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Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
Calpis Co., Ltd., Tokyo 150-0021, Japan
a r t i c l e
i n f o
Article history:
Received 12 January 2013
Received in revised form 3 May 2013
Accepted 25 June 2013
Available online 18 July 2013
Keywords:
Bacillus subtilis
Cytokine expression
Autochthonous microbiota
Hybrid tilapia
a b s t r a c t
Bacillus spp. are widely used in aquafeeds as probiotics for enhanced growth performance, innate immune
responses, and disease resistance. The present study aimed to evaluate the effects of low doses of dietary
Bacillus subtilis C-3102 on the production, intestinal cytokine expression and adhesive bacteria of hybrid tilapia Oreochromis niloticus Oreochromis aureus . Juvenile hybrid tilapia were fed B. subtilis C-3102 at
doses of 0 (CK), 2.5 105 (Ta) and 5.0 105 (Tb) CFU g1 of diets for 56 days. The results showed that different diets had no effects on sh growth performance. The B. subtilis supplement was detected in the gut
wall of sh treated with 5.0 105 CFU g1 at 28 days and 56 days by both plate culture and PCR-DGGE
methods, while it was only identied in sh treated with 2.5 105 CFU g1 at 56 days by PCR-DGGE. Supplementation of dietary B. subtilis C-3102 altered the autochthonous gut bacterial communities, signicantly
increased (P b 0.05) the total amounts of adhesive viable bacteria, induced upregulation of intestinal cytokine expression (IL-1b, TGF- and TNF-) and downregulation of intestinal HSP70. Thus dietary supplement
of B. subtilis C-3102 at low dose (105 CFU g1) is benecial to tilapia health.
2013 Elsevier B.V. All rights reserved.
1. Introduction
With the development of commercial-scale aquaculture, disease has
been one of the signicant limiting factors and causes of severe economic loss (Gomez-Gil et al., 2000; Sun et al., 2010). Probiotics are live microorganisms that provide benet to the host animals, such as enhanced
growth performance, innate immune responses, and resistance against
disease, so are widely used in aquaculture (Apn-Molina et al., 2009;
Denev et al., 2009; Nayak, 2010). In sh, Bacillus has been identied to
be probiotics in many studies (Gnther and Jimenez-Montealegre,
2004; He et al., 2011; Nayak, 2010; Sun et al., 2010).
The dose of probiotics is a key factor for optimal benecial effects
on host animals (Minelli and Benini, 2008; Nayak, 2010). It is not
only required for the colonization and subsequent proliferation in
host intestines, but also exerts various benecial effects including
immunostimulatory activity. In previous studies, the variable doses
of Bacillus (1061012 CFU g1) have been tested in aquaculture, and
Bacillus supplement of more than 108 CFU g1 was found to be
Corresponding authors at: Key Laboratory for Feed Biotechnology of the Ministry of
Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12
Zhongguancun South Street, Beijing 100081, PR China. Tel.: +86 10 82106073; fax: +86
10 82106054.
E-mail addresses: zhou_zg@msn.com (Z. Zhou), binyao@caas.cn (B. Yao).
0044-8486/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2013.06.028
126
Table 1
Formulations and chemical compositions of the experimental diets.
Ingredients
Percentages (%)
CK
Ta
Tb
18.00
0.30
30.53
32.00
12.27
4.00
2.20
0.10
0.20
0.20
0.20
0.00
18.00
0.295
30.53
32.00
12.27
4.00
2.20
0.10
0.20
0.20
0.20
0.005
18.00
0.29
30.53
32.00
12.27
4.00
2.20
0.10
0.20
0.20
0.20
0.01
36.00
6.00
36.00
6.00
36.00
6.00
Table 2
Primer sequences for intestinal cytokine analysis by qPCR.
Genes
Primer sequences
Reference genes
IL-
F: TGCACTGTCACTGACAGCCAA
R: ATGTTCAGGTGCACTTTGCGG
F: CTTCCCATAGACTCTGAGTAGCG
R:GAGGCCAACAAAATCATCATCCC
F: TGCCTTTGTCCAGACCGTAG
R: GTGTCCAACGCTGTCATCAC
F: TGCGGCACCCAATCACACAAC
R: GTTAGCATAGTAACCCGTTGGC
F: GCTACTCCTTCACCACCACAG
R: CGTCAGGCAGCTCGTAACTC
236
JF957365
183
JF957367
114
JF957370
190
JF957373
233
JF957365
Fish were batch weighed from each tank at the initial stage, and at
day 56 after probiotic feeding. Production was assessed in terms of
weight gain (WG, %), feed conversion ratio (FCR) and survival rate
(SR, %). The calculations were performed using the following formulae:
WG (%) = 100 (Wt W0) / W0, FCR = FI / (Wt W0), and SR
(%) = (100 number of nal sh number) / total sh number where
HSP70
TGF-
Actin
127
PCR detection system (Bio-Rad, Hercules, CA). The total volume of PCR
reaction was 20 L and consisted of 10 L of SYBR Green Premix Ex
Taq II (2), 1 L primer of each, 2 L cDNA and 6 L ddH2O. The cycling
conditions were as follows: 95 C for 3 min and then 40 cycles of 95 C
for 20 s and 55 C for 20 s and 72 C for 20 s. All real time-PCRs were
performed at least in triplicate. Data analysis was conducted using the
2CT method (Livak and Schmittgen, 2001), and -actin was included
as an internal reference for normalization of gene expression data.
