Dietary supplementation of Bacillus subtilis and fructooligosaccharide enhance the growth, non-specific immunity of juvenile ovate pompano, Trachinotus ovatus and its disease resistance against Vibrio vulnificus

Fish & Shellfish Immunology 38 (2014) 7e14

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Dietary supplementation of Bacillus subtilis and fructooligosaccharide enhance the growth, non-specific immunity of juvenile ovate pompano, Trachinotus ovatus and its disease resistance against Vibrio vulnificus Qin Zhang a, Hairui Yu b, *, Tong Tong a, Wanping Tong a, Lanfang Dong a, Mingzhu Xu a, Zhicheng Wang a a

Key Laboratory of Marine Biotechnology of Guangxi, Guangxi Institute of Oceanology, 92 Changqing Road East, Beihai, Guangxi 536000, PR China Key Laboratory of Biochemistry and Molecular Biology in Universities of Shandong (Weifang University), College of Biological and Agricultural Engineering, Weifang University, Tianrun Bioengineering R & D Center of Weifang New and High-tech Zone, Weifang 261061, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 October 2013 Received in revised form 6 February 2014 Accepted 12 February 2014 Available online 12 March 2014

A feeding trial was conducted to investigate the effects of dietary administration of probiotic Bacillus subtilis and prebiotic fructooligosaccharide (FOS) on growth performance, immune responses and disease resistance of juvenile ovate pompano, Trachinotus ovatus. One thousand six hundred and twenty individuals (initial body weight: 10.32
0.46 g, mean
S.E) were fed nine practical diets according to a 3  3 factorial design: the basal diet as the control diet supplemented with three levels of B. subtilis (0, 1.05  107 or 5.62  107 CFU g1 diet), crossed with 0, 0.2% or 0.4% FOS. After an 8-week feeding experimental period, six fish per cage were sampled for immunity determination. Then 18 fish of each cage left were challenged by Vibrio vulnificus. The results showed that fish fed with 5.62  107 CFU B. subtilis g1 in combination with 0.2% FOS produced the highest specific growth rate, and were significantly higher than the groups fed with 0 and 0.2% FOS without B. subtilis supplementation (P < 0.05). Feed efficiency ratio significantly increased with the increasing doses of dietary FOS without B. subtilis added (P < 0.05). The immune assay showed that fish fed with the control diet produced the lowest respiratory burst activity and was significantly different from the groups fed the diets containing 0.2% FOS at each B. subtilis level and containing 0.4% FOS single (P < 0.05). Phagocytic activity was significantly decreased with the increasing doses of dietary B. subtilis at 0.4% FOS level (P < 0.05). Alternative complement pathway activity of the fish fed with 0.2% FOS single was significantly lower than those fed with 5.62  107 CFU B. subtilis g1 diet supplemented at each FOS level (P < 0.05). Fish fed with the control diet had the lowest lysozyme activity, and were significantly different from those fed with 0.2 or 0.4% FOS at 1.05 and 5.62  107 CFU B. subtilis g1 diet level. Moreover, fish fed with diets supplemented with 0.2% and 0.4% FOS at each B. subtilis level had notably lower cumulative mortality after 10 days following V. vulnificus infection (P < 0.05). Under the experimental conditions, dietary B. subtilis and FOS had a significant interaction on enhancing the immune responses and disease resistance of juvenile ovate pompano (P < 0.05). Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Trachinotus ovatus Probiotic Prebiotic Immunity Vibrio vulnificus

1. Introduction The ovate pompano, Trachinotus ovatus is an economically important warm-water farmed marine fish species in the world. Especially in recent years, ovate pompano aquaculture has

* Corresponding author. Tel./fax: þ86 536 8785288. E-mail addresses: [email protected], [email protected] (H. Yu). http://dx.doi.org/10.1016/j.fsi.2014.02.008 1050-4648/Ó 2014 Elsevier Ltd. All rights reserved.

developed rapidly and widely along the southern coast of China in tropical and subtropical climates, such as Guangdong, Guangxi and Hainan. As intensive aquaculture expanded and culture density increased, diseases occurred more frequently, especially between May and October each year, causing considerable economic losses [1]. Moreover, the application of antibiotics and chemotherapeutics to control these diseases caused many other problems such as the spread of drug resistant pathogens, suppression of aquatic animal’s immune system, environmental hazards and food

