1 Introduction

As a commercially important echinoderm species, the production of sea cucumber, Apostichopus japonicus has been increased from 53315 metric tons in 2004 (Fishery Bureau, Ministry of Agriculture, China, 2004) to 200969 metric tons in 2014 in China (Fishery Bureau, Ministry of Agriculture, China, 2015). However, the rapidly increased farming of sea cucumber has resulted in serious diseases such as skin ulceration syndrome, peristome tumescence and viscera ejection syndrome (Wang et al., 2007; Li et al.,2008; Deng et al., 2009; Li et al., 2016; Sun et al., 2016).Although antibiotics are effective to dealing with some bacterial diseases in aquatic animals, the abuse of antibiotics in aquaculture has caused many problems (Shah et al.,2014; Yano et al., 2014; Chen et al., 2015; Stalin and Srinivasan, 2016), which leads to the strict regulations on the use of antibiotics in aquaculture. Therefore, many researchers (Liu et al., 2012; Zhao et al., 2012; Ma et al.,2013; Chi et al., 2014; Yan et al., 2014; Yang et al., 2014;Li et al., 2015; Wang etal., 2015; Yang et al., 2015; Yang et al., 2016; Zhao et al., 2016) have emphasized on the study and application of probiotics to confer better health of farmed sea cucumber.

Probiotics in aquaculture are live, or dead, or an element of microbe cells that can increase the disease resistance, health standards, growth performance, feed optimization and stress resistance of the aquatic animals(Ibrahem, 2015). This may be achieved by increasing the microbic balance of the hosts or the close surroundings(Ibrahem, 2015). The genus Bacillus is one of the extensively used probiotics in aquaculture, which has been proved to improve growth, immunity and disease resistance in sea cucumber (Zhao et al., 2012; Li et al., 2015;Yang et al., 2015; Zhao et al., 2015; Zhao etal., 2016).The effects of Bacillus sp. on the digestive enzymes in sea cucumber were evaluated in recent studies, which indicated that a specific dosage of Bacillus sp. BC26, B.cereus BC-01 and BC-02 could increase the intestinal amylase, trypin and lipase activities in sea cucumber (Liu et al., 2013; Tian et al., 2015; Zhao et al., 2015). However, the effects of the probiotics including genus Bacillus on the intestinal microbiota of sea cucumber are scarce.Up to date, the effect of Bacillus on the intestinal microbiota of sea cucumber has been documented only by Zhang et al. (2010) using traditional culture-dependent method and Wang et al. (2016) via polymerase chain reaction denaturing gradient gel electrophoresis (PCRDGGE). Furthermore, autochthonous probiotics isolated from animals themselves or their rearing environments have attracted more and more attention on their effects on the aquatic animals including sea cucumber (Liu et al.,2012; Zhao et al., 2012; Ma et al., 2013; Chi et al., 2014;Yan etal., 2014; Yang et al., 2014; Li et al., 2015; Wang etal., 2015; Yang etal., 2015; Yang etal., 2016; Zhao et al., 2015; Zhao et al., 2016). These autochthonous probiotics have adapted to the same ecological niche and have higher probability of competitive exclusion (Lalloo et al., 2010; De et al., 2014).

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The present study was conducted to investigate the effects of a new autochthonous probiotic Bacillussubtilis 2-1 from the intestine of healthy sea cucumber on the growth, the intestinal digestive enzymes and intestinal microbiota of sea cucumber, A. japonicus.

2 Materials and Methods

2.1 Bacterial Strain

The bacterial strain 2-1 was isolated from the intestine of healthy sea cucumber in this study, which was identified as B. subtilis by cluster analysis based on the sequence of 16S rRNA gene. B. subtilis 2-1 was confirmed to be able to inhibit Vibrio splendius which caused the skin ulceration syndrome disease in sea cucumber. The safety of B. subtilis 2-1 was tested by immersing sea cucumber (0.24 ± 0.01 g) in the suspension of B. subtilis 2-1 at final concentrations of 0, 103, 105 and 107 CFU mL−1(Verschuere et al., 2000). Fresh suspension of B. subtilis 2-1 was added into each aquarium again after seawater was changed fully every 24 h. The disease symptoms and mortality of sea cucumber were monitored for 7 days(Zhou et al., 2010). The safety test showed that B. subtilis 2-1 did not induce disease symptoms and mortality in sea cucumber.