2.7. Statistical analysis
Results were expressed as the mean S.D. Difference between
groups were determined using a one-way analysis of variance
(ANOVA) with the statistical software package SPSS 17.0. Signicant
and very signicant differences were accepted at P b 0.05 and P b 0.01,
respectively.
3. Results
3.1. Production of tilapia
After 56-day-feeding, no signicant difference of FCR, SR, WG and
SGR (P N 0.05) was observed in the sh fed diet Ta and Tb by comparison with the sh fed the control diet (Table 3).
3.2. DGGE ngerprints of autochthonous gut bacteria in tilapia
The 16S rDNA V3 PCR-DGGE ngerprints of the autochthonous intestinal bacterial communities showed that dietary B. subtilis had effects
on tilapia intestinal bacterial communities (Fig. 1). A total of 18 unique
bands, i.e. 18 bacteria representatives, were retrieved from the ngerprints. Their taxa and closest relatives are shown in Table 4. Of them,
12 OTUs were Proteobacteria, 2 were Fusobacteria, 1 was Firmicutes,
and the rest were uncultured bacteria. Eleven of them were detected
in all gut samples, and Bacillus was only detected in the sh fed
5 108 CFU kg1 B. subtilis at day 28 and the sh fed B. subtilis of
both doses at day 56. Some autochthonous gut bacteria changed over
time; for example, bands 5, 11 and 17 only appeared at day 28 and
band 12 was only detected at day 56. The cluster analysis of the band
patterns is displayed in Fig. 2. We found that sh fed different diets
but sampled at the same time point are more similar than that sampled
at different times but fed the same diet. The similarity between CK and
Ta (2.5 105 CFU g1 B. subtilis) was more close than CK and Tb
(5 105 CFU g1 B. subtilis). It indicated that autochthonous bacterial
communities are dynamic, and the effects of probiotic are long-lasting
and dose dependent.
3.3. Total aerobic bacterial and B. subtilis counts determined by plate
culture
After 56-day feeding, the total counts of allochthonous aerobic bacteria in tilapia gut of Ta were signicantly greater (P b 0.05) than those
autochthonous ones in sh fed CK or Tb diets. The total aerobic bacteria
of autochthonous or allochthonous in tilapia fed dietary probtiotics
showed an increase compared to the CK diet. B. subtilis was not detected
in sh fed CK diet. Autochthonous Bacillus was detected in sh fed
higher dose of B. subtilis but not at low dose. Allochthonous Bacillus
Table 3
Effects of low doses of B. subtilis C-3102 (2.5 105 and 5.0 105 CFU g1) on the growth, diet conversion and survival rates of hybrid tilapia after 56-day-feeding (mean S.D.).
Treatments
IBW (g)
FBW (g)
WG (%)
FCR
SR (%)
CK
Ta
Tb
0.99 0.01
1.00 0.03
1.00 0.01
18.19 1.31
16.85 0.72
17.42 0.60
1731.78 142.24
1579.52 46.78
1634.19 71.52
1.12 0.08
1.05 0.06
1.05 0.02
72.50 10.90
70.00 12.25
85.00 5.00
128
Table 4
Representative and distributiona of autochthonous bacteria isolated from tilapia gut based on the 16S rDNA V3 DGGE ngerprints.
Phylum
Band no.
Identity (%)
CK1
Ta1
Tb1
CK2
Ta2
Tb2
Proteobacteria
4
7
17
8
12
2
13
16
18
9
10
14
1
5
6
11
15
100
100
100
100
100
100
100
100
99
100
100
100
100
99
100
99
100
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Fusobacteria
Firmicutes
Uncultured
a
+: If the value of a specic band intensity to the total band intensity percentage N1% mean exist, : If the value of a specic band intensity to the total band intensity percentage
b1% mean non-existent.
methods, both autochthonous and allochthonous B. subtilis could be detected in tilapia intestines over the whole feeding trial at the dose of
5.0 105 CFU g1. The counts of allochthonous and autochthonous
Bacillus were determined to be 104105 and 102 CFU g1, respectively.