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Q. Zhang et al. / Fish & Shellfish Immunology 38 (2014) 7e14

safety problems. Increasing with the demand for environment friendly aquaculture, works on finding out the alternatives for the antibiotics are of most urgently important. Probiotics, prebiotics and synbiotics, which have various health promoting properties used as immunostimulants, have received increasing scientific and commercial interest in aquaculture. Probiotics, defined as live microorganisms which confer a health benefit on the host, when administered in adequate amounts [2]. Previous studies have reported that dietary probiotics effectively improved the feed utilization, modulated intestinal microflora, enhanced the immunity and also improved the antagonism to pathogens [3-7]. Probiotics Bacillus subtilis as one of the genus Bacillus that can be resistant to high temperature and high pressure in the pelleting and resistant to the acid conditions in gastric area [8] has been supplemented extensively in fish diets especially in recent five years [9e15]. Prebiotics have been defined as “non-digestible food ingredients which beneficially affect the host by selectively stimulating the growth and/or activity of health-promoting bacteria in the intestinal tract” [16]. Previous research efforts have demonstrated the benefits of prebiotics on growth, physiological status and immune responses (for review see Refs. [6,17]). Fructooligosaccharide (FOS) as one of prebiotics has been used extensively in different fish species such as turbot (Psetta maxima) [18], red drum (Sciaenops ocellatus) [19], large yellow croaker (Larimichthys crocea) [20], Japanese flounder (Paralichthys olivaceus) [21], Caspian roach (Rutilus rutilus) [22], stellate sturgeon (Acipenser stellatus) [23] and triangular bream (Megalobrama terminalis) [24]. The synbiotics are classified as a mixture of probiotics and prebiotics that beneficially affects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract [25]. Limited data is available regarding the application of synbiotics in fish [20,21,24,26,27]. However, despite recent advances on the administration of probiotics, prebiotics and synbiotics on other species, there is currently no data available on the efficacy of any probiotics, prebiotics or synbiotics for ovate pompano. Hence, the present study was conducted to determine the effects of dietary B. subtilis and FOS on growth, non-specific immunity of juvenile ovate pompano, T. ovatus and its disease resistance against Vibrio vulnificus.

Table 1 Ingredients and proximate composition of the basal diets. Ingredients

(%)

Proximate composition

(% Air-dry basis)

White fish meala Soybean meal Wheat meal Fish oil Soybean oil Lecithinb Vitamin mixb Mineral mixb Calcium biphosphateb Mold inhibitorc

44 18 27.4 2.5 2.5 1 1.5 1.5 1.5 0.1

Crude protein Crude lipid Ash Energy (MJ kg1)

43.93 8.85 12.53 18.86

a Imported from Japan: crude protein 69.5% (dry weight basis), crude lipid 7.6% (dry weight basis). b Kindly provided by Weifang Conqueren Bioscience & Technology Co. Ltd., China. c Contained 50% calcium propionic acid and 50% fumaric acid.

5.62  107 CFU g1 diet) and at each B. subtilis level one of three supplemental levels of FOS (0, 0.2, 0.4%) were included (3  3 factorial experiment). Graded doses of B. subtilis or FOS were added into the basal diet followed by mixing manually. Ingredients were ground into fine powder through a 200 mm mesh sieve. The test diets were prepared by thoroughly mixing the dry ingredients with fish oil and soybean oil and then adding cold water until a stiff dough produced. The dough was then pelleted with an experimental feed mill and dried in a ventilated oven at 40  C. After drying, the diets were broken up and sieved into the appropriate pellet size, and were stored at 20  C until use. Fish diets were analyzed for total bacterial counts by spreading plate method of Nikoskelainen et al. [28] and Zhang et al. [29] with slight modification. Each diet was analyzed by vortexing 0.5 g of diet in 4.5 ml of sterile saline and preparing serial dilution (from 103 to 107), whereupon 0.1 ml portion was spread onto triplicate plates of nutrient agar (NA) agar. The colony count was determined after incubation at 28  C for 48 h. Ten colonies were randomly picked for further identification after colony counting. And B. subtilis bacteria were confirmed. Then calculate the B. subtilis bacteria within the plate by the following formula:

A ¼ B

C f 10

2. Materials and methods 2.1. B. subtilis and FOS B. subtilis used in the study was kindly provided by Weifang Conqueren Bio-Tech Co., Ltd (China). The spores content of bacteria is approximate 1.5  1010 CFU g1 bacterial lyophilized powder. FOS was kindly provided by Shandong Baolingbao Bio-Tech Co., Ltd (China). According to method of the China Fermentation Industry Association (QB2581-2003), the ingredients in FOS 900P used in the present study were determined by high performance liquid chromatography (HPLC, HP 1100, USA) and expressed in percentage as follows: kestose, 28.65%; nystose, 52.66%; fructofuranosyl nystose, 14.08%; others, 4.61%. In other words, the purity of FOS 900P, which was made up of kestose, nystose and fructofuranosyl nystose, was 95.39%. 2.2. Diet preparation The basal diet used in this study (Table 1) was formulated to contain approximately 43.93% crude protein and 8.85% lipid, which have been shown to be sufficient to support the optimal growth of ovate pompano. This basal diet (used as the control) was supplemented with one of three levels of B. subtilis (0, 1.05  107,

