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In aquaculture, infectious diseases are the major cause of economic losses. Probiotic supplementation may change the microbiota of the digestive tract and modulate the immune defences and nutritional performance. This study was conducted to evaluate the dietary supplementation of multi-species (A: Bacillus sp., Pedicoccus sp., Enterococcus sp., Lactobacillus sp.) and single-species probiotics (B: Pediococcus acidilactici) on growth performance and gut microbiota of rainbow trout (Oncorhynchus mykiss). A basal diet (46% crude protein, 16% crude lipid, 21kJ g-1 gross energy) were supplemented with probiotic A or B, at two concentrations each (A1: 1.5 g kg-1 and A2: 3.0 g kg-1; B1: 0.1 g kg-1 and B2: 0.2 g kg-1). Diets were distributed to 30 groups (6 groups/treatment), of 20 fish each (16.4 0.5 g), 3 times a day until apparent satiety. The gut microbiota was analysed from ten fish per treatment at the end of the feeding trial (96 days) with 16S rDNA denaturing gradient gel electrophoresis (16S-DGGE). Changes in gut microbial community were assessed by Shannon index (H) and richness (R). After 56 days of the feeding trial, weight gain was significantly improved in fish fed diet A1 when compared to the control group (89.5 3.7 g versus 82.3 3.0 g). Dietary probiotic supplementation changed the gut microbial composition. Gut microbiota from fish fed diet A and diet B were separated in two robust clusters. R was higher in fish fed A1 (multi-species at lower concentration) than in control group, while H was higher in fish fed A1, B1 and B2. Our study showed that dietary probiotic treatment modulated gut microbiota of rainbow trout and in one of the treatments growth performance was significantly improved.
In intensive fish production, large quantities of disinfectants and drugs are released into the rearing water, which may affect the integrity of the gastrointestinal microbiota , reducing growth and immune competence . To date, antibiotics have been the most employed agents in the treatment of fish diseases; however, the environmental and food contamination resulting from its use, as well as the development of pathogens, increased antimicrobial resistance [3-6]. Vaccination can significantly reduce the incidence of many important diseases, although immunity is generally specific against a particular pathogen. In addition, several organisms may not respond to vaccination and new diseases or pathogen variants are constantly evolving .
In the past decade, the reduction of chemical and drug use in aquaculture to obtain more ecological and acceptable fish to the consumer has been the target . Nutritional and management approaches to disease prevention and health enhancement have been under research and development . Microbial manipulations of the microbiota present in the fish gastrointestinal tract and live feed, is one way to reduce the incidence of opportunistic pathogens [10, 11]. Many studies have pointed out that probiotics in fish diet and rearing water improved the resistance to colonization by pathogenic bacteria [12-14], and improved growth performance and nutrient utilization [15, 16]. Probiotics are live microorganisms that confer health benefits to the host, when administered in adequate amounts . The enhancement of animal health status can be a result from changes in the mucosal microbiota [10, 18] through competitive exclusion [19, 20], competition for nutrients , production of antimicrobial substances [22-24], and/or immunomodulatory effects [25-27]. Moreover, probiotic supplementation may provide vitamins, short chain fatty acids and/or digestive enzymes, and therefore may also contribute to host nutrition [28-30].
A large range of microorganisms are already commercially available worldwide as probiotics for use in aquaculture, including single species and multi species products . So far only one probiotic was authorised for use in aquaculture in the European Union, namely Pediococcus acidilactici CNCM MA18/5M, a member of the lactic acid bacteria . Probiotic efficacy is associated with presence or multiplication of the probiotics in the environment and/or host . Potential colonization and replication within the host is considered an important probiotic property [18, 34] and studies of the composition of the dominant microbiota are an essential part in probiotic fish research . The application of culture-independent molecular techniques is growing to evaluate the microbial community in fish gut as a result of probiotic supplementation . The methods rely on the 16S rRNA gene, using primers to determine dominant populations from amplified DNA product, followed by sequencing .