HER-2为人表皮生长因子受体家族成员之一，它是其它家族成员的共受体，可形成同源或异源二聚体，从而启动一系列信号转导通路，激活酪氨酸激酶活性启动一系列信号转导通路介导细胞的增殖、分化、抗凋亡等过程从而参与多种肿瘤的发生，这一论点已被国内外广泛公认 [6]。目前已知Her-2基因扩增和蛋白过表达能帮助判断HER-2阳性乳腺癌、胃癌患者的预后情况[7]。有研究表明，Her-2可作为卵巢癌的1个独立预后因素，Her-2的高表达与卵巢癌的预后呈负相关[8]。但也有研究表明Her-2的过表达与卵巢癌的预后无相关性[9]。本研究可进一步证实Her-2与卵巢癌预后的关系。

2.2 Experimental Diets

The powder of seaweed (Sargassum thunbergii), the palatable food for sea cucumber, was used as the basal diet. The experimental diets were prepared by supplementing graded doses of B. subtilis 2-1 at 0, 105, 107 and 109 CFU g−1 feed, respectively. S. thunbergii powder was sifted through a 149 μm mesh. B. subtilis 2-1 was cultured in trypticase soy broth (TSB) with 1.5% NaCl for 24 h at 28℃. The bacteria were collected by centrifugation at 5000 g and 4℃ for 10 min. Then they were washed twice with sterile saline and the concentration of the final suspension was adjusted to 1×1010 CFU mL−1 in sterile saline(Zhao et al., 2016). Graded doses of B. subtilis 2-1 in moderate sterile saline were supplemented into the S.thunbergii powder and the mixture was made into dough.Then the dough was separated into many small balls manually, and the balls with a diameter of about 1.00–3.35 mm were selected by meshes. The experimental diets were prepared every day to guarantee the vitality of B.subtilis 2-1.

The sequencing of prominent DGGE bands was performed as described by Zhou et al. (2013). Briefly, the DNA of prominent DGGE bands was retrieved and used as the template for amplification with primers 338F without GC-clamp and 518R. The PCR products were purified, cloned and sequenced. The resulting 16S rRNA genes sequences (about 200 bp) were analyzed in the GenBank database by BLAST sequence algorithm (www.ncbi.nlm.nih.gov/BLAST). The phylogenetic analysis of all sequences was performed by constructing a neighbor-joining phylogenetic tree, which was constructed via p-distance and was assessed by a bootstrap analysis with 1000 replicates with software MEGA 4.0.

2.3 Feeding Experiment

Healthy juvenile sea cucumber individuals were obtained from Shandong Oriental Ocean Sci-Tech Co., Ltd.(Yantai, China). After they were acclimated to basal diet and the rearing conditions for 3 weeks, the sea cucumber with a similar size (4.51 ± 0.04 g) was randomly distributed into 12 aquaria. Each treatment was conducted with three replicates. Each aquarium (40 cm × 30 cm × 30 cm)was stocked with 30 sea cucumber individuals in 30 L seawater. During the 56-day feeding trial, all experimental animals were fed diets twice (06:00 and 18:00) a day at a feeding rate of 2% of the body weight, and 50% seawater in each aquarium was replaced with the fresh before feeding. Water temperature, salinity and acidity were maintained at 16–18℃, 28–31 and pH 7.8–8.2, respectively. Dissolved oxygen (DO) was no less than 5 mg L−1 which was maintained by continuous aeration.

2.4 Growth Performance

All sea cucumber individuals in each aquarium were weighed at the end of the experiment and the average final weight was calculated.