Although strain C-3102 was detected in the gut of tilapia, whether
B. subtilis C-3102 have colonized the tilapia gut still needs further studies.
Treatment of rainbow trout with Bacillus results in the signicantly increased counts of bacteria associated with the intestine (P b 0.05)
(Bagheri et al., 2008). Dietary Bacillus can regulate the gut microbiota of
grouper, selectively stimulate various potentially benecial Enterococcus
sp.-like and B. pumilus-like bacteria, and depress some potential
harmful species like Staphylococcus and Vibrio ponticus (Yang et al.,
2012). B. subtilis under study not only increased the total number of
viable bacteria but also modulated the predominant bacteria in gut
(Tables 4 and 5, Fig. 1). Compared with our previous study that
B. subtilis C-3102 at 108 CFU g1 affected the intestinal microbiota community of koi carp at the early stage and became weaker in the later
stages (He et al., 2011), B. subtilis C-3102 at lower dose also inuenced
the gut microbiota at the early stage, but strengthened its effects over
time. This difference might be attributed to the supplement level of
B. subtilis. On the other hand, B. subtilis might modulate the intestinal
microbiota by producing a number of substances with biocontrol activities, such as turins and cyclic lipoproteins (Rosovitz et al., 1998; Sugita
et al., 1998).
The gut is the organ where probiotics establish and execute functions. Cytokines that regulate innate immunity are produced in response to microbial antigens or compounds released from damaged
cells. IL-1 is an important pro-inammatory cytokine, TNF- is an
interferon tumor necrosis factor, and TGF- is a trans-forming growth
Table 5
The total aerobic bacteria and B. subtilis counts (CFU g1) of tilapia gut determined by
agar culture method at day 56.a
Fig. 2. Cluster analysis of the intestinal autochthonous microbiota of hybrid tilapia fed
B. subtilis C-3102 based on 16S rDNA V3-DGGE. CK1 13: the triplicates of the control
at the end of 28 feeding days; Ta1 13: the triplicates of the B. subtilis C-3102 2.5 105
treatment at the end of 28 feeding days; Tb1 13: the triplicates of the B. subtilis C-310
5 105 treatment at the end of 28 feeding days; CK2 13: the triplicates of the control
at the end of 56 feeding days; Ta2 13: the triplicates of the B. subtilis C-3102 2.5 105
treatment at the end of 56 feeding days; Tb2 13: the triplicates of the B. subtilis C-310
5 105 treatment at the end of 56 feeding days.
Total autochthonous
bacterial count
Total allochthonous
bacterial count
Autochthonous
Bacillus count
Allochthonous
Bacillus count
CK
Ta
Tb
(4.64 3.46)
107b
(3.70 0.70)
108b
Undetected
(3.70 2.80)
108a
(8.00 3.00)
108a
Undetected
Undetected
(5.00 0.20)
104b
(3.05
107b
(6.50
108ab
(1.00
102
(1.40
105a
0.15)
1.00)
0.20)
0.18)
a
Data in the same column that shared a common superscript means no signicant
differences existed (P 0.05).
129
Fig. 3. Effects of dietary CALSPORIN on the immune responses of tilapia after 56 days feeding. CK, the control group; Ta, the B. subtilis C-310 2.5 105 treatment; Tb, the B. subtilis
C-3102 5 105 treatment. P b 0.01, P b 0.05.
factor. The transcript levels of all these genes relative to the housekeeping gene -actin were signicantly higher in the gut tissue of
sh fed Bacillus diet than that in CK sh (P b 0.05) (Fig. 2). Some studies indicate that a number of probiotics can effectively modulate the
production of proinammatory cytokines including IL-1, TNF-, and
TGF- (Awad et al., 2011; Nayak, 2010; Panigrahi et al., 2007;
Prez-Snchez et al., 2011; Wang et al., 2010). Bacillus can modify
the immune response of the host by interacting with epithelial cells
and by modulating the secretion of anti-inammatory cytokines, consequently resulting in a reduction of inammation (Nayak, 2010). The
70 kDa heat shock protein HSP70 has a number of functions, including the maintenance of cellular homeostasis and the protection of
the individual following pathogenic stress (Liu et al., 2012; Nayak,
2010). Levels of HSP70 are usually induced by some pathogens or disadvantageous conditions (Liu et al., 2012). The downregulated HSP70
in the present study indicates that B. subtilis C-3102 had positive effects on tilapia intestinal cells. The immunostimulatory effects of
Bacillus may ascribe to its production of -glucan and bacteriocins
(Gullian et al., 2004; Rengpipat et al., 2000).
In conclusion, probiotic B. subtilis C-3102 at low dose had no
effects on sh production, but modulated the intestinal microbiota
of tilapia and induced the upregulation of innate cellular cytokines
and downregulation of HSP70. Administration of low-level B. subtilis
130
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