between strain CIGBC-232 treated with strain CIGBC-232 were significantly
(0.73 lg animal
1 day
1, r2 = 0.9618) and EPI- (P < 0.05) higher than in control postlarvae, by
CIN 3W (0.74 lg animal
1 day
1, r2 = 0.9328) 44% (Experiment I). SOD activity decreased from
treatments (Fig. 2). PZ-I to PL7 in both treated groups of experiment
II. No significant differences in SOD activity among
Influence of probiotic treatment on larval and CIGBC-232 and EPICIN 3W were observed at any
postlarval immunity developmental stage (Fig. 5).

PO specific activity Peroxidase specific activity

In the first experiment, the PO activity of postlar- Significant increase (21%; P < 0.05) in peroxidase
vae treated with the strain CIGBC-232 was signifi- activities was observed in postlarvae treated with
cantly higher (31.8%) compared to the control. In strain CIGBC-232 compared to the control group
experiment II, PO activity decreased during growth (experiment I). In experiment II, a similar peroxi-
up to the PL-7 stage in both treated groups. In PZ- dase activity was obtained from stage PZ-I to PL3.
I, PZ-II, M-II, M-III and PL2-PL7, the treatment At stage PL3 and up, peroxidase activity showed
with strain CIGBC-232 resulted in a 30–40% differences between groups receiving strain CIGBC-
higher PO activity compared to the treatment with 232 and EPICIN 3W. However, no significant
EPICIN 3W. For the PZ-III, M-I and PL1 stages, no differences were found at any stage among
significant difference in PO activity was detected experimental groups (Fig. 6).
among the treatments (Fig. 3).
Presence of lectins and agglutinins
Respiratory burst
The capacity of the samples, in all shrimp stages,
In the second experiment, the respiratory burst and in both groups of experiment II, to agglutinate
showed fluctuations during growth up to PL1, rabbit blood cells, was calculated as % efficacy,

(a) (b)
Experiment I
Soluble protein content (µg animal–1)

Soluble protein content (µg animal–1)

Experiment II
160
b 10
140 CIGBC-232 a
120 EPICIN 3W
8 b
a Linear (CIGBC-232)
100 Linear (EPICIN 3W)
80 6

60
4
40
20 2

0
Control CIGBC-232 0
PZ-I PZ-II PZ-III M-I M-II M-III PL1 PL2 PL3 PL4 PL5 PL6 PL7
Experimental groups
Stages during the ontogeny of shrimp L.vannamei

Figure 2 Soluble protein content (lg animal
1) in L. vannamei postlarvae cultivated with (■) and without ( ) pro-
biotic CIGBC-232 in experiment I, n = 50 (a). The samples were taken from the stages: protozoeas (PZ-I, -II and -III
stages), mysis (M-I, -II and -III stages) and postlarvae (PL-1 to PL-7), n = 8 (b). Data represents mean  SE. Bar T
indicates the SE. Different letters indicate significant differences among groups (P < 0.05).

6 © 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15

Aquaculture Research, 2016, 1–15 Two probiotics enhancing production of L. vannamei R Franco et al.

(a) Experimet I (b) Experiment II

0.020 a
2.4E–03 0.018
2.1E–03 b CIGBC-232
0.016

PO activity (U µg–1)
PO activity (U mg–1) 1.8E–03 0.014 EPICIN 3W
a 0.012 a
1.5E–03
0.010
1.2E–03 b a
0.008 a
9.2E–04 a a a
0.006 a a a
6.2E–04 a a a
0.004 b a a
3.2E–04 0.002
b b a b b b b b b
2.0E–05 0.000
Control CIGBC-232 PZ-I PZ-II PZ-III M-I M-II M-III PL1 PL2 PL3 PL4 PL5 PL6 PL7
Experimental groups Stages during the ontogeny of white shrimp L. vannamei

Figure 3 Specific activity of phenoloxidase (U mg
1) in L. vannamei postlarvae cultivated with (■) and without ( )
probiotic CIGBC-232 in experiment I, n = 50 (a). PO specific activity (U lg
1) of L. vannamei larvae and postlarvae
treated with probiotic CIGBC-232 and EPICIN 3W in experiment II (b). The samples were taken from the stages:
protozoeas (PZ-I, -II and -III stages), mysis (M-I, -II and -III stages) and postlarvae (PL-1 to PL-7), n = 8. Data from
both experiment represents mean  SE. Bar T indicates the SE. Different letters indicate significant differences
among groups (P < 0.05).