The V3 region of 16S rRNA gene was amplified using the primers 338F including 40 bases of GC-clamp at the 5’ end and 518R (338F: 5’-ACT CCT ACG GGA GGC AGC AG-3’; 518R: 5’-ATT ACC GCG GCT GCT GG-3’)(Muyzer et al., 1993). The PCR mixture contained 5 μL 10×PCR buffer, 200 μmol L−1 dNTP (each), 0.4 μmol L−1 primers (each direction), 1.25 U rTaq DNA polymerase(TaKaRa, Japan), 2.5 ng DNA, and sterile water to a final volume of 50 μL. The PCR included 1 cycle at 94℃ for 10 min; 30 cycles of amplification at 94℃ for 1 min, at 55℃ for 1 min after decreasing the temperature by 0.1℃per cycle, and at 72℃ for 1 min; a final extension step for 10 min at 72℃. PCR product was analyzed by electrophoresis in 1.0% agarose gel stained with ethidium bromide.

where Wt and W0 were average final and initial weights of sea cucumber in each aquarium, respectively.

2.5 Intestinal Digestive Enzyme Activity Assay

At the termination of the feeding trial, after sea cucumber was fasted for 24 h, six individuals each tank were sampled randomly for determining the activities of intestinal digestive enzymes including amylase, trypsin and lipase. Sea cucumber was dissected immediately and the whole intestines were removed by incising at the esophagus and cloaca. The intestines were blotted dry with filter paper. Then the intestine samples were weighed and homogenized in ice-cold 0.85% NaCl using a manual glass homogenizer on ice. The number of milliliters of NaCl was nine times of the intestine weight in grams. The homogenates were then centrifuged (4000 r min−1, 10 min,4℃) and the supernatants were transferred into clean tubes. Then the enzyme activities were analyzed within 12 h.

The PCR-DGGE fingerprints showed that dietary B.subtilis 2-1 obviously influenced the dominant intestinal bacterial community in a dosage dependent way (Figs.1,2). To better define the effect of B. subtilis 2-1 on the dominant intestinal microbial community in sea cucumber,prominent DGGE bands (Fig.1; 18 bands) were excised and sequenced. Their taxa and closest relatives were shown in Table 3. Compared with the control, the genera Vibrio (bands 1, 4, 5, 8, 10), Psychrobacter (band 3) and Photobacterium (band 9) were not dominant intestinal bacteria in at least two replicates of groups fed with dietary B. subtilis 2-1 at 105 and 107 CFU g−1 (Fig.1; Table 3)while genera Psychrobacter (band 3), Vibrio (band 4) and Bacillus (band 13) were the dominant in at least two replicates of group fed with dietary B. subtilis 2-1 at 109 CFU g−1 (Fig.1; Table 3). The cluster analysis of the band patterns (Fig.2) showed that sea cucumber fed with B. subtilis 2-1 at 109 CFU g−1 were more similar to the control

The activities of amylase in intestinal supernatants were determined by iodine spectrophotometry (Gao et al.,2009) with a commercial kit (Nanjing Jiancheng BioEngineering Institute, China). The optical density was measured at 660 nm. One unit of amylase activity was defined as the amount required for hydrolyzing 10 mg soluble starch in 30 min at 37℃ in per mg protein of intestinal supernatant.

The activity of trypsin in intestinal supernatants was determined according to the method described by Holm et al. (1988) with a commercial kit (Nanjing Jiancheng Bioengineering Institute, China). The reaction was based on trypsin’s ability to hydrolyze l-arginine ethyl ester,which resulted in an increase in optical density when measured at 253 nm. One unit of trypsin activity was defined as the amount of enzyme causing an increase in absorbance of 0.003 per min per mg protein in intestinal supernatants at 37℃ and pH 8.0.

The activity of lipase in intestinal supernatants was measured according to the method described by Shihabi and Bishop (1971) with a commercial kit (Nanjing Jiancheng Bioengineering Institute, China). The reaction was based on lipase’s ability to hydrolyze triglyceride in stabilized emulsion of olive oil, which resulted in a decrease in optical density when measured at 420 nm. One unit of lipase activity was defined as 1μmol substrate consumed per min per g protein in intestinal supernatants at 37℃.

Dietary B. subtilis 2-1 at 105 or 107 CFU g−1 had no significant effect on the growth of sea cucumber (P ＞ 0.05;Table 1); while sea cucumber fed with B. subtilis 2-1 at 109 CFU g−1 had significantly higher growth performance than those fed with control diet (P ＜ 0.05; Table 1).