AdDetermination of colonies of B. subtilis BdTotal number of plate colonies on NA agar CdThe identified and recognized colonies of bacteria B. subtilis from 10 colonies fdDilution

2.3. Feeding trial Healthy ovate pompano juveniles were obtained from a commercial hatchery (Beihai, China). The juveniles were reared in 5 floating sea cages (4.0  4.0  2.5 m), and fed with the basal diet for 2 weeks prior to the feeding trial to make the animals acclimate the experimental diet and environment. At the start of the experiment, the fish were fasted for 24 h and weighed after being anesthetized with 0.01% MS-222 (Sigma, USA). Fish of similar sizes (initial mean body weight 10.32
0.46 g) were randomly distributed into 27 sea cages (2.0  2.0  1.5 m) and each cage was stocked with 60 fish. Each diet was randomly assigned to triplicate cages. Animals were fed to apparent satiation twice daily at 05:00 and 18:00 for 8 weeks. During the experimental period, water temperature ranged from

Q. Zhang et al. / Fish & Shellfish Immunology 38 (2014) 7e14

28.5 to 32  C, salinity from 20.9 to 25.2, the dissolved oxygen was no less than 6 mg l1 and the mortality was recorded. 2.4. Sample collection After being fasted for 24 h, the fish were anesthetized with MS222 (Sigma, USA) before sample collection. Blood samples from the caudal vein of six fish per cage by a 1 ml syringe were pooled with the aim of obtaining enough samples to reduce analytic error, and allowed to clot at room temperature for 2 h and for 4e6 h in the cold. The clot was removed and residual blood cells separated from the straw-colored serum by centrifugation (3000  g, 10 min, 4  C). The serum was frozen at 80  C until use. Head kidney macrophages from six fish in each cage were isolated as described by Secombes [30] with some modifications. Briefly, the head kidney was excised, cut into small fragments and transferred to RPMI-1640 (Gibco, USA) medium supplemented with 10 IU ml1 heparin (Sigma, USA), 100 IU ml1 penicillin (Amresco, USA), 100 IU ml1 streptomycin (Amresco, USA) and 2% fetal calf serum (FCS) (Gibco, USA). Cell suspensions were prepared by forcing the head kidney through a 100 mm steel mesh. The resultant cell suspensions were enriched by centrifugation (1600  g for 20 min at 4  C) on 34%/51% Percoll (Pharmacia, USA) density gradient. The cells were collected at the 34e51% interface and washed twice. Cell viability was determined by the trypan blue (0.1%) and the cell density was determined in a hemocytometer. Then additional RPMI 1640 medium was added to adjust the cell concentration (1  107 ml1) for analysis. 2.5. Challenge test A bacterial pathogen, V. vulnificus was isolated from diseased T. ovatus in cage mariculture. A 10-day LD50 (V. vulnificus dose that killed 50% of the test ovate pompano) was determined before challenge test and the result showed that the LD50 on day 10 was 106 CFU ml1. V. vulnificus was grown in marine bacteria 2216E Zobell Marine broth for 24 h at 28  C to yield a cell concentration of approximately 107 CFU ml1. At the end of the feeding experiment, 18 fish of each cage were individually injected intraperitoneally with 0.2 ml 1.5% saline containing 1.9  106 CFU bacteria. The cumulative mortality was calculated for 10 days. 2.6. Respiratory burst (RB) activity The respiratory burst activity of head kidney macrophages was carried out by nitroblue tetrazolium (NBT, Sigma, USA) following the method of Secombes [30] with some modifications. A 100 ml cell suspension was stained with 100 ml 0.3% NBT and 100 ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) (1 mg ml1) for 40 min. Absolute methanol was added to terminate the staining. Each tube was washed three times with 70% methanol and airdried. Then 120 ml 2 M KOH and 140 ml dimethyl sulfoxide (DMSO, Sigma, USA) were added and the color was subsequently measured at 630 nm with a spectrophotometer using KOH/DMSO as a blank. Respiratory burst was expressed as NBT-reduction in 100 ml of cell suspension.