The present study was designed to evaluate the influence of dietary probiotic supplementation in gut microbiota profile of rainbow trout (Oncorhynchus mykiss), a salmonid species with economic importance worldwide . Two commercial probiotics were tested in two doses, and fish intestine were analysed by denaturing gradient gel electrophoresis (DGGE), using the 16S rDNA sequences.
Material and Methods
Fish and husbandry conditions
Six hundred rainbow trout juveniles (initial body weight: 16.4 0.5 g each) were evenly distributed into 30 square fibre glass tanks with 20 fish per tank. Fish were reared in a semi-closed recirculating freshwater system, stocked at an initial density of 6.6 kg/m3 at a constant water temperature of 17 C. All tanks were cleaned every two to three days with a 30% water renewal.
Diet preparation and feeding trial
A commercial diet (46% protein, 16% lipid, 21kJ g-1 gross energy; dry matter basis) was grounded and the defined quantities of the lyophilized probiotic commercial preparations A (1x109 CFU g-1 of dry powder) and B (1x1010 CFU g-1 of dry powder) were added to the basal diet (Table 1). After homogenization, the mixture was pelleted (2.4mm diameter) with 10% water incorporation, dried in a ventilation oven (48h, at 40C) and maintained at 4C in vacuum bags. During the trial, diets were had-fed to apparent satiety for 96 days.
Experimental design, sampling and growth performance
After 21 days adaptation to experimental rearing conditions, each of the five experimental diets was randomly attributed to the six tanks. Thereafter, fish growth was monitored for 56 days. The fish were bulk weighed at the beginning of the experiment and after 28 and 56 days of probiotic feeding. Weight gain was calculated as the difference between final and initial biomass. Specific growth rate (SGR) was calculated as [(ln Final weight) - (ln Initial weight)/days] ? 100. Diets with probiotic supplements were fed for a total of 96 days. At day 96, 10 fish per diet were sacrificed with anaesthetics overdose (ethylene glycol monophenyl ether, 1mL L-1) after a 12h fasting period. The whole gut was aseptically excised, immediately kept in liquid N and maintained at -80C until DNA extraction procedures.
DNA extraction from trout intestinal tissue
Total genomic DNA was extracted from 300 mg intestinal tissue (hind gut) as described in Griffith et al . In brief, tissue was aseptically transferred into bead beating tubes containing a 1:1 mixture of glass beads and ceramic beads with a diameter of 0.5 mm and 1.4 mm, respectively (PEQlab). To isolate DNA, samples were incubated with 0.5 ml hexadecyltrimethylammonium bromide (CTAB) buffer and 0.1 ml lysozyme (125mg ml-1) for 2 hours at 37C. Then, 0.5 ml phenol-chloroform-isoamylalcohol (25:24:1; pH8.0; Sigma Aldrich) were added and the samples were lysed for 30s in a bead beater (Precellys PEQlabs) with a speed setting of 5.5 ms-1. The aqueous phase was separated by centrifugation (16.000xg) for 10 min at 4C and treated with 40l proteinaseK (25mg ml-1) and 10l RNaseA (10mg ml-1) for 30 min at 37C. Phenol remnants were removed by centrifugation with a corresponding volume of chloroform: isoamylalcohol (24:1). DNA was precipitated from the aqueous phase with 0.6 vol isopropanol and 0.1 vol 3M sodium acetate (pH5.2) for two hours at room temperature and subsequent centrifugation (14.000xg) for 30 min at 4C. The DNA pellet was washed twice with 70% ethanol and dried prior to re-suspension in 60l bi-distilled water. Quality and quantity of the extracted nucleic acids was determined by gel electrophoresis and concentration measurements with the Nanodrop photometer. DNA from two to four fish per dietary group was pooled to counteract individual variations within a dietary group.
Amplification of bacterial 16S rDNA V3 regions
The variable region V3 of the 16S rDNA was amplified with KAPA 2G Robust polymerase (PEQlab) and universal primers 518r and 341f-GC . The PCR reaction (50l) contained 0.1 mM of each deoxynucleotide, 125 nM of each primer, 1x buffer B, 100 ng genomic DNA and, 0.02 U KAPA 2G polymerase. Enhancer 1 was added to improve reaction efficiency and specificity according to the manufacturers instructions. The PCR program started with an initial denaturing step of 95 C for 5 min, followed by 29 cycles of 95 C for 30 s, 56 C for 20 s and 72 C for 40 s and final elongation at 72 C for 7 min.