2.6 Intestinal Microbiota Analysis

At the termination of the feeding trial, sea cucumber was fasted for 24 h. The entire intestinal tract was collected and excised with sterile forceps and scissors. Then the intestinal tracts were frozen in the liquid nitrogen. To avoid inter-individual variation, intestinal tract of eight sea cucumber each aquarium was pooled for microbiota analysis. The pooled intestinal tract was ground into powder with liquid nitrogen. The total genomic DNA was isolated from the samples using a PowerFecal® DNA Isolation Kit (Mobio, USA) according to the manufacturer’s instructions. All DNA was stored at −20℃.

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Denaturing gradient gel electrophoresis (DGGE) of PCR products was conducted with the Bio-rad Dcode™mutation detection system (Bio-rad, USA). Approximately equal amount of PCR product was loaded per sample in a final volume of 40 μL into 8% (weight in volume)polyacrylamide (37.5:1 acrylamide/bisacrylamide) gel with a denaturing gradient ranging from 30% to 60%. Electrophoresis was then conducted at a constant voltage of 150 V at 60℃ for about 7 h. The gel was stained with AgNO3 (Sanguinetti et al., 1994), visualized and photographed. The band scan software (version 5.0) was used to analyze the DGGE band profiles. The densities and migration patterns of the bands were calculated. Cluster analysis of DGGE patterns was achieved by constructing dendrograms using the unweighted pair groups method with arithmetic averages (UPGMA) and the dendrograms were completed by software PHYLIP 4.0 and MEGA 4.0.

(1)单个资料可以预测地质浅层，但是准确度不够。多种资料相互结合，通过多种特征判断浅层的位置，有利于提高预测精度，降低钻井风险。

2.7 Statistical Analysis

The results were presented as mean ± SE (standard error) and were analyzed by SPSS 16.0 for windows (Chicago, IL, USA). The homogeneity of variance of all data was tested by Levene’s test and the normality of residuals was tested by Shapiro-Wilk test. Statistical analysis was performed by one-way analysis of variance (ANOVA)followed by Duncan’s multiple range test. Difference between groups was considered significant if P ＜ 0.05.

3 Results

3.1 Growth Performance

Total protein of the intestinal supernatants was determined according to Bradford (1976) using bovine serum albumin (BSA) as the standard.

3.2 Intestinal Digestive Enzymes

Sea cucumber fed with B. subtilis 2-1 at 109 CFU g−1 had significantly higher amylase activity than those fed with B. subtilis 2-1 at 0, 105 and 107 CFU g−1 (P ＜ 0.05;Table 2). Trypsin activity in sea cucumber fed with B.subtilis 2-1 at 107 and 109 CFU g−1 was significantly increased in comparison with that of sea cucumber fed with B. subtilis 2-1 at 0 and 105 CFU g−1 (P ＜ 0.05; Table 2).Moreover, sea cucumber fed with B. subtilis 2-1 at 109 CFU g−1 showed a significantly higher trypsin activity than those fed with B. subtilis 2-1 at 107 CFU g−1 (P ＜ 0.05;Table 2). However, dietary B. subtilis 2-1 had no significant influence on the intestinal lipase activity in sea cucumber (P ＞ 0.05; Table 2).

Table 1 Effects of dietary Bacillus subtilis 2-1 for 8 weeks on growth performance of sea cucumber

Notes: The values and standard errors are means of three replicates (mean ± SE, n = 3). Every replicate has 30 animals. Means in the same column with different superscript letters are significantly different by Duncan’s test (P ＜ 0.05). Levene’s test was used to test the homogeneity of variances of all data (P ＜ 0.05). Shapiro-Wilk test was used to test the normality of residuals of all data (P ＜ 0.05). ANOVA: One way analysis of variance.