9

2.7. Phagocytic activity (PA) Phagocytic activity for six fish in each tank was determined by a modified method of Pulsford et al. [31]. The 100 ml cell suspension of head kidney leucocytes (1  107 cells ml1) was placed into a sterile slide and the cells allowed to attach for 30 min at 25  C. Following attachment, 100 ml yeast suspension (Bakers yeast, Type II, Sigma, USA, 1  108 cells ml1) was added to the cell monolayer, and the slide was incubated for 45 min at 25  C. Then unattached cells were washed off with 0.05 M sodium phosphate buffer (PBS, pH 7.0). After air-drying, the slides were fixed in methanol, redried and stained with Giemsa solution (Sigma, St. Louis, MO, USA). Slides were viewed under oil immersion at 100. Approximately 200 cells were counted at random and Phagocytic activity (PA) was expressed as:

PA ¼ ðNumber of phagocytic cells with engulfed yeast =Number of total cellsÞ  100

2.8. Alternative complement pathway (ACP) activity Serum ACP activity was assayed according to Yano [32]. Briefly, a series of volumes of the diluted serum ranging from 0.1 to 0.25 ml were dispensed into test tubes and the total volume made up to 0.25 ml with barbitone buffer in the presence of ethyleneglycol-bis (2-aminoethoxy)-tetraacetic acid (EGTA) and Mg2þ, then 0.1 ml of rabbit red blood cells (RaRBC) was added to each tube. After incubation for 90 min at 22  C, 3.15 ml 0.9% NaCl was added. Following this, the sample was centrifuged at 836  g for 5 min at 4  C to eliminate unlysed RaRBC. The optical density of the supernatant was measured at 414 nm. The volume of serum producing 50% hemolysis (ACH50) was determined and the number of ACH50 U ml1 was obtained for each group. 2.9. Lysozyme activity Serum lysozyme activity was determined by turbidimetric assay according to the method described by Ellis [33]. Briefly, test serum (0.1 ml) was added to 1.9 ml of a suspension of Micrococcus lysodeikticus (Sigma) (0.2 mg ml1) in a 0.05 M PBS (pH 6.2). The reaction was carried out at 25  C and absorbance was measured at 530 nm after 0.5 and 4.5 min in a spectrophotometer. One unit of lysozyme activity was defined as the amount of sample causing a reduction in absorbance of 0.001 min1. 2.10. Calculations and statistical analysis

Specific growth rateðSGRÞ ¼ ðLn Wt  Ln W0 Þ  100=t Feed efficiency ratioðFERÞ ¼ Wet weight gaining =dry feed feeding Survival rate % ¼ Nt  100=N0

Relative percentage survivalðRPSÞ ¼ 1  ½Percent mortality in treated group=Percent mortality in control group  100Þ

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Q. Zhang et al. / Fish & Shellfish Immunology 38 (2014) 7e14

Table 2 Growth performance of juvenile ovate pompano (Trachinotus ovatus) fed the diets with graded levels of dietary Bacillus subtilis and fructooligosaccharide (FOS) (means
S.E.).* Diet NO. (B. subtilis/FOS supplementation level  107 CFU g1/%)

B. subtilis levels (CFU g1)

FOS levels (% d.w.)

SGR (% d1)

Diet 1 (0/0) Diet 2 (0/0.2) Diet 3 (0/0.4) Diet 4 (1.05/0) Diet 5 (1.05/0.2) Diet 6 (1.05/0.4) Diet 7 (5.62/0) Diet 8 (5.62/0.2) Diet 9 (5.62/0.4) One-way ANOVA P-value Two-way ANOVA P-value (B. Subtilis) P-value (FOS) P-value (B. subtilis  FOS)

0 0 0 1.05 1.05 1.05 5.62 5.62 5.62

0 0.2 0.4 0 0.2 0.4 0 0.2 0.4

1.96 1.98 2.09 2.06 2.11 2.13 2.08 2.21 2.13

     

107 107 107 107 107 107












0.04a 0.04a 0.04ab 0.02ab 0.06ab 0.03ab 0.05ab 0.03b 0.04ab

FER 0.54 0.55 0.67 0.60 0.62 0.69 0.68 0.68 0.73

Survival rate










0.03a 0.04a 0.01bc 0.02ab 0.03ab 0.06bc 0.01bc 0.03bc 0.03c

83.89 85.00 85.56 86.11 86.67 88.33 84.44 90.56 87.22

0.012

0.000

0.840

0.003 0.046 0.307

0.000 0.000 0.433

0.518 0.512 0.861












3.64 3.47 3.09 2.94 1.92 3.47 3.09 2.78 1.11

*In the same column, values with no letter or the same letter superscripts mean no significant difference (P > 0.05), while with different small letter superscripts mean significant difference (P < 0.05).