Experiment II
1.6E–05

1.4E–05
CiGBC-232
Respiratory burst (U µg–1)

a
1.2E–05
EPICIN 3W
Figure 4 Respiratory burst (U lg
1) 1.0E–05
in experiment II. The samples were
8.0E–06 a
taken from the stages: protozoeas a
a
(PZ-I, -II and -III stages), mysis (M-I, 6.0E–06
a a
a a
-II and -III stages) and postlarvae 4.0E–06 a a
(PL-1 to PL-7). Data represents a a b b
a a
mean  SE, n = 8. Bar T indicates 2.0E–06 a b b b
a b
a a a
the SE. Different letters indicate sig- a
0.0E+00
nificant differences among groups PZ-I PZ-II PZ-III M-I M-II M-III PL1 PL2 PL3 PL4 PL5 PL6 PL7

(P < 0.05). Stages during the ontogeny of white shrimp L. vannamei

which is presented in Fig. 6. In both cases, the per survival rate (90%) after a 1-h immersion in fresh
cent of samples able to agglutinate rabbit blood water, as compared to the control group (55%).
cells was similar from stage PZ-I to stage PL5. A For experiment II, the survival per cent after the
significant reduction was observed in stages PL1, osmotic stress test was 93.3% for CIGBC-232 and
PL6 and PL7 in the group treated with EPICIN 90% for EPICIN 3W. No significant differences
3W as compared to the strain CIGBC-232 group were detected among treatments (v2 = 0.182)
(Fig. 7). (Table 2).
The group subjected to the CIGBC-232 treat-
ment was significantly higher than the untreated
Quality evaluation of postlarvae
control (experiment I) in terms of weight (95.7%
At the end of each experiment, the quality of post- increased weight) and length (20.1% increased
larvae was evaluated measuring the resistance to length), respectively (Fig. 8a, b). Also in experi-
osmotic stress and the parameters of length, ment II, the treatment of postlarvae with strain
weight (in experiment I and II), survival and gill CIGBC-232 enhanced the length and the weight
branch number (in experiment II). (104.6% and 113.6% respectively) as compared
In experiment I, postlarvae treated with probi- with postlarvae that received treatment with EPI-
otic CIGBC-232 showed a significantly superior CIN 3W (Fig. 8c, d). Furthermore, at the PL7

© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15 7

Two probiotics enhancing production of L. vannamei R Franco et al. Aquaculture Research, 2016, 1–15

(a) Experiment I (b) Experiment II

1.30 b 1.4E–02
1.20 CIGBC-232
1.2E–02
1.10
SOD activity (Umg–1)

a EPICIN 3W

SOD activity (Uµg–1)

1.0E–02
1.00
0.90 a
8.0E–03
a
a
0.80
6.0E–03 a
0.70 a
4.0E–03 a
0.60 a a
a a
a
0.50 2.0E–03 a a
a a a
a a a
0.40 a a a a a a
Control CIGBC-232 0.0E+00 a
PZ-I PZ-II PZ-III M-I M-II M-III PL1 PL2 PL3 PL4 PL5 PL6 PL7
Experimental groups Stages during the ontogeny of white shrimp L. vannamei

Figure 5 Specific activity of superoxide dismutase activity (U mg
1) in L. vannamei postlarvae cultivated with (■)
and without ( ) probiotic CIGBC-232 in experiment I, n = 50 (a). SOD specific activity (U lg
1) of L. vannamei lar-
vae and postlarvae treated with probiotic CIGBC-232 and EPICIN 3W in experiment II. The samples were taken
from the stages: protozoeas (PZ-I, -II and -III stages), mysis (M-I, -II and -III stages) and postlarvae (PL-1 to PL-7),
n = 8 (b). Data from both experiments represents mean  SE. Bar T indicates the SE. Different letters indicate signif-
icant differences among groups (P < 0.05).