Bacillus level (CFU g−1) Initial body weight (g) Final body weight (g) Weight gain rate (%)0 4.40 ± 0.09 8.46 ± 0.21a 92.67 ± 8.56a 105 4.58 ± 0.04 8.72 ± 0.19a 90.42 ± 2.60a 107 4.50 ± 0.07 9.30 ± 0.57ab 106.57 ± 9.96ab 109 4.56 ± 0.07 10.16 ± 0.26b 122.81 ± 6.08b Pooled SE 0.04 0.25 5.01 P Value of Levene’s test 0.648 0.364 0.286 P Value of Shapiro-Wilk test 0.667 0.087 0.348 ANOVA F Value 1.382 4.809 4.133 P Value 0.317 0.034 0.048

Table 2 Effects of dietary Bacillus subtilis 2-1 for 8 weeks on the intestinal digestive enzymes of sea cucumber

Notes: Same as those of Table 1.

Bacillus level (CFU g−1) Amylase (U mg−1 protein) Trypsin (U mg−1 protein) Lipase (U g−1 protein)0 0.25 ± 0.01a 70.81 ± 2.04a 5.00 ± 0.24 105 0.25 ± 0.00a 78.94 ± 5.70a 6.30 ± 0.16 107 0.24 ± 0.01a 105.59 ± 4.86b 6.31 ± 0.39 109 0.27 ± 0.00b 137.10 ± 11.32c 5.75 ± 0.62 Pooled SE 0.01 8.35 0.23 P Value of Levene’s testb 0.330 0.089 0.294 P Value of Shapiro-Wilk testc 0.738 0.261 0.344 ANOVAd F Value 6.889 19.038 2.508 P Value 0.013 0.001 0.133

3.3 Intestinal Microbiota Analysis

Fig.1 DGGE fingerprints of the V3 regions of 16S rRNA genes in intestinal bacterial communities of sea cucumber,Apostichopus japonicus fed with different dietary Bacillus subtilis 2-1 for 8 weeks (Control: 0 CFU g−1 [S1, S2, S3];105 CFU g−1 [S4, S5, S6]; 107 CFU g−1 [S7, S8, S9]; 109 CFU g−1 [S10, S11, S12]).

Fig.2 Cluster analysis of DGGE patterns of the intestinal microbiota of sea cucumber, Apostichopus japonicus fed with different dietary Bacillus subtilis 2-1 for 8 weeks(Control: 0 CFU g−1 [S1, S2, S3]; 105 CFU g−1 [S4, S5, S6];107 CFU g−1 [S7, S8, S9]; 109 CFU g−1 [S10, S11, S12]).

在高速公路桥梁施工伸缩缝结构处理的前期，施工技术部门要结合施工图纸和具体操作流程进行系统化技术交底，并且要对伸缩缝异行边缘的顺直程度以及平整度等进行统筹分析，关注缝隙之间的间隙结构，从而提升施工流程的基本效果。最重要的是，在施工过程中，技术部门要对机器进行集中筛查，合理提升设备配备的完整性，也为公路桥梁质量管理优质性奠定基础。

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than those fed with B. subtilis 2-1 at 105 or 107 CFU g−1.However, compared with the control, the genus Vibrio(bands 1, 2 and10) was not dominant intestinal bacteria in sea cucumber fed with B. subtilis 2-1 at 109 CFU g−1(Fig.1; Table 3). Moreover, feeding sea cucumber with B.subtilis 2-1 at 109 CFU g−1 had increased the intensity of genera Psychrobacter (band 3) and Bacillus (band 13)compared with other groups (Fig.1; Table 3). The phylogenetic distributions of the bacterial operational taxonomic units (OTUs) showed that 8 OTUs were Proteobacteria, 3 OTUs were Actinobacteria and 1 OTU was Firmicutes (Fig.3; Table 3).

2）受冻部位。冻害大多以主干冻伤为主，主要在地面以上30～50 cm，最高可达100 cm，一般树体受冻部位多在树干西北方向，向南方向冻害轻或未受冻。徐香等品种嫁接口及以上部位受冻严重。

Table 3 DGGE bands and strains identified in this study by means of 16S rRNA gene sequencing

Notes: † The number of 11-1, 11-2 and 11-3 represent the first, second and third clone of band 11, respectively. +: If the value of a specific band intensity to the total band intensity percentage ＞ 1% means exist, which ‘+’, ‘++’ and ‘+++’ represent a specific band was showed in one replicate, two replicates and three replicates of each treatment, respectively; −: If the value of a specific band intensity to the total band intensity percentage ＜ 1% means non-existent.