where Wt and W0 were final and initial weight of fish, respectively; Nt and N0 were final and initial number of fish, respectively; t is duration of experimental days. All data were analyzed by SPSS 15.0 for windows. One-way analysis of variance (One-way ANOVA) was used to determine whether significant variation existed between the treatments. When overall differences were found, differences between means were determined and compared by Tukey’s honest significant difference post hoc test. Two-way ANOVA was used for analyzing the synergistic effects between B. subtilis and FOS on growth performance, immunity and disease resistance of juvenile T. ovatus. All differences were considered significant at P < 0.05. All data were presented as means
S.E.M (standard error of the mean) of three replications.

significantly different from those fed with Diet 8 (B. subtilis at dose of 5.62  107 CFU g1 diet in combination with 0.2% FOS levels) (P < 0.05). Fish fed with diet 9 (B. subtilis at dose of 5.62  107 CFU g1 diet in combination with 0.4% FOS levels) produced the highest FER, and were significantly higher than the groups fed with 0 or 1.05  107 CFU g1 B. subtilis at 0 and 0.2% FOS levels (P < 0.05). Moreover, FER significantly increased with the increasing doses of dietary FOS without B. subtilis added (P < 0.05). During the whole experimental period, the survival rates of fish fed with graded levels of dietary B. subtilis and FOS ranged from 83.89 to 90.56%. No significant difference in survival rate was observed among dietary treatments (P > 0.05).

3.2. Mortality after challenge Mortalities of fish challenged with V. vulnificus are shown in Table 3. A two-way ANOVA test showed that the cumulative percent mortalities were significantly affected by both B. subtilis and FOS (P < 0.05), and a significant interaction was found between B. subtilis and FOS (P < 0.05). The fish fed with control diet produced the highest cumulative percent mortality than the other groups except the one fed with Diet 2 (0.2% FOS) (P < 0.05). The cumulative percent mortalities decreased significantly with the increasing doses of dietary B. subtilis at 0 or 0.2% FOS level (P < 0.05).

3. Results 3.1. Growth performance The SGR, FER and survival rate of ovate pompano juveniles fed the experimental diets for 8 weeks are provided in Table 2. A twoway ANOVA test showed that SGR and FER were significantly affected by B. subtilis and FOS supplementation (P < 0.05). However, no interaction was found between the parameters (P > 0.05). Fish fed with the control diet obtained the lowest SGR, and were

Table 3 Cumulative mortality following a 10 day Vibiro vulnificus challenge of juvenile ovate pompano (Trachinotus ovatus) fed the experimental diets with graded levels of dietary Bacillus subtilis and fructooligosaccharide (FOS) (means
S.E.).* Diet NO. (B. subtilis/FOS supplementation level  107 CFU g1/%)

B. subtilis levels (CFU g1)

FOS levels (% d.w.)

Cumulative mortality (%)

Diet 1 (0/0) Diet 2 (0/0.2) Diet 3 (0/0.4) Diet 4 (1.05/0) Diet 5 (1.05/0.2) Diet 6 (1.05/0.4) Diet 7 (5.62/0) Diet 8 (5.62/0.2) Diet 9 (5.62/0.4) One-way ANOVA P-value Two-way ANOVA P-value (B. Subtilis) P-value (FOS) P-value (B. subtilis  FOS)

0 0 0 1.05 1.05 1.05 5.62 5.62 5.62

0 0.2 0.4 0 0.2 0.4 0 0.2 0.4

75.93 62.96 51.85 51.85 57.41 61.11 40.74 29.63 33.33

     

7

10 107 107 107 107 107












1.85d 1.85cd 3.70bc 1.85bc 1.85c 3.21c 3.70ab 1.85a 3.21a

Relative percent survival (%) 0 17.07 31.71 31.71 24.39 19.51 46.34 60.97 56.10











2.43 4.88 2.44 2.44 4.22 4.88 2.44 4.22

0.002 0.000 0.000 0.021

*In the same column, values with no letter or the same letter superscripts mean no significant difference (P > 0.05), while with different small letter superscripts mean significant difference (P < 0.05).

Q. Zhang et al. / Fish & Shellfish Immunology 38 (2014) 7e14

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Table 4 Immune response of juvenile ovate pompano (Trachinotus ovatus) fed the experimental diets with graded levels of dietary Bacillus subtilis and fructooligosaccharide (FOS) (means
S.E.).* Diet NO. (B. subtilis/FOS supplementation level  107 CFU g1/%)

B. subtilis levels (CFU g1)

FOS levels (% d.w.)