Denaturing gradient gel electrophoresis (DGGE) and cluster analysis
DGGE was performed with the INGENYphorU system (Goes, The Netherlands). PCR products were separated for 16 hours at 60C in a 30% - 60% gradient, 8% (w/v) polyacrylamide gel containing 32% formamide and 5.6M urea. The gel was stained with Sybr Green I Nucleic Acid Gel Stain (Sigma Aldrich) diluted 1:10.000 in 0.5x TAE buffer after electrophoresis. DGGE fingerprints (gel picture) were documented with Bio-Vision system and Vision-cap software (PeqLab) and assessed with the Gel compare II software version 6.0 (Applied Maths NV, Sint-Martens-Latem, Belgium). The gels were normalized to a reference sample that consists of 16S rDNA V3 fragments amplified from genomic DNA of various bacteria with different G/C contents. The Pearsons correlation describes the association between two samples and was thus used as a similarity matrix. The Unweight Pair Group Method with Arithmetic Mean (UPGMA), a hierarchical method, was applied to create a dendogram. Cophenetic correlation was included to measure the reliability of the dendogram.
Statistical analyses were carried out using STATISTICA program (StatSoft, Inc., 2008, version 8). Data was submitted to Kolmogorov-Smirnov and Levene tests, to verify normal data distribution and homogeneity of variances, respectively. Then data were submitted to a one-way Anova. Post-hot Tuckey test was used when anova showed significance, to determine significant differences between means. Changes in diversity of the microbial community were assessed with Shannon index (H) and richness (R). The Shannon index H was calculated with the formula H= - ?_(i=1)^S?Pi*log??(Pi)? ?, where S is the number of bands and Pi the proportion of a species in the sample. Pi was calculated by dividing the height of a peak with the all peaks in the sample (Pi= ni/N ). The richness was calculated as the sum of all bands in a sample. Statistical analysis of H and R was performed in Origin 6.1 with a t-test. Significant differences were considered when P < 0.05. Results are presented as mean standard deviation.
Results and Discussion
In this study, two commercial probiotics were added to fish diet at two concentrations. The multi-species probiotic (A) consists of strains belonging to the genera: Bacillus sp., Pedicoccus sp., Enterococcus sp., Lactobacillus sp. The single-species probiotic (B), consists of the lactic acid bacterium P. acidilactici (Table 1).
Growth performance was monitored for 56 days after feeding diets with probioticsAfter 28 days, no significant differences in weight gain and growth rate were observed, regardless of the diet. After 56 days of feeding the different diets, only fish fed with diet A1 gained significantly more weight compared to the control fish (P < 0.05). Weight gain of fish fed with diet A2, B1 or B2 did not differ from the control group (Figure 1). Similar to weight gain, the specific growth rate of fish fed with diet A1 was significantly better than fish fed with the control diet (Figure 2). These results indicate that diet A1, containing the multi-species probiotic at 1.5 g kg-1 positively contributes to growth performance. The positive effect may dependent on the dose, since growth performance of fish fed with diet A2 (multi-species probiotic at 3 g kg-1 diet) did not differ from fish fed with control diet.
The single-species probiotic, P. acidilactici (diet B1 and B2) did not affect weight gain or specific growth rate. In previous studies, P. acidilactici did not affect weight gain of juvenile rainbow trout fed for ten weeks  and for five months .
Other commercial probiotics that contain P. acidilactici or E. faecium did not improve growth performance of young channel catfish. In fact, fish fed for 56 days with a combination of P. acidilactici and E. faecium at a 106 CFU/g diet, showed decreased weight gain compared to unsupplemented (control) group and to fish fed with either P. acidilactici or E. faecium .
Similarly, addition of E. faecium to the diet of Nile-tilapia larvae did not affect weight gain of the larvae, but addition of P. acidilactici, to the diet significantly decreased weight gain of the larvae .