Phylum Band no. Nearest type strain (Accession no.) Identity (%) 0 105 107 109 Bacillus level (CFU g−1)1 Vibrio sp. (KC207077.1) 100 ++ – – –2 Vibrio sp. R-66650 (KT185186.1) 100 ++ – ++ –3 Psychrobacter sp. ( KR023910.1) 98 ++ – + +++4 Vibrio rumoiensis (KJ616367.1) 100 ++ + + +++5 Vibrio tasmaniensis (KR232925.1) 100 +++ – – –6 Vibrio rumoiensis (KP860596.1) 97 – – – +++7 Vibrio sp. QY107 (KC207077.1) 99 – – + –8 Vibrio splendidus (KP204158.1) 100 +++ + + –9 Photobacterium sp. (AB819694.1) 100 + – – –10 Vibrio sp. 7G03 (AY836897.1) 99 +++ + – –11-1 Novosphingobium pentaromativorans (KP721487.1) 100 ++ – ＋ +++Sphingomonas aquatilis(KX110354.1) 100 11†11-2 Proteobacterium (KP204144.1) 100 Psychromonas sp.( AB893933.1) 100 11-3 Uncultured Roseovarius sp. ( KJ513741.1) 100 12 Sphingopyxis sp. (HE716931.1) 100 +++ +++ +++ +++14 Vibrio rumoiensis (KJ616367.1) 97 – – – +15 Novosphingobium pentaromativorans (KP721487.1) 100+++ +++ +++ +++Sphingomonas aquatilis(KX110354.1) 100 13 Bacillus amyloliquefaciens (JQ775387.1) 100 + + + +++16 Rhodococcus sp. (KF715428.1) 98 – – + + 17 Curtobacterium sp. (KR094812.1) 100 +++ +++ +++ +++Proteobacteria Firmicutes Actinobacteria 18 Rhodococcus erythropolis (KM047507.1) 98 +++ +++ +++ +++

Fig.3 Phylogenetic tree derived from 16S rRNA gene sequences (about 200 bp) of predominant bands in DGGE gels. The tree was constructed with the neighbor-joining method of MEGA 4.0 program package. The number in each tree entry represented the band excised from DGGE gels. The number of 11-1, 11-2 and 11-3 represented the first, second and third clone of band 11, respectively.

4 Discussion

The present study showed that dietary indigenous B.subtilis 2-1 at 109 CFU g−1 could significantly improve the growth performance of sea cucumber. This growth-improving effect of B. subtilis 2-1 on sea cucumber was coordinated with previous studies which had proved that dietary Bacillus sp. such as indigenous B. subtilis T13(Zhao et al., 2012), indigenous B. cereus (Tian et al.,2015; Yang et al., 2015; Zhao et al., 2015) and commercial B. subtilis (Zhang et al., 2010) could improve the growth performance of sea cucumber at suitable doses.However, the precise mechanism by which Bacillus sp.increase the growth of sea cucumber is not clear. This study showed that dietary B. subtilis 2-1 at 109 CFU g−1 significantly increased the amylase and trypsin activities in sea cucumber. The higher amylase and trypin activities could promote the decomposition of carbohydrate and protein, which may lead to improving diet digestion and absorption. This may partially explain why the growth of sea cucumber was significantly increased by dietary B.subtilis 2-1. Moreover, some previous studies (Zhang et al.,2010; Zhao et al., 2012; Li et al., 2015; Yang et al., 2015;Tian et al., 2015; Zhao et al., 2015; Zhao et al., 2016)confirmed that dietary Bacillus sp. at suitable doses could increase the innate immunity and disease resistance of sea cucumber, which probably made sea cucumber more healthy thus gain much more weight. Therefore, it is necessary to conduct studies to elucidate the effect of B. subtilis 2-1 on the innate immunity and disease resistance of sea cucumber in future.