RB (OD630)

Diet 1 (0/0) Diet 2 (0/0.2) Diet 3 (0/0.4) Diet 4 (1.05/0) Diet 5 (1.05/0.2) Diet 6 (1.05/0.4) Diet 7 (5.62/0) Diet 8 (5.62/0.2) Diet 9 (5.62/0.4) One-way ANOVA P-value Two-way ANOVA P-value (B. Subtilis) P-value (FOS) P-value (B. subtilis  FOS)

0 0 0 1.05 1.05 1.05 5.62 5.62 5.62

0 0.2 0.4 0 0.2 0.4 0 0.2 0.4

0.13 0.28 0.27 0.20 0.23 0.19 0.27 0.29 0.18

     

107 107 107 107 107 107












0.01a 0.03d 0.01cd 0.02abc 0.04bcd 0.02 ab 0.01d 0.05d 0.01ab

PA (%)

37.91 43.29 48.42 39.87 48.26 47.17 42.24 49.17 33.78












2.62ab 2.68ab 3.55b 2.32ab 1.27b 3.37b 2.44ab 2.15b 2.05a

ACP3 (ACH350 units ml1)

Lysozyme (units ml1)

73.87
1.56a 73.43
2.69a 85.09
1.77abc 77.96
1.80abc 78.59
2.59abc 77.17
3.02ab 102.11
3.71d 91.17
2.18cd 89.78
3.99bcd

53.22
3.95a 87.78
4.84bc 84.22
5.02abc 69.96
6.94ab 64.89
5.92ab 96.57
10.25bc 110.44
5.58c 105.56
10.60c 92.20
4.43bc

0.000

0.004

0.008

0.004

0.008 0.000 0.002

0.298 0.014 0.008

0.000 0.252 0.015

0.000 0.084 0.007

* In the same column, values with no letter or the same letter superscripts mean no significant difference (P > 0.05), while with different small letter superscripts mean significant difference (P < 0.05).

Fish fed with Diet 8 (B. subtilis at dose of 5.62  107 CFU g1 diet in combination with 0.2% FOS) produced the highest relative percentage survival (RPS) (Table 3), followed by those fed with Diet 9 (B. subtilis at dose of 5.62  107 CFU g1 diet in combination with 0.4% FOS), RPS of them were 60.97% and 56.10%, respectively.

3.3. Immune parameters RB activity of head kidney macrophage was significantly affected by both B. subtilis and FOS (P < 0.05; Table 4). RB activity of fish was significantly affected by the interaction of dietary B. subtilis and FOS (P < 0.05). Fish fed with the control diet produced the lowest RB activity and were significantly different from the groups fed the diets containing 0.2% FOS at each B. subtilis level and containing 0.4% FOS without B. subtilis added (P < 0.05). PA of head kidney macrophage was significantly affected by dietary FOS levels (P < 0.05; Table 4), but not by B. subtilis levels (P > 0.05). There was significant interaction between the two parameters (P < 0.05). PA showed increasing tendency with the increasing administration doses of B. subtilis in diets with 0 or 0.2% FOS. However, it was significantly decreased with the increasing doses of dietary B. subtilis at 0.4% FOS level (P < 0.05). ACP activity in fish serum was significantly affected by dietary B. subtilis levels (P < 0.05; Table 4), but not by FOS levels (P > 0.05). There was significant interaction between the two parameters (P < 0.05). Fish fed with Diet 2 (0.2% FOS) produced the lowest ACP activity, followed by fish fed with the control diet, and they were significantly lower than the groups fed with 5.62  107 CFU B. subtilis g1 supplemented at each FOS level (P < 0.05). Lysozyme activity in fish serum was significantly affected by dietary B. subtilis levels (P < 0.05; Table 4), but not by FOS levels (P > 0.05). There was significant interaction between the two parameters (P < 0.05). Fish fed with the control diet produced the lowest lysozyme activity, and were significantly different from those fed with Diet 2 (0.2% FOS), Diet 6 (B. subtilis at dose of 1.05  107 CFU g1 diet in combination with 0.4% FOS), Diet 7 (B. subtilis at dose of 5.62  107 CFU g1 diet), Diet 8 (B. subtilis at dose of 5.62  107 CFU g1 diet in combination with 0.2% FOS) and Diet 9 (B. subtilis at dose of 5.62  107 CFU g1 diet in combination with 0.4% FOS) (P < 0.05). In addition, lysozyme activity was significantly increased with the increasing doses of dietary B. subtilis without FOS supplementation (P < 0.05).