(a) Experiment I (b) Experiment II

2.1E–04 6.0E–04 a
EPICIN 3W
b
Peroxidase activity (U µg–1)
Peroxidase activity (Umg–1)

2.0E–04 CIGBC-232
5.0E–04 a
1.9E–04 a
a
1.8E–04
4.0E–04 a
1.7E–04 a
a
1.6E–04 a a
3.0E–04 a a
1.5E–04 a a a
a a
a
1.4E–04 2.0E–04 a
a a
1.3E–04 a a a
a a a a
1.2E–04 1.0E–04
Control CIGBC-232 PZ-I PZ-2 PZ-3 M-I M-II M-III PL1 PL2 PL3 PL4 PL5 PL6 PL7
Experimental groups Stages during the ontogeny of white shrimp L. vannamei

Figure 6 Specific peroxidase activity (U mg
1) in L. vannamei postlarvae cultivated with (■) and without ( ) probi-
otic CIGBC-232 in experiment I, n = 50 (a). Specific peroxidase activity (U lg
1) of L. vannamei larvae and postlar-
vae treated with probiotic CIGBC-232 and EPICIN 3W in experiment II. The samples were taken from the stages:
protozoeas (PZ-I, -II and -III stages), mysis (M-I, -II and -III stages) and postlarvae (PL-1 to PL-7), n = 8 (b). Data
from both experiments represents mean  SE. Bar T indicates the SE. Different letters indicate significant differences
among groups (P < 0.05).

stage in experiment II, postlarvae receiving stage PZ-I up to stage M-I. Later, it began to
probiotic strain CIGBC-232 had a significantly decline up to the PL7 stage, while the bacterial
(P < 0.05) higher number of branchial ramifica- shrimp population showed increment-reduction
tions (4–6) than animals treated with probiotic cycles during the experimental evaluation. More-
EPICIN 3W (2–4). On the other hand, no signifi- over, treatment with strain CIGBC-232 had a
cant differences in survival were detected among higher action in diminishing presumptive Vibrio
the treatments at the end of experiment II (Fig. 8). count in the tank water and inside the animals
than treatment with EPICIN 3W. Except in the
stages PZ-I, PL4 and PL5, significant differences
Bacterial content
(P < 0.05) in presumptive Vibrio count for pond
In experiment II, presumptive Vibrio counts in water treated with strain CIGBC-232 and EPICIN
pond water of both treatments increased from 3W were recorded. While in shrimp, significant

8 © 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15

Aquaculture Research, 2016, 1–15 Two probiotics enhancing production of L. vannamei R Franco et al.

Experiment II
120

100
aa aa aa aa aa
Figure 7 Per cent of samples able

Haemagglutination (%)
aa
to agglutinate rabbit blood cells in
L. vannamei larvae and postlarvae 80 a aa
treated with probiotic CIGBC-232 b
( ) and EPICIN 3W ( ) in experi- 60
aa aa
ment II. The samples were taken
from the stages: protozoeas (PZ-I, 40
-II and -III stages), mysis (M-I, -II aa a a
and -III stages) and postlarvae (PL- 20
b b
1 to PL-7). Data represents
mean  SE, n = 8. Different letters 0
indicate significant differences PZ-I PZ-II PZ-III M-I M-II M-III PL1 PL2 PL3 PL4 PL5 PL6 PL7