The activities of intestinal digestive enzymes including amylase and trypsin were significantly improved by dietary B. subtilis 2-1 as was observed in this study, which was in agreement with the results of Tian et al. (2015)and Zhao et al. (2015). They found that feeding B. cereus BC-01 or BC-02 could increase the intestinal amylase and trypin activities in sea cucumber. These improving effects of Bacillus sp. on the intestinal amylase and trypsin activities in sea cucumber may partly contribute to exogenous enzymes produced by Bacillus sp. Previous studies have indicated that Bacillus sp. could produce proteolytic enzymes, amylase and cellulase in moderate quantities(Mondal et al., 2010; Yang et al., 2013; Li et al., 2016).However, the previous studies (Yang et al., 2013; Ma et al.,2014) confirmed that probiotic Pseudoalteromonas sp.BC228 could not increase intestinal amylase activity in sea cucumber even though BC228 could secrete extracellular amylase, which suggested that the exogenous enzyme produced by the probiotics would contribute at best only a little to the total intestinal enzyme activities in sea cucumber (Ma et al., 2014). Probably, the dietary probiotics including B. subtilis 2-1 could stimulate sea cucumber to secrete more endogenous intestinal enzymes.Therefore, further studies should be conducted to understand how probiotics including B. subtilis 2-1 could increase the intestinal digestive enzymes in sea cucumber.

The PCR-DGGE and sequencing analysis showed that dietary B. subtilis 2-1 had important effect on the intestinal bacterial communities in sea cucumber, which was related to the dosage of B. subtilis 2-1. Compared with the control, dietary B. subtilis 2-1 at 105 and 107 CFU g−1 disturbed the dominant intestinal bacterial communities largely in at least two replicates each treatment, which caused that Proteobacteria including genus Vibrio was not the dominant intestinal bacteria. This disturbance was probably not good to sea cucumber because Proteobacteria members were the main metabolically active microbial populations in the intestine of A. japonicus (Enomoto et al.,2012). Insteadly, the effect of dietary B. subtilis 2-1 at 109 CFU g−1 on the dominant intestinal bacterial communities of sea cucumber were mild and consistent in three replicates. Dietary B. subtilis 2-1 at 109 CFU g−1 not only decreased the species of genus Vibrio, but also increased the intensities of genera Psychrobacter and Bacillus. Psychrobacter sp., which exhibited antagonistic activity against pathogenic Vibrio parahaemolyticus, Vibrio harveyi, Vibrio metschnikovi and Staphyloccocus aureus, was the dominant bacteria in the intestine of fast growing Epinephelus coioides (Sun et al., 2009; Yang et al., 2012).This may be the reason of improved growth of sea cucumber fed with B. subtilis 2-1 at 109 CFU g−1 in the present study. Moreover, it has been reported that Bacillus sp.could increase the growth, the digestive enzymes activities, the innate immunity and resistance against V. splendidus in sea cucumber (Zhao et al., 2012; Li et al., 2015;Tian et al., 2015; Yang et al., 2015; Zhao et al., 2015;Zhao et al., 2016). The findings in the present study suggested that B. subtilis 2-1 obviously changed the dominant intestinal microbiota of sea cucumber. However, the role that individual microbes play in the health and nutrition of sea cucumber is still poorly understood. Therefore,it is necessary to further investigate the intestinal microbiota in sea cucumber.

In conclusion, under the experimental conditions, dietary potential probiotic B. subtilis 2-1 at 109 CFU g−1 could significantly increase the growth and the activities of intestinal digestive enzymes including amylase and trypsin. The present study also confirmed that dietary B.subtilis 2-1 could modulate the dominant intestinal microbial community of sea cucumber, which was related to the dosage of B. subtilis 2-1. Dietary B. subtilis 2-1 at 105 and 107 CFU g−1 made the number of dominant species of intestinal microbiota in sea cucumber to be more less,although it decreased the abundance and species of genusVibrio. Dietary B. subtilis 2-1 at 109 CFU g−1 could modulate the dominant intestinal microbial community of sea cucumber by decreasing the abundance and species of genus Vibrio and increasing the abundance of genera Psychrobacter and Bacillus.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Nos. 31202004, 31402304),PhD Programs Foundation of Ludong University (No.LY2012009), and the Open Fund of the Key Laboratory of Marine Biotech of Guangxi (No. GLMBT-201205).

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