4. Discussion 4.1. Effects of probiotic B. subtilis on juvenile ovate pompano In the present work, SGR and FER of the junvenile ovate pompano were significantly affected by probiotic B. subtilis. The growth improving property of B. subtilis has previously been reported in other aquatic animals such as white shrimp (Litopenaeus vannamei) [34], tilapia (Oreochromis niloticus) [9], large yellow croaker [20] and sea cucumber (Apostichopus japonicus) [35,36]. Evidence is available that indicates probiotic bacteria take part in the decomposition of nutrients, improve digestive activity by enhancing the synthesis of vitamins, enzymatic activity [37-40], and thus facilitate feed utilization and digestion. This may account for the enhanced FER by dietary B. subtilis supplementation in this study. The similar result was found in the study of Ai et al. [20], who reported that dietary supplementation with B. subtilis resulted in enhanced FER and growth response of large yellow croaker. However, the growth and feed utilization of tilapia and freshwater prawn (Macrobrachium rosenbergii) were independent of dietary B. subtilis levels under laboratory conditions [41]. The difference may be caused mainly by different aquatic animal species coupled with dietary different experimental conditions, such as water quality, hardness, dissolved oxygen, temperature, pH, osmotic pressure, mechanical friction and the environmental microflora. These environmental factors influence the probiotics in the digestive tract chiefly on their establishment, proliferation and function [42,43]. The role of probiotics in modulating the immune system has been studied in many aquatic animals. Earlier research showed that oral administration of probiotic B. subtilis could enhance the innate immune responses of Labeo rohita (Ham.) [10]. Lin et al. [27] reported that the koi fed with diets containing B. subtilis exhibited a significant increase in total leukocyte count, respiratory burst activity, phagocytic activity, lysozyme activity and superoxide dismutase activity compared to the control. Similar findings have also been reported in gilthead seabream (Sparus aurata L.) with the dietary supplementation of probiotic Lactobacillus delbrüeckii and B. subtilis, single or combined [44]. In the present study, the fish fed with diets containing B. subtilis exhibited a significant increase in RB activity, ACP activity and lysozyme activity. All of these results confirmed that non specific immunity improved in aquatic animals fed with diets containing B. subtilis. Also, the current literature is

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indicative of dietary probiotics supplementation effectively improving disease resistance of fish [6,7]. Previous studies demonstrated that oral administration of Bacillus sp. (strain S11) to tiger shrimp (Penaeus monodon) enhanced the disease resistance by activating both cellular and humoral immune defenses [45]. Balcázar [46] demonstrated that the administration of a mixture of bacterial strains (Bacillus and Vibrios sp.) positively influenced the growth and survival of juvenile white shrimp and presented a protective effect against the pathogens Vibrio harveyi and white spot syndrome virus by increasing phagocytosis and antibacterial activity. Newaj-Fyzul et al. [47] mentioned that B. subtilis AB1 stimulated both cellular and humoral immune responses by increasing the number of leucocytes as well as enhancing respiratory burst and phagocytic activity, and thus provided the rainbow trout with adequate protection to survive the challenge by the highly virulent Aeromonas sp. In the present study, dietary B. subtilis supplementation elevated non-specific immunity and the resistance to V. vulnificus infection. Moreover, the increase in resistance against V. vulnificus in fish fed with B. subtilis can be possibly explained on the basis of increased non specific immune response. 4.2. Effects of prebiotic FOS on juvenile ovate pompano Influences of dietary FOS supplementation on growth have been evaluated with several aquacultured species with varied results. In the present study, dietary supplementation of FOS (0.2% or 0.4% of dry weight) exerted beneficial effects on the growth performance and feed utilization. This result was in agreement with results from studies on turbot larvae (P. maxima) [18], hybrid tilapia (O. niloticus\  Oreochromis aureus_) [48], juvenile white shrimp (L. vannamei) [49] and sea cucumber (A. japonicus) [35], but in contrast to studies on Pacific white shrimp (L. vannamei) [50], Atlantic salmon (Salmo salar) [51] and large yellow croaker [20]. The majority of the beneficial effects claimed by the prebiotics FOS are associated with the following aspects, such as an increase in beneficial bacteria (bifidobacteria and lactobacilli), an inhibition various pathogenic bacteria strains, an enhancement in the immunity, an increase digestibility of feed [52]. It may also be the reason for the growth promoted by FOS in the present study. Regarding the health promoting effects of dietary FOS, dietary FOS has been reported to stimulate the innate immune responses, such as serum lysozyme activity and head kidney macrophage intracellular superoxide anion production of red drum [19]. Soleimani et al. [22] reported that Caspian roach (R. rutilus) fry fed 2% and 3% FOS obtained an improved immunity by increasing serum total immunoglobulin serum lysozyme activity and serum alternative complement activity (ACH50). Other studies on allogynogenetic silver crucian carp (Carassius auratus) [53] and large yellow croaker (L. crocea) [20] also confirmed the beneficial effects of FOS on fish innate immune responses. Different results were found in the studies on Atlantic salmon (S. salar) [51] showed blood neutrophil oxidative radical production and serum lysozyme remained unaffected after feeding 10 g FOS kg1 for 4 months. In the present study, substantial increment of RB activity, lysozyme activity and PA and substantial reduction in cumulative mortality were observed at 0.2 and 0.4% FOS incorporation compared to the FOS-deficient treatment when B. subtilis was not supplemented. However, no significant effects of dietary FOS incorporation were observed on ACP. Data from experiments of dietary FOS effect is inconsistent, and its probable reasons may be due to dietary FOS contents and levels, aquatic animals as well as culture conditions. Benefits of FOS supplementation are presumably conferred through