In contrast to the studies above, significant improvement were observed on weight gain in rainbow trout juveniles fed for ten weeks with Bacillus licheniformis or Bacillus subtilis (BioPlus2B ?) or one of them in combination with E. faecium [16, 45]. Also, addition of B. subtilis to the diet improved weight gain in first-feeding trout larvae .
In tilapia larvae, addition of B. licheniformis and B. subtilis at106 CFU g-1 per diet for 39 days did not affect final weight. Interestingly at 108 CFU g-1 per diet for 56 days weight gain was reduced .
In juvenile catfish, the addition of B. licheniformis and B. subtilis or Lactobacillus spp. to the diet had no effect on weight gain .
Our study is in agreement with previous findings, which indicate the potential use of dietary probiotic supplements as growth promoters and evidenced the importance of probiotic incorporation level to fish diet.
DGGE analysis of intestinal microbiota
The variable region V3 of the 16S rDNA was amplified from genomic DNA extracted from intestinal content and tissue of the hindgut and then PCR products were separated on a DGGE (Fig 3).
All samples contained three to four dominant species and several minor species in their DGGE fingerprints (Figure 3, on the right). Changes in the species composition of the intestinal microbiota were visualized with the dendogram (Figure 3, left side). The dendogram consists of two clusters. Cluster one consists of samples from fish fed with control diet, diet B1, or diet B2, while cluster two contains all samples from fish fed with diet A1 or A2 (multi-species probiotics) as well as samples from fish fed with diet B2, (single-species probiotic), and some control samples. Within cluster two, the high dose of the multi-species probiotic is well separated from the low dose of the multi-species probiotic. The first makes up sub cluster 2b, whereas the latter makes up sub cluster 2a. Consequently, not only the composition of the probiotic, but also the dosage does influence the intestinal micro flora of rainbow trout. Separation of clusters is robust, as indicated by cophenetic correlation values at the nodes, which range from 84 through 100.
To determine whether dietary probiotic supplementation may alter the number of species in the gut, the richness R was calculated based on the DGGE finger prints. For each sample, the number of bands should reflect the number of detectable species (Figure 4). In the current study, only fish fed diet A1 showed an increased richness.
Not only richness, but also diversity is important for intestinal microbiota. Thus, Shannon index H was calculated, as a diversity index based on the presence and relative microorganisms abundance (Figure 5). Three out of four diets with dietary probiotic supplementation had an increased species diversity. However, samples from fish fed with A1 diet showed a a higher increase in diversity from 2.8 (control) to 3.7 (A1) compared to the other dietary groups. Similar to richness (Figure 4), these data suggest that the multi-species probiotic at a dose of 1.5 g kg-1 significantly affected the microbiota in the hindgut.
The presence of a probiotic in the diet likely affects the gut microbiota of the fish and can help to improve growth performance [10, 18]. Here, we evaluated the effects of a multi-species probiotic and a single-species probiotic on growth performance and intestinal microbiota, when these probiotics were added to the diet at two concentrations. The multi-species probiotic that contains Bacillus sp., Pedicoccus sp., Enterococcus sp., Lactobacillus sp., improved weight gain and specific growth rate in juvenile rainbow trout. Interestingly, not only the composition of the probiotic, but also the concentration of the probiotic was important. When fish were fed 3 g kg-1 multi-species probiotic (diet A2), changes were less apparent in specific growth rate and diverstiy of the microbiota, when compared to fish fed 1.5 g kg-1 multi-species probiotic (diet A1).
Even though, A2 and B1 diets could increase diversity of the microbiota in the intestine compared to the control (Figure 5), these changes were not translated into increased weight gain or specific growth rate. Thus, changes in the diversity or richness of the gut microbiota may not per se reflect improvements in fish performance. It is important to analyse, growth performance data and gut microbiota to determine the action of a given probiotic product.
Our study showed that probiotic A supplemented at 1.5 g kg-1 diet modulated gut microbiota and significantly improved growth performance of juvenile rainbow trout.