among groups (P < 0.05). Stages during the ontogeny of white shrimp L. vannamei

Table 2 Survival of L. vannamei postlarvae treated with
CIGBC-232, untreated control group (experiment I) and
EPICIN 3W (experiment II) after being abrupt changed to
fresh water for 1 h
Live
Treatments N animals
Experiment I CIGBC-232 60 57
Control 60 33
Experiment II CIGBC-232 60 56
EPICIN 3W 60 54
Different letters indicate significant differences among groups
(P < 0.05). N: number of animals in each group.
differences (P < 0.05) were detected from PZ-I up
to PL2 but not in later stages (Fig. 9).
Discussion
Probiotics are able to improve the chemical and
microbiological water quality, and the survival,
growth, immune response and general health of
shrimp (Moriarty 1998). Bacillus spp. are among
the most widely used probiotics in aquaculture
(Nakayama, Lu & Nomura 2009; Ninawe & Selvin
2009), because they secrete a wide range of
exoenzymes and antimicrobial compounds (Mori-
arty 1998).
In this study, a candidate probiotic was isolated
from the intestine of healthy L. vannamei shrimp,
which showed growth inhibitory activity against
shrimp pathogens such as V. harveyi. The isolated
strain CIGBC-232 was identified as B. licheniformis.
Various species of genus Bacillus have shown
antagonist activity against shrimp pathogens
(NavinChandran et al. 2014).
Although antagonism is considered an impor-
tant feature of aquaculture probiotics and is thus
commonly used to select probiotics, further in vivo
confirmation is needed (Balc
azar, Blas, Ruiz-Zar-
zuela, Cunningham, Vendrell & M
uzquiz 2006).
Survival
Herein, the probiotic B. licheniformis strain CIGBC-
(%)
232 was applied to L. vannamei larval and early
95.0a postlarval stages under laboratory and farm-pro-
55.0b duction conditions.
93.3a
It is well known that a sustained increment in
90.0a
soluble protein content is a good nutritional and
health indicator in white shrimp Litopenaeus seti-
ferus (Brito, Chimal, Gaxiola & Rosas 2000) and
Litopenaeus schmitti (Martin, Arenal, Fajardo,
Pimentel, Hidalgo, Pacheco, Garcia & Santiesteban
2006). In this study, a higher protein content was
observed in postlarvae treated with CIGBC-232
(experiment I). During the ontogeny of L. van-
namei, soluble protein content showed similar con-
stant increases in the two probiotic treatments
(experiment II). This result gives evidence of a
high rate of growth and a good state of nutrition
and health (Martin et al. 2006).
The PO enzymatic cascade has a key role in the
innate immune response in invertebrates, produc-
ing melanin, which activates phagocytosis and
cell-to-cell adhesion, etc. Its multiplicity of direct
or indirect effects have made it one of the principal
factors within the crustacean immune system to
be targeted for up-regulation by probiotic and
other externally administered stimulants
(Amparyup, Charoensapsri & Tassanakajon 2013;
Iwanaga & Lee 2005; Smith et al. 2003).
Groups treated with B. licheniformis strain
CIGBC-232 had significantly greater PO activity as
compared with untreated control and EPICIN 3W

© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15 9

Two probiotics enhancing production of L. vannamei R Franco et al. Aquaculture Research, 2016, 1–15

(a) (b)
Experiment I Experiment I
10.00
b 7.0 b
9.50 6.5
6.0
Length (mm)

9.00 5.5

weight (mg)
5.0
8.50 4.5
a 4.0
8.00 a
3.5
7.50 3.0
2.5
7.00 2.0
Control CIGBC-232 Control CIGBC-232
Experimental groups Experimental groups
(c) (d)
Experiment II Experiment II
6.6 1.00
b
6.5 0.95
0.90 b
6.4
Length (mm)

Weight (mg)

0.85
6.3
0.80 a
6.2
a
0.75
6.1 0.70
6.0 0.65
Figure 8 Quality evaluation of
5.9 0.60
EPICIN 3W CIGBC-232 EPICIN 3W CIGBC-232 postlarvae. Results of from both
Experimental groups Experimental groups experiments I and II are shown.
(e) (f) Means of Length (a) and weight
Experiment Il Experiment Il
54 160
(b) of L. vannamei treated with
a a CIGBC-232 (■) and untreated con-
52 140
120 b trol group ( ), N = 50. Length (c),
50
Survival (%)

Frequency

a weight (d) and survival of L. van-

100
48
80 namei postlarvae at stage PL7 trea-
46 ted with CIGBC-232 ( ) and
60
44
40 EPICIN 3W ( ), n = 8. (e). Gill
42 20 branch numbers in L. vannamei
40 0 postlarvae (PL7), n = 400 (f). Data
EPICIN 3W CIGBC-232 2 3 4 5 6 in a, b, c and d represents
Experimental groups Gill branch number mean  SE, bar T indicates the SE.

(experiments I and II), which provides evidence for present there is not much knowledge regarding
immune modulation at larval stages by the admin- the metamorphosis mechanism in penaeid shrimp
istered probiotic. Bacillus subtilis E20 has previ- larvae.
ously been observed to modulate PO activity in In crustaceans, after contact between haemocyte
juveniles of L. vannamei (Tseng, Ho, Huang, and pathogen is established, the NADPH oxidase
Cheng, Shiu, Chiu & Liu 2009). In addition, signif- system is activated to generate ROS (reactive oxy-
icant differences in PO activity were observed dur- gen species), which have a potent cytotoxic effect
ing metamorphosis of PZ-III to M-I and (Roch 1999) and is an important microbicidal
metamorphosis of M-III to PL1. In peneid shrimp, activity (Campa-C
ordova, Hern
andez-Saavedra,
larval metamorphosis is a critical phase in which Aguirre-Guzm
an & Ascencio 2005; Ji, Yao &
larvae are affected by diseases such as protozoea Wang 2011). During the metamorphosis of PL-2
syndrome and bacterial infections. In fact, a signif- to PL-7 the group treated with B. licheniformis
icant reduction in PO activity during the proto- strain CIGBC-232 showed higher respiratory burst
zoea-mysis and mysis-postlarvae metamorphoses activity. We consider that the increment in the
was recently observed in an ontogenetic study of respiratory burst could be due to a better meta-
immune response (Mart
ın et al. 2012). Currently, bolic rate in postlarvae treated with probiotic
the molting cycle of crustaceans is fairly well B. licheniformis strain CIGBC-232, which can be
described (Chang & Mykles 2011). Nevertheless, at translated into the better growth shown by this

10 © 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15

Aquaculture Research, 2016, 1–15 Two probiotics enhancing production of L. vannamei R Franco et al.

(a) Experiment II
1.0E+07
a a a a a a
a a

Content bacterial water

1.0E+06 a b
a b b b a
b a

Log (CFU mL–1)

b
1.0E+05 a b b a
b
a a
1.0E+04 a
1.0E+03

1.0E+02

1.0E+01
PZ-I PZ-II PZ-III M-I M-II M-III PL1 PL2 PL3 PL4 PL5 PL6 PL7
Stages during the ontogeny of white shrimp L. vannamei
(b)
Figure 9 Bacterial count (Vibrio 1.0E+05
spp. and Pseudomonas spp.) in water

Bacterial shrimp content

of ponds (a), and L. vannamei lar- a a a
a a a
1.0E+04

Log (CFU animal–1)

vae and postlarvae (b) in experi- a a b a
a a
ment II. ( ) ponds treated with b a a b
a a a
CIGBC-232 (B. licheniformis) ( ) 1.0E+03 a b b b
ponds treated with EPICIN 3W. PZ b a
(protozoea), M (mysis), PL (Postlar- b
1.0E+02
vae). Data represents mean  SE,
n = 8. Bar T indicates the SE. Dif-
ferent letters indicate significant 1.0E+01
differences among groups (P < PZ-I PZ-II PZ-III M-I M-II M-III PL1 PL2 PL3 PL4 PL5 PL6 PL7

0.05). Stages during the ontogeny of white shrimp L. vannamei

group. A direct effect of respiratory burst activity
on survival was observed when shrimp were chal-
lenged artificially with V. alginolyticus (Tseng et al.
2009).
It has been shown that SOD actively neutralizes
O2
, thereby efficiently avoiding the accumulation
of such ROS during the immune response and
metabolism (Campa-C
ordova et al. 2005). The
effect of Bacillus species on SOD activity has been
well demonstrated in various studies. Bacillus spp.
was able to increase SOD activity in postlarvae of
L. vannamei, Palaemonetes argentinus (Neves, Santos
& Bainy 2000) and juveniles of Palaemonetes pugio
(Downs, Fauth & Woodley 2001).
In this study, improvement of SOD and peroxi-
dase activities was observed in postlarvae treated
with B. licheniformis strain CIGBC-232 compared
to the untreated control group (experiment I).
These results suggest that B. licheniformis strain
CIGBC-232 may enhance postlarvae antioxidant
status. In experiment II, no significant difference
in modulation of SOD activity by probiotic treat-
ment using B. licheniformis strain CIGBC-232 or
EPICIN 3W was observed at any developmental
stages of the shrimp. This fact is very interesting,
because a substantially elevated SOD activity
should be observed in postlarvae (PL2 to PL7)
treated with B. licheniformis strain CIGBC-232 as
consequence of the significant level of O2
pro-
duced by these animals. On the other hand, both
probiotic treatments showed a similar capacity in
modulation of peroxidase activity. We assumed
that peroxidase performance was due to a lack of
accumulation of H2O2 by the action of SOD. Today
it is a well-established fact that a considerable
H2O2 quantity is derived from O2
by SOD action
(Holmblad & S€ oderh€all 1999). However, B. subtilis
(E23) was not able to stimulate peroxidase activity
in juveniles of L. vannamei (Gullian, Thompson &
Rodriguez 2004).
Lectins and agglutinins are proteins involved in
the recognition of non-selfmatter in the humoral
response of the immune system in crustaceans
(Iwanaga & Lee 2005; Vazquez, Alpuche, Maldon-
ado, Agundis, Pereyra-Morales & Zenteno 2009).
Modulation of lectins may result in protecting lar-
vae and postlarvae from natural infections. In this
study, both probiotic treatments had similar abili-
ties to modulate lectin titre in the majority of
stages evaluated, with the exception that an incre-
ment was observed for probiotic B. licheniformis
strain CIGBC-232 treatment in stages PL1, PL6

© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15 11

Two probiotics enhancing production of L. vannamei R Franco et al.
and PL7. The reduction in lectins achieved by
both probiotic treatments from the M-III stage, is
in accordance with our previous observation in
characterizing lectins/agglutinins titres in ontoge-
netic development of L. vannamei. Such behaviour
was attributed to the hardness of physical barriers
such as the exoskeleton of postlarvae which is able
to prevent the passing of potentially pathogenic
microorganisms (Mart
ın et al. 2012).
Various parameters are used by breeders to eval-
uate postlarval quality. Generally, these include
survival, weight, size, count of the number of gill
branches and evaluation of stress resistance
(Racotta, Palacios & Ibarra 2003).
When the osmotic stress resistance test is made,
at least 80% of postlarvae should be able to sur-
vive in freshwater for 30 min to consider that ani-
mals have good health and quality (Dhert, Lavens
& Sorgeloos 1992). Also, some authors have
shown that survival during an osmotic stress test
depends on the content of polyunsaturated fatty
acids present in postlarvae, whose intake is
through the feed (Rees, Cur
e, Piyatiratitivorakul,
Menasveta & Sorgeloos 1994). Liu et al. (2010)
found that L. vannamei postlarvae treated with
bacillus E-20 showed greater salinity stress toler-
ance. In this study, a higher survival after an
osmotic stress test was observed in postlarvae trea-
ted with probiotic B. licheniformis strain CIGBC-
232 in experiment I. In experiment II a similar
survival response to osmotic stress was observed
among the groups. This results suggest that the
nutritional state of postlarvae treated with CIGBC-
232 was improved in experiment I and that nutri-
tional requirements of postlarvae were satisfied in
both groups during experiment II. The growth
observed in animals treated with some probiotic
has been attributed to mechanisms such as: estab-
lishment of the probiotic in the host gastrointesti-
nal tract, which can improve the nutrient
absorption (Gatesoupe 2008), production of diges-
tive enzymes (proteases and amylases) and synthe-
sis of vitamins and cofactors (Ninawe & Selvin
2009). In this study, The probiotic B. licheniformis
strain CIGBC-232 significantly increased the size
and weight of the shrimp postlarvae as compared
either with the untreated control (experiment I) or
the EPICIN 3W group (experiment II). Further-
more, animals treated with B. licheniformis strain
CIGBC-232 showed a greater number of gill
branches than postlarvae treated with EPICIN 3W.
A high-protease-producing probiotic, B. subtilis,
12
Aquaculture Research, 2016, 1–15
was demonstrated to be beneficial to growth
performance of white shrimp, L. vannamei, by
improving its food digestion and nutrient absorp-
tion (Liu et al. 2009). Moreover, a-amylase activ-
ity and protease activity were increased in
Fenneropenaeus indicus through addition of a com-
mercial probiotic containing various Bacillus spe-
cies that included B. licheniformis (Ziaei-Nejad,
Rezaei, Takami, Lovett, Mirvaghefi & Shakouri
2006). More recently, application of a probiotic
containing multiple species of Bacillus, in sea
bream larviculture, improved growth and activa-
tion in the transcription of the IGF-1 gene (Avella,
Gioacchini, Decamp, Makridis, Bracciatelli & Car-
nevali 2010).
In experiment II, similar survival was obtained
for both probiotic treatments. Probiotic bacterial
strains belonging to the genus Bacillus have been
shown to improve survival in P. monodon (Rengpi-
pat et al. 1998). Recently, juveniles of L. vannamei
treated with Bacillus subtilis E20 showed higher
survival than the control and this increment was
dose-dependent (Liu et al. 2009). We used a single
dose in both probiotic treatments. For the EPICIN
we used the dose recommended by the manufac-
turer. The use of different doses than those applied
in the B. licheniformis strain CIGBC-232 treatment
could vary survival results.
Except for stages PL4, PL5, PL7, a greater
reduction in the count of presumptive Vibrio in the
culture water, as well as inside the animals was
found in shrimp treated with probiotic B. licheni-
formis strain CIGBC-232 as compared with ani-
mals treated with EPICIN 3W (Fig. 9). In contrast,
the number of presumptive Vibrios inside the ani-
mals was significantly reduced by probiotic
B. licheniformis strain CIGBC-232 from stage PZ-I
up to PL2. From this stage forward in develop-
ment, both probiotic treatments had a similar
effect on the presumptive Vibrio count. This evi-
dence suggests that antibacterial action of
B. licheniformis strain CIGBC-232 is better than
that observed for EPICIN 3W under these experi-
mental conditions. Probiotics have several mecha-
nisms to induce antagonism with other bacteria in
water or within the animal’s gastrointestinal tract,
these mechanisms refer to: production of antimi-
crobial agents such as bacteriocins and sidero-
phores (Jenssen, Hamill & Hancock 2006;
Kesarcodi-Watson et al. 2008; Ninawe & Selvin
2009) and competition for nutrients or for adhe-
sion sites in the gastrointestinal tract (Moriarty
© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15
Aquaculture Research, 2016, 1–15
1998). It is already well known that bacteria
belonging to the genus Bacillus have the ability to
produce bacteriocins (Barbosa, Serra, La Ragione,
Woodward & Henriques 2005; Cherif, Ouzari, Daf-
fonchio, Cherif, Ben Slama, Hassen, Jaoua & Boud-
abous 2001; Duc, Hong, Barbosa, Henriques &
Cutting 2004). Also, bacitracin production by
B. licheniformis is well documented (Guo, Yu, Xie
& Zhang 2012; Haddar, Aziz & Al-Gelawi 2007).
Although a lower presumptive Vibrio count was
observed in animals treated with B. licheniformis
strain CIGBC-232, this result did not influence the
survival between the treatments. Generally, Vibrios
are opportunists that only cause mortality when
the host organisms are physiologically stressed,
which is often attributed to adverse growing con-
ditions (Alderman & Hastings 1998). For that rea-
son, it must be considered that similar survival
among treatments during the culture up to to PL7
may be due to lack of physiological stress by the
maintenance of optimal environmental conditions
during the experiment.
Multispecies probiotics are more effective and
consistent than their mono-species counterparts
since mixed cultures may exhibit synergistic proper-
ties (Nayak 2010). However, B. licheniformis strain
CIGBC-232, which is a monospecies probiotic, per-
formed notably better as a probiotic than EPICIN
3W, which is a multispecies probiotic, with regard
to immune modulation and postlarval quality.
In summary, we isolated a strain CIGBC-232,
from the gut of healthy shrimp. The strain CIGBC-
232 was identified as Bacillus licheniformis by bio-
chemical testing and was further confirmed by
16S rDNA gene sequence. As a water additive it
improved the weight, length, total protein content,
osmotic stress resistance and immunity of L. van-
namei postlarvae. Under farming conditions, the
monospecies probiotic, B. licheniformis strain
CIGBC-232, modulated the immunological oxida-
tive response better than the multispecies probiotic
EPICIN 3W in larval and postlarval stages, in con-
trast to that observed for the antioxidant response,
where both probiotics performed similarly. In addi-
tion, a superior animal quality and lowered Vibrio
sp. load in postlarvae and water for larvae treated
with probiotic CIGBC-232 were demonstrated.
Acknowledgments
We thank Dr. John van der Meer (Pan-American
Marine Biotechnology Association) for advice on
© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–15
Two probiotics enhancing production of L. vannamei R Franco et al.
English improvement for the manuscript. We also
thank all the technicians and workers at the
Yag€uacam shrimp hatchery, Cumanayag€ ua, Cien-
fuegos, Cuba, for their collaboration and excellent
technical assistance.
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