intestinal microbial changes [20]. Besides, oligosaccharides can be selectively used by bifidobacterium to reproduce probiotic bacteria and restrain the adherence and colonization of pathogenic microorganism [54], Mahious et al. [18] reported that FOS stimulated the growth of Bacillus sp. in the intestine of larval turbot, which could use FOS as a single source of carbon. They also inferred that the stimulation of FOS on Bacillus sp. might play a role in the beneficial effect of FOS on turbot growth, since Bacillus sp. have been documented as probiotics in fish. Similarly, Li et al. [50] found that FOS could selectively support growth of certain bacterial species in the gastrointestinal tract of white shrimp; however, the mechanism for the microbial shifts affected the host was still uncertain. In addition, some studies have reported that oligosaccharide effectively enhanced the immunity of animals and thus improved the resistance to pathogenic bacteria infection [20,35,55]. The increase in resistance against V. vulnificus in fish fed with FOS can be possibly explained on the basis of promoted the growth of B. subtilis, which can increase the non specific immune response of fish. 4.3. Effects of probiotic B. subtilis in combination with FOS on juvenile ovate pompano Gibson et al. [16] suggested that a prebiotic could be selectively fermented by the intestinal microbiota and stimulate selectively the growth and/or activity of intestinal bacteria. It was demonstrated that probiotics given in the form of synbiotics to the host yielded significantly better results than that given in individual form [26]. Prebiotics in the synbiotics can enhance the survival of probiotics in gastrointestinal tract [56,57], and thus improving the quick reproducibility of probiotics in vivo and perform beneficial effects. Especially in recent years, researchers showed great interested in the interactions, especially synergistic actions between prebiotics and probiotics. Synergistic actions between isomaltooligosaccharides and Bacillus OJ, mannan oligosaccharides and Bacillus sp., FOS and B. subtilis, chitosan oligosaccharides and Bacillus coagulans, FOS and Bacillus licheniformis have been revealed in studies on shrimp (L. vannamei) [58], European lobster larvae (Homarus gammarus L.) [59], sea cucumber (A. japonicus) [35], koi (Cyprinus carpio koi) [27] and triangular bream (M. terminalis) [24], respectively. The similar significant interactions between B. subtilis and FOS on non-specific immunity and disease resistance were observed in the present study. Since production of specific antibody in most fish need long-term adaptation and formed by producing memory, the improvement in RPS in the present study caused by B. subtilis and FOS in combination can be attributed to the enhancement of non-specific immunity, thereby enhancing the disease resistance of fish. However, the relation and mechanism of probiotic B. subtilis and prebiotic FOS with the significant increase of non-specific immunity and disease resistance in juvenile ovate pompano need to be determined further. 5. Conclusion In conclusion, under the experimental conditions, dietary B. subtilis and FOS can significantly improve disease resistance by enhancing immunity of fish. The present work is the first study demonstrated that dietary B. subtilis and FOS had a significant interaction on enhancing the immune responses and disease resistance of juvenile ovate pompano. This may be of great interest to those involved in aquaculture research and the fish farming industry.

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Acknowledgments This work was financially supported by the Natural Science Fund of Guangxi (2012GXNSFDA053013), Marine Public Welfare Industry Special Funds for Scientific Research (201205028), Scientific Research and Technology Development Program of Guangxi (1222013-2) and Open Fund of the Key Laboratory of Marine Biotechnology of Guangxi (GLMBT-201303